CN105671000A - Recombinant mesenchymal stem cells, preparation method and application thereof - Google Patents
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Abstract
The invention discloses recombinant mesenchymal stem cells, a preparation method and application thereof. A first messenger RNA (ribonucleic acid) for expressing P lectin glycoprotein ligand-1, a second messenger RNA for expressing sialated Lewis oligosaccharide-X antigen and a third messenger RNA for expressing interleukin-10 are transfected into mesenchymal stem cells to obtain the recombinant mesenchymal stem cells.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of restructuring mescenchymal stem cell and its preparation method and application.
Background technology
Immune disease (immunediseases) refers to immunomodulating disequilibrium, affects the immunne response of body and the disease that causes. The immune disease of broad sense includes exception in the immune system structure that congenital or posteriority reason causes or functionally. Wherein multiple sclerosis (multiplesclerosis, MS) is a kind of common central nervous system's inflammatory demyelinating disease mediated by autoimmune response. Focus is mainly in the white matter of brain and spinal cord, invades profit, demyelination and neuronal damage in multifocal inflammatory cell, may result in many-sided dysfunctions such as sensation, motion, consciousness and neuro-cognitive. It is high that this disease is mainly in the between twenty and fifty people of 20～40 years old, relapse rate and disability rate, is the disabled modal reason of the between twenty and fifty non-traumatic nerve of people, also cannot cure completely up to now.
Wherein with mescenchymal stem cell (Mesenchymalstemcell, MSCs) transplantation treatment multiple sclerosis (multiplesclerosis, MS), inflammatory bowel, the immunoinflammatory disorders such as rheumatoid arthritis, achieve and significantly treat effect. Mesenchyme is dry carefully refers to have self-renewal capacity and can be divided into the cell mass of one group of mesoderma origin of bone, cartilage and adipose cell. Many scholars find that MSCs not only has Multidirectional Differentiation ability in recent years, and MSCs can also very effectively regulate immune function, and Just because of this, MSCs has wide treatment use prospect for the disease that autoimmunity is relevant with inflammation.
Current vein transplantation stem cell is the most commonly used and safe ready transplanting approach, but, after vein transplantation mescenchymal stem cell, owing to MSCs goes back to the nest the inefficiency of disease or inflammatory lesions, even and if a small amount of MSCs is after reaching disease location, the uncontrollability of the anti-inflammatory molecules of its secretion, greatly have impact on MSCs and treats the efficiency of immunoinflammatory disorders. This is also why be complete now or the result of also afoot hundreds of clinical trials has often drawn the result of feminine gender. Therefore, how developing to be directed to increases MSCs and goes back to the nest the position of disease or inflammation damnification and how to improve MSCs and secrete the effective ways of ability of anti-inflammatory molecules at disease location, will promotion MSCs application clinically be had great importance.
In sum, traditional mesenchymal stem cell homing is inefficient to disease or inflammatory lesions, the ability of secretion anti-inflammatory molecules is relatively low.
Summary of the invention
Based on this, it is necessary to provide a kind of disease or inflammatory lesions efficiency is higher, the higher restructuring mescenchymal stem cell of ability of secretion anti-inflammatory molecules and its preparation method and application of going back to the nest.
A kind of restructuring mescenchymal stem cell, has transfected first messenger RNA, second message,second messenger RNA and third messenger RNA in described restructuring mescenchymal stem cell;
Described first messenger RNA is used for expressing P and selects element glycoprotein ligand-1, and described second message,second messenger RNA is used for expressing sialyl Lewis oligosaccharide-X antigen, and described third messenger RNA is used for expressing IL-10 INTERLEUKIN-10.
The preparation method of a kind of mescenchymal stem cell of recombinating, comprises the steps:
First messenger RNA, second message,second messenger RNA and third messenger RNA are provided, described first messenger RNA is used for expressing P and selects element glycoprotein ligand-1, described second message,second messenger RNA is used for expressing sialyl Lewis oligosaccharide-X antigen, and described third messenger RNA is used for expressing IL-10 INTERLEUKIN-10;
Thering is provided mescenchymal stem cell, described mescenchymal stem cell is cultivated in cell culture fluid; And
Transfection reagent, described first messenger RNA, described second message,second messenger RNA and described third messenger RNA are added in the described cell culture fluid of the described mescenchymal stem cell of described cultivation and carry out gene transfection, obtain described restructuring mescenchymal stem cell.
The targeting vector of a kind of medicine, described targeting vector includes the restructuring mescenchymal stem cell described in any of the above-described item.
Above-mentioned restructuring mescenchymal stem cell, it is used for expressing P by transfection in mescenchymal stem cell and selects element glycoprotein ligand-1 (P-selectinglycoproteinligand1, PSGL-1) first messenger RNA, it is used for expressing sialyl Lewis oligosaccharide-X antigen (SialylLewis-X, SLeX) second message,second messenger RNA and be used for expressing IL-10 INTERLEUKIN-10 (Interleukin-10, IL-10) third messenger RNA, obtains restructuring mescenchymal stem cell. Test result indicate that, this restructuring mescenchymal stem cell can expression-secretion PSGL-1, SLeX and IL-10, secretion anti-inflammatory molecules ability higher. Promotive factor PSGL-1 and SLeX that go back to the nest is remarkably improved restructuring mescenchymal stem cell targeting and goes back to the nest the efficiency of disease or inflammatory lesions, in conjunction with anti-inflammatory factors IL-10, can increase the inflammation rejection ability of restructuring mescenchymal stem cell.
Accompanying drawing explanation
Fig. 1 is the flow chart of the preparation method of the restructuring mescenchymal stem cell of an embodiment;
Fig. 2 a is the result figure of the expression of the PSGL-1 of Immunofluorescence test restructuring mescenchymal stem cell;
Fig. 2 b is the result figure of the expression of the SLeX of Immunofluorescence test restructuring mescenchymal stem cell;
Fig. 2 c is the result figure of the expression of the IL-10 in Elisa detection restructuring mescenchymal stem cell supernatant;
Fig. 3 is the result figure that flow cytomery restructuring mescenchymal stem cell suppresses the lymphocytic proliferative conditions of T;
Fig. 4 a is flow cavity 1dyn/cm2Under condition, detection has transfected the result figure of the adhesive capacity of Human Umbilical Vein Endothelial Cells between mescenchymal stem cell that recombinates of PSGL-1 and SLeX;
Fig. 4 b is flow cavity 1dyn/cm2The result figure of the adhesive capacity of Human Umbilical Vein Endothelial Cells between the mescenchymal stem cell of untransfected is detected under condition;
Fig. 4 c is flow cavity 1dyn/cm2The result figure of the adhesive capacity of HL-60 iuntercellular Human Umbilical Vein Endothelial Cells is detected under condition;
Fig. 4 d is flow cavity 2dyn/cm2Under condition, detection has transfected the result figure of the adhesive capacity of Human Umbilical Vein Endothelial Cells between mescenchymal stem cell that recombinates of PSGL-1 and SLeX;
Fig. 4 e is flow cavity 2dyn/cm2The result figure of the adhesive capacity of Human Umbilical Vein Endothelial Cells between the mescenchymal stem cell of untransfected is detected under condition;
Fig. 4 f is flow cavity 2dyn/cm2The result figure of the adhesive capacity of HL-60 iuntercellular Human Umbilical Vein Endothelial Cells is detected under condition;
Fig. 4 g is flow cavity 5dyn/cm2The result figure of the adhesive capacity of Human Umbilical Vein Endothelial Cells between the restructuring mescenchymal stem cell of PSGL-1 and SLeX has been transfected under detection under condition;
Fig. 4 h is flow cavity 5dyn/cm2The result figure of the adhesive capacity of Human Umbilical Vein Endothelial Cells between the mescenchymal stem cell of untransfected is detected under condition;
Fig. 4 i is flow cavity 5dyn/cm2The result figure of the adhesive capacity of HL-60 iuntercellular Human Umbilical Vein Endothelial Cells is detected under condition;
Fig. 4 j is flow cavity 10dyn/cm2Under condition, detection has transfected the result figure of the adhesive capacity of Human Umbilical Vein Endothelial Cells between mescenchymal stem cell that recombinates of PSGL-1 and SLeX;
Fig. 4 k is flow cavity 10dyn/cm2The result figure of the adhesive capacity of Human Umbilical Vein Endothelial Cells between the mescenchymal stem cell of untransfected is detected under condition;
Fig. 4 l is flow cavity 10dyn/cm2The result figure of the adhesive capacity of HL-60 iuntercellular Human Umbilical Vein Endothelial Cells is detected under condition;
Fig. 4 m is the statistical result figure of Fig. 4 a～Fig. 4 l;
Fig. 5 a is the fluorescence microscope detection restructuring mesenchymal stem cell homing result figure to the cell quantity in spinal cord;
Fig. 5 b is the mesenchymal stem cell homing result figure to the cell quantity in spinal cord of fluorescence microscope detection untransfected;
Fig. 5 c is the statistical result figure of Fig. 5 a and Fig. 5 b;
Fig. 6 is the result figure of the symptom scores of the MS animal model of restructuring mesenchymal stem cell transplantation;
Fig. 7 is the result figure of the degree of inflammation of the EAE animal model of Immunofluorescence test restructuring mesenchymal stem cell transplantation;
Fig. 8 is the result figure of the demyelination degree of the spinal cord of the EAE animal model of Lxuol speed indigo plant dyeing detection restructuring mesenchymal stem cell transplantation.
Detailed description of the invention
Mainly in combination with drawings and the specific embodiments, restructuring mescenchymal stem cell and its preparation method and application is further explained explanation below.
A kind of restructuring mescenchymal stem cell, has transfected first messenger RNA, second message,second messenger RNA and third messenger RNA in this restructuring mescenchymal stem cell. First messenger RNA is used for expressing P and selects element glycoprotein ligand-1 (P-selectinglycoproteinligand1, PSGL-1), second message,second messenger RNA is used for expressing sialyl Lewis oligosaccharide-X antigen (SialylLewis-X, SLeX), third messenger RNA is used for expressing IL-10 INTERLEUKIN-10 (Interleukin-10, IL-10).
Messenger RNA (messageRNA, mRNA) carries hereditary information, serves as template when protein synthesis, it is easy to be transfected in cell.
In one embodiment, the gene order of first messenger RNA includes: the nucleotide sequence shown in (a), SEQIDNo.1; B the nucleotide sequence shown in () and SEQIDNo.1 has the nucleotide sequence of at least 98% homology; Or the nucleotide sequence shown in (c), SEQIDNo.1, wherein one or more bases are lacked, substituted or are increased the nucleotide sequence obtained.
The gene order of second message,second messenger RNA includes: the nucleotide sequence shown in (a), SEQIDNo.2; B the nucleotide sequence shown in () and SEQIDNo.2 has the nucleotide sequence of at least 98% homology; Or the nucleotide sequence shown in (c), SEQIDNo.2, wherein one or more bases are lacked, substituted or are increased the nucleotide sequence obtained.
The gene order of third messenger RNA includes: the nucleotide sequence shown in (a), SEQIDNo.3; B the nucleotide sequence shown in () and SEQIDNo.3 has the nucleotide sequence of at least 98% homology; Or the nucleotide sequence shown in (c), SEQIDNo.3, wherein one or more bases are lacked, substituted or are increased the nucleotide sequence obtained.
It is appreciated that, owing to the coding amino acid whose codon of same has multiple, the polymorphism of the coded sequence of albumen and variation, with nucleotide sequence, there is the nucleotide sequence of at least 98% homology or at least one change (in the coded sequence of the protein disappearance of one or more bases, insert or replace, or the aminoacid sequence of protein has one or more aminoacid deletion, insert or replace) corresponding gene order, if being transfected in mescenchymal stem cell, obtain the restructuring mescenchymal stem cell restructuring mescenchymal stem cell with the application without obvious function difference, should also be as being included within the scope of the invention.
By transfection in mescenchymal stem cell for expressing the first messenger RNA of PSGL-1, for expressing the second message,second messenger RNA of SLeX and for expressing the third messenger RNA of IL-10, obtain restructuring mescenchymal stem cell. Test result indicate that, this restructuring mescenchymal stem cell can expression-secretion PSGL-1, SLeX and IL-10, secretion anti-inflammatory molecules ability higher. Promotive factor PSGL-1 and SLeX that go back to the nest can significantly improve restructuring mescenchymal stem cell targeting and go back to the nest the efficiency of disease or inflammatory lesions, in conjunction with anti-inflammatory factors IL-10, increase the inflammation rejection ability of restructuring mescenchymal stem cell, thus increasing the effect for the treatment of.
Additionally, as it is shown in figure 1, the preparation method of a kind of mescenchymal stem cell of recombinating, including step S110～S130.
S110, offer first messenger RNA, second message,second messenger RNA and third messenger RNA, first messenger RNA is used for expressing P and selects element glycoprotein ligand-1, second message,second messenger RNA is used for expressing sialyl Lewis oligosaccharide-X antigen, and third messenger RNA is used for expressing IL-10 INTERLEUKIN-10.
First messenger RNA, second message,second messenger RNA and third messenger RNA can according to corresponding protein sequences, it is provided that triphosphoric acid ribonucleotide, RNA polymerase, Mg2+And Mn2+Deng raw material, synthesized by engineered method.
In one embodiment, the gene order of first messenger RNA includes: the nucleotide sequence shown in (a), SEQIDNo.1; B the nucleotide sequence shown in () and SEQIDNo.1 has the nucleotide sequence of at least 98% homology; Or the nucleotide sequence shown in (c), SEQIDNo.1, wherein one or more bases are lacked, substituted or are increased the nucleotide sequence obtained.
The gene order of second message,second messenger RNA includes: the nucleotide sequence shown in (a), SEQIDNo.2; B the nucleotide sequence shown in () and SEQIDNo.2 has the nucleotide sequence of at least 98% homology; Or the nucleotide sequence shown in (c), SEQIDNo.2, wherein one or more bases are lacked, substituted or are increased the nucleotide sequence obtained.
The gene order of third messenger RNA includes: the nucleotide sequence shown in (a), SEQIDNo.3; B the nucleotide sequence shown in () and SEQIDNo.3 has the nucleotide sequence of at least 98% homology; Or the nucleotide sequence shown in (c), SEQIDNo.3, wherein one or more bases are lacked, substituted or are increased the nucleotide sequence obtained.
S120, offer mescenchymal stem cell, mescenchymal stem cell is cultivated in cell culture fluid.
Mescenchymal stem cell can derive from bone marrow, Cord blood and umbilical cord tissue, placenta tissue, fatty tissue etc., can subculture preserving after extraction, it is also possible to buy from ScienCell research laboratory or other biological reagent company. The cell culture fluid cultivating mescenchymal stem cell can be DMEM culture medium.
Concrete, mescenchymal stem cell is human marrow mesenchymal stem cell or human umbilical cord mesenchymal stem cells.
The method preparing human marrow mesenchymal stem cell may comprise steps of: extracts people's bone marrow 8mL～12mL, add PBS (phosphate buffered saline(PBS)) 8mL～12mL, centrifugal (800g～1200g, 18 DEG C～22 DEG C, 18min～22min), removes supernatant. Then same method PBS washes once again. Afterwards with the PBS re-suspended cell of 8mL～12mL. Cell is added in the Ficoll (proportion is 1.073, TBD companies) of 18mL～22mL, centrifugal (1000g～1200g, 18 DEG C～22 DEG C, 25min～30min).Obtain milky fine hair shape cellular layer. Cell is sucked in the PBS of 8mL～12mL, centrifugal collecting cell (800g～1000g, 18 DEG C～22 DEG C, 18min～22min). Then with 2mL culture medium re-suspended cell, counting, with 1 × 105/cm2～5 × 105/cm2Density be inoculated in culture bottle, obtain human marrow mesenchymal stem cell after cultivation.
The method preparing human umbilical cord mesenchymal stem cells may comprise steps of: takes healthy fetal cord 4cm～5cm under aseptic condition, fully washs with PBS, wash away umbilical vein and endarterial remained blood. Separate and remove umbilical cord adventitial tissue and vascular tissue, the glue tissue of umbilical cord can be obtained. Glue tissue is cut to 0.5cm3～1cm3The piece of tissue of size, resuspended by DMEM culture medium, obtain human umbilical cord mesenchymal stem cells after cultivation.
Preferably, carry out the 0.5h～2h before the operation of gene transfection, also include the cell culture fluid changing mescenchymal stem cell. It is specially the cell culture fluid removing mescenchymal stem cell, adds fresh cell culture fluid. So can ensure that the activity of mescenchymal stem cell, improve the efficiency of transfection.
S130, by transfection reagent, first messenger RNA, second message,second messenger RNA and third messenger RNA add cultivate mescenchymal stem cell cell culture fluid in carry out gene transfection, obtain restructuring mescenchymal stem cell.
Concrete, the ratio of the addition of first messenger RNA and the cell density of mescenchymal stem cell is 0.01 μ g/cm2～1 μ g/cm2, the ratio of the addition of second message,second messenger RNA and the cell density of mescenchymal stem cell is 0.01 μ g/cm2～1 μ g/cm2, the ratio of the addition of third messenger RNA and the cell density of mescenchymal stem cell is 0.01 μ g/cm2～1 μ g/cm2. Owing to mescenchymal stem cell is the cell of adherent growth, therefore cell density can according to the area reckoning of culture dish.
Concrete, transfection reagent can be RNAiMAX transfection reagent. It is appreciated that other can promote that the reagent of gene transfection can also as the transfection reagent of the present invention.
The consumption of the transfection reagent Dosage calculation according to the expressed sequence to transfect, concrete, the mass volume ratio of first messenger RNA and transfection reagent is 0.1 μ g/ μ L～1 μ g/ μ L, the mass volume ratio that the mass volume ratio of second message,second messenger RNA and transfection reagent is 0.1 μ g/ μ L～1 μ g/ μ L, third messenger RNA and transfection reagent is 0.1 μ g/ μ L～1 μ g/ μ L. Be equivalent to 1 μ g messenger RNA and use the transfection reagent of 1 μ L～10 μ L. More specifically, 1 μ g messenger RNA uses the RNAiMAX transfection reagent of 3 μ L～5 μ L.
In one embodiment, before carrying out the operation of gene transfection, offer is also provided and subtracts serum cell culture fluid, first messenger RNA, second message,second messenger RNA and third messenger RNA are mixed with subtracting serum cell culture fluid, adds in the cell culture fluid cultivating mescenchymal stem cell afterwards and carry out gene transfection. Subtract serum cell culture fluid and refer to add the culture fluid of serum, for instance Opti-MEM culture fluid etc. Expressed sequence is dissolved mixing and is subtracting serum cell culture fluid, then carry out the efficiency that gene transfection is conducive to raising to transfect.
Subtract the consumption of the serum cell culture fluid Dosage calculation according to the expressed sequence to transfect, concrete, first messenger RNA and the mass volume ratio subtracting serum cell culture fluid are 0.01 μ g/ μ L～1 μ g/ μ L, second message,second messenger RNA and the mass volume ratio subtracting serum cell culture fluid are 0.01 μ g/ μ L～1 μ g/ μ L, third messenger RNA is 0.01 μ g/ μ L～1 μ g/ μ L with the mass volume ratio subtracting serum cell culture fluid.Being equivalent to 1 μ g messenger RNA uses 1 μ L～100 μ L's to subtract serum cell culture fluid. More specifically, 1 μ g messenger RNA uses 30 μ L～60 μ L's to subtract serum cell culture fluid.
In present embodiment, before carrying out the operation of gene transfection, also include mixing transfection reagent, first messenger RNA, second message,second messenger RNA and third messenger RNA, stand 10min～30min. Stand a period of time after transfection reagent and the expressed sequence mixing to transfect, be conducive to improving the efficiency of transfection.
Concrete, after carrying out the operation of gene transfection, also include the mescenchymal stem cell having transfected first messenger RNA, second message,second messenger RNA and third messenger RNA is cultivated 2h～6h, remove cell conditioned medium liquid, add fresh cell culture fluid and carry out cell cultivation. After cultivating 2h～6h, it is ensured that expressed sequence has transfected in entrance cell, now removes cell conditioned medium liquid and unnecessary transfection reagent or expressed sequence can be avoided to affect the growth of cell. Add fresh cell culture fluid afterwards, continue to cultivate the mescenchymal stem cell of restructuring. Namely can detect that in the mescenchymal stem cell of restructuring have secreting, expressing PSGL-1, SLeX and IL-10 after general continuation cultivation 12h～48h.
The operation such as passage, cell preservation can be carried out after being appreciated that the mescenchymal stem cell obtaining restructuring. Can directly take the mescenchymal stem cell of the restructuring preserved to carry out cell recovery cultivation when needing to use later.
In one embodiment, after carrying out the operation of gene transfection, also include detection P and select the expression contents of element glycoprotein ligand-1, sialyl Lewis oligosaccharide-X antigen and IL-10 INTERLEUKIN-10. Concrete, it is possible to it is respectively adopted the expression of PSGL-1 antibody, the SLeX antibody expression by the PSGL-1 of the mescenchymal stem cell of the method detection restructuring of Immunofluorescence test and SLeX. IL-10 secretion concentration in the culture supernatant of the mescenchymal stem cell of restructuring is detected by ELISA (MBP enzyme linked immuno-adsorbent assay).
It is shown that PSGL-1 or SLeX can be expressed in the surface of the mescenchymal stem cell of restructuring more than 90%, after transfecting seven days, the secretory volume of IL-10 may also reach up 10ng/ ten thousand cell, and the ability of expression-secretion anti-inflammatory molecules is strong.
The preparation method operating procedure of above-mentioned restructuring mescenchymal stem cell is simply and efficiently, utilize mRNA transfection method, PSGL-1 and SLeX can improve the ability that mescenchymal stem cell cell-targeting in transplantation treatment is gone back to the nest, and mescenchymal stem cell of recombinating can have the IL-10 molecule of Effective Anti inflammatory effect in inflammation part secretion, improves therapeutic effect.
Additionally, also provide for the targeting vector of a kind of medicine, targeting vector includes the restructuring mescenchymal stem cell described in any of the above-described item.
When this restructuring mescenchymal stem cell is used for medicine or the targeting vector treating multiple sclerosis and Other diseases, the effect for the treatment of can be increased, provide a kind of brand-new efficient medicine for treatment immune disease.
It is specific embodiment part below.
In following example, if no special instructions, the experimental technique of unreceipted actual conditions, the method that generally conventionally condition or test kit manufacturer are recommended realizes. What reagent used in experiment did not indicate especially is all purchased from singma company.
Embodiment 1 prepares people's bone marrow restructuring mescenchymal stem cell
(1) it is respectively synthesized a mRNA, the 2nd mRNA and the 3rd mRNA by engineered method, obtains selecting a mRNA of element glycoprotein ligand-1 for expressing P, for expressing the 2nd mRNA of sialyl Lewis oligosaccharide-X antigen and for expressing the 3rd mRNA of described IL-10 INTERLEUKIN-10.
(2) prepare human marrow mesenchymal stem cell, 1h before gene transfection will be carried out, change the fresh culture medium (DMEM) having been warmed up.
(3) prepare to subtract blood serum medium (Opti-MEM, it is purchased from GibcoLifeTechnology) and RNAiMAX transfection reagent (being purchased from Lipofectamine), first messenger RNA, second message,second messenger RNA and third messenger RNA being dissolved in subtracts in blood serum medium, add RNAiMAX transfection reagent afterwards, stand 15min after mixing, obtain transfection cocktail. Subtracting the consumption Dosage calculation according to mRNA of blood serum medium, what every microgram mRNA used 50 microlitres subtracts blood serum medium. RNAiMAX transfection reagent consumption is also the Dosage calculation according to mRNA, and every microgram mRNA uses 4 microlitre RNAiMAX.
(4) transfection cocktail prepared in step (3) is added in the cell culture fluid of cultivation mescenchymal stem cell and carry out gene transfection. The consumption of mRNA can calculate according to the cell density of mescenchymal stem cell (cell density can according to cultivate mescenchymal stem cell culture dish floor space estimation, each mRNA, every 10 square centimeters of use 1 microgram mRNA. 4h after transfection, removes the supernatant of mescenchymal stem cell, adds normal incubation medium and continues to cultivate, and obtains expressing people's bone marrow restructuring mescenchymal stem cell of PSGL-1, SLeX and IL-10.
Embodiment 2 prepares recombined human umbilical cord mesenchymal stem cells
The cell of transfection is human umbilical cord mesenchymal stem cells, and all the other conditions are identical with embodiment 1, obtains expressing people's umbilical cord restructuring mescenchymal stem cell of PSGL-1, SLeX and IL-10.
Embodiment 3PSGL-1, SLeX and IL-10 expression detection
Adopt the preparation method in embodiment 1 to obtain restructuring mescenchymal stem cell, after gene transfection 24h, be separately added into PSGL-1 antibody and SLeX antibody, by the expression of Immunofluorescence test PSGL-1 and SLeX. As shown in Figure 2 a, the expression of Immunofluorescence test SLeX is as shown in Figure 2 b for the result of the expression of Immunofluorescence test PSGL-1. Result shows surface expression PSGL-1 or SLeX of the restructuring mescenchymal stem cell more than 90%. By the IL-10 result in the restructuring mescenchymal stem cell supernatant after Elisa detection transfection 24h as shown in Figure 2 c, the IL-10 factor of the high concentration that discovery restructuring mescenchymal stem cell can be secreted, after transfecting seven days, IL-10 secretory volume may also reach up 10ng/ ten thousand cell.
Embodiment 4 recombinate mescenchymal stem cell suppress the lymphocytic propagation of T
(1) preparation method in embodiment 1 is adopted to obtain restructuring mescenchymal stem cell.
(2) making of EAE (encephalomyelitis) mouse model: after taking female C57BL/6 mouse anesthesia, back part subcutaneous injection myelin oligodendrocyte glycoprotein emulsion (MOG35-55), 200 μ g/ only, inject both sides, 100 μ g/ sides, induce MS animal model. The same day and 48 is pertussis toxin, PT solution (400ng/ time) of each lumbar injection as a child. EAE mice is after immunity 14 days, single cell suspension is made with 70 μm of cell strainer (BDFalcon) extruding, filtration spleen tissue, after the mononuclearcell of separating spleen separates, with the good splenocyte of Green fluorescent dye CFSE preliminary making that can analyze cell proliferation division number of times, cultivate and add in the culture medium that 10%FBS and 1% is dual anti-at RPMI-1640, obtain T lymphocyte. Unactivated T lymphocyte is as blank group. Stimulate by CD3 antibody (1ng/ul), CD28 antibody (1ng/ul), IL-2 antibody (2ng/ul) cytokine in vitro, obtain the T lymphocyte activated, the T lymphocyte activated and the mescenchymal stem cell (MSCs) of untransfected co-culture 6 days, and this is experiment contrast group.The T lymphocyte activated and the preparation method in embodiment 1 obtain restructuring mescenchymal stem cell and co-culture 6 days, and this is experimental group. Flow cytometer is utilized to detect the lymphocytic proliferative conditions of T of blank group, experiment contrast group and experimental group respectively, result is as shown in Figure 3, it is shown that restructuring mescenchymal stem cell is compared experiment contrast group and can significantly more be suppressed the lymphocytic propagation of T (P < 0.01).
Embodiment 5PSGL-1, SLeX transfection significantly improves the adhesive capacity of mescenchymal stem cell Human Umbilical Vein Endothelial Cells
External FlowChamber (flow cavity) is the conventional method analyzing leukocyte adhesion vascular endothelial cell ability. In order to detect the adhesive capacity of mescenchymal stem cell Human Umbilical Vein Endothelial Cells, cultivate people source brain microvessel endothelial cells in vitro (BMECs) in vitro, after 100% fusion, stimulate to simulate the blood vessel endothelium under inflammatory environment with the tumor necrosis factor (TNF-alpha) of 50ng/mL, then allow the culture medium of mescenchymal stem cell (1dyn/cm at different flow rates on BMECs by Flowchamber method2, 2dyn/cm2, 5dyn/cm2Or 10dyn/cm2) pass through. Experimental group is the restructuring mescenchymal stem cell of PSGL-1 and SLeX transfection. HL-60 cell is front myeloid-lineage leukocytes system, has very strong endotheliocyte and adheres to ability, is positive control in this experiment. The mescenchymal stem cell of untransfected is as experiment contrast group. When altogether by after 100,000 mescenchymal stem cell cells, experiment with computing matched group, experimental group and positive controls stick to the quantity of the mescenchymal stem cell cell on BMECs cell monolayer respectively. Such as Fig. 4 a～Fig. 4 c respectively 1dyn/cm2Under condition experimental group, matched group and positive controls adhesion number, Fig. 4 d～Fig. 4 f respectively 2dyn/cm2Under condition experimental group, matched group and positive controls adhesion number, Fig. 4 g～Fig. 4 i respectively 5dyn/cm2Under condition experimental group, matched group and positive controls adhesion number, Fig. 4 j～Fig. 4 l respectively 10dyn/cm2Under condition experimental group, matched group and positive controls adhesion number. Fig. 4 m be Fig. 4 a～Fig. 4 l statistical result figure. Result shows that the quantity that the restructuring mescenchymal stem cell that PSGL-1, SLeX transfection obtains sticks on BMECs is all significantly more than the mescenchymal stem cell of untransfected under flow condition four kinds different, and its difference has significant statistical significance.
Embodiment 6 is recombinated the mesenchymal stem cell homing efficiency to EAE mouse spinal cord inflammation part
(1) preparation method in embodiment 1 is adopted to obtain restructuring mescenchymal stem cell.
(2) EAE mouse immune is after 14 days, and the mescenchymal stem cell (matched group) of recombinated by tail vein injection mescenchymal stem cell or untransfected, dosage is 1 × 106A cell/mice. Cell is used that silver photoinitiator dye DiD preliminary making before injection, after injection 24 hours, take the spinal cord (L3-L5) of mice, prepare frozen section 10 microns thick, after fixing with the paraformaldehyde of 4% and embedding mounting with glycerol, being placed in fluorescence microscopy Microscopic observation and count the cell quantity going back to the nest in spinal cord in each treated animal, the result of restructuring mescenchymal stem cell is as shown in Figure 5 a, as shown in Figure 5 b, wherein Fig. 5 c is the statistical result figure of Fig. 5 a and Fig. 5 b to matched group result. Result shows, restructuring mescenchymal stem cell be can be observed, and (white fluorescent is the MSCs transplanted, the blue nucleus for all cells) quantity of spinal cord of going back to the nest is significantly more than the quantity of the mesenchymal stem cell homing of matched group untransfected, and its difference is pointed out significantly (P < 0.01) after statistical analysis.
Embodiment 7 is recombinated the animal model of mesenchymal stem cell transplantation MS (metabolism syndrome) and EAE (experimental autoimmune encephalomyelitis)
(1) foundation of MS animal model and bone marrow or umbilical cord derived neural stem cellular transplantation therapy: take female C57BL/6 mice (8 weeks～12 weeks), often group 10, after anesthesia, back part subcutaneous injection myelin oligodendrocyte glycoprotein emulsion (MOG35-55) 200 μ g/ is only, injection both sides, 100 μ g/ sides, induce MS animal model. The same day and 48 is pertussis toxin, PT solution (400ng/ time) of each lumbar injection as a child. The mescenchymal stem cell each 1 × 10 of the MOG33-35 immunity restructuring mescenchymal stem cell that after 14 days, tail vein injection (i.v.) adopts the method for embodiment 1 to prepare respectively and matched group6Individual cell/only. The cell that a part of mice accepts is with after different fluorescent dye spikes, and 24 hours post analysis are gone back to the nest the cell quantity in mouse spinal cord and ratio, and another part mice is for observing, record the change of clinical score. The change of the symptom scores of every 2 days record mices after cell transplantation. Mark is more high, and surface animal symptom is more serious. Standards of grading are as follows, normal: 0 point, flaccid tail: 0.5 point, tail paralysis: 1 point, it is unable that tail paralysis adds unilateral hindlimb: 1.5 points, unilateral hindlimb is paralysed: 2 points, bilateral hind limb weakness: 2.5 points, bilateral hind limb paralysis: 3 points, bilateral hind limb paralysis adds front myasthenia of limbs: 3.5 points, and quadriplegia is dying or dead: 4 points. As shown in Figure 6, PBS control group (placebo) presents typical disease process, and animal clinical symptom does not significantly alleviate. And mesenchymal stem cell homing of recombinating is significantly more than experiment contrast group to the quantity in MS mouse spinal cord. Illustrate that restructuring mescenchymal stem cell is more more significantly than the mescenchymal stem cell of matched group to the inhibitory action of disease. (P value that wherein PBS control group compares with the mescenchymal stem cell of experiment contrast group < and 0.05, and PBS control group recombinate with experimental group P value that mescenchymal stem cell compares < 0.01).
(2) inflammation is suppressed in restructuring mescenchymal stem cell body, promote myelin reparation and regeneration: after stem cell transplantation 4 weeks, take each group of mouse spinal cord, make frozen section to adopt, immunofluorescence dyeing agent CD45 monoclonal antibody fluorescence staining cell, detection spinal cord inflammatory cell infiltration degree, calculates inflammation area and accounts for the ratio of the substantia alba medullae spinalis gross area. Result as it is shown in fig. 7, wherein WT be normal mouse matched group, its spinal cord does not have inflammation damnification. Relative to PBS placebo group, the mescenchymal stem cell of untransfected can reduce the degree of inflammation of EAE mice. And the restructuring mescenchymal stem cell that PSGL-1/SLeX/IL-10 transfection obtains further alleviates the inflammation damnification degree of spinal cord.
(3) EAE mice accepts to transplant PBS (placebo group), the mescenchymal stem cell (experiment contrast group) of untransfected or PSGL-1/SLeX/IL-10 in immunity respectively after 14 days and transfects the restructuring mescenchymal stem cell (experimental group) obtained, after 3 weeks, take the spinal cord (L3-L5) of mice, by the demyelination degree of Lxuol speed blue (LFB) dyeing detection EAE mouse spinal cord after fixing, and calculate demyelinating area and account for the ratio of the substantia alba medullae spinalis gross area. As shown in Figure 8, wherein WT is normal mouse matched group, and its spinal cord does not have demyelination to damage. Relative to PBS placebo group, the mescenchymal stem cell of untransfected can reduce EAE mice demyelinating area. And the restructuring mescenchymal stem cell that PSGL-1/SLeX/IL-10 transfection obtains further alleviates the demyelination degree of spinal cord.
Embodiment described above only have expressed one or more embodiments of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention. It should be pointed out that, for the person of ordinary skill of the art, without departing from the inventive concept of the premise, it is also possible to making some deformation and improvement, these broadly fall into protection scope of the present invention. Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (18)
1. a restructuring mescenchymal stem cell, it is characterised in that transfected first messenger RNA, second message,second messenger RNA and third messenger RNA in described restructuring mescenchymal stem cell;
Described first messenger RNA is used for expressing P and selects element glycoprotein ligand-1, and described second message,second messenger RNA is used for expressing sialyl Lewis oligosaccharide-X antigen, and described third messenger RNA is used for expressing IL-10 INTERLEUKIN-10.
2. restructuring mescenchymal stem cell according to claim 1, it is characterised in that the gene order of described first messenger RNA includes: the nucleotide sequence shown in (a), SEQIDNo.1; B the nucleotide sequence shown in () and SEQIDNo.1 has the nucleotide sequence of at least 98% homology; Or the nucleotide sequence shown in (c), SEQIDNo.1, wherein one or more bases are lacked, substituted or are increased the nucleotide sequence obtained.
3. restructuring mescenchymal stem cell according to claim 1, it is characterised in that the gene order of described second message,second messenger RNA includes: the nucleotide sequence shown in (a), SEQIDNo.2; B the nucleotide sequence shown in () and SEQIDNo.2 has the nucleotide sequence of at least 98% homology; Or the nucleotide sequence shown in (c), SEQIDNo.2, wherein one or more bases are lacked, substituted or are increased the nucleotide sequence obtained.
4. restructuring mescenchymal stem cell according to claim 1, it is characterised in that the gene order of described third messenger RNA includes: the nucleotide sequence shown in (a), SEQIDNo.3; B the nucleotide sequence shown in () and SEQIDNo.3 has the nucleotide sequence of at least 98% homology; Or the nucleotide sequence shown in (c), SEQIDNo.3, wherein one or more bases are lacked, substituted or are increased the nucleotide sequence obtained.
5. the preparation method of a mescenchymal stem cell of recombinating, it is characterised in that comprise the steps:
First messenger RNA, second message,second messenger RNA and third messenger RNA are provided, described first messenger RNA is used for expressing P and selects element glycoprotein ligand-1, described second message,second messenger RNA is used for expressing sialyl Lewis oligosaccharide-X antigen, and described third messenger RNA is used for expressing IL-10 INTERLEUKIN-10;
Thering is provided mescenchymal stem cell, described mescenchymal stem cell is cultivated in cell culture fluid; And
Transfection reagent, described first messenger RNA, described second message,second messenger RNA and described third messenger RNA are added in the described cell culture fluid of the described mescenchymal stem cell of described cultivation and carry out gene transfection, obtain described restructuring mescenchymal stem cell.
6. the preparation method of restructuring mescenchymal stem cell according to claim 5, it is characterised in that be additionally included in and carry out 0.5h～2h before described gene transfection, changes the cell culture fluid cultivating described mescenchymal stem cell.
7. the preparation method of restructuring mescenchymal stem cell according to claim 5, it is characterized in that, it is additionally included in before carrying out described gene transfection, offer subtracts serum cell culture fluid, and with described, described first messenger RNA, described second message,second messenger RNA and described third messenger RNA are subtracted the mixing of serum cell culture fluid;
Described first messenger RNA and the described mass volume ratio subtracting serum cell culture fluid are 0.01 μ g/ μ L～1 μ g/ μ L, described second message,second messenger RNA and the described mass volume ratio subtracting serum cell culture fluid are 0.01 μ g/ μ L～1 μ g/ μ L, described third messenger RNA is 0.01 μ g/ μ L～1 μ g/ μ L with the described mass volume ratio subtracting serum cell culture fluid.
8. the preparation method of restructuring mescenchymal stem cell according to claim 5, it is characterised in that the ratio of the addition of described first messenger RNA and the cell density of described mescenchymal stem cell is 0.01 μ g/cm2～1 μ g/cm2, the ratio of the addition of described second message,second messenger RNA and the cell density of described mescenchymal stem cell is 0.01 μ g/cm2～1 μ g/cm2, the ratio of the addition of described third messenger RNA and the cell density of described mescenchymal stem cell is 0.01 μ g/cm2～1 μ g/cm2。
9. the preparation method of restructuring mescenchymal stem cell according to claim 5, it is characterized in that, it is additionally included in before carrying out described gene transfection, described transfection reagent, described first messenger RNA, described second message,second messenger RNA and described third messenger RNA are mixed, stand 10min～30min.
10. the preparation method of restructuring mescenchymal stem cell according to claim 5, it is characterised in that described transfection reagent is RNAiMAX transfection reagent.
11. the preparation method of restructuring mescenchymal stem cell according to claim 5, it is characterized in that, the mass volume ratio of described first messenger RNA and described transfection reagent is 0.1 μ g/ μ L～1 μ g/ μ L, the mass volume ratio that the mass volume ratio of described second message,second messenger RNA and described transfection reagent is 0.1 μ g/ μ L～1 μ g/ μ L, described third messenger RNA and described transfection reagent is 0.1 μ g/ μ L～1 μ g/ μ L.
12. the preparation method of restructuring mescenchymal stem cell according to claim 5, it is characterized in that, it is additionally included in after carrying out described gene transfection, the described mescenchymal stem cell having transfected described first messenger RNA, described second message,second messenger RNA and described third messenger RNA is cultivated 2h～6h, remove cell conditioned medium liquid, add fresh cell culture fluid and carry out cell cultivation.
13. the preparation method of restructuring mescenchymal stem cell according to claim 5, it is characterized in that, it is additionally included in after carrying out described gene transfection, detects described P and select the expression contents of element glycoprotein ligand-1, described sialyl Lewis oligosaccharide-X antigen and described IL-10 INTERLEUKIN-10.
14. the preparation method of restructuring mescenchymal stem cell according to claim 5, it is characterised in that the gene order of described first messenger RNA includes: the nucleotide sequence shown in (a), SEQIDNo.1; B the nucleotide sequence shown in () and SEQIDNo.1 has the nucleotide sequence of at least 98% homology; Or the nucleotide sequence shown in (c), SEQIDNo.1, wherein one or more bases are lacked, substituted or are increased the nucleotide sequence obtained.
15. the preparation method of restructuring mescenchymal stem cell according to claim 5, it is characterised in that the gene order of described second message,second messenger RNA includes: the nucleotide sequence shown in (a), SEQIDNo.2; B the nucleotide sequence shown in () and SEQIDNo.2 has the nucleotide sequence of at least 98% homology; Or the nucleotide sequence shown in (c), SEQIDNo.2, wherein one or more bases are lacked, substituted or are increased the nucleotide sequence obtained.
16. the preparation method of restructuring mescenchymal stem cell according to claim 5, it is characterised in that the gene order of described third messenger RNA includes: the nucleotide sequence shown in (a), SEQIDNo.3;B the nucleotide sequence shown in () and SEQIDNo.3 has the nucleotide sequence of at least 98% homology; Or the nucleotide sequence shown in (c), SEQIDNo.3, wherein one or more bases are lacked, substituted or are increased the nucleotide sequence obtained.
17. the preparation method of restructuring mescenchymal stem cell according to claim 5, it is characterised in that described mescenchymal stem cell is human marrow mesenchymal stem cell or human umbilical cord mesenchymal stem cells.
18. the targeting vector of a medicine, it is characterised in that described targeting vector includes the restructuring mescenchymal stem cell according to any one of Claims 1 to 5.
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