WO2017068226A1 - Method for the identification, isolation and/or selection of urethral tissue - Google Patents

Method for the identification, isolation and/or selection of urethral tissue Download PDF

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Publication number
WO2017068226A1
WO2017068226A1 PCT/ES2016/070752 ES2016070752W WO2017068226A1 WO 2017068226 A1 WO2017068226 A1 WO 2017068226A1 ES 2016070752 W ES2016070752 W ES 2016070752W WO 2017068226 A1 WO2017068226 A1 WO 2017068226A1
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WIPO (PCT)
Prior art keywords
tissue
cell
cells
urethra
expression
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PCT/ES2016/070752
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Spanish (es)
French (fr)
Inventor
Bernardo HERRERA IMBRODA
Francisco Javier MACHUCA SANTA CRUZ
María Isabel HIERRO MARTÍN
María Fernanda LARA CABANÁS
Martina ÁLVAREZ PÉREZ
Miguel Alaminos Mingorance
Antonio CAMPOS MUÑOZ
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Universidad De Granada
Servicio Andaluz De Salud
Universidad De Málaga
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Publication of WO2017068226A1 publication Critical patent/WO2017068226A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals

Definitions

  • the present invention is within the field of biomedicine, and more specifically, within tissue engineering and histology. Specifically, it refers to a method for selecting a cell, a cell culture or a tissue and the use of this biological material selected by said method to increase, restore or partially or totally replace the functional activity of a damaged tissue.
  • the urethra is a tubular structure that runs from the bladder neck to the urinary meatus.
  • the male urethra has a length of approximately 18-20 cm and is divided into an anterior portion (penile and bulbar) and a posterior portion (membranous and prosthetic).
  • the female urethra is only approximately 4 cm and is divided into proximal (near the bladder), middle and distal (near the meatus) (Noah S Schenkman, MAP, Department of Urology, University of Virginia School of Medicine, Female Urethra Anatomy, Medscape; Arthur S Schenkman, MAP, Department of Urology, University of Virginia School of Medicine, Male Urethra Anatomy Medscape; del Pozo-Jimenez, G, et al., 2014. Arch. Esp. Urol., 67 (1): p. 5-1 1).
  • Urethral bioengineering would offer the advantage of generating functional autologous tissue that reproduces all the characteristics of a native urethra, since "the urethra itself will be the best substitute"; however, it is not known today what the correct histological and functional composition of the artificial tissue to generate to maximize the clinical outcome should be; Therefore, a correct characterization of the native human urethra is essential from a clinical point of view and as a quality control for the generation of artificial substitutes.
  • the invention aims to contribute to a better knowledge of the adult urethra with a broad characterization of the male and female urethra using histological, histochemical and immunohistochemical methods. The results could be useful for a more suitable selection of replacement tissues for urethral replacement.
  • the male urethra is composed of three layers of cells: epithelium, muscular layer consisting mainly of smooth muscle cells (SMC) and spongy tissue (from Pozo-Jimenez, et al., 2014. Arch. Esp. Uroi, 67 (1) : p. 5-1 1).
  • the epithelium changes along the length of the urethra. Studies conducted so far suggest that the lining epithelium of the male urethra varies between different regions of the urethra, with more distal areas showing a stratified squamous epithelium consisting of numerous layers of cells, and the most proximal areas are more similar to the bladder epithelium.
  • a first aspect of the invention relates to a method of identification, isolation and / or selection of cell (s), cell culture (s) or tissue (s), hereinafter method of the invention, useful ( es) to increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue, which comprises: a) identifying the cytoplasmic and / or nuclear expression of the markers CK34BE12, CKCAM 5.2, CKAE1 / AE3, CK5 / 6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrine, Uroplachin III and / or Ki67, in basal and suprabasal cells in said cell culture (s) or tissue (s); and b) select the cell (s), the cell culture (s) or the tissue (s) of step a) to increase, restore or partially or totally replace the functional activity of the urethra male prostate if it has a profile that consists of: i) cytoplasmic
  • the identification of the cytoplasmic and / or nuclear expression of the markers of step a) is performed by immunostaining.
  • the cell (s), the cell culture (s) or (a) tissue (s) identified in step (a) come from from the list consisting of: prepucial mucosa, genital and extragenital skin, oral mucosa, lingual mucosa, submucosa of the small intestine, bladder mucosa or any combination thereof.
  • the cell (s), the cell culture (s) or (a) tissue (s) identified in step (a) come from of donors who have suffered cardiac arrest or brain or brain death.
  • a second aspect of the invention relates to the use of the cell (s), the cell culture (s) or (a) tissue (s) having at least one profile identified by the method of The invention, in medicine.
  • a third aspect of the invention relates to the use of the cell (s), the cell culture (s) or (a) tissue (s) according to the second aspect of the invention, for the preparation of a medicament to increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue.
  • the different portions of urethral tissue are selected from the list consisting of: male prostatic urethral tissue, tissue male membranous urethral, male bulbar urethral tissue, male penile urethral tissue and female urethral tissue
  • the urethra is diseased or damaged as a result of a dysfunction, an injury or a disease selected from the list comprising: a benign or malignant neoplasm, an infection, a trauma, a congenital malformation or a stricture.
  • a fourth aspect of the invention relates to the use of the cell (s), the cell culture (s) or (a) tissue (s) having at least one profile identified by the method of the invention, for the evaluation of a pharmacological and / or chemical product.
  • a fifth aspect of the invention relates to a kit or device, hereafter kit or device of the invention, comprising the elements necessary to identify the cytoplasmic and / or nuclear expression of the markers CK34BE12, CKCAM 5.2, CKAE1 / AE3 , CK5 / 6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrine, Uroplachin III and / or Ki67.
  • the kit or device of the invention further comprises the elements necessary to perform the identification of the markers by immunostaining.
  • it comprises anti-CK34BE12, anti-CKCAM 5.2, anti-CKAE1 / AE3, anti-CK5 / 6, anti-CK7, anti-CKI O, anti-CK13, anti- CK14, anti-CK17, anti-CK18, anti-CK19, anti-lnvolucrine, anti-Uroplaquina III and / or anti-K ⁇ 67.
  • the antibodies are modified.
  • a sixth aspect of the invention relates to the use of the kit or device of the invention for the identification, isolation and / or selection of cell (s), cell culture (s) or tissue (s) useful for increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue.
  • a seventh aspect of the invention relates to a composition, hereinafter composition of the invention, comprising the cell (s), the cell culture (s) or the tissue (s) ) identified (s), isolated (s) or selected (s) by the method of the invention.
  • the composition of the invention is a pharmaceutical composition.
  • composition of the invention further comprises a pharmaceutically acceptable carrier. In another preferred embodiment of this aspect of the invention, the composition of the invention further comprises another active ingredient.
  • FIG. 1A Histological analysis of the human urethra. H&E staining of the sections corresponding to the male prostate urethra (i), membranous (ii), bulbar (iii) showing the differences in the number of cell layers. Scale bar: 100 ⁇ .
  • Figure 1 B Histological analysis of the human urethra. H&E staining of the sections corresponding to the male urethra of the penis (iv) and the female urethra (v) showing the differences in the number of cell layers. Scale bar: 100 ⁇ .
  • FIG. 2A Histochemical analysis of the connective and muscular layers of human urethra, (a) Masson's trichrome method was used in sections corresponding to the male and female urethra, (b) PAS staining shows glycoprotein content (c) Immunohistochemical staining against actin The stained sections correspond to the male urethra of prostate (i), membranous (ii), bulbar (iii). Scale bar: 100 ⁇ .
  • Figure 2B Histochemical analysis of the connective and muscular layers of human urethra, (a) Masson's trichrome method was used in sections corresponding to the male and female urethra, (b) PAS staining shows glycoprotein content (c) Immunohistochemical staining against actin The stained sections correspond to the male urethra of the penis (iv) and the female urethra (v). Scale bar: 100 ⁇ .
  • FIG. 3A Immunohistochemical analysis of cytokeratins of the epithelial layer of the human urethra.
  • the immunodetection of a cytokeratin battery was performed in tissue sections of the male urethral prostate (i), membranous (ii), bulbar (iii) and penis (iv) and female urethra.
  • Cytokeratin complex broad-spectrum antibodies against CK34BE12 (a), CKCAM 5.2 (b) and AE1 / 3 (c) showed cytoplasmic staining in all layers.
  • Specific antibodies against cytokeratin CK7 (d) showed specific spatial expression along the length of the urethra and the differences between the male and female urethra.
  • FIG. 1 Figure 3B Immunohistochemical analysis of cytokeratins of the epithelial layer of the human urethra. Specific antibodies against cytokeratin CK18 (e), CK5 / 6 (f), CK13 (g), CK14 (h), showed specific spatial expression along the length of the urethra and the differences between the male and female urethra. Scale bar: 100 ⁇ .
  • FIGS. 4A and 4B Immunohistochemical analysis of epithelial, urothelial and proliferation differentiation markers, (a) The analysis of the implcrine epithelial differentiation marker showed weak positive expression in the upper cell layers of the male and female urethra, (b) Staining of uroplachin III shows staining Slight positive in the nucleus of cells dispersed in all layers of the male and female urethra, (c) Staining for the Ki67 proliferation marker in the nucleus of the most basal layers of all regions of the male and female urethra. Scale bar: 100 ⁇ .
  • the authors of the present invention have studied the importance of cytokeratins, implicacrine, uroplachin III and Ki67 in the identification of cell (s), cell culture (s) or useful tissue (s) for increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue.
  • a first aspect of the invention relates to a method of identification, isolation and / or selection of cell (s), cell culture (s) or tissue (s), hereinafter method of the invention, useful ( es) to increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue, which comprises: a) identifying the cytoplasmic and / or nuclear expression of the markers CK34BE12, CKCAM 5.2, CKAE1 / AE3, CK5 / 6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrine, Uroplachin III and / or Ki67, in basal and suprabasal cells in said cell culture (s) or tissue (s); Y b) select the cell (s), the cell culture (s) or the tissue (s) of step (a) to increase, restore or partially or totally replace the functional activity of the Male prostatic urethra if it has a profile consisting of: i) cytoplasm
  • the identification of the cytoplasmic and / or nuclear expression of the markers of step a) is performed by immunostaining.
  • Immunostaining is understood herein as an antibody-based method for detecting a specific protein in a sample. Immunostaining encompasses a wide range of techniques that are used in histology, cell biology, and molecular biology that use antibody-based staining methods.
  • the cells identified by the method of the invention can be analyzed by flow cytometry.
  • the cell (s), the cell culture (s) or (a) tissue (s) identified in step (a) come from from the list consisting of: prepucial mucosa, genital and extragenital skin, oral mucosa, lingual mucosa, submucosa of the small intestine, bladder mucosa or any combination thereof.
  • the cell (s), the cell culture (s) or (a) tissue (s) identified in step (a) come from of donors who have suffered cardiac arrest or brain or brain death.
  • the "urethra” is the conduit through which urine circulates in the final phase of the urinary process. Specifically, it is the conduit that connects the neck of the urinary bladder to the outside of the body during urination. There are differences in the urethra depending on sex:
  • the male urethra has different portions that are:
  • Prosthetic urethra It runs through the prosthetic gland, where the ejaculatory ducts shed its contents.
  • Membranous urethra it is the portion distal to the prosthetic one, or two cm, and in this location is the external sphincter or rhabdosphincter, striated musculature, formed by muscle fibers that run from the pelvic floor musculature.
  • Penile or spongy urethra It is called that because it is inside the spongy body of the penis, an erectile sheath that runs along the entire ventral side of the penis. It reaches the glans and opens at the meatus. It has a length of about 9-11 centimeters.
  • the bulbar urethra can also be seen: which is the portion between the membranous (proximal) and the penile (distal) urethra and where the thickness of the spongy is the largest.
  • the female urethra is short and flows into the vulva between the clitoris and the vaginal introite. The short size of the female urethra explains the increased susceptibility of urinary infections in women.
  • basic cell is understood, but not limited to, the cell located at the base of the urethral epithelium.
  • suprabasal cell is understood, but not limited to, the cells located in the apical or luminal area of the urethral epithelium.
  • cell culture refers, but not limited to, in vitro cell cultures consisting of a system formed by cells from an organ or tissue, normal or tumor, maintained in culture media of defined chemical composition and under controlled temperature, pH, aeration and humidity conditions. In this way they ensure their survival and multiplication, maintaining all their metabolic functions in a manner similar to those they had in the host.
  • This invention is not limited to any culture mode and contemplates the different variants as to the way in which the culture that can be carried out, for example the term “additional culture” refers to culturing a cell or tissue up to a certain phase of growth to then use another culture method to make said cell or tissue reach another stage of growth.
  • a “monolayer culture” refers to a culture in which cells multiply in a suitable medium while remaining attached to each other and to a substrate.
  • a "suspension culture” refers to a culture in which cells multiply, in suspension in a suitable medium.
  • a “continuous flow culture” refers to the culture of cells or explants in a continuous flow of fresh medium to maintain cell growth, for example, viability.
  • conditioned media refers to the supernatant, for example, free of cell / tissue cultures, resulting after a period of time in contact with cultured cells such that the media has been modified to include certain paracrine factors. and / or autocrine produced by the cells and secreted in the culture.
  • a "confluent culture” is a culture of cells in which all cells are in contact and therefore the entire surface of the culture vessel is covered, and implies that the cells have reached their maximum density, although confluence does not necessarily mean that the cell division ceases or the population stops increasing in size.
  • tissue refers to those structures constituted by an organized set of cells and distributed regularly, with a coordinated physiological behavior.
  • immunohistochemistry means the histopathological procedure that is based on the use of antibodies that specifically bind to a substance to be identified (primary antibody). These antibodies may have an enzyme bound or it may be bound to a secondary antibody that recognizes and binds to the primary. Applied to an organic tissue, the primary antibody specifically binds to the substrate and enzymatic activity is used to visualize binding. In this way a substrate-antibody-enzyme complex is attached to the place where the substrate is located and by activating the enzyme with the addition of its substrate an identifiable product is generated where the complex is located.
  • This technique allows to identify the location of a specific substance allowing to identify its tissue or cytological location, in this way you can identify the characteristic antigen markers of a cell line, identify cells that secrete a protein, membrane receptors, tissue concentration gradients or cells that have responded to a hormone (with antibodies specific for intracellular signaling pathways).
  • cytokeratins or “tonofibrils” means those fibrous proteins that form the intermediate filaments of the intracellular cytoskeleton in particular of epithelial cells (including mucous membranes and glands), as well as in the nails, hair and feathers of the animals.
  • Cytokeratin monomers are linked to each other by phylagrin molecules. Its main function is the organization of the internal three-dimensional structure of the cell (for example, they are part of the nuclear envelope and the sarcomeres), they form a rigid barrier which prevents the entry of microorganisms. That same barrier fulfills the vital function of retaining water within the cells. They also participate in some intercellular junctions (desmosomes). Certain irregularities in the expression of cytokeratins are the basis of some atypical conditions, such as eczema, ichthyosis, psoriasis and certain types of metastatic cancer.
  • Cytokeratins There are two types of cytokeratins: the basic type I cytokeratins and the type II cytokeratins of acidic or neutral. Cytokeratins are generally found in pairs that comprise a type I cytokeratin and another type II cytokeratin. Basic or neutral cytokeratins include CK1, CK2, CK3, CK4, CK5, CK6, CK7 and CK8. The acid cytokeratins are CK9, CK10, CK12, CK13, CK14, CK16, CK17, CK18, CK19 and CK20. Cytokeratins can be divided according to whether their molecular weight is high or low.
  • CK7 is normally expressed in the ductal epithelium of the genitourinary tract (GU) and CK20 more frequently in the gastrointestinal tract (Gl).
  • cytokeratins that express epithelial cells depend mainly on the type of epithelium, the time in the course of terminal differentiation and the stage of development. In this way, the specific cytokeratin fingerprint allows the classification of all epithelia on its cytokeratin expression profile.
  • the study of the cytokeratin profile by immunohistochemical techniques is a tool of immense value used for the diagnosis of tumors and characterization of surgical pathology.
  • Cytokeratins are encoded by a family that encompasses 30 genes. Among them, 20 are epithelial genes and the remaining 10 are specific for tricocysts.
  • All cytokeratin chains are composed of a central domain rich in a-helices (with a sequence identity of 50-90% among cytokeratins of the same type and about 30% between cytokeratins of different types) with N- terminal domains and C-non-helical.
  • the ⁇ -helical domain has 310-150 amino acids and comprises four segments in which a pattern of seven residues is repeated. In this repeated pattern, the first and fourth residues are hydrophobic and the charged residues show positive and negative polarity alternately, as a result the polar residues are located on one side of the helix.
  • This central domain of the chain provides molecular alignment in the keratin structure and causes the chains to form spiral dimers in solution.
  • the end-of-domain sequences of type I and II contain cytokeratin chains on both sides of the rod of the subdomains of domain V1 and V2, which have sequence and variable size.
  • Type II also has conserved H1 and H2 subdomains, covering 36 and 20 residues, respectively.
  • Subdomains V1 and V2 contain residues enriched by glycine and / or serine, the former providing the cytokeratin chain with a strong insoluble character and facilitating interaction with other molecules.
  • These terminal domains are also important in defining the function of the cytokeratin chain characteristic of a particular type of epithelial cell. Groups of two cytokeratin dimers form a keratin tetramer by binding antiparallel construction.
  • This cytokeratin tetramer is considered to be the main building block of the cytokeratin chain.
  • the protofilaments originate, which at their Once they are intertwined in pairs to form protofibrils.
  • Four protofi shines give rise to a cytokeratin filament.
  • the keratin filaments form a complex network that extends from the surface of the nucleus to the cell membrane. Numerous accessory proteins are involved in the genesis and maintenance of said structure.
  • cytokeratin networks undergo rapid phosphate exchanges mediating depolymerization, with important implications in more dynamic cellular processes such as mitosis and post-mitotic period, cell movement and differentiation.
  • Cytokeratins generally interact with desmosomes and hemidesmosomes, thus contributing to cell-cell adhesion and basal connection of underlying connective tissue cells.
  • the intermediate filaments of the eukaryotic cytoskeleton, of which cytokeratins are one of its three components, have been probed to also associate with the protein network of the ankyrin complex and spectrin that underlies the cell membrane.
  • the cytokeratins used as markers in the method of the invention are: CK34BE12, CKCAM 5.2, CKAE1 / AE3, CK5 / 6, CK7 (NCBI Gene ID: 3855), CK10 (NCBI Gene ID: 3858), CK13 (NCBI Gene ID: 3860), CK14 (NCBI Gene ID: 3861), CK17 (NCBI Gene ID: 3872), CK18 (NCBI Gene ID: 3875) and / or CK19 (NCBI Gene ID: 3880).
  • the method of the invention also uses:
  • “Invite” (NCBI Gene ID: 3713) is a component of the cross-linked keratinocyte envelope, found in the cytoplasm and bound to membrane proteins by transglutaminase. The chromosomal location of this gene is 1q21.
  • “Uroplachin III” (NCBI Gene ID: 7380) belongs to a group of transmembrane proteins that form complexes on the apical surface of the bladder epithelium. Mutations in this gene may be associated with renal adisplasia. / ' -67 (NCBI Gene ID: 4288) is a gene encoding a nuclear protein that in an associated manner may be necessary for cell proliferation. Variants have been described in the alternatively spliced transcription. There is a pseudogen on the related X chromosome.
  • a second aspect of the invention relates to the use of the cell (s), the / the cell culture (s) or (a) tissue (s) having at least one profile identified by the method of the invention, in medicine.
  • a third aspect of the invention relates to the use of the cell (s), the cell culture (s) or (a) tissue (s) according to the second aspect of the invention, for the preparation of a medicament to increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue.
  • the different portions of urethral tissue are selected from the list consisting of: male prosthetic urethral tissue, male membranous urethral tissue, male bulbar urethral tissue, male penile urethral tissue and female urethral tissue
  • the urethra is diseased or damaged as a result of a dysfunction, an injury or a disease selected from the list comprising: a benign or malignant neoplasm, an infection, a trauma, a congenital malformation or a stricture.
  • a fourth aspect of the invention relates to the use of the cell (s), the cell culture (s) or (a) tissue (s) having at least one profile identified by the method of the invention, for the evaluation of a pharmacological and / or chemical product.
  • a fifth aspect of the invention relates to a kit or device, hereinafter kit or device of the invention, comprising the elements necessary to identify the cytoplasmic and / or nuclear expression of the markers CK34BE12, CKCAM 5.2, CKAE1 / AE3, CK5 / 6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrine, Uroplachin III and / or Ki67.
  • the kit or device of the invention further comprises the elements necessary to perform the identification of the markers by immunostaining.
  • it comprises anti-CK34BE12, anti-CKCAM 5.2, anti-CKAE1 / AE3, anti-CK5 / 6, anti-CK7, anti-CKI O, anti-CK13, anti- CK14, anti-CK17, anti-CK18, anti-CK19, anti-lnvolucrine, anti-Uroplaquina III and / or anti-K ⁇ 67.
  • the antibodies used in the invention are selected from the list consisting of: actin (Alpha-Sr-1), CK34BE12, CK19 (RCK108) from Master Diagnostic (Granada, Spain): CK 20 (Ks20.8), CAM 5.2 (clone CAM 5.2), CKAE1 / AE3 (clone Y85), cytokeratin 5 and 6 (D5 / 16B4), CK7 (OV-TL 12/30), CK10 (SP99), CK13 (KS-1A3), CK14 (LL002) , CK17 (E3), CK18 (DC-10), implicacrine (SY5), uroplachin III (SP73), Ki-67 (clone SP6).
  • the antibodies are modified.
  • the antibody is human, humanized or synthetic.
  • the antibody is monoclonal and / or is labeled with a fluorochrome.
  • the flurochrome is selected from the list comprising Fluorescein (FITC), Tetramethylrodamine and derivatives, Phycoerythrin (PE), PerCP, Cy5, Texas, allophycocyanin, or any combination thereof.
  • the kit may also contain, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc.
  • the kit or device of the invention can include all the supports and containers necessary for its implementation and optimization.
  • the kit further comprises instructions for carrying out the method of the invention.
  • the kit or device of the invention comprises oligonucleotides.
  • the oligonucleotides have modifications in some of their nucleotides, such as, but not limited to, nucleotides having any of their atoms with a radioactive isotope, usually 32 P or tritium, immunologically labeled nucleotides, such as with a molecule of digoxigenin, and / or immobilized in a membrane.
  • a sixth aspect of the invention relates to the use of the kit or device of the invention for the identification, isolation and / or selection of cell (s), cell culture (s) or tissue (s) useful for increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue.
  • a seventh aspect of the invention relates to a composition, hereinafter composition of the invention, comprising the cell (s), the cell culture (s) or the tissue (s) ) identified (s), isolated (s) or selected (s) by the method of the invention.
  • the composition of the invention is a pharmaceutical composition.
  • composition of the invention further comprises a pharmaceutically acceptable carrier.
  • composition of the invention further comprises another active ingredient.
  • pharmaceutically acceptable vehicle refers to a vehicle that must be approved by a federal government regulatory agency or a state government or listed in the United States Pharmacopoeia or European Pharmacopoeia, or other pharmacopoeia generally recognized for use in animals, and more specifically in humans.
  • vehicle refers to a diluent, adjuvant, excipient or carrier with which they should be administered to the cell culture (s) or tissue (s) of the invention or of said composition comprising cell culture (s) (s) or fabric (s) of the invention selected by the method of the invention; obviously, said vehicle must be compatible with said cells.
  • Illustrative, non-limiting examples of said vehicle include any physiologically compatible vehicle, for example, isotonic solutions (eg, sterile saline (0.9% NaCI), phosphate buffered saline (PBS), Ringer-lactate solution, etc.
  • cell culture media e.g., DMEM, etc.
  • a solid, semi-solid, gelatinous or viscous support medium such as collagen, collagen-glycosamino-glycan, fibrin, polyvinyl chloride, polyamino acids, such as polylysine, or polynithine, hydrogels, agarose, silicone dextran sulfate.
  • the support medium may, in specific embodiments, contain growth factors or other agents. If the support is solid, semi-solid, or gelatinous, the cells can be introduced into a liquid phase of the vehicle that is subsequently treated in such a way that it becomes a more solid phase.
  • the pharmaceutical composition of the invention may also contain, when necessary, additives to increase, control or otherwise direct the therapeutic effect.
  • desired of the cell culture (s) or tissue (s), which comprise said pharmaceutical composition and / or auxiliary substances or pharmaceutically acceptable substances, such as buffering agents, surfactants, co-solvents, preservatives, etc.
  • metal chelators to stabilize the cell suspension, it is possible to add metal chelators.
  • the stability of the cell culture (s) or tissue (s) in the liquid medium of the pharmaceutical composition of the invention can be improved by the addition of additional substances, such as, for example, aspartic acid, glutamic acid, etc. .
  • Such pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known to those skilled in the art and are normally used in the preparation of cellular compositions.
  • suitable pharmaceutical vehicles are described, for example, in "Remington's Pharmaceutical Sciences” by EW Martin. Additional information on these vehicles can be found in any pharmaceutical technology manual (Galenic Pharmacy).
  • the pharmaceutical composition of the invention will contain a therapeutically effective amount of the cell culture (s) or tissue (s) of the invention, to provide the desired therapeutic effect.
  • the term "therapeutically or prophylactically effective amount” refers to the amount of cell culture (s) or tissue (s) of the invention contained in the pharmaceutical composition that is capable of producing the desired therapeutic effect and, in general, will be determined, among other factors, by the characteristics of the cell culture (s) or tissue (s) of the invention and the desired therapeutic effect that is sought.
  • the therapeutically effective amount of cell culture (s) or tissue (s) of the invention to be administered will depend, among other factors, on the subject's own characteristics, the severity of the disease, the manner of administration. , etc.
  • the pharmaceutical composition of the invention will be formulated according to the chosen form of administration.
  • the pharmaceutical composition of the invention can be stored until the moment of its application by conventional procedures known to those skilled in the art.
  • This pharmaceutical composition can also be stored together with additional medications, useful in the treatment of diseases, in an active form comprising a combination therapy.
  • the pharmaceutical composition of the invention can be stored at or below room temperature in a sealed container complementing it or not with a nutrient solution.
  • Medium term storage (less than 48 hours) is done preferably at 2-8 ° C, and the pharmaceutical composition of the invention will include an iso-osmotic and buffered solution in a container composed of, or coated with, material that prevents cell adhesion.
  • Longer term storage is preferably carried out by means of adequate cryopreservation and storage under conditions that promote retention of cellular function.
  • the cells, cell culture or tissue of the invention can be genetically modified by any conventional method including, by way of illustration, not limitation, transgenesis processes, deletions or insertions in your genome that modify the expression of genes that are important for its basic properties (proliferation, migration, transdifferentiation, etc.).
  • microarray hereafter referred to as a microarray of the invention, comprising oligonucleotides or single channel microarrays designed from a known sequence or mRNA of any of the markers in step a).
  • oligonucleotide sequences can be constructed on the surface of a chip by sequential elongation of a growing chain with a single nucleotide using photolithography.
  • the oligonucleotides are anchored at the 3 'end by a method of selective activation of nucleotides, protected by a photolabile reagent, by the selective incidence of light through a photomask.
  • the photomask can be physical or virtual.
  • oligonucleotide probes can be between 10 and 100 nucleotides, more preferably, between 20 and 70 nucleotides, and even more preferably, between 24 and 30 nucleotides.
  • Synthesis in situ on a solid support could be done using ink-jet technology, which requires longer probes.
  • the supports could be, but not limited to, filters or membranes of NC or nylon (charged), silicon, or glass slides for microscopes covered with aminosilanes, polylysine, aldehydes or epoxy.
  • the probe is each of the chip samples.
  • the target is the sample to be analyzed: messenger RNA, total RNA, a PCR fragment, etc.
  • a protein microarray hereafter referred to as a protein microarray of the invention, comprising antibodies or fragments thereof specific against any of the markers of step a) or against amino acid sequences that present a degree of identity to the amino acid sequences of said markers of at least 85%, typically of at least 90%, preferably, at least 95%, more preferably, at least 98%, even more preferably, at least 99%.
  • the probes are antibodies fixed to glass slides and the targets are samples of cells, cell cultures and / or tissues.
  • Another aspect of the invention relates to the use of the microarray of the invention or the protein microarray of the invention, for the identification, isolation and / or selection of cell (s), cell culture (s) or tissue (s). ) useful (s) to increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue.
  • the microarray of the invention or the protein microarray of the invention are inserted into an automated microscopy and / or immunohistochemical device for reading.
  • Another aspect of the invention relates to a computer program comprising program instructions to make a computer carry out the method according to the method of the invention.
  • the invention encompasses computer programs arranged on or within a carrier.
  • the carrier can be any entity or device capable of supporting the program.
  • the carrier may be constituted by said cable or other device or means.
  • the carrier could be an integrated circuit in which the program is included, the integrated circuit being adapted to execute, or to be used in the execution of, the corresponding processes.
  • the programs could be incorporated into a storage medium, such as a ROM, a CD ROM or a semiconductor ROM, a USB memory, or a magnetic recording medium, for example, a floppy disk or a disk Lasted.
  • a storage medium such as a ROM, a CD ROM or a semiconductor ROM, a USB memory, or a magnetic recording medium, for example, a floppy disk or a disk Lasted.
  • the programs could be supported on a transmissible carrier signal.
  • it could be an electrical or optical signal that could be transported through an electrical or optical cable, by radio or by any other means.
  • the invention also extends to computer programs adapted so that any processing means can carry out the method of the invention.
  • Such programs may have the form of source code, object code, an intermediate source of code and object code, for example, as in partially compiled form, or in any other form suitable for use in the implementation of the processes according to the invention .
  • Computer programs also cover cloud applications based on that procedure.
  • Another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of causing a computer to carry out the steps of the methods of the invention.
  • Another aspect of the invention relates to a transmissible signal comprising program instructions capable of causing a computer to carry out the steps of the methods of the invention.
  • urethral tissue samples were obtained from cadaveric donors. This study has been approved by the Autonomous Coordination of Transplants, the Ethics Committee of the Hospital of Malaga and the Biobank of Andalusia. Informed consent was approved by donor relatives. To obtain urethra samples, cystoprostatectomy (men) or cystectomy (in women) with urethrectomy were performed. Once the piece was removed, the urethra was dissected in its anatomical regions, serial cross sections were made every cm and fixed with formaldehyde for histological and immunohistochemical analysis.
  • cytokeratin and proliferation markers were carried out in formalin-fixed urethra tissues (FFPE).
  • FFPE formalin-fixed urethra tissues
  • the 3 ⁇ tissue sections were incubated with a primary antibody and stained according to the EnVision TM FLEX standard, high pH (K8010, Dako, Agilent Tecnologies, Denmark) working procedures using the Dako Autostainer automatic immunotoner (Dako).
  • Immunohistochemical staining was performed with ready-to-use antibodies from Dako: actin (Alpha-Sr-1), CK34BE12, CK19 (RCK108), and from the Diagnostic Master (Granada, Spain): CAM 5.2 (CAM 5.2 clone), CKAE1 / AE3 (Y85 clone), cytokeratin types 5 and 6 (D5 / 16B4), CK7 (OV-TL 12/30), CK10 (SP99), CK13 (KS-1A3), CK14 (LL002), CK17 (E3), CK18 (DC-10), implicacrine (SY5), uroplaquine III (SP73), Ki-67 (SP6 clone).
  • Image analysis Microscopic images at 200X magnification were transformed to 8 bits with the Image J program (Schneider et al., 2012. Nat. Methods, 9 (7): p. 671-5) and the gray intensity was measured in a scale from 0 to 255 in black and white to quantify staining intensity.
  • Image J was also used to count nuclear staining and the total number of nuclei per layer in tissue sections stained with uroplachin III or Ki 67. Results
  • H&E histological analysis of the human urethra by staining with H&E
  • the proximal region (prosthetic and membranous) of the male urethra had few layers of epithelial cells (4.26 ⁇ 0.88), while the distal (bulbar and penis) urethra showed a greater number of cell layers (6.9 ⁇ 1 ) ( Figure 1; i-iv).
  • the female urethra has a thickness comparable to the male distal urethra (7.6 ⁇ 1.8 layers of cells) (Fig. 1).
  • the broad spectrum anti-cytokeratin antibodies CK34BE12 (which recognizes human cytokeratins 1, 5, 10 and 14), CKCAM5.2 (anti-CK7 and CK8) and CKAE1 / AE3 (mixture of pan-cytokeratin antibodies that reacts with 14 CKs from both families A and B) revealed strong cytoplasmic staining in all layers along the male urethra and also in the female urethra ( Figure 3, ac) with the only exception of some layer cells suprabasal of the prosthetic urethra that appeared negative for CK34BE12 (Fig. 3a).
  • CK7 and CK18 staining was generally observed in all layers in male prosthetic, membranous and urethra Bulbar epithelium ( Figure 3 of). Sporadic cells of the male prosthetic and membranous urethra showed no expression (Fig. 3 of). In the urethra of the penis, CK7 was observed in suprabasal layers but not in the first basal layer (Fig. 3d), and CK18 was expressed more intensely in suprabasal layers (Fig. 3e).
  • the female urethra showed very light staining, both for cytokeratins and only in sporadic cells of all layers (Fig. 3, DE).
  • stained CK5 / 6 antibodies were used to reveal intense cytoplasmic staining in all samples (Fig. 3F), although restricted to the basal layers in the prosthetic urethra (Fig. 3f).
  • Fig. 3f stained CK5 / 6 antibodies were used to reveal intense cytoplasmic staining in all samples
  • Fig. 3f A very similar profile was found for CK13 expression, which is located primarily in the basal cell layer of the prosthetic urethra and expanded to all layers once the urethra moves away from the prostate (Fig. 3g).
  • the female urethra had a similar staining profile for CK5 / 6 and CK13 of bulbar and urethra penis (Fig. 3f, g).
  • CK14 specific basal cell is expressed only by the basal cells of the bulbar and urethra of the penis, and also the female urethra, but not the prostate and the membranous urethra (Fig. 3h).
  • CK17 is expressed only by distinct basal layer cells in all areas of the male urethra and in the female urethra (Fig. 3i).
  • CK19 we found a strong cytoplasmic staining in all layers of the male prosthetic and membranous urethra (Fig.
  • the CKI O suprabasal layer marker was completely absent in the prostate and membranous urethra, but was intensely present in all suprabasal layers of the bulbar urethra and penis (Fig. 3k).
  • the female urethra showed only some positive cells dispersed in suprabasal layers (Fig. 3k).
  • the immunohistochemical analysis of the expression of cytokeratin in the human male (prosthetic, membranous, bulbar and penis) and of the female urethra revealed important differences between the different samples.
  • an epithelial differentiation marker showed slightly positive expression in the male suprabasal and female urethra cell layers, but not in the basal layers (Fig. 4a; Table 1-2).
  • a Umbrella urothelial cell marker mild positive staining was present in the nucleus of cells dispersed in all layers of the male and female urethra (Fig. 4b; Table 1-2).
  • Ki67 proliferation marker was present in the basal layers of all regions of the male urethra and in the female urethra, and the number of positive cells was higher in areas where the epithelium had a greater number of layers of cells (2 ⁇ 0.9%; 6 ⁇ 1%; 6 ⁇ 0.4%; 40 ⁇ 1% and 48 ⁇ 3% of Ki67 positive cells in the prostate, membranous, bulbar, penis and female urethra, respectively ) ( Figure 4c; Table 1-2).
  • Type of tissue sample male and female human urethra.
  • the intensity of staining was based on a gray scale from 0 to 255 using (black-white) the Image J program. Values are represented ⁇ the standard deviation.
  • uroplakin III the ratio of percentage of stained cells in the apical cell layers / the percentage of stained cells in basal layers was calculated in three different samples.
  • Ki67 the percentage of stained cells was calculated by counting staining cells and the total number of cells per optical area. Three different optical zones were used in three samples. Data are presented as mean ⁇ standard deviation in each case.

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Abstract

The invention relates to a method for the identification, isolation and/or selection of one or more cells, cell cultures or tissues that can be used to increase, restore or substitute, partially or fully, the functional activity of different portions of urethral tissue. The invention also relates to the use of said cell(s), cell culture(s) or tissue(s), to a kit or device, to the pharmaceutical composition, and uses of same.

Description

Método de identificación, aislamiento y/o selección de tejido uretral CAMPO DE LA INVENCIÓN  Method of identification, isolation and / or selection of urethral tissue FIELD OF THE INVENTION
La presente invención se encuentra dentro del campo de la biomedicina, y más específicamente, dentro de la ingeniería tisular y la histología. Específicamente, se refiere a un método para seleccionar una célula, un cultivo celular o un tejido y al uso de este material biológico seleccionado por dicho método para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de un tejido dañado. The present invention is within the field of biomedicine, and more specifically, within tissue engineering and histology. Specifically, it refers to a method for selecting a cell, a cell culture or a tissue and the use of this biological material selected by said method to increase, restore or partially or totally replace the functional activity of a damaged tissue.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
La uretra es una estructura tubular que discurre desde el cuello vesical hasta el meato urinario. La uretra masculina tiene una longitud de aproximadamente 18-20 cm y se divide en una porción anterior (peneana y bulbar) y una posterior (membranosa y prostética). La uretra femenina es sólo aproximadamente de 4 cm y se divide en proximal (cerca de la vejiga), media y distal (cerca del meato) (Noah S Schenkman, M.A.P., Departamento de Urología, Facultad de Medicina de la Universidad de Virginia, Female Urethra Anatomy. Medscape; Noah S Schenkman, M.A.P., Departamento de Urología, Facultad de Medicina de la Universidad de Virginia, Male Urethra Anatomy Medscape; del Pozo-Jimenez, G, et al., 2014. Arch. Esp. Urol., 67(1): p. 5-1 1). The urethra is a tubular structure that runs from the bladder neck to the urinary meatus. The male urethra has a length of approximately 18-20 cm and is divided into an anterior portion (penile and bulbar) and a posterior portion (membranous and prosthetic). The female urethra is only approximately 4 cm and is divided into proximal (near the bladder), middle and distal (near the meatus) (Noah S Schenkman, MAP, Department of Urology, University of Virginia School of Medicine, Female Urethra Anatomy, Medscape; Noah S Schenkman, MAP, Department of Urology, University of Virginia School of Medicine, Male Urethra Anatomy Medscape; del Pozo-Jimenez, G, et al., 2014. Arch. Esp. Urol., 67 (1): p. 5-1 1).
El tratamiento de las enfermedades que afectan a la uretra ocasionando estenosis complejas que requieren de tejido de sustitución continúan siendo un reto quirúrgico y un problema, en ocasiones, difícil de resolver. Las estrategias actuales se basan mayoritariamente en la utilización de tejido de sustitución a modo de colgajos pediculados (en uretra peneana) o injertos libres autólogos (peneanos, peno-bulbar y bulbar; McAninch, 2014. Urotrauma guidelines. J. Urol. 192(2): p. 336). Se han propuesto diversos tejidos tales como mucosa prepucial, la piel genital y extragenital, mucosa bucal, mucosa lingual, submucosa del intestino delgado y la mucosa de la vejiga para la reconstrucción uretral (Hampson et al., 2014. Nat. Rev. Urol. 11 (1): p. 43-50). Sin embargo existen varios problemas cuando se utiliza tejido de donante: 1) suministro de tejido del donante limitado, 2) problemas de morbilidad en el área de la toma de tejido y daños en el tejido a ser injertado, 3) Deterioro del tejido injertado a largo plazo, 4) integración y supervivencia del tejido donante en el lecho receptor (Mundy, 1995. Br. J. Urol. 75(1): p. 59-61 ; Dublin y Stewart, 2004. BJU Int. 94(6): p. 867-9; Barbagli et ai, 2006. Contemporary Urology 18(3): p. 25-33). Encontrar un tejido ideal susceptibles de ser utilizados en la uretra humana y el reciente desarrollo de sustitutos uretrales de ingeniería tisular requiere un profundo conocimiento de las características histológicas y moleculares de la uretra humana nativa. The treatment of diseases that affect the urethra causing complex strictures that require replacement tissue continue to be a surgical challenge and a problem, sometimes difficult to solve. Current strategies are mostly based on the use of replacement tissue as pedicle flaps (in penile urethra) or autologous free grafts (penis, peno-bulbar and bulbar; McAninch, 2014. Urotrauma guidelines. J. Urol. 192 (2 ): p. 336). Various tissues such as prepucial mucosa, genital and extragenital skin, buccal mucosa, lingual mucosa, submucosa of the small intestine and bladder mucosa have been proposed for urethral reconstruction (Hampson et al., 2014. Nat. Rev. Urol. 11 (1): p. 43-50). However, there are several problems when donor tissue is used: 1) limited donor tissue supply, 2) morbidity problems in the area of tissue collection and tissue damage to be grafted, 3) Graft tissue deterioration a long-term, 4) integration and survival of donor tissue in the recipient bed (Mundy, 1995. Br. J. Urol. 75 (1): p. 59-61; Dublin and Stewart, 2004. BJU Int. 94 (6) : p. 867-9; Barbagli et ai, 2006. Contemporary Urology 18 (3): p. 25-33). Find an ideal tissue that can be used in the human urethra and the Recent development of tissue engineering urethral substitutes requires a thorough knowledge of the histological and molecular characteristics of the native human urethra.
La bioingeniería uretral ofrecería la ventaja de generar tejido autólogo funcional que reproduzca todas las características de una uretra nativa, ya "que el mejor sustituto de la uretra será la propia uretra"; sin embargo, a día de hoy no se conoce cuál debe ser la correcta composición histológica y funcional del tejido artificial a generar para maximizar el resultado clínico; por ello, una caracterización correcta de la uretra humana nativa es esencial desde un punto de vista clínico y como control de calidad para la generación de sustitutos artificiales. La invención tiene como objetivo contribuir a un mejor conocimiento de la uretra adulta con una amplia caracterización de la uretra masculina y femenina utilizando métodos histológicos, histoquímicos e inmunohistoquímicos. Los resultados podrían ser útiles para una selección más adecuada de los tejidos de sustitución para el reemplazo uretral. Urethral bioengineering would offer the advantage of generating functional autologous tissue that reproduces all the characteristics of a native urethra, since "the urethra itself will be the best substitute"; however, it is not known today what the correct histological and functional composition of the artificial tissue to generate to maximize the clinical outcome should be; Therefore, a correct characterization of the native human urethra is essential from a clinical point of view and as a quality control for the generation of artificial substitutes. The invention aims to contribute to a better knowledge of the adult urethra with a broad characterization of the male and female urethra using histological, histochemical and immunohistochemical methods. The results could be useful for a more suitable selection of replacement tissues for urethral replacement.
La uretra masculina está compuesta por tres capas de células: epitelio, capa muscular que consiste principalmente en células musculares lisas (SMC) y tejido esponjoso (del Pozo- Jimenez, et al., 2014. Arch. Esp. Uroi, 67(1): p. 5-1 1). El epitelio cambia a lo largo de la longitud de la uretra. Los estudios realizados hasta el momento sugieren que el epitelio de revestimiento de la uretra masculina varía entre las diferentes regiones de la uretra, con zonas más distales que muestran un epitelio escamoso estratificado que consta de numerosas capas de células, y las zonas más proximales son más similares al epitelio de la vejiga. En el caso femenino, parece que toda la uretra es muy homogénea, aunque distintos autores indican que 2/3 proximales de la uretra está revestida por un epitelio de transición similar, mientras 1/3 distal está revestida por un epitelio escamoso estratificado (Michael H. Ross, G.I.K., Wojciech Pawlina, Histology: A Text and Atlas: With Cell and Molecular Biology. Wolters Kluwer). En consecuencia, cada área de la uretra puede expresar diferentes marcadores moleculares que permiten que el tejido ejerza adecuadamente su función. Las citoqueratinas son filamentos intermedios característicos para los tejidos epiteliales. Las combinaciones específicas de expresión de citoqueratinas están relacionados con el tipo de célula epitelial específico o con condiciones patológicas [Paramio, 2006. Intermedíate filaments.: Springer; Molí, R., 1993. Veroff Pathol. 142: p. 1- 197). Aunque varios investigadores han identificado marcadores espaciotemporales en la uretra tanto masculina (Pechriggl et al., 2013. Ann. Anat. 195(3): p. 260-71), como femenina (Pechriggl et al., 2013. Ann. Anat. 195(6): p. 586-95) durante el desarrollo, en la actualidad no existe una caracterización molecular completa de la uretra humana, y tan sólo se ha publicado, y hace ya unos años, una caracterización bioquímica de los polipéptidos de citoqueratina de los distintos epitelios del tracto urogenital masculino humano (Quinlan et al., 1985. Ann. N. Y. Acad. Sci. 455: p. 282-306). Por esta razón, en el presente trabajo hemos analizado y caracterizado la uretra masculina y femenina utilizando métodos histológicos y diversos marcadores para determinar la estructura de este órgano. Curiosamente, cada zona de la uretra masculina mostró diferentes perfiles de componentes epiteliales y estromales que pueden reflejar las funciones específicas de tejido. La identificación de perfiles histoquímicos e inmunohistoquímicos específicos de cada área de la uretra puede contribuir a la búsqueda de fuentes de tejidos más adecuados para la reparación de la uretra. The male urethra is composed of three layers of cells: epithelium, muscular layer consisting mainly of smooth muscle cells (SMC) and spongy tissue (from Pozo-Jimenez, et al., 2014. Arch. Esp. Uroi, 67 (1) : p. 5-1 1). The epithelium changes along the length of the urethra. Studies conducted so far suggest that the lining epithelium of the male urethra varies between different regions of the urethra, with more distal areas showing a stratified squamous epithelium consisting of numerous layers of cells, and the most proximal areas are more similar to the bladder epithelium. In the female case, it appears that the entire urethra is very homogeneous, although different authors indicate that 2/3 proximal urethra is lined by a similar transitional epithelium, while 1/3 distal is lined by a stratified squamous epithelium (Michael H Ross, GIK, Wojciech Pawlina, Histology: A Text and Atlas: With Cell and Molecular Biology. Wolters Kluwer). Consequently, each area of the urethra can express different molecular markers that allow the tissue to exercise its function properly. Cytokeratins are characteristic intermediate filaments for epithelial tissues. Specific combinations of cytokeratin expression are related to the specific epithelial cell type or pathological conditions [Paramio, 2006. Intermediate filaments .: Springer; Molí, R., 1993. Veroff Pathol. 142: p. 1- 197). Although several researchers have identified spacetime markers in both the male urethra (Pechriggl et al., 2013. Ann. Anat. 195 (3): p. 260-71), and female (Pechriggl et al., 2013. Ann. Anat. 195 (6): p. 586-95) during development, there is currently no complete molecular characterization of the human urethra, and only published, and a few years ago, a biochemical characterization of the cytokeratin polypeptides of the different epithelia of the human male urogenital tract (Quinlan et al., 1985. Ann. NY Acad. Sci. 455: p. 282-306). For this reason, in the present work we have analyzed and characterized the male and female urethra using histological methods and various markers to determine the structure of this organ. Interestingly, each area of the male urethra showed different profiles of epithelial and stromal components that may reflect specific tissue functions. The identification of histochemical and immunohistochemical profiles specific to each area of the urethra may contribute to the search for more suitable tissue sources for urethral repair.
BREVE DESCRIPCIÓN DE LA INVENCIÓN BRIEF DESCRIPTION OF THE INVENTION
Un primer aspecto de la invención se refiere a un método de identificación, aislamiento y/o selección de célula(s), cultivo(s) celular(es) o tejido(s), de ahora en adelante método de la invención, útil(es) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de distintas porciones del tejido uretral, que comprende: a) identificar la expresión citoplasmática y/o nuclear de los marcadores CK34BE12, CKCAM 5.2, CKAE1/AE3, CK5/6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrina, Uroplaquina III y/o Ki67, en células básales y suprabasales en dicho cultivo(s) celular(es) o tejido(s); y b) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra prostática masculina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK5/6, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK18, CK19 y CK13; y ii) expresión citoplasmática en células suprabasales de CKCAM 5.2, CKAE1/AE3,A first aspect of the invention relates to a method of identification, isolation and / or selection of cell (s), cell culture (s) or tissue (s), hereinafter method of the invention, useful ( es) to increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue, which comprises: a) identifying the cytoplasmic and / or nuclear expression of the markers CK34BE12, CKCAM 5.2, CKAE1 / AE3, CK5 / 6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrine, Uroplachin III and / or Ki67, in basal and suprabasal cells in said cell culture (s) or tissue (s); and b) select the cell (s), the cell culture (s) or the tissue (s) of step a) to increase, restore or partially or totally replace the functional activity of the urethra male prostate if it has a profile that consists of: i) cytoplasmic expression in basal cells of CK5 / 6, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK18, CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CKCAM 5.2, CKAE1 / AE3,
CK34BE12, CK7, CK18, CK19, CK13 e involucrina; y iii) expresión nuclear en células suprabasales de uroplaquina; y iv) expresión nuclear en células básales de Ki67, y/o, c) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra membranosa masculina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK5/6, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK18, CK19 y CK13; y ii) expresión citoplasmática en células suprabasales de CK5/6, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK18, CK19, CK13 e involucrina; y iii) expresión nuclear en células suprabasales de uroplaquina; y iv) expresión nuclear en células básales de Ki67, y/o, d) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra bulbar masculina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK14, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK5/6, CK18, CK19 y CK13; y ii) expresión citoplasmática en células suprabasales de CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK5/6, CK18, CK19 y CK13, CK10 e involucrina; y iii) expresión nuclear en células suprabasales de uroplaquina; y iv) expresión nuclear en células básales de Ki67, y/o, e) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra peneana masculina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK14, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK5/6, CK18, CK19 y CK13; y ii) expresión citoplasmática en células suprabasales de CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK5/6, CK18, CK19, CK13, CK10 e involucrina; y iii) expresión nuclear en células suprabasales de uroplaquina; y iv) expresión nuclear en células básales de K¡67, y/o, f) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra femenina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK14, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK5/6, CK18, CK19 y CK13; y ii) expresión citoplasmática en células suprabasales de CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK5/6, CK18, CK19, CK13, CK10 e involucrina; y iii) expresión nuclear en células suprabasales de uroplaquina; y iv) expresión nuclear en células básales de Ki67. CK34BE12, CK7, CK18, CK19, CK13 and entacrine; and iii) nuclear expression in suprabasal uroplachin cells; and iv) nuclear expression in basal cells of Ki67, and / or, c) select the cell (s), the cell culture (s) or the tissue (s) of step a) to increase, restore or partially or totally replace the functional activity of the urethra male membranous if it has a profile consisting of: i) cytoplasmic expression in basal cells of CK5 / 6, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK18, CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CK5 / 6, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK18, CK19, CK13 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; and iv) nuclear expression in basal cells of Ki67, and / or, d) select the cell (s), the cell culture (s) or the tissue (s) of step a) to increase, restore or partially or totally replace the functional activity of the male bulbar urethra if it has a profile consisting of: i) cytoplasmic expression in basal cells of CK14, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK5 / 6, CK18, CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK5 / 6, CK18, CK19 and CK13, CK10 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; and iv) nuclear expression in basal cells of Ki67, and / or, e) select the cell (s), the cell culture (s) or the tissue (s) of step a) to increase, restore or partially or totally replace the functional activity of the male penile urethra if it has a profile that consists of: i) cytoplasmic expression in basal cells of CK14, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK5 / 6, CK18, CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK5 / 6, CK18, CK19, CK13, CK10 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; Y iv) nuclear expression in basal cells of K¡67, and / or, f) select the cell (s), the cell culture (s) or the tissue (s) of step a ) to increase, restore or partially or totally replace the functional activity of the female urethra if it has a profile consisting of: i) cytoplasmic expression in basal cells of CK14, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK5 / 6, CK18, CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK5 / 6, CK18, CK19, CK13, CK10 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; and iv) nuclear expression in basal cells of Ki67.
En una realización preferida de este aspecto de la invención, la identificación de la expresión citoplasmática y/o nuclear de los marcadores del paso a) se realiza mediante inmunotinción. In a preferred embodiment of this aspect of the invention, the identification of the cytoplasmic and / or nuclear expression of the markers of step a) is performed by immunostaining.
En otra realización preferida de este aspecto de la invención, la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) identificado(s) en el paso (a) provienen de la lista que consiste en: mucosa prepucial, piel genital y extragenital, mucosa bucal, mucosa lingual, submucosa del intestino delgado, mucosa de la vejiga o cualquiera de sus combinaciones. In another preferred embodiment of this aspect of the invention, the cell (s), the cell culture (s) or (a) tissue (s) identified in step (a) come from from the list consisting of: prepucial mucosa, genital and extragenital skin, oral mucosa, lingual mucosa, submucosa of the small intestine, bladder mucosa or any combination thereof.
En otra realización preferida de este aspecto de la invención, la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) identificado(s) en el paso (a) provienen de donantes que han sufrido parada cardiorrespiratoria o muerte encefálica o cerebral. In another preferred embodiment of this aspect of the invention, the cell (s), the cell culture (s) or (a) tissue (s) identified in step (a) come from of donors who have suffered cardiac arrest or brain or brain death.
Un segundo aspecto de la invención se refiere al uso de la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) que presente al menos un perfil identificado por el método de la invención, en medicina. A second aspect of the invention relates to the use of the cell (s), the cell culture (s) or (a) tissue (s) having at least one profile identified by the method of The invention, in medicine.
Un tercer aspecto de la invención se refiere al uso de la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) según el segundo aspecto de la invención, para la elaboración de un medicamento para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de distintas porciones del tejido uretral. A third aspect of the invention relates to the use of the cell (s), the cell culture (s) or (a) tissue (s) according to the second aspect of the invention, for the preparation of a medicament to increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue.
En una realización preferida de este aspecto de la invención, las distintas porciones de tejido uretral se seleccionan de la lista que consiste en: tejido uretral prostático masculino, tejido uretral membranoso masculino, tejido uretral bulbar masculino, tejido uretral peneano masculino y tejido uretral femenino In a preferred embodiment of this aspect of the invention, the different portions of urethral tissue are selected from the list consisting of: male prostatic urethral tissue, tissue male membranous urethral, male bulbar urethral tissue, male penile urethral tissue and female urethral tissue
En una realización preferida de este aspecto de la invención, la uretra está enferma o dañada como consecuencia de una disfunción, una lesión o una enfermedad seleccionada de la lista que comprende: una neoplasia benigna o maligna, una infección, un traumatismo, una malformación congénita o una estenosis. In a preferred embodiment of this aspect of the invention, the urethra is diseased or damaged as a result of a dysfunction, an injury or a disease selected from the list comprising: a benign or malignant neoplasm, an infection, a trauma, a congenital malformation or a stricture.
Un cuarto aspecto de la invención se refiere al uso de la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) que presente al menos un perfil identificado por el método de la invención, para la evaluación de un producto farmacológico y/o químico. Un quinto aspecto de la invención se refiere a un kit o dispositivo, de ahora en adelante kit o dispositivo de la invención, que comprende los elementos necesarios para identificar la expresión citoplasmática y/o nuclear de los marcadores CK34BE12, CKCAM 5.2, CKAE1/AE3, CK5/6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrina, Uroplaquina III y/o Ki67. En una realización preferida de este aspecto de la invención, el kit o dispositivo de la invención además comprende los elementos necesarios para realizar la identificación de los marcadores mediante inmunotinción. A fourth aspect of the invention relates to the use of the cell (s), the cell culture (s) or (a) tissue (s) having at least one profile identified by the method of the invention, for the evaluation of a pharmacological and / or chemical product. A fifth aspect of the invention relates to a kit or device, hereafter kit or device of the invention, comprising the elements necessary to identify the cytoplasmic and / or nuclear expression of the markers CK34BE12, CKCAM 5.2, CKAE1 / AE3 , CK5 / 6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrine, Uroplachin III and / or Ki67. In a preferred embodiment of this aspect of the invention, the kit or device of the invention further comprises the elements necessary to perform the identification of the markers by immunostaining.
En otra realización preferida de este aspecto de la invención, comprende los anticuerpos anti-CK34BE12, anti-CKCAM 5.2, anti-CKAE1/AE3, anti-CK5/6, anti-CK7, anti-CKI O, anti- CK13, anti-CK14, anti-CK17, anti-CK18, anti-CK19, anti-lnvolucrina, anti-Uroplaquina III y/o anti-K¡67. In another preferred embodiment of this aspect of the invention, it comprises anti-CK34BE12, anti-CKCAM 5.2, anti-CKAE1 / AE3, anti-CK5 / 6, anti-CK7, anti-CKI O, anti-CK13, anti- CK14, anti-CK17, anti-CK18, anti-CK19, anti-lnvolucrine, anti-Uroplaquina III and / or anti-K¡67.
En otra realización preferida de este aspecto de la invención, los anticuerpos están modificados. In another preferred embodiment of this aspect of the invention, the antibodies are modified.
Un sexto aspecto de la invención se refiere al uso del kit o dispositivo de la invención para la identificación, aislamiento y/o selección de célula(s), cultivo(s) celular(es) o tejido(s) útil(es) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de distintas porciones del tejido uretral. A sixth aspect of the invention relates to the use of the kit or device of the invention for the identification, isolation and / or selection of cell (s), cell culture (s) or tissue (s) useful for increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue.
Un séptimo aspecto de la invención se refiere a una composición, de ahora en adelante composición de la invención, que comprende la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) identificado(s), aislado(s) o seleccionado(s) por el método de la invención. En una realización preferida de este aspecto de la invención, la composición de la invención es una composición farmacéutica. A seventh aspect of the invention relates to a composition, hereinafter composition of the invention, comprising the cell (s), the cell culture (s) or the tissue (s) ) identified (s), isolated (s) or selected (s) by the method of the invention. In a preferred embodiment of this aspect of the invention, the composition of the invention is a pharmaceutical composition.
En otra realización preferida de este aspecto de la invención, la composición de la invención además comprende un vehículo farmacéuticamente aceptable. En otra realización preferida de este aspecto de la invención, la composición de la invención además comprende otro principio activo. In another preferred embodiment of this aspect of the invention, the composition of the invention further comprises a pharmaceutically acceptable carrier. In another preferred embodiment of this aspect of the invention, the composition of the invention further comprises another active ingredient.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Figura 1A. Análisis histológico de la uretra humana. Tinción H&E de las secciones correspondientes a la uretra masculina próstática (i), membranosa (ii), bulbar (iii) que muestra las diferencias en el número de capas de células. Barra de escala: 100 μηι. Figure 1A Histological analysis of the human urethra. H&E staining of the sections corresponding to the male prostate urethra (i), membranous (ii), bulbar (iii) showing the differences in the number of cell layers. Scale bar: 100 μηι.
Figura 1 B. Análisis histológico de la uretra humana. Tinción H&E de las secciones correspondientes a la uretra masculina del pene (iv) y a la uretra femenina (v) que muestra las diferencias en el número de capas de células. Barra de escala: 100 μηι. Figure 1 B. Histological analysis of the human urethra. H&E staining of the sections corresponding to the male urethra of the penis (iv) and the female urethra (v) showing the differences in the number of cell layers. Scale bar: 100 μηι.
Figura 2A. Análisis histoquímico de las capas de conectivo y musculares de uretra humana, (a) El método tricrómico de Masson se utilizó en secciones correspondientes a la uretra masculina y femenina, (b) la tinción de PAS muestra el contenido de glicoproteínas (c) Tinción inmunohistoquímica contra actina. Las secciones teñidas corresponden a la uretra masculina de próstata (i), membranosa (ii), bulbar (iii). Barra de escala: 100 μηι. Figure 2A Histochemical analysis of the connective and muscular layers of human urethra, (a) Masson's trichrome method was used in sections corresponding to the male and female urethra, (b) PAS staining shows glycoprotein content (c) Immunohistochemical staining against actin The stained sections correspond to the male urethra of prostate (i), membranous (ii), bulbar (iii). Scale bar: 100 μηι.
Figura 2B. Análisis histoquímico de las capas de conectivo y musculares de uretra humana, (a) El método tricrómico de Masson se utilizó en secciones correspondientes a la uretra masculina y femenina, (b) la tinción de PAS muestra el contenido de glicoproteínas (c) Tinción inmunohistoquímica contra actina. Las secciones teñidas corresponden a la uretra masculina de del pene (iv) y a la uretra femenina (v). Barra de escala: 100 μηι. Figure 2B Histochemical analysis of the connective and muscular layers of human urethra, (a) Masson's trichrome method was used in sections corresponding to the male and female urethra, (b) PAS staining shows glycoprotein content (c) Immunohistochemical staining against actin The stained sections correspond to the male urethra of the penis (iv) and the female urethra (v). Scale bar: 100 μηι.
Figura 3A. Análisis inmunohistoquímico de citoqueratinas de la capa epitelial de la uretra humana. La inmunodetección de una batería de citoqueratinas se realizó en secciones de tejido de la uretra masculina de próstata (i), membranosa (ii), bulbar (iii) y del pene (iv) y de uretra femenina. Los anticuerpos de amplio espectro complejos citoqueratina contra CK34BE12 (a), CKCAM 5.2 (b) y AE1/3 (c) mostraron tinción citoplásmica en todas las capas. Los anticuerpos específicos contra citoqueratina CK7 (d), mostraron expresión espacial específica a lo largo la longitud de la uretra y las diferencias entre la uretra masculina y femenina. Barra de escala: 100 μηι. Figura 3B. Análisis inmunohistoquímico de citoqueratinas de la capa epitelial de la uretra humana. Los anticuerpos específicos contra citoqueratina CK18 (e), CK5/6 (f), CK13 (g), CK14 (h), mostraron expresión espacial específica a lo largo la longitud de la uretra y las diferencias entre la uretra masculina y femenina. Barra de escala: 100 μηι. Figura 3B. Análisis inmunohistoquímico de citoqueratinas de la capa epitelial de la uretra humana. Los anticuerpos específicos contra citoqueratina CK17 (i), CK19 (j) y CK10 (k), mostraron expresión espacial específica a lo largo la longitud de la uretra y las diferencias entre la uretra masculina y femenina. Barra de escala: 100 μηι. Figure 3A Immunohistochemical analysis of cytokeratins of the epithelial layer of the human urethra. The immunodetection of a cytokeratin battery was performed in tissue sections of the male urethral prostate (i), membranous (ii), bulbar (iii) and penis (iv) and female urethra. Cytokeratin complex broad-spectrum antibodies against CK34BE12 (a), CKCAM 5.2 (b) and AE1 / 3 (c) showed cytoplasmic staining in all layers. Specific antibodies against cytokeratin CK7 (d), showed specific spatial expression along the length of the urethra and the differences between the male and female urethra. Scale bar: 100 μηι. Figure 3B Immunohistochemical analysis of cytokeratins of the epithelial layer of the human urethra. Specific antibodies against cytokeratin CK18 (e), CK5 / 6 (f), CK13 (g), CK14 (h), showed specific spatial expression along the length of the urethra and the differences between the male and female urethra. Scale bar: 100 μηι. Figure 3B Immunohistochemical analysis of cytokeratins of the epithelial layer of the human urethra. Specific antibodies against cytokeratin CK17 (i), CK19 (j) and CK10 (k), showed specific spatial expression along the length of the urethra and the differences between the male and female urethra. Scale bar: 100 μηι.
Figura 4A y 4B. Análisis inmunohistoquímico de marcadores de diferenciación epitelial, uroteliales y de proliferación, (a) El análisis del marcador de diferenciación epitelial involucrina mostró expresión positiva débil en las capas celulares superiores de la uretra masculina y femenina, (b) La tinción de uroplaquina III muestra tinción positiva leve en el núcleo de células dispersas en todas las capas de la uretra masculina y femenina, (c) La tinción para el marcador de proliferación Ki67 en el núcleo de las capas más básales de todas las regiones de la uretra masculina y femenina. Barra de escala: 100 μηι. Figure 4A and 4B. Immunohistochemical analysis of epithelial, urothelial and proliferation differentiation markers, (a) The analysis of the implcrine epithelial differentiation marker showed weak positive expression in the upper cell layers of the male and female urethra, (b) Staining of uroplachin III shows staining Slight positive in the nucleus of cells dispersed in all layers of the male and female urethra, (c) Staining for the Ki67 proliferation marker in the nucleus of the most basal layers of all regions of the male and female urethra. Scale bar: 100 μηι.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DETAILED DESCRIPTION OF THE INVENTION
Los autores de la presente invención han estudiado la importancia de las citoqueratinas, la involucrina, la uroplaquina III y la Ki67 en la identificación de célula(s), cultivo(s) celular(es) o tejido(s) útil(es) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de distintas porciones del tejido uretral. The authors of the present invention have studied the importance of cytokeratins, implicacrine, uroplachin III and Ki67 in the identification of cell (s), cell culture (s) or useful tissue (s) for increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue.
MÉTODO DE LA INVENCIÓN METHOD OF THE INVENTION
Un primer aspecto de la invención se refiere a un método de identificación, aislamiento y/o selección de célula(s), cultivo(s) celular(es) o tejido(s), de ahora en adelante método de la invención, útil(es) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de distintas porciones del tejido uretral, que comprende: a) identificar la expresión citoplasmática y/o nuclear de los marcadores CK34BE12, CKCAM 5.2, CKAE1/AE3, CK5/6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrina, Uroplaquina III y/o Ki67, en células básales y suprabasales en dicho cultivo(s) celular(es) o tejido(s); y b) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso (a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra prostática masculina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK5/6, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK18, CK19 y CK13; y ii) expresión citoplasmática en células suprabasales de CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK18, CK19, CK13 e involucrina; y iii) expresión nuclear en células suprabasales de uroplaquina; y iv) expresión nuclear en células básales de Ki67, y/o, c) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra membranosa masculina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK5/6, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK18, CK19 y CK13; y ii) expresión citoplasmática en células suprabasales de CK5/6, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK18, CK19, CK13 e involucrina; y iii) expresión nuclear en células suprabasales de uroplaquina; y iv) expresión nuclear en células básales de Ki67, y/o, d) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra bulbar masculina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK14, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK5/6, CK18, CK19 y CK13; y ii) expresión citoplasmática en células suprabasales de CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK5/6, CK18, CK19 y CK13, CK10 e involucrina; y iii) expresión nuclear en células suprabasales de uroplaquina; y iv) expresión nuclear en células básales de K¡67, y/o, e) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra peneana masculina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK14, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK5/6, CK18, CK19 y CK13; y ii) expresión citoplasmática en células suprabasales de CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK5/6, CK18, CK19, CK13, CK10 e involucrina; y iii) expresión nuclear en células suprabasales de uroplaquina; y iv) expresión nuclear en células básales de Ki67, y/o, f) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra femenina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK14, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK5/6, CK18, CK19 y CK13; y ii) expresión citoplasmática en células suprabasales de CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK5/6, CK18, CK19, CK13, CK10 e involucrina; y iii) expresión nuclear en células suprabasales de uroplaquina; y iv) expresión nuclear en células básales de Ki67. A first aspect of the invention relates to a method of identification, isolation and / or selection of cell (s), cell culture (s) or tissue (s), hereinafter method of the invention, useful ( es) to increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue, which comprises: a) identifying the cytoplasmic and / or nuclear expression of the markers CK34BE12, CKCAM 5.2, CKAE1 / AE3, CK5 / 6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrine, Uroplachin III and / or Ki67, in basal and suprabasal cells in said cell culture (s) or tissue (s); Y b) select the cell (s), the cell culture (s) or the tissue (s) of step (a) to increase, restore or partially or totally replace the functional activity of the Male prostatic urethra if it has a profile consisting of: i) cytoplasmic expression in basal cells of CK5 / 6, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK18, CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK18, CK19, CK13 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; and iv) nuclear expression in basal cells of Ki67, and / or, c) select the cell (s), the cell culture (s) or the tissue (s) of step a) to increase, restore or partially or totally replace the functional activity of the male membranous urethra if it has a profile consisting of: i) cytoplasmic expression in basal cells of CK5 / 6, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK18, CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CK5 / 6, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK18, CK19, CK13 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; and iv) nuclear expression in basal cells of Ki67, and / or, d) select the cell (s), the cell culture (s) or the tissue (s) of step a) to increase, restore or partially or totally replace the functional activity of the male bulbar urethra if it has a profile consisting of: i) cytoplasmic expression in basal cells of CK14, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK5 / 6, CK18, CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK5 / 6, CK18, CK19 and CK13, CK10 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; Y iv) nuclear expression in basal cells of K¡67, and / or, e) select the cell (s), the cell culture (s) or the tissue (s) of step a ) to increase, restore or partially or totally replace the functional activity of the male penile urethra if it has a profile consisting of: i) cytoplasmic expression in basal cells of CK14, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK5 / 6 , CK18, CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK5 / 6, CK18, CK19, CK13, CK10 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; and iv) nuclear expression in basal cells of Ki67, and / or, f) select the cell (s), the cell culture (s) or the tissue (s) of step a) to increase, restore or partially or totally replace the functional activity of the female urethra if it presents a profile consisting of: i) cytoplasmic expression in basal cells of CK14, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK5 / 6, CK18 , CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK5 / 6, CK18, CK19, CK13, CK10 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; and iv) nuclear expression in basal cells of Ki67.
En una realización preferida de este aspecto de la invención, la identificación de la expresión citoplasmática y/o nuclear de los marcadores del paso a) se realiza mediante inmunotinción. In a preferred embodiment of this aspect of the invention, the identification of the cytoplasmic and / or nuclear expression of the markers of step a) is performed by immunostaining.
En la presente memoria se entiende por "inmunotinción" a un método basado en anticuerpos para detectar una proteína específica en una muestra. La inmunotinción abarca una amplia gama de técnicas que se utilizan en la histología, biología celular, y biología molecular que utilizan métodos de tinción basados en anticuerpos. Las células identificadas por el método de la invención pueden ser analizadas por citometría de flujo. En otra realización preferida de este aspecto de la invención, la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) identificado(s) en el paso (a) provienen de la lista que consiste en: mucosa prepucial, piel genital y extragenital, mucosa bucal, mucosa lingual, submucosa del intestino delgado, mucosa de la vejiga o cualquiera de sus combinaciones. En otra realización preferida de este aspecto de la invención, la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) identificado(s) en el paso (a) provienen de donantes que han sufrido parada cardiorrespiratoria o muerte encefálica o cerebral. "Immunostaining" is understood herein as an antibody-based method for detecting a specific protein in a sample. Immunostaining encompasses a wide range of techniques that are used in histology, cell biology, and molecular biology that use antibody-based staining methods. The cells identified by the method of the invention can be analyzed by flow cytometry. In another preferred embodiment of this aspect of the invention, the cell (s), the cell culture (s) or (a) tissue (s) identified in step (a) come from from the list consisting of: prepucial mucosa, genital and extragenital skin, oral mucosa, lingual mucosa, submucosa of the small intestine, bladder mucosa or any combination thereof. In another preferred embodiment of this aspect of the invention, the cell (s), the cell culture (s) or (a) tissue (s) identified in step (a) come from of donors who have suffered cardiac arrest or brain or brain death.
La "uretra" es el conducto por donde circula la orina en la fase final del proceso urinario. Concretamente, es el conducto que conecta el cuello de la vejiga urinaria hasta el exterior del cuerpo durante la micción. Existen diferencias en la uretra dependiendo del sexo: The "urethra" is the conduit through which urine circulates in the final phase of the urinary process. Specifically, it is the conduit that connects the neck of the urinary bladder to the outside of the body during urination. There are differences in the urethra depending on sex:
• Tiene función secretora en el sexo femenino y masculino, y además reproductora en el masculino. • It has a secretory function in the female and male sex, and also reproductive in the male.
• En las mujeres mide aproximadamente entre 3,5-4 cm de longitud y se abre al exterior del cuerpo justo encima de la vagina. En los varones, la uretra mide cerca de 18-20 cm de largo, pasa por la glándula prostética y luego a través del pene al exterior del cuerpo.  • In women, it measures approximately 3.5-4 cm in length and opens to the outside of the body just above the vagina. In men, the urethra is about 18-20 cm long, passes through the prosthetic gland and then through the penis outside the body.
En cuanto a la anatomía de la uretra, también son diferentes en función del sexo: la uretra masculina tiene distintas porciones que son: As for the anatomy of the urethra, they are also different depending on sex: the male urethra has different portions that are:
• Uretra prostética: Discurre a través de la glándula prostética, donde los conductos eyaculadores vierten su contenido. • Prosthetic urethra: It runs through the prosthetic gland, where the ejaculatory ducts shed its contents.
• Uretra membranosa: es la porción distal a la prostética, de uno o dos cm, y en esta localización se encuentra el esfínter externo o rabdoesfínter, musculatura estriada, formado por fibras musculares que discurren desde la musculatura del suelo pélvico. • Membranous urethra: it is the portion distal to the prosthetic one, or two cm, and in this location is the external sphincter or rhabdosphincter, striated musculature, formed by muscle fibers that run from the pelvic floor musculature.
• Uretra peneana o esponjosa: Se llama así porque se encuentra en el interior del cuerpo esponjoso del pene, una vaina eréctil que recorre toda la cara ventral del pene. Llega al glande y se abre en el meato. Tiene una longitud de unos 9-11 centímetros. • Penile or spongy urethra: It is called that because it is inside the spongy body of the penis, an erectile sheath that runs along the entire ventral side of the penis. It reaches the glans and opens at the meatus. It has a length of about 9-11 centimeters.
• Se puede contemplar también la uretra bulbar: que es la porción que está entre la uretra membranosa (proximal) y la peneana (distal) y donde el espesor del esponjoso es el mayor. Por otro lado, la uretra femenina es corta y desemboca en la vulva entre el clítoris y el introito vaginal. El tamaño tan corto de la uretra femenina explica la mayor susceptibilidad de infecciones urinarias en las mujeres. • The bulbar urethra can also be seen: which is the portion between the membranous (proximal) and the penile (distal) urethra and where the thickness of the spongy is the largest. On the other hand, the female urethra is short and flows into the vulva between the clitoris and the vaginal introite. The short size of the female urethra explains the increased susceptibility of urinary infections in women.
En el contexto de la presente invención, se entiende por "célula basal", aunque sin limitarnos, a la célula ubicada en la base del epitelio uretral. In the context of the present invention, "basal cell" is understood, but not limited to, the cell located at the base of the urethral epithelium.
En el contexto de la presente invención, se entiende por "célula suprabasal", aunque sin limitarnos, a las células ubicadas en la zona apical o luminal del epitelio uretral. In the context of the present invention, "suprabasal cell" is understood, but not limited to, the cells located in the apical or luminal area of the urethral epithelium.
El término "cultivo celular" se refiere, aunque sin limitarnos, a los cultivos de células in vitro que consisten en un sistema formado por células provenientes de un órgano o un tejido, normal o tumoral, mantenidas en medios de cultivo de composición química definida y en condiciones de temperatura, pH, aireación y humedad controladas. De esta forma se aseguran su supervivencia y multiplicación, manteniendo todas sus funciones metabólicas de una manera semejante a las que tenían en el huésped. The term "cell culture" refers, but not limited to, in vitro cell cultures consisting of a system formed by cells from an organ or tissue, normal or tumor, maintained in culture media of defined chemical composition and under controlled temperature, pH, aeration and humidity conditions. In this way they ensure their survival and multiplication, maintaining all their metabolic functions in a manner similar to those they had in the host.
Esta invención no se limita a ningún modo de cultivo y contempla las distintas variantes en cuanto a la manera que el cultivo que pueden llevarse a cabo, por ejemplo el término "cultivo adicional" se refiere a cultivar una célula o tejido hasta una determinada fase de crecimiento para, a continuación, utilizar otro método de cultivo para hacer que dicha célula o tejido alcance otra etapa de crecimiento. Un "cultivo en monocapa" se refiere a un cultivo en el que las células se multiplican en un medio adecuado mientras permanecen unidas entre sí y a un sustrato. Además, un "cultivo en suspensión" se refiere a un cultivo en el que las células se multiplican, en suspensión en un medio adecuado. Del mismo modo, un "cultivo de flujo continuo" se refiere al cultivo de células o explantes en un flujo continuo de medio fresco para mantener el crecimiento celular, por ejemplo, viabilidad. El término "medios condicionados" se refiere al sobrenadante, por ejemplo, libre de los cultivos de células/tejido, resultando después de un período de tiempo en contacto con las células cultivadas de tal manera que los medios han sido modificado para incluir determinados factores paracrinos y/o autocrinos producidos por las células y que se secretan en el cultivo. Un "cultivo confluente" es un cultivo de células en las que todas las células están en contacto y por lo tanto toda la superficie del recipiente de cultivo está cubierta, e implica que las células hayan alcanzado su máxima densidad, aunque confluencia no significa necesariamente que la división celular cese o que la población deje de aumentar de tamaño. This invention is not limited to any culture mode and contemplates the different variants as to the way in which the culture that can be carried out, for example the term "additional culture" refers to culturing a cell or tissue up to a certain phase of growth to then use another culture method to make said cell or tissue reach another stage of growth. A "monolayer culture" refers to a culture in which cells multiply in a suitable medium while remaining attached to each other and to a substrate. In addition, a "suspension culture" refers to a culture in which cells multiply, in suspension in a suitable medium. Similarly, a "continuous flow culture" refers to the culture of cells or explants in a continuous flow of fresh medium to maintain cell growth, for example, viability. The term "conditioned media" refers to the supernatant, for example, free of cell / tissue cultures, resulting after a period of time in contact with cultured cells such that the media has been modified to include certain paracrine factors. and / or autocrine produced by the cells and secreted in the culture. A "confluent culture" is a culture of cells in which all cells are in contact and therefore the entire surface of the culture vessel is covered, and implies that the cells have reached their maximum density, although confluence does not necessarily mean that the cell division ceases or the population stops increasing in size.
El término "tejido" se refiere a aquellas estructuras constituidas por un conjunto organizado de células y distribuidas regularmente, con un comportamiento fisiológico coordinado. En el contexto de la presente invención, se entiende por "inmunohistoquímica" al procedimiento histopatológico que se basa en la utilización de anticuerpos que se unen específicamente a una sustancia que se quiere identificar (anticuerpo primario). Estos anticuerpos pueden tener unida una enzima o esta puede encontrarse unida a un anticuerpo secundario que reconoce y se une al primario. Aplicado a un tejido orgánico, el anticuerpo primario se une específicamente al sustrato y se aprovecha la actividad enzimática para visualizar la unión. De esta manera se consigue un complejo sustrato-anticuerpos-enzima unido al lugar donde se encuentre el sustrato y mediante la activación de la enzima con la adición de su sustrato se genera un producto identificable donde se encuentre el complejo Esta técnica permite identificar la localización de una sustancia específica permitiendo identificar su localización tisular o citológica, de esta manera se pueden identificar los marcadores antigénicos característicos de una línea celular, identificar células que secretan una proteína, receptores de membrana, gradientes de concentración tisulares o células que han respondido a una hormona (con anticuerpos específicos para las vías de señalización intracelular). The term "tissue" refers to those structures constituted by an organized set of cells and distributed regularly, with a coordinated physiological behavior. In the context of the present invention, "immunohistochemistry" means the histopathological procedure that is based on the use of antibodies that specifically bind to a substance to be identified (primary antibody). These antibodies may have an enzyme bound or it may be bound to a secondary antibody that recognizes and binds to the primary. Applied to an organic tissue, the primary antibody specifically binds to the substrate and enzymatic activity is used to visualize binding. In this way a substrate-antibody-enzyme complex is attached to the place where the substrate is located and by activating the enzyme with the addition of its substrate an identifiable product is generated where the complex is located. This technique allows to identify the location of a specific substance allowing to identify its tissue or cytological location, in this way you can identify the characteristic antigen markers of a cell line, identify cells that secrete a protein, membrane receptors, tissue concentration gradients or cells that have responded to a hormone ( with antibodies specific for intracellular signaling pathways).
En esta memoria se entiende por "citoqueratinas" o "tonofibrillas" a aquellas proteínas fibrosas que forman los filamentos intermedios del citoesqueleto intracelular en particular de células epiteliales (incluyendo mucosas y glándulas), así como en las uñas, pelo y en las plumas de los animales. Los monómeros de citoqueratina están ligados los unos a los otros por moléculas de filagrina. Su función principal es la organización de la estructura tridimensional interna de la célula (por ejemplo, forman parte de la envuelta nuclear y de los sarcómeros), forman una barrera rígida la cual evita la entrada de microorganismos. Esa misma barrera cumple la vital función de retener agua dentro de las células. También participan en algunas uniones intercelulares (desmosomas). Determinadas irregularidades en la expresión de las citoqueratinas son la base de algunas condiciones atípicas, tales como eccema, ictiosis, soriasis y ciertos tipos de cáncer metastático. In this report, "cytokeratins" or "tonofibrils" means those fibrous proteins that form the intermediate filaments of the intracellular cytoskeleton in particular of epithelial cells (including mucous membranes and glands), as well as in the nails, hair and feathers of the animals. Cytokeratin monomers are linked to each other by phylagrin molecules. Its main function is the organization of the internal three-dimensional structure of the cell (for example, they are part of the nuclear envelope and the sarcomeres), they form a rigid barrier which prevents the entry of microorganisms. That same barrier fulfills the vital function of retaining water within the cells. They also participate in some intercellular junctions (desmosomes). Certain irregularities in the expression of cytokeratins are the basis of some atypical conditions, such as eczema, ichthyosis, psoriasis and certain types of metastatic cancer.
Hay dos tipos de citoqueratinas: las citoqueratinas de tipo I básicas y las citoqueratinas de tipo II de ácidas o neutras. Las citoqueratinas se encuentran generalmente en pares que comprenden una citoqueratina de tipo I y otra citoqueratina de tipo II. Las citoqueratinas básicas o neutras incluyen CK1 , CK2, CK3, CK4, CK5, CK6, CK7 y CK8. Las citoqueratinas ácidas son CK9, CK10, CK12, CK13, CK14, CK16, CK17, CK18, CK19 y CK20. Las citoqueratinas se pueden dividir en función si su peso molecular es alto o bajo. La expresión de estas citoqueratinas es frecuentemente órgano o tejido específico. Por ejemplo, CK7 se expresa normalmente en el epitelio ductal del tracto genitourinario (GU) y CK20 más frecuentemente en el tracto gastrointestinal (Gl). There are two types of cytokeratins: the basic type I cytokeratins and the type II cytokeratins of acidic or neutral. Cytokeratins are generally found in pairs that comprise a type I cytokeratin and another type II cytokeratin. Basic or neutral cytokeratins include CK1, CK2, CK3, CK4, CK5, CK6, CK7 and CK8. The acid cytokeratins are CK9, CK10, CK12, CK13, CK14, CK16, CK17, CK18, CK19 and CK20. Cytokeratins can be divided according to whether their molecular weight is high or low. The expression of these cytokeratins is often a specific organ or tissue. For example, CK7 is normally expressed in the ductal epithelium of the genitourinary tract (GU) and CK20 more frequently in the gastrointestinal tract (Gl).
Los subconjuntos de citoqueratinas que expresan las células epiteliales dependen principalmente del tipo de epitelio, el momento en el curso de la diferenciación terminal y la etapa de desarrollo. De esta manera, la huella digital citoqueratina específica permite la clasificación de todos los epitelios sobre su perfil de expresión de citoqueratina. El estudio del perfil de citoqueratinas por técnicas de inmunohistoquímica es una herramienta de inmenso valor utilizada para el diagnóstico de tumores y caracterización de la patología quirúrgica. Las citoqueratinas están codificadas por una familia que abarca 30 genes. Entre ellos, 20 son genes epiteliales y los restantes 10 son específicos para tricocistos. The subsets of cytokeratins that express epithelial cells depend mainly on the type of epithelium, the time in the course of terminal differentiation and the stage of development. In this way, the specific cytokeratin fingerprint allows the classification of all epithelia on its cytokeratin expression profile. The study of the cytokeratin profile by immunohistochemical techniques is a tool of immense value used for the diagnosis of tumors and characterization of surgical pathology. Cytokeratins are encoded by a family that encompasses 30 genes. Among them, 20 are epithelial genes and the remaining 10 are specific for tricocysts.
Todas las cadenas de citoqueratina se componen de un dominio central rica en a-hélices (con una identidad de secuencia de 50-90% entre las citoqueratinas del mismo tipo y alrededor de 30% entre las citoqueratinas de distinto tipo) con dominios terminales N- y C- no-a-helicoidales. El dominio α-helicoidal tiene 310-150 aminoácidos y comprende cuatro segmentos en los que se repite un patrón de siete residuos. En este patrón repetido, el primer y el cuarto residuo son hidrófobos y los residuos cargados muestran polaridad positiva y negativa de forma alterna, como resultado los residuos polares se sitúan en un lado de la hélice. Este dominio central de la cadena proporciona la alineación molecular en la estructura de la queratina y hace que las cadenas formen dímeros en espiral en disolución. All cytokeratin chains are composed of a central domain rich in a-helices (with a sequence identity of 50-90% among cytokeratins of the same type and about 30% between cytokeratins of different types) with N- terminal domains and C-non-helical. The α-helical domain has 310-150 amino acids and comprises four segments in which a pattern of seven residues is repeated. In this repeated pattern, the first and fourth residues are hydrophobic and the charged residues show positive and negative polarity alternately, as a result the polar residues are located on one side of the helix. This central domain of the chain provides molecular alignment in the keratin structure and causes the chains to form spiral dimers in solution.
Las secuencias de fin de dominio de tipo I y II contienen cadenas de citoqueratina en ambos lados de la varilla de los subdominios del dominio V1 y V2, que tienen secuencia y tamaño variable. El tipo II también presenta subdominios H1 y H2 conservados, que abarca 36 y 20 residuos, respectivamente. Los subdominios V1 y V2 contienen residuos enriquecidos por glicinas y/o serinas, el ex proporcionando la cadena de citoqueratina un carácter insoluble fuerte y facilitando la interacción con otras moléculas. Estos dominios terminales también son importantes en la definición de la función de la característica de la cadena de citoqueratina de un tipo de célula epitelial particular. Los grupos de dos dímeros de citoqueratinas forman un tetrámero queratina por construcción antiparalela vinculante. Este tetrámero de citoqueratina se considera que es el principal bloque de construcción de la cadena de citoqueratina. Por la vinculación de la cabeza a la cola de los tetrámeros citoqueratina, los protofilamentos se originan, que a su vez se entrelazan en pares para formar protofibrillas. Cuatro protofi brillas dan lugar a un filamento de citoqueratina. The end-of-domain sequences of type I and II contain cytokeratin chains on both sides of the rod of the subdomains of domain V1 and V2, which have sequence and variable size. Type II also has conserved H1 and H2 subdomains, covering 36 and 20 residues, respectively. Subdomains V1 and V2 contain residues enriched by glycine and / or serine, the former providing the cytokeratin chain with a strong insoluble character and facilitating interaction with other molecules. These terminal domains are also important in defining the function of the cytokeratin chain characteristic of a particular type of epithelial cell. Groups of two cytokeratin dimers form a keratin tetramer by binding antiparallel construction. This cytokeratin tetramer is considered to be the main building block of the cytokeratin chain. By linking the head to the tail of the cytokeratin tetramers, the protofilaments originate, which at their Once they are intertwined in pairs to form protofibrils. Four protofi shines give rise to a cytokeratin filament.
En el citoplasma, los filamentos de queratina conforman una compleja red que se extiende desde la superficie del núcleo a la membrana celular. Numerosas proteínas accesorias están implicadas en la génesis y el mantenimiento de dicha estructura. In the cytoplasm, the keratin filaments form a complex network that extends from the surface of the nucleus to the cell membrane. Numerous accessory proteins are involved in the genesis and maintenance of said structure.
Esta asociación entre la membrana plasmática y la superficie nuclear proporciona importantes implicaciones para la organización del citoplasma y mecanismos de comunicación celular. Aparte de las funciones relativamente estáticas previstas en términos de apoyo del núcleo y proporcionar resistencia a la tracción a la célula, las redes de citoqueratina experimentan intercambios de fosfato rápidos mediando la despolimerización, con importantes implicaciones en los procesos celulares más dinámicos como la mitosis y el período post-mitótico, célula movimiento y diferenciación. This association between the plasma membrane and the nuclear surface provides important implications for the organization of the cytoplasm and cellular communication mechanisms. Apart from the relatively static functions envisaged in terms of core support and providing tensile strength to the cell, cytokeratin networks undergo rapid phosphate exchanges mediating depolymerization, with important implications in more dynamic cellular processes such as mitosis and post-mitotic period, cell movement and differentiation.
Las citoqueratinas de manera general, interactúan con desmosomas y hemidesmosomas, colaborando así a la adhesión célula-célula y la conexión basal de células de tejido conectivo subyacente. Los filamentos intermedios del citoesqueleto eucariota, de los cuales las citoqueratinas son uno de sus tres componentes, se han sondeado para asociar también con red de proteínas del complejo ankirina y espectrina que subyace a la membrana celular. Cytokeratins generally interact with desmosomes and hemidesmosomes, thus contributing to cell-cell adhesion and basal connection of underlying connective tissue cells. The intermediate filaments of the eukaryotic cytoskeleton, of which cytokeratins are one of its three components, have been probed to also associate with the protein network of the ankyrin complex and spectrin that underlies the cell membrane.
Las citoqueratinas usadas como marcadores en el método de la invención son: CK34BE12, CKCAM 5.2, CKAE1/AE3, CK5/6, CK7 (NCBI Gene ID: 3855), CK10 (NCBI Gene ID: 3858), CK13 (NCBI Gene ID: 3860), CK14 (NCBI Gene ID: 3861), CK17 (NCBI Gene ID: 3872), CK18 (NCBI Gene ID: 3875) y/o CK19 (NCBI Gene ID: 3880). The cytokeratins used as markers in the method of the invention are: CK34BE12, CKCAM 5.2, CKAE1 / AE3, CK5 / 6, CK7 (NCBI Gene ID: 3855), CK10 (NCBI Gene ID: 3858), CK13 (NCBI Gene ID: 3860), CK14 (NCBI Gene ID: 3861), CK17 (NCBI Gene ID: 3872), CK18 (NCBI Gene ID: 3875) and / or CK19 (NCBI Gene ID: 3880).
Por otro lado el método de la invención también utiliza: On the other hand, the method of the invention also uses:
La "involucrina" (NCBI Gene ID: 3713) es un componente de la envoltura reticulada de queratinocitos, se encuentra en el citoplasma y unidas a proteínas de la membrana por transglutaminasa. La localización cromosómica de este gen es 1q21. "Invite" (NCBI Gene ID: 3713) is a component of the cross-linked keratinocyte envelope, found in the cytoplasm and bound to membrane proteins by transglutaminase. The chromosomal location of this gene is 1q21.
La "uroplaquina III" (NCBI Gene ID: 7380) pertenece a un grupo de proteínas transmembrana que forman complejos en la superficie apical del epitelio de la vejiga. Las mutaciones en este gen pueden estar asociadas con adisplasia renal. /'-67(NCBI Gene ID: 4288) es un gen codifica una proteína nuclear que de manera asociada puede ser necesario para la proliferación celular. Se han descrito variantes en la transcripción alternativamente empalmados. Existe un pseudogen en el cromosoma X relacionado. "Uroplachin III" (NCBI Gene ID: 7380) belongs to a group of transmembrane proteins that form complexes on the apical surface of the bladder epithelium. Mutations in this gene may be associated with renal adisplasia. / ' -67 (NCBI Gene ID: 4288) is a gene encoding a nuclear protein that in an associated manner may be necessary for cell proliferation. Variants have been described in the alternatively spliced transcription. There is a pseudogen on the related X chromosome.
USOS DE LAS CÉLULA(S), CULTIVO(S) CULTIVO(S) O TEJIDO(S) IDENTIFICADOS POR EL MÉTODO DE LA INVENCIÓN Un segundo aspecto de la invención se refiere al uso de la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) que presente al menos un perfil identificado por el método de la invención, en medicina. USES OF THE CELL (S), CULTURE (S) CULTURE (S) OR FABRIC (S) IDENTIFIED BY THE METHOD OF THE INVENTION A second aspect of the invention relates to the use of the cell (s), the / the cell culture (s) or (a) tissue (s) having at least one profile identified by the method of the invention, in medicine.
Un tercer aspecto de la invención se refiere al uso de la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) según el segundo aspecto de la invención, para la elaboración de un medicamento para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de distintas porciones del tejido uretral. A third aspect of the invention relates to the use of the cell (s), the cell culture (s) or (a) tissue (s) according to the second aspect of the invention, for the preparation of a medicament to increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue.
En una realización preferida de este aspecto de la invención, las distintas porciones de tejido uretral se seleccionan de la lista que consiste en: tejido uretral prostético masculino, tejido uretral membranoso masculino, tejido uretral bulbar masculino, tejido uretral peneano masculino y tejido uretral femenino In a preferred embodiment of this aspect of the invention, the different portions of urethral tissue are selected from the list consisting of: male prosthetic urethral tissue, male membranous urethral tissue, male bulbar urethral tissue, male penile urethral tissue and female urethral tissue
En una realización preferida de este aspecto de la invención, la uretra está enferma o dañada como consecuencia de una disfunción, una lesión o una enfermedad seleccionada de la lista que comprende: una neoplasia benigna o maligna, una infección, un traumatismo, una malformación congénita o una estenosis. Un cuarto aspecto de la invención se refiere al uso de la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) que presente al menos un perfil identificado por el método de la invención, para la evaluación de un producto farmacológico y/o químico. In a preferred embodiment of this aspect of the invention, the urethra is diseased or damaged as a result of a dysfunction, an injury or a disease selected from the list comprising: a benign or malignant neoplasm, an infection, a trauma, a congenital malformation or a stricture. A fourth aspect of the invention relates to the use of the cell (s), the cell culture (s) or (a) tissue (s) having at least one profile identified by the method of the invention, for the evaluation of a pharmacological and / or chemical product.
KIT O DISPOSITIVO DE LA INVENCIÓN Y USO KIT OR DEVICE OF THE INVENTION AND USE
Un quinto aspecto de la invención se refiere a un kit o dispositivo, de ahora en adelante kit o dispositivo de la invención, que comprende los elementos necesarios identificar la expresión citoplasmática y/o nuclear de los marcadores CK34BE12, CKCAM 5.2, CKAE1/AE3, CK5/6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrina, Uroplaquina III y/o Ki67. A fifth aspect of the invention relates to a kit or device, hereinafter kit or device of the invention, comprising the elements necessary to identify the cytoplasmic and / or nuclear expression of the markers CK34BE12, CKCAM 5.2, CKAE1 / AE3, CK5 / 6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrine, Uroplachin III and / or Ki67.
En una realización preferida de este aspecto de la invención, el kit o dispositivo de la invención además comprende los elementos necesarios para realizar la identificación de los marcadores mediante inmunotinción. En otra realización preferida de este aspecto de la invención, comprende los anticuerpos anti-CK34BE12, anti-CKCAM 5.2, anti-CKAE1/AE3, anti-CK5/6, anti-CK7, anti-CKI O, anti- CK13, anti-CK14, anti-CK17, anti-CK18, anti-CK19, anti-lnvolucrina, anti-Uroplaquina III y/o anti-K¡67. Preferiblemente los anticuerpos usados en la invención se seleccionan de la lista que consiste en: actina (Alpha-Sr-1), CK34BE12, CK19 (RCK108) de Master Diagnóstica (Granada, Spain): CK 20 (Ks20.8), CAM 5.2 (clon CAM 5.2), CKAE1/AE3 (clon Y85), citoqueratina 5 y 6 (D5/16B4), CK7 (OV-TL 12/30), CK10 (SP99), CK13 (KS-1A3), CK14(LL002), CK17 (E3), CK18 (DC-10), involucrina (SY5), uroplaquina III (SP73), Ki-67 (clon SP6). En otra realización preferida de este aspecto de la invención, los anticuerpos están modificados. In a preferred embodiment of this aspect of the invention, the kit or device of the invention further comprises the elements necessary to perform the identification of the markers by immunostaining. In another preferred embodiment of this aspect of the invention, it comprises anti-CK34BE12, anti-CKCAM 5.2, anti-CKAE1 / AE3, anti-CK5 / 6, anti-CK7, anti-CKI O, anti-CK13, anti- CK14, anti-CK17, anti-CK18, anti-CK19, anti-lnvolucrine, anti-Uroplaquina III and / or anti-K¡67. Preferably the antibodies used in the invention are selected from the list consisting of: actin (Alpha-Sr-1), CK34BE12, CK19 (RCK108) from Master Diagnostic (Granada, Spain): CK 20 (Ks20.8), CAM 5.2 (clone CAM 5.2), CKAE1 / AE3 (clone Y85), cytokeratin 5 and 6 (D5 / 16B4), CK7 (OV-TL 12/30), CK10 (SP99), CK13 (KS-1A3), CK14 (LL002) , CK17 (E3), CK18 (DC-10), implicacrine (SY5), uroplachin III (SP73), Ki-67 (clone SP6). In another preferred embodiment of this aspect of the invention, the antibodies are modified.
En otra realización preferida de este aspecto de la invención, el anticuerpo es humano, humanizado o sintético. En otra realización preferida, el anticuerpo es monoclonal y/o se encuentra marcado con un fluorocromo. Preferiblemente, el flurocromo se selecciona de la lista que comprende Fluoresceína (FITC), Tetrametilrodamina y derivados, Ficoeritrina (PE), PerCP, Cy5, Texas, aloficocianina, o cualquiera de sus combinaciones. In another preferred embodiment of this aspect of the invention, the antibody is human, humanized or synthetic. In another preferred embodiment, the antibody is monoclonal and / or is labeled with a fluorochrome. Preferably, the flurochrome is selected from the list comprising Fluorescein (FITC), Tetramethylrodamine and derivatives, Phycoerythrin (PE), PerCP, Cy5, Texas, allophycocyanin, or any combination thereof.
El kit además puede contener, sin ningún tipo de limitación, tampones, soluciones de extracción de proteínas, agentes para prevenir la contaminación, inhibidores de la degradación de las proteínas, etc. Por otro lado, el kit o dispositivo de la invención puede incluir todos los soportes y recipientes necesarios para su puesta en marcha y optimización. Preferiblemente, el kit comprende además las instrucciones para llevar a cabo el método de la invención. The kit may also contain, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc. On the other hand, the kit or device of the invention can include all the supports and containers necessary for its implementation and optimization. Preferably, the kit further comprises instructions for carrying out the method of the invention.
En una realización preferida el kit o dispositivo de la invención comprende oligonucleótidos. En otra realización preferida, los oligonucleótidos presentan modificaciones en alguno de sus nucleótidos, como por ejemplo, pero sin limitarnos a, nucleótidos que tengan alguno de sus átomos con un isótopo radiactivo, normalmente 32P o tritio, nucleótidos marcados inmunológicamente, como por ejemplo con una molécula de digoxigenina, y/o inmovilizadas en una membrana. Varias posibilidades son conocidas en el estado de la técnica. In a preferred embodiment the kit or device of the invention comprises oligonucleotides. In another preferred embodiment, the oligonucleotides have modifications in some of their nucleotides, such as, but not limited to, nucleotides having any of their atoms with a radioactive isotope, usually 32 P or tritium, immunologically labeled nucleotides, such as with a molecule of digoxigenin, and / or immobilized in a membrane. Several possibilities are known in the state of the art.
Un sexto aspecto de la invención se refiere al uso del kit o dispositivo de la invención para la identificación, aislamiento y/o selección de célula(s), cultivo(s) celular(es) o tejido(s) útil(es) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de distintas porciones del tejido uretral. COMPOSICIÓN DE LA INVENCIÓN A sixth aspect of the invention relates to the use of the kit or device of the invention for the identification, isolation and / or selection of cell (s), cell culture (s) or tissue (s) useful for increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue. COMPOSITION OF THE INVENTION
Un séptimo aspecto de la invención se refiere a una composición, de ahora en adelante composición de la invención, que comprende la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) identificado(s), aislado(s) o seleccionado(s) por el método de la invención. En una realización preferida de este aspecto de la invención, la composición de la invención es una composición farmacéutica. A seventh aspect of the invention relates to a composition, hereinafter composition of the invention, comprising the cell (s), the cell culture (s) or the tissue (s) ) identified (s), isolated (s) or selected (s) by the method of the invention. In a preferred embodiment of this aspect of the invention, the composition of the invention is a pharmaceutical composition.
En otra realización preferida de este aspecto de la invención, la composición de la invención además comprende un vehículo farmacéuticamente aceptable. In another preferred embodiment of this aspect of the invention, the composition of the invention further comprises a pharmaceutically acceptable carrier.
En otra realización preferida de este aspecto de la invención, la composición de la invención además comprende otro principio activo. In another preferred embodiment of this aspect of the invention, the composition of the invention further comprises another active ingredient.
El término "vehículo aceptable farmacéuticamente" se refiere a un vehículo que debe estar aprobado por una agencia reguladora del gobierno federal o un gobierno estatal o enumerado en la Farmacopea Estadounidense o la Farmacopea Europea, u otra farmacopea reconocida generalmente para su uso en animales, y más concretamente en humanos. The term "pharmaceutically acceptable vehicle" refers to a vehicle that must be approved by a federal government regulatory agency or a state government or listed in the United States Pharmacopoeia or European Pharmacopoeia, or other pharmacopoeia generally recognized for use in animals, and more specifically in humans.
El término "vehículo" se refiere a un diluyente, coadyuvante, excipiente o portador con el que se deben administrar al cultivo(s) celular(es) o tejido(s) de la invención o de dicha composición que comprende cultivo(s) celular(es) o tejido(s) de la invención seleccionadas por el método de la invención; obviamente, dicho vehículo debe ser compatible con dichas células. Ejemplos ilustrativos, no limitativos, de dicho vehículo incluyen cualquier vehículo fisiológicamente compatible, por ejemplo, soluciones isotónicas (e.g., solución salina estéril (0,9% NaCI), solución salina tamponada con fosfatos (PBS), solución Ringer-lactato, etc.), opcionalmente suplementadas con suero, preferiblemente con suero autólogo; medios de cultivo celular (e.g., DMEM, etc.); o, alternativamente, un medio soporte sólido, semisólido, gelatinoso o viscoso, tal como colágeno, colagen-glicosamino-glicano, fibrina, cloruro de polivinilo, poliaminoácidos, tales como polilisina, o poliornitina, hidrogeles, agarosa, sulfato de dextrano silicona. Asimismo, si se desea, el medio de soporte puede, en realizaciones específicas, contener factores de crecimiento u otros agentes. Si el soporte es sólido, semisólido, o gelatinoso, las células pueden ser introducidas en una fase líquida del vehículo que es tratada posteriormente de forma tal que se convierte en una fase más sólida. The term "vehicle" refers to a diluent, adjuvant, excipient or carrier with which they should be administered to the cell culture (s) or tissue (s) of the invention or of said composition comprising cell culture (s) (s) or fabric (s) of the invention selected by the method of the invention; obviously, said vehicle must be compatible with said cells. Illustrative, non-limiting examples of said vehicle include any physiologically compatible vehicle, for example, isotonic solutions (eg, sterile saline (0.9% NaCI), phosphate buffered saline (PBS), Ringer-lactate solution, etc. ), optionally supplemented with serum, preferably with autologous serum; cell culture media (e.g., DMEM, etc.); or, alternatively, a solid, semi-solid, gelatinous or viscous support medium, such as collagen, collagen-glycosamino-glycan, fibrin, polyvinyl chloride, polyamino acids, such as polylysine, or polynithine, hydrogels, agarose, silicone dextran sulfate. Also, if desired, the support medium may, in specific embodiments, contain growth factors or other agents. If the support is solid, semi-solid, or gelatinous, the cells can be introduced into a liquid phase of the vehicle that is subsequently treated in such a way that it becomes a more solid phase.
La composición farmacéutica de la invención, si se desea, puede contener también, cuando sea necesario, aditivos para aumentar, controlar o dirigir de otro modo el efecto terapéutico deseado de el cultivo(s) celular(es) o tejido(s), los cuales comprenden dicha composición farmacéutica, y/o sustancias auxiliares o sustancias farmacéuticamente aceptables, tales como agentes tamponantes, tensioactivos, codisolventes, conservantes, etc. También, para estabilizar la suspensión celular, es posible añadir quelantes de metales. La estabilidad del el cultivo(s) celular(es) o tejido(s) en el medio líquido de la composición farmacéutica de la invención puede mejorarse mediante la adición de sustancias adicionales, tales como, por ejemplo, ácido aspártico, ácido glutámico, etcétera. Dichas sustancias farmacéuticamente aceptables que pueden usarse en la composición farmacéutica de la invención son conocidas, en general, los técnicos en la materia y se usan normalmente en la elaboración de composiciones celulares. Ejemplos de vehículos farmacéuticos adecuados se describen, por ejemplo, en "Remington's Pharmaceutical Sciences", de E.W. Martin. Puede encontrarse información adicional sobre dichos vehículos en cualquier manual de tecnología farmacéutica (Farmacia Galénica). The pharmaceutical composition of the invention, if desired, may also contain, when necessary, additives to increase, control or otherwise direct the therapeutic effect. desired of the cell culture (s) or tissue (s), which comprise said pharmaceutical composition, and / or auxiliary substances or pharmaceutically acceptable substances, such as buffering agents, surfactants, co-solvents, preservatives, etc. Also, to stabilize the cell suspension, it is possible to add metal chelators. The stability of the cell culture (s) or tissue (s) in the liquid medium of the pharmaceutical composition of the invention can be improved by the addition of additional substances, such as, for example, aspartic acid, glutamic acid, etc. . Such pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known to those skilled in the art and are normally used in the preparation of cellular compositions. Examples of suitable pharmaceutical vehicles are described, for example, in "Remington's Pharmaceutical Sciences" by EW Martin. Additional information on these vehicles can be found in any pharmaceutical technology manual (Galenic Pharmacy).
La composición farmacéutica de la invención contendrá una cantidad terapéuticamente efectiva del cultivo(s) celular(es) o tejido(s) de la invención, para proporcionar el efecto terapéutico deseado. Tal como se usa en la presente descripción, el término "cantidad terapéutica o profilácticamente efectiva" se refiere a la cantidad de cultivo(s) celular(es) o tejido(s) de la invención contenida en la composición farmacéutica que es capaz de producir el efecto terapéutico deseado y, en general, se determinará, entre otros factores, por las propias características del cultivo(s) celular(es) o tejido(s) de la invención y el efecto terapéutico deseado que se persigue. En general, la cantidad terapéuticamente efectiva de cultivo(s) celular(es) o tejido(s) de la invención que debe administrarse dependerá, entre otros factores, de las propias características del sujeto, la gravedad de la enfermedad, la forma de administración, etc. La composición farmacéutica de la invención se formulará de acuerdo con la forma de administración elegida. The pharmaceutical composition of the invention will contain a therapeutically effective amount of the cell culture (s) or tissue (s) of the invention, to provide the desired therapeutic effect. As used herein, the term "therapeutically or prophylactically effective amount" refers to the amount of cell culture (s) or tissue (s) of the invention contained in the pharmaceutical composition that is capable of producing the desired therapeutic effect and, in general, will be determined, among other factors, by the characteristics of the cell culture (s) or tissue (s) of the invention and the desired therapeutic effect that is sought. In general, the therapeutically effective amount of cell culture (s) or tissue (s) of the invention to be administered will depend, among other factors, on the subject's own characteristics, the severity of the disease, the manner of administration. , etc. The pharmaceutical composition of the invention will be formulated according to the chosen form of administration.
La composición farmacéutica de la invención, si se desea, se puede almacenar hasta el momento de su aplicación mediante los procedimientos convencionales conocidos por los técnicos en la materia. Esta composición farmacéutica también se puede almacenar junto a medicamentos adicionales, útiles en el tratamiento en enfermedades, en una forma activa que comprende una terapia combinada. Para el almacenamiento a corto plazo (menos de 6 horas), la composición farmacéutica de la invención puede almacenarse a temperatura ambiente o por debajo de ésta en un recipiente sellado complementándola o no con una solución nutriente. El almacenamiento a medio plazo (menos de 48 horas) se realiza preferentemente a 2-8°C, y la composición farmacéutica de la invención incluirá una solución iso-osmótica y tamponada en un contenedor compuesta de, o revestida de, material que previene la adhesión celular. El almacenamiento a más largo plazo se lleva a cabo preferentemente por medio de crioconservación adecuada y almacenamiento en condiciones que promueven la retención de la función celular. The pharmaceutical composition of the invention, if desired, can be stored until the moment of its application by conventional procedures known to those skilled in the art. This pharmaceutical composition can also be stored together with additional medications, useful in the treatment of diseases, in an active form comprising a combination therapy. For short-term storage (less than 6 hours), the pharmaceutical composition of the invention can be stored at or below room temperature in a sealed container complementing it or not with a nutrient solution. Medium term storage (less than 48 hours) is done preferably at 2-8 ° C, and the pharmaceutical composition of the invention will include an iso-osmotic and buffered solution in a container composed of, or coated with, material that prevents cell adhesion. Longer term storage is preferably carried out by means of adequate cryopreservation and storage under conditions that promote retention of cellular function.
Si se desea, las células, el cultivo celular o tejido de la invención puede ser modificados genéticamente por cualquier método convencional incluyendo, a modo ilustrativo, no limitativo, procesos de transgénesis, deleciones o inserciones en su genoma que modifiquen la expresión de genes que sean importantes para sus propiedades básicas (proliferación, migración, transdiferenciación, etc.). If desired, the cells, cell culture or tissue of the invention can be genetically modified by any conventional method including, by way of illustration, not limitation, transgenesis processes, deletions or insertions in your genome that modify the expression of genes that are important for its basic properties (proliferation, migration, transdifferentiation, etc.).
Otro aspecto de la invención se refiere a un microarray, de ahora en adelante microarray de la invención, que comprende oligonucleótidos o microarreglos de canal único diseñados a partir de una secuencia conocida o ARNm de cualquiera de los marcadores del paso a). Another aspect of the invention relates to a microarray, hereafter referred to as a microarray of the invention, comprising oligonucleotides or single channel microarrays designed from a known sequence or mRNA of any of the markers in step a).
Así, por ejemplo, las secuencias de oligonucleótidos pueden ser construidas en la superficie un chip mediante el elongamiento secuencial de una cadena en crecimiento con un sólo nucleótido utilizando fotolitografía. Así, los oligonucleótidos son anclados por el extremo 3' mediante un método de activación selectiva de nucleótidos, protegidos por un reactivo fotolábil, mediante la incidencia selectiva de luz a través de una fotomáscara. La fotomáscara puede ser física o virtual. Así, las sondas de oligonucleótidos pueden ser de entre 10 y 100 nucleótidos, más preferiblemente, de entre 20 y 70 nucleótidos, y aún más preferiblemente, de entre 24 y 30 nucleótidos. Thus, for example, oligonucleotide sequences can be constructed on the surface of a chip by sequential elongation of a growing chain with a single nucleotide using photolithography. Thus, the oligonucleotides are anchored at the 3 'end by a method of selective activation of nucleotides, protected by a photolabile reagent, by the selective incidence of light through a photomask. The photomask can be physical or virtual. Thus, oligonucleotide probes can be between 10 and 100 nucleotides, more preferably, between 20 and 70 nucleotides, and even more preferably, between 24 and 30 nucleotides.
La síntesis in situ sobre un soporte sólido (por ejemplo, vidrio), podría hacerse mediante tecnología chorro de tinta (ink-jet), lo que requiere sondas más largas. Los soportes podrían ser, pero sin limitarse a, filtros o membranas de NC o nylon (cargadas), silicio, o Portas de vidrio para microscopios cubiertos con aminosilanos, polilisina, aldehidos o epoxy. La sonda es cada una de las muestras del chip. El target es la muestra a analizar: RNA mensajero, RNA total, un fragmento de PCR, etc. Synthesis in situ on a solid support (for example, glass) could be done using ink-jet technology, which requires longer probes. The supports could be, but not limited to, filters or membranes of NC or nylon (charged), silicon, or glass slides for microscopes covered with aminosilanes, polylysine, aldehydes or epoxy. The probe is each of the chip samples. The target is the sample to be analyzed: messenger RNA, total RNA, a PCR fragment, etc.
Otro aspecto de la invención se refiere a un microarray de proteínas, de ahora en adelante microarray de proteínas de la invención, que comprende anticuerpos o fragmentos de los mismos específicos frente a cualquiera de los marcadores del paso a) o frente a secuencias aminoacídicas que presenten un grado de identidad a las secuencias aminoacídicas de dichos marcadores de, al menos del 85%, típicamente de, al menos del 90%, preferiblemente de, al menos del 95%, más preferiblemente de, al menos del 98%, aún más preferiblemente de, al menos del 99%. Las sondas son anticuerpos fijados a portaobjetos de vidrio y los blancos son muestras de células, cultivos celulares y/o tejidos. Another aspect of the invention relates to a protein microarray, hereafter referred to as a protein microarray of the invention, comprising antibodies or fragments thereof specific against any of the markers of step a) or against amino acid sequences that present a degree of identity to the amino acid sequences of said markers of at least 85%, typically of at least 90%, preferably, at least 95%, more preferably, at least 98%, even more preferably, at least 99%. The probes are antibodies fixed to glass slides and the targets are samples of cells, cell cultures and / or tissues.
Otro aspecto de la invención se refiere al uso del microarray de la invención o el microarray de proteínas de la invención, para la identificación, aislamiento y/o selección de célula(s), cultivo(s) celular(es) o tejido(s) útil(es) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de distintas porciones del tejido uretral. Another aspect of the invention relates to the use of the microarray of the invention or the protein microarray of the invention, for the identification, isolation and / or selection of cell (s), cell culture (s) or tissue (s). ) useful (s) to increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue.
En otra realización preferida de este aspecto de la invención, el microarray de la invención o el microarray de proteínas de la invención, se insertan en un dispositivo de microscopía y/o inmunohistoquímica automatizada para su lectura. In another preferred embodiment of this aspect of the invention, the microarray of the invention or the protein microarray of the invention are inserted into an automated microscopy and / or immunohistochemical device for reading.
Otro aspecto de la invención se refiere a un programa de ordenador que comprende instrucciones de programa para hacer que un ordenador lleve a la práctica el procedimiento de acuerdo con el método de la invención. Another aspect of the invention relates to a computer program comprising program instructions to make a computer carry out the method according to the method of the invention.
En particular, la invención abarca programas de ordenador dispuestos sobre o dentro de una portadora. La portadora puede ser cualquier entidad o dispositivo capaz de soportar el programa. Cuando el programa va incorporado en una señal que puede ser transportada directamente por un cable u otro dispositivo o medio, la portadora puede estar constituida por dicho cable u otro dispositivo o medio. Como variante, la portadora podría ser un circuito integrado en el que va incluido el programa, estando el circuito integrado adaptado para ejecutar, o para ser utilizado en la ejecución de, los procesos correspondientes. In particular, the invention encompasses computer programs arranged on or within a carrier. The carrier can be any entity or device capable of supporting the program. When the program is incorporated into a signal that can be directly transported by a cable or other device or medium, the carrier may be constituted by said cable or other device or means. As a variant, the carrier could be an integrated circuit in which the program is included, the integrated circuit being adapted to execute, or to be used in the execution of, the corresponding processes.
Por ejemplo, los programas podrían estar incorporados en un medio de almacenamiento, como una memoria ROM, una memoria CD ROM o una memoria ROM de semiconductor, una memoria USB, o un soporte de grabación magnética, por ejemplo, un disco flexible o un disco duro. Alternativamente, los programas podrían estar soportados en una señal portadora transmisible. Por ejemplo, podría tratarse de una señal eléctrica u óptica que podría transportarse a través de cable eléctrico u óptico, por radio o por cualesquiera otros medios. For example, the programs could be incorporated into a storage medium, such as a ROM, a CD ROM or a semiconductor ROM, a USB memory, or a magnetic recording medium, for example, a floppy disk or a disk Lasted. Alternatively, the programs could be supported on a transmissible carrier signal. For example, it could be an electrical or optical signal that could be transported through an electrical or optical cable, by radio or by any other means.
La invención se extiende también a programas de ordenador adaptados para que cualquier medio de procesamiento pueda llevar a la práctica el método de la invención. Tales programas pueden tener la forma de código fuente, código objeto, una fuente intermedia de código y código objeto, por ejemplo, como en forma parcialmente compilada, o en cualquier otra forma adecuada para uso en la puesta en práctica de los procesos según la invención. Los programas de ordenador también abarcan aplicaciones en la nube basadas en dicho procedimiento. The invention also extends to computer programs adapted so that any processing means can carry out the method of the invention. Such programs may have the form of source code, object code, an intermediate source of code and object code, for example, as in partially compiled form, or in any other form suitable for use in the implementation of the processes according to the invention . Computer programs also cover cloud applications based on that procedure.
Por tanto, otro aspecto de la invención se refiere a un medio de almacenamiento legible por un ordenador que comprende instrucciones de programa capaces de hacer que un ordenador lleve a cabo los pasos del métodos de la invención. Therefore, another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of causing a computer to carry out the steps of the methods of the invention.
Otro aspecto de la invención se refiere a una señal transmisible que comprende instrucciones de programa capaces de hacer que un ordenador lleve a cabo los pasos del métodos de la invención. Another aspect of the invention relates to a transmissible signal comprising program instructions capable of causing a computer to carry out the steps of the methods of the invention.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. EJEMPLO DE LA INVENCIÓN Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. EXAMPLE OF THE INVENTION
Materiales y métodos Materials and methods
Muestras de tejido uretral humano Samples of human urethral tissue
Las muestras de tejido uretral humano se obtuvieron de donantes cadavéricos. Este estudio ha sido aprobado por la Coordinación Autonómica de Trasplantes, el Comité de Ética del Hospital de Málaga y el Biobanco de Andalucía. El consentimiento informado fue aprobado por los familiares de los donantes. Para obtener muestras de uretra se realizaron cistoprostatectomía (varones) o cistectomía (en las mujeres) con uretrectomía. Una vez extirpada la pieza, la uretra se disecó en sus regiones anatómicas, se realizaron cortes seriados transversales cada cm y se fijaron con formaldehído para el análisis histológico e inmunohistoquímico. Human urethral tissue samples were obtained from cadaveric donors. This study has been approved by the Autonomous Coordination of Transplants, the Ethics Committee of the Hospital of Malaga and the Biobank of Andalusia. Informed consent was approved by donor relatives. To obtain urethra samples, cystoprostatectomy (men) or cystectomy (in women) with urethrectomy were performed. Once the piece was removed, the urethra was dissected in its anatomical regions, serial cross sections were made every cm and fixed with formaldehyde for histological and immunohistochemical analysis.
Análisis histológicos Histological analysis
Las muestras de tejidos de uretra, recogidas después de la cirugía, se fijaron con formaldehído al 4% y se incrustaron en parafina. Las muestras se cortaron a 3μηι y se utilizaron las tinciones de hematoxilina/eosina, tricrómico de Masson y PAS para determinar la histología de la uretra con microscopía de luz. Estudios de inmunohistoquímica Urethra tissue samples, collected after surgery, were fixed with 4% formaldehyde and embedded in paraffin. Samples were cut at 3μηι and hematoxylin / eosin stains, Masson's trichrome and PAS were used to determine the histology of the urethra with light microscopy. Immunohistochemical studies
La evaluación de la expresión de marcadores de citoqueratinas y de proliferación se llevó a cabo en los tejidos de la uretra fijados en formol incluidos en parafina (FFPE). Las secciones de tejido de 3 μηι se incubaron con un anticuerpo primario y se tiñeron según el estándar EnVision™ FLEX, pH alto (K8010, Dako, Agilent Tecnologies, Dinamarca) procedimientos de trabajo utilizando el inmunoteñidor automático Dako Autostainer (Dako). Evaluation of the expression of cytokeratin and proliferation markers was carried out in formalin-fixed urethra tissues (FFPE). The 3 μηι tissue sections were incubated with a primary antibody and stained according to the EnVision ™ FLEX standard, high pH (K8010, Dako, Agilent Tecnologies, Denmark) working procedures using the Dako Autostainer automatic immunotoner (Dako).
La tinción inmunohistoquímica se realizó con anticuerpos listos para usar de Dako: actina (Alpha-Sr-1), CK34BE12, CK19 (RCK108), y de la Master Diagnóstica (Granada, España): CAM 5.2 (clon CAM 5.2), CKAE1/AE3 (Y85 clon), tipos de citoqueratina 5 y 6 (D5/16B4), CK7 (OV-TL 12/30), CK10 (SP99), CK13 (KS-1A3), CK14 (LL002), CK17 (E3), CK18 (DC - 10), involucrina (SY5), uroplaquina III (SP73), Ki-67 (SP6 clon). Immunohistochemical staining was performed with ready-to-use antibodies from Dako: actin (Alpha-Sr-1), CK34BE12, CK19 (RCK108), and from the Diagnostic Master (Granada, Spain): CAM 5.2 (CAM 5.2 clone), CKAE1 / AE3 (Y85 clone), cytokeratin types 5 and 6 (D5 / 16B4), CK7 (OV-TL 12/30), CK10 (SP99), CK13 (KS-1A3), CK14 (LL002), CK17 (E3), CK18 (DC-10), implicacrine (SY5), uroplaquine III (SP73), Ki-67 (SP6 clone).
Los anticuerpos primarios se incubaron durante 20 minutos a temperatura ambiente. La unión del anticuerpo se detectó por EnVision FLEX / HRP (20 minutos) (Dako). El revelado del color se realizó con tetrahidrocloruro 3,3 'diaminobencidina como cromógeno (Dako). Las láminas se contratiñeron con hematoxilina, se deshidrataron y montaron. Primary antibodies were incubated for 20 minutes at room temperature. Antibody binding was detected by EnVision FLEX / HRP (20 minutes) (Dako). The color development was performed with 3,3'-diaminobenzidine tetrahydrochloride as chromogen (Dako). The sheets were counterstained with hematoxylin, dehydrated and mounted.
Como controles positivos se utilizaron secciones de tejido conocidos que expresan las citoqueratinas investigadas. Se llevaron a cabo de forma rutinaria controles negativos mediante la sustitución del anticuerpo primario por IgG no específico. En paralelo al mismo tiempo que las muestras de estudio se trataron las secciones control. Los resultados se analizaron independientemente por 3 investigadores. Los niveles de expresión de citoqueratinas de membrana y/o citoplásmica y la expresión nuclear de de Ki- 67 y uroplaquina, se registraron y puntuaron teniendo en cuenta la intensidad de la tinción como bajo (+), moderada (++) o alto (+++) y la zona ampliada (superficial o basal). As positive controls, known tissue sections expressing the cytokeratins investigated were used. Negative controls were routinely carried out by replacing the primary antibody with non-specific IgG. In parallel at the same time as the study samples, the control sections were treated. The results were analyzed independently by 3 researchers. Expression levels of membrane and / or cytoplasmic cytokeratins and nuclear expression of Ki- 67 and uroplachin were recorded and scored taking into account the intensity of staining as low (+), moderate (++) or high ( +++) and the enlarged area (superficial or basal).
Análisis de imagen Imágenes microscópicas a magnificación 200X se transformaron a 8 bits con el programa Image J (Schneider et al., 2012. Nat. Methods, 9(7): p. 671-5) y la intensidad de color gris se midió en una escala de 0 a 255 en blanco y negro para cuantificar intensidad de la tinción. El image J también se utilizó para contar la tinción nuclear y el número total de núcleos por capa en secciones de tejidos teñidos con uroplaquina III o Ki 67. Resultados Image analysis Microscopic images at 200X magnification were transformed to 8 bits with the Image J program (Schneider et al., 2012. Nat. Methods, 9 (7): p. 671-5) and the gray intensity was measured in a scale from 0 to 255 in black and white to quantify staining intensity. Image J was also used to count nuclear staining and the total number of nuclei per layer in tissue sections stained with uroplachin III or Ki 67. Results
Análisis histológico de la uretra humana por tinción con H&E Para caracterizar el espesor del epitelio a lo largo del tracto uretral masculino, las secciones histológicas se evaluaron por tinción hematoxilina y eosina (H & E). La región proximal (prostética y membranosa) de la uretra masculina tenía pocas capas de células epiteliales (4,26 ± 0,88), mientras que la distal (bulbar y pene) uretra mostró mayor número de capas celulares (6,9 ± 1) (Figura 1 ; i-iv ). La uretra femenina presenta un espesor comparable a la uretra distal masculina (7,6 ± 1 ,8 capas de células) (Fig. 1). Estos resultados demuestran que la histología de la uretra masculina es altamente dependiente de la zona donde se toma la muestra a lo largo de su longitud desde la vejiga hasta el meato. Histological analysis of the human urethra by staining with H&E To characterize the thickness of the epithelium along the male urethral tract, histological sections were evaluated by hematoxylin and eosin staining (H&E). The proximal region (prosthetic and membranous) of the male urethra had few layers of epithelial cells (4.26 ± 0.88), while the distal (bulbar and penis) urethra showed a greater number of cell layers (6.9 ± 1 ) (Figure 1; i-iv). The female urethra has a thickness comparable to the male distal urethra (7.6 ± 1.8 layers of cells) (Fig. 1). These results demonstrate that the histology of the male urethra is highly dependent on the area where the sample is taken along its length from the bladder to the meatus.
Análisis histoquímico de las capas musculares y conectiva de la uretra humana Para entender las variaciones en la arquitectura del tejido a lo largo de la uretra masculina, se realizaron tinciones específicas para las capas conectivo y muscular de la uretra. Cuando las secciones de tejido se tiñeron por el método de tricrómico de Masson (Figura 2a), se encontró que todas las muestras eran ricas en colágeno del tejido conectivo (Fig. 2a). Como se determinó por tinción con PAS (figura 2b) los mayores contenidos de glicoproteínas se observaron en las capas de células epiteliales apicales de la uretra femenina en comparación con uretra masculina (0, 1 ±5,5; 0±3,8; 0,3±9; 0,4±10,7 y 31 ,6±84 medido en una escala de grises en la uretra de próstata, membranosa, bulbar, peneana y femenina, respectivamente) (Fig. 2b). Tinción inmunohistoquímica músculo específica para actina sarcomérica puso de manifiesto que todas las regiones de la uretra masculina tenían una capa muscular (Fig.2c). Histochemical analysis of the muscular and connective layers of the human urethra To understand the variations in tissue architecture along the male urethra, specific stains were made for the connective and muscular layers of the urethra. When the tissue sections were stained by Masson's trichrome method (Figure 2a), all samples were found to be rich in connective tissue collagen (Fig. 2a). As determined by staining with PAS (Figure 2b), the highest glycoprotein contents were observed in the apical epithelial cell layers of the female urethra compared to the male urethra (0.1 ± 5.5; 0 ± 3.8; 0 , 3 ± 9; 0.4 ± 10.7 and 31, 6 ± 84 measured on a gray scale in the prostate, membranous, bulbar, penile and female urethra, respectively) (Fig. 2b). Immunohistochemical staining specific muscle for sarcomeric actin showed that all regions of the male urethra had a muscular layer (Fig. 2c).
Análisis inmunohistoquímico de la capa epitelial con anticuerpos anti-citoqueratina Immunohistochemical analysis of the epithelial layer with anti-cytokeratin antibodies
Para caracterizar adicionalmente las diferencias aparentes en las capas epiteliales de la uretra masculina y femenina, se empleó un panel integral de anticuerpos citoqueratina específicos (Figura 3) y sus patrones de reactividad analizada de una manera semicuantitativa (Tabla 1-2 2). Como era de esperar, los anticuerpos de amplio espectro anti-citoqueratina CK34BE12 (que reconoce citoqueratinas humanos 1 , 5, 10 y 14), CKCAM5.2 (anti-CK7 y CK8) y CKAE1/AE3 (mezcla de anticuerpos pan-citoqueratina que reacciona con 14 CKs tanto de las familias A y B) revelaron una fuerte tinción citoplásmica en todas las capas a lo largo de la uretra masculina y también en la uretra femenina (Figura 3, a-c) con la única excepción de algunas células de la capa suprabasal de la uretra prostética que apareció negativo para CK34BE12 (Fig. 3a). Para caracterizar la expresión de los epitelios simples y marcadores de citoqueratina urotelio específicos, se llevó a cabo una detección inmunológica de citoqueratinas 7 y 18. De manera generalizada se observaron tinción de CK7 y CK18 en todas las capas en masculino prostética, membranosa y la uretra bulbar epitelio (Figura 3 d-e). Células esporádicas de la uretra prostética y membranosa masculina no mostraron expresión (Fig. 3 d-e). En la uretra del pene, CK7 se observó en capas suprabasales pero no en la primera capa basal (Fig. 3d), y CK18 se expresó más intensamente en capas suprabasales (Fig. 3e). La uretra femenina mostró tinción muy ligera, tanto para citoqueratinas y sólo en células esporádicas de todas las capas (Fig. 3, DE). Para caracterizar la expresión de CK5 y CK6, los anticuerpos CK5/6 teñidos se utilizaron para revelar una intensa tinción citoplásmica en todas las muestras (Fig. 3F), aunque restringido a las capas básales en la uretra prostética (Fig. 3f). Un perfil muy similar se encontró para la expresión CK13, que se localiza principalmente en la capa de células básales de la uretra prostética y se expandió a todas las capas una vez que la uretra se aleja de la próstata (Fig. 3g). La uretra femenina tenía un perfil de tinción similar para CK5/6 y CK13 de bulbar y peneana uretra (Fig. 3f, g). Curiosamente, CK14 específico de célula basal se expresa sólo por las células básales de la bulbar y la uretra del pene, y también la uretra femenina, pero no el de próstata y la uretra membranosa (Fig. 3h). Sorprendentemente CK17 se expresa sólo por las células de la capa basal distintas en todas las áreas de la uretra masculina y en la uretra femenina (Fig. 3i). En cuanto a la CK19, encontramos una fuerte tinción citoplásmica en todas las capas de la uretra prostética y membranosa masculina (Fig. 3j). Sin embargo, se observó una notable diferencia en el patrón de CK19 del epitelio bulbar (que era intensamente positiva en todas las capas) y la uretra del pene (negativo en capas suprabasales; la abrupta transición de una zona a la otra puede verse claramente en el panel iii de la Fig. 3j). La uretra femenina tuvo una fuerte tinción CK19 en la capa basal y una tinción esporádica muy ligera en algunas células suprabasales (Fig. 3j). La inmunotinción CK20 resultó negativa para todas las muestras (datos no mostrados). Finalmente, el marcador de capa suprabasal CKI Oestaba completamente ausente en la próstata y la uretra membranosa, pero era intensamente presente en todas las capas suprabasales de la uretra bulbar y del pene (Fig. 3k). La uretra femenina mostró sólo algunas células positivas dispersas en capas suprabasales (Fig. 3k). En resumen, el análisis inmunohistoquímico de la expresión de citoqueratina en el varón humano (prostética, membranosa, bulbar y pene) y de la uretra femenina reveló diferencias importantes entre las diferentes muestras. To further characterize the apparent differences in the epithelial layers of the male and female urethra, an integral panel of specific cytokeratin antibodies was used (Figure 3) and their reactivity patterns analyzed in a semi-quantitative manner (Table 1-2 2). As expected, the broad spectrum anti-cytokeratin antibodies CK34BE12 (which recognizes human cytokeratins 1, 5, 10 and 14), CKCAM5.2 (anti-CK7 and CK8) and CKAE1 / AE3 (mixture of pan-cytokeratin antibodies that reacts with 14 CKs from both families A and B) revealed strong cytoplasmic staining in all layers along the male urethra and also in the female urethra (Figure 3, ac) with the only exception of some layer cells suprabasal of the prosthetic urethra that appeared negative for CK34BE12 (Fig. 3a). To characterize the expression of simple epithelia and specific urothelial cytokeratin markers, immunological detection of cytokeratins 7 and 18 was carried out. CK7 and CK18 staining was generally observed in all layers in male prosthetic, membranous and urethra Bulbar epithelium (Figure 3 of). Sporadic cells of the male prosthetic and membranous urethra showed no expression (Fig. 3 of). In the urethra of the penis, CK7 was observed in suprabasal layers but not in the first basal layer (Fig. 3d), and CK18 was expressed more intensely in suprabasal layers (Fig. 3e). The female urethra showed very light staining, both for cytokeratins and only in sporadic cells of all layers (Fig. 3, DE). To characterize the expression of CK5 and CK6, stained CK5 / 6 antibodies were used to reveal intense cytoplasmic staining in all samples (Fig. 3F), although restricted to the basal layers in the prosthetic urethra (Fig. 3f). A very similar profile was found for CK13 expression, which is located primarily in the basal cell layer of the prosthetic urethra and expanded to all layers once the urethra moves away from the prostate (Fig. 3g). The female urethra had a similar staining profile for CK5 / 6 and CK13 of bulbar and urethra penis (Fig. 3f, g). Interestingly, CK14 specific basal cell is expressed only by the basal cells of the bulbar and urethra of the penis, and also the female urethra, but not the prostate and the membranous urethra (Fig. 3h). Surprisingly CK17 is expressed only by distinct basal layer cells in all areas of the male urethra and in the female urethra (Fig. 3i). As for CK19, we found a strong cytoplasmic staining in all layers of the male prosthetic and membranous urethra (Fig. 3j). However, a notable difference was observed in the CK19 pattern of the bulbar epithelium (which was intensely positive in all layers) and the urethra of the penis (negative in suprabasal layers; the abrupt transition from one area to the other can be clearly seen in panel iii of Fig. 3j). The female urethra had a strong CK19 staining in the basal layer and a very slight sporadic staining in some suprabasal cells (Fig. 3j). CK20 immunostaining was negative for all samples (data not shown). Finally, the CKI O suprabasal layer marker was completely absent in the prostate and membranous urethra, but was intensely present in all suprabasal layers of the bulbar urethra and penis (Fig. 3k). The female urethra showed only some positive cells dispersed in suprabasal layers (Fig. 3k). In summary, the immunohistochemical analysis of the expression of cytokeratin in the human male (prosthetic, membranous, bulbar and penis) and of the female urethra revealed important differences between the different samples.
Análisis inmunohistoquímico de la capa epitelial con marcadores de proliferación y diferenciación Immunohistochemical analysis of the epithelial layer with proliferation and differentiation markers
Como sería de esperar, involucrina, un marcador de diferenciación epitelial mostró expresión ligeramente positivo en las capas de células suprabasal masculina y de la uretra femenina, pero no en las capas básales (Fig. 4a;. Tabla 1-2). Con respecto a uroplaquina III, un marcador para las células uroteliales paraguas, tinción positiva leve estaba presente en el núcleo de células dispersas en todas las capas del macho y la uretra femenina (Fig. 4b;. Tabla 1-2). Notablemente, el número de células teñidas fueron mayores en las capas de células apicales (Ratio de porcentaje de células teñidas en las capas de células apicales / porcentaje de células teñidas en capas básales = 17, 1 ± 5, 1 ; 7,8 ± 2,5; 10, 1 ± 2,7; 3,5 ± 0,9 y 3,8 ± 0,8 en uretra prostética, membranosa, bulbar, de pene y femenina, respectivamente). Finalmente, el marcador de proliferación Ki67 estuvo presente en las capas básales de todas las regiones de la uretra masculina y en la uretra femenina, y el número de células positivas fue mayor en las zonas en las que el epitelio tenían un mayor número de capas de células (2 ± 0,9% ; 6 ± 1 %; 6 ± 0,4%; 40 ± 1 % y 48 ± 3% de células positivas Ki67 en la próstata, membranosa, bulbar, de pene y la uretra femenina, respectivamente) (figura 4c;. tabla 1-2). As would be expected, involving, an epithelial differentiation marker showed slightly positive expression in the male suprabasal and female urethra cell layers, but not in the basal layers (Fig. 4a; Table 1-2). With respect to uroplaquine III, a Umbrella urothelial cell marker, mild positive staining was present in the nucleus of cells dispersed in all layers of the male and female urethra (Fig. 4b; Table 1-2). Notably, the number of stained cells were higher in the apical cell layers (Ratio of percentage of stained cells in the apical cell layers / percentage of stained cells in basal layers = 17, 1 ± 5, 1; 7.8 ± 2 , 5; 10, 1 ± 2.7; 3.5 ± 0.9 and 3.8 ± 0.8 in prosthetic, membranous, bulbar, penile and female urethra, respectively). Finally, the Ki67 proliferation marker was present in the basal layers of all regions of the male urethra and in the female urethra, and the number of positive cells was higher in areas where the epithelium had a greater number of layers of cells (2 ± 0.9%; 6 ± 1%; 6 ± 0.4%; 40 ± 1% and 48 ± 3% of Ki67 positive cells in the prostate, membranous, bulbar, penis and female urethra, respectively ) (Figure 4c; Table 1-2).
Tabla 1. Tinción inmunohistoquímica Table 1. Immunohistochemical staining
Figure imgf000028_0001
Tabla 2. Cuantificación de la intensidad de tinción inmunohistoquímica mediante programa image J.
Figure imgf000028_0001
Table 2. Quantification of immunohistochemical staining intensity by image J program.
Uretra Uretra Uretra Uretra Uretra Límite prostética membranosa bulbar del pene femenina Urethra Urethra Urethra Urethra Urethra Membranous prosthetic boundary of the female penis bulbar
CK34BE120 32,3±5,7 32,1 ±5,7 35,4±4,3 33,3±5,3 33,1 ±5,4 0-40 CK34BE120 32.3 ± 5.7 32.1 ± 5.7 35.4 ± 4.3 33.3 ± 5.3 33.1 ± 5.4 0-40
CKCAM 5.2 31 ,8±8,9 41 ,4±5,8 41 ,0±6,3 44,7±4,1 43,2±5,1 CKCAM 5.2 31, 8 ± 8.9 41, 4 ± 5.8 41, 0 ± 6.3 44.7 ± 4.1 43.2 ± 5.1
0-49 0-49
CKAE1/AE3 43,5±9,7 46,8±8,4 37,4±9,5 44,4±8,6 43,2±9,2 0-61CKAE1 / AE3 43.5 ± 9.7 46.8 ± 8.4 37.4 ± 9.5 44.4 ± 8.6 43.2 ± 9.2 0-61
CK5/6 58,4±9 56,0±9,0 65,2±5,1 59,8±7,8 53,8±9,7 0-71CK5 / 6 58.4 ± 9 56.0 ± 9.0 65.2 ± 5.1 59.8 ± 7.8 53.8 ± 9.7 0-71
CK7 43,1 ±13,1 54,8±13,2 51 ,8±13,6 59,8±17,5 85,6±8,6 0-96CK7 43.1 ± 13.1 54.8 ± 13.2 51, 8 ± 13.6 59.8 ± 17.5 85.6 ± 8.6 0-96
CK10 133,1 ±10,9 1 18,0±1 ,9 35,2±2,9 34,3±3,7 35,0±2,6 0-68CK10 133.1 ± 10.9 1 18.0 ± 1, 9 35.2 ± 2.9 34.3 ± 3.7 35.0 ± 2.6 0-68
CK13 73,1 ±1 1 ,5 80,9±6,3 61 ,1 ±13,3 50,5±13,4 41 ,6±14,8 0-88CK13 73.1 ± 1 1, 5 80.9 ± 6.3 61, 1 ± 13.3 50.5 ± 13.4 41, 6 ± 14.8 0-88
CK14 87,4±10,9 94,9±4 74,0±13,8 66,7±19,5 85,5±10,6 0-98CK14 87.4 ± 10.9 94.9 ± 4 74.0 ± 13.8 66.7 ± 19.5 85.5 ± 10.6 0-98
CK17 100,4±7 90,5±13,9 96,8±10,2 92,3±13,6 97,4±1 1 ,3 0-108CK17 100.4 ± 7 90.5 ± 13.9 96.8 ± 10.2 92.3 ± 13.6 97.4 ± 1 1, 3 0-108
CK18 81 ,9±9,5 89,2±4,8 83,0±10,9 83,3±10,8 86,0±6,9 0-94CK18 81, 9 ± 9.5 89.2 ± 4.8 83.0 ± 10.9 83.3 ± 10.8 86.0 ± 6.9 0-94
CK19 42,3±1 1 ,1 49,3±9,0 42,5±9,9 53,4±7,9 48,4±10,5 0-64CK19 42.3 ± 1 1, 1 49.3 ± 9.0 42.5 ± 9.9 53.4 ± 7.9 48.4 ± 10.5 0-64
INVOLUCRINA 71 ,9±9,0 72,2±9,6 78,5±3,7 73,5±8,0 79,4±5,5 0-82INVOLUCRINE 71, 9 ± 9.0 72.2 ± 9.6 78.5 ± 3.7 73.5 ± 8.0 79.4 ± 5.5 0-82
UROPLAQUINAIII 17,1 ±5,1 7,8±2,5 10,1 ±2,7 3,5±0,9 3,8±0,8 UROPLAQUINAIII 17.1 ± 5.1 7.8 ± 2.5 10.1 ± 2.7 3.5 ± 0.9 3.8 ± 0.8
KÍ67 2± 0,9% 6± 1 % 6± 0,4% 40± 1 % 48± 3%  KI67 2 ± 0.9% 6 ± 1% 6 ± 0.4% 40 ± 1% 48 ± 3%
Tipo de muestra de tejido: uretra humana masculina y femenina. Para cada CK, la intensidad de la tinción se basó en una escala de grises de 0 a 255 utilizando (negro-blanco) el programa Image J. Los valores se representan ± la desviación estándar. Para uroplakina III la relación de porcentaje de células teñidas en las capas de células apicales / se calculó el porcentaje de células teñidas en capas básales en tres muestras diferentes. Para Ki67 el porcentaje de células teñidas se calculó contando células de la tinción y el número total de células por área óptica. Se utilizaron tres zonas ópticas diferentes en tres muestras. Los datos se presentan como media ± desviación estándar en cada caso.  Type of tissue sample: male and female human urethra. For each CK, the intensity of staining was based on a gray scale from 0 to 255 using (black-white) the Image J program. Values are represented ± the standard deviation. For uroplakin III the ratio of percentage of stained cells in the apical cell layers / the percentage of stained cells in basal layers was calculated in three different samples. For Ki67, the percentage of stained cells was calculated by counting staining cells and the total number of cells per optical area. Three different optical zones were used in three samples. Data are presented as mean ± standard deviation in each case.

Claims

REIVINDICACIONES
1.- Un método de identificación, aislamiento y/o selección de célula(s), cultivo(s) celular(es) o tejido(s) útil(es) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de distintas porciones del tejido uretral, que comprende: a) identificar la expresión citoplasmática y/o nuclear de los marcadores CK34BE12, CKCAM 5.2, CKAE1/AE3, CK5/6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrina, Uroplaquina III y/o Ki67, en células básales y suprabasales en dicho cultivo(s) celular(es) o tejido(s); y b) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra prostática masculina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK5/6, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK18, CK19 y CK13; y ii) expresión citoplasmática en células suprabasales de CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK18, CK19, CK13 e involucrina; e iii) expresión nuclear en células suprabasales de uroplaquina; y iv) expresión nuclear en células básales de Ki67, y/o, c) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra membranosa masculina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK5/6, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK18, CK19 y CK13; e ii) expresión citoplasmática en células suprabasales de CK5/6, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK18, CK19, CK13 e involucrina; e iii) expresión nuclear en células suprabasales de uroplaquina; e iv) expresión nuclear en células básales de Ki67, y/o, d) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra bulbar masculina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK14, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK5/6, CK18, CK19 y CK13; e ii) expresión citoplasmática en células suprabasales de CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK5/6, CK18, CK19 y CK13, CK10 e involucrina; e iii) expresión nuclear en células suprabasales de uroplaquina; e iv) expresión nuclear en células básales de Ki67, y/o, e) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra peneana masculina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK14, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK5/6, CK18, CK19 y CK13; e ii) expresión citoplasmática en células suprabasales de CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK5/6, CK18, CK19, CK13, CK10 e involucrina; e iii) expresión nuclear en células suprabasales de uroplaquina; e iv) expresión nuclear en células básales de Ki67, y/o, f) seleccionar la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) del paso a) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de la uretra femenina si presenta un perfil que consista en: i) expresión citoplasmática en células básales de CK14, CK17, CKCAM 5.2, CKAE1/AE3, CK34BE12, CK5/6, CK18, CK19 y CK13; e ii) expresión citoplasmática en células suprabasales de CKCAM 5.2, CKAE1/AE3, CK34BE12, CK7, CK5/6, CK18, CK19, CK13, CK10 e involucrina; e iii) expresión nuclear en células suprabasales de uroplaquina; e iv) expresión nuclear en células básales de K¡67. 1.- A method of identification, isolation and / or selection of cell (s), cell culture (s) or tissue (s) useful to increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue, comprising: a) identify the cytoplasmic and / or nuclear expression of the markers CK34BE12, CKCAM 5.2, CKAE1 / AE3, CK5 / 6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrine, Uroplaquina III and / or Ki67, in basal and suprabasal cells in said cell culture (s) or tissue (s); and b) select the cell (s), the cell culture (s) or the tissue (s) of step a) to increase, restore or partially or totally replace the functional activity of the urethra male prostate if it has a profile that consists of: i) cytoplasmic expression in basal cells of CK5 / 6, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK18, CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK18, CK19, CK13 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; and iv) nuclear expression in basal cells of Ki67, and / or, c) select the cell (s), the cell culture (s) or the tissue (s) of step a) to increase, restore or partially or totally replace the functional activity of the male membranous urethra if it has a profile consisting of: i) cytoplasmic expression in basal cells of CK5 / 6, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK18, CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CK5 / 6, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK18, CK19, CK13 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; and iv) nuclear expression in basal cells of Ki67, and / or, d) select the cell (s), the cell culture (s) or the tissue (s) of step a) to increase, restore or partially or totally replace the functional activity of the urethra male bulbar if it has a profile consisting of: i) cytoplasmic expression in basal cells of CK14, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK5 / 6, CK18, CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK5 / 6, CK18, CK19 and CK13, CK10 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; and iv) nuclear expression in basal cells of Ki67, and / or, e) select the cell (s), the cell culture (s) or the tissue (s) of step a) to increase, restore or partially or totally replace the functional activity of the male penile urethra if it has a profile that consists of: i) cytoplasmic expression in basal cells of CK14, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK5 / 6, CK18, CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK5 / 6, CK18, CK19, CK13, CK10 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; and iv) nuclear expression in basal cells of Ki67, and / or, f) select the cell (s), the cell culture (s) or the tissue (s) of step a) to increase, restore or partially or totally replace the functional activity of the female urethra if it presents a profile consisting of: i) cytoplasmic expression in basal cells of CK14, CK17, CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK5 / 6, CK18 , CK19 and CK13; and ii) cytoplasmic expression in suprabasal cells of CKCAM 5.2, CKAE1 / AE3, CK34BE12, CK7, CK5 / 6, CK18, CK19, CK13, CK10 and implicacrine; and iii) nuclear expression in suprabasal uroplachin cells; and iv) nuclear expression in basal cells of K67.
2. - El método según la reivindicación anterior, donde la identificación de la expresión citoplasmática y/o nuclear de los marcadores del paso a) se realiza mediante inmunotinción. 2. - The method according to the preceding claim, wherein the identification of the cytoplasmic and / or nuclear expression of the markers of step a) is carried out by immunostaining.
3. - El método según cualquiera de las reivindicaciones 1-2, donde la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) identificado(s) en el paso (a) provienen de la lista que consiste en: mucosa prepucial, piel genital y extragenital, mucosa bucal, mucosa lingual, submucosa del intestino delgado, mucosa de la vejiga o cualquiera de sus combinaciones. 3. - The method according to any of claims 1-2, wherein the cell (s), the cell culture (s) or (a) tissue (s) identified in the step (a) come from the list consisting of: prepucial mucosa, genital and extragenital skin, oral mucosa, lingual mucosa, submucosa of the small intestine, bladder mucosa or any combination thereof.
4. - El método según cualquiera de las reivindicaciones 1-3, donde la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) identificado(s) en el paso (a) provienen de donantes que han sufrido parada cardiorrespiratoria o muerte encefálica o cerebral. 4. - The method according to any of claims 1-3, wherein the cell (s), the cell culture (s) or (a) tissue (s) identified in the step (a) come from donors who have suffered cardiac arrest or brain or brain death.
5. - Uso de la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) que presente al menos un perfil identificado por el método según cualquiera de las reivindicaciones 1-4, en medicina. 5. - Use of the cell (s), the cell culture (s) or (a) tissue (s) having at least one profile identified by the method according to any of claims 1- 4, in medicine.
6. - Uso de la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) según la reivindicación anterior, para la elaboración de un medicamento para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de distintas porciones del tejido uretral. 6. - Use of the cell (s), the cell culture (s) or (a) tissue (s) according to the preceding claim, for the preparation of a medicament to increase, restore or replace partially or totally the functional activity of different portions of the urethral tissue.
7. - El uso de la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) según cualquiera de las reivindicaciones 5-6, donde las distintas porciones de tejido uretral se seleccionan de la lista que consiste en: tejido uretral prostético masculino, tejido uretral membranoso masculino, tejido uretral bulbar masculino, tejido uretral peneano masculino y tejido uretral femenino 7. - The use of the cell (s), the cell culture (s) or (a) tissue (s) according to any of claims 5-6, wherein the different portions of urethral tissue are selected from the list consisting of: male prosthetic urethral tissue, male membranous urethral tissue, male bulbar urethral tissue, male penile urethral tissue and female urethral tissue
8. - El uso de la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) según cualquiera de las reivindicaciones 5-7, donde la uretra está enferma o dañada como consecuencia de una disfunción, una lesión o una enfermedad seleccionada de la lista que comprende: una neoplasia benigna o maligna, una infección, un traumatismo, una malformación congénita o una estenosis. 8. - The use of the cell (s), the cell culture (s) or (a) tissue (s) according to any of claims 5-7, wherein the urethra is diseased or damaged as a consequence of a dysfunction, an injury or a disease selected from the list comprising: a benign or malignant neoplasm, an infection, a trauma, a congenital malformation or a stricture
9. - Uso de la(s) célula(s), el/los cultivo(s) celular(es) o (un) tejido(s) que presente al menos un perfil identificado por el método según cualquiera de las reivindicaciones 1-4, para la evaluación de un producto farmacológico y/o químico. 9. - Use of the cell (s), the cell culture (s) or (a) tissue (s) having at least one profile identified by the method according to any of claims 1- 4, for the evaluation of a pharmacological and / or chemical product.
10. - Un kit o dispositivo que comprende los elementos necesarios identificar la expresión citoplasmática y/o nuclear de los marcadores CK34BE12, CKCAM 5.2, CKAE1/AE3, CK5/6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrina, Uroplaquina III y/o Ki67. 10. - A kit or device comprising the necessary elements to identify the cytoplasmic and / or nuclear expression of the markers CK34BE12, CKCAM 5.2, CKAE1 / AE3, CK5 / 6, CK7, CK10, CK13, CK14, CK17, CK18, CK19, Involucrine, Uroplaquina III and / or Ki67.
1 1. - El kit o dispositivo según la reivindicación anterior, que además comprende los elementos necesarios para realizar la identificación de los marcadores mediante inmunotinción. 1 1. - The kit or device according to the preceding claim, which further comprises the elements necessary to perform the identification of the markers by immunostaining.
12. - El kit o dispositivo según cualquiera de las reivindicaciones 8-9, que comprende los anticuerpos CK34BE12, CKCAM 5.2, CKAE1/AE3, CK5/6, CK7, CK10, CK13, CK14, anti- CK17, CK18, CK19, Involucrina, Uroplaquina III y/o Ki67. 12. - The kit or device according to any of claims 8-9, comprising the antibodies CK34BE12, CKCAM 5.2, CKAE1 / AE3, CK5 / 6, CK7, CK10, CK13, CK14, anti-CK17, CK18, CK19, Involucrine , Uroplaquina III and / or Ki67.
13.- El kit o dispositivo según la reivindicación anterior, donde los anticuerpos están modificados. 13. The kit or device according to the preceding claim, wherein the antibodies are modified.
14. - Uso del kit o dispositivo tal y como se define en las reivindicaciones 10-13, para la identificación, aislamiento y/o selección de célula(s), cultivo(s) celular(es) o tejido(s) útil(es) para incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de distintas porciones del tejido uretral. 14. - Use of the kit or device as defined in claims 10-13, for the identification, isolation and / or selection of cell (s), cell culture (s) or useful tissue (s) ( es) to increase, restore or partially or totally replace the functional activity of different portions of the urethral tissue.
15. - Una composición que comprende la(s) célula(s), el/los cultivo(s) celular(es) o el/los tejido(s) identificado(s), aislado(s) o seleccionado(s) por el método según cualquiera de las reivindicaciones 1-4. 15. - A composition comprising the cell (s), the cell culture (s) or the tissue (s) identified, isolated (s) or selected by the method according to any of claims 1-4.
16. - La composición según la reivindicación anterior, que es una composición farmacéutica. 16. - The composition according to the preceding claim, which is a pharmaceutical composition.
17.- La composición según cualquiera de las reivindicaciones 15-16, que además comprende un vehículo farmacéuticamente aceptable. 17. The composition according to any of claims 15-16, which further comprises a pharmaceutically acceptable carrier.
18.- La composición según cualquiera de las reivindicaciones 15-17, que además comprende otro principio activo. 18. The composition according to any of claims 15-17, which further comprises another active ingredient.
PCT/ES2016/070752 2015-10-22 2016-10-24 Method for the identification, isolation and/or selection of urethral tissue WO2017068226A1 (en)

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