CN101224312B - Tissue-engineered androgen secretory tissue implant - Google Patents
Tissue-engineered androgen secretory tissue implant Download PDFInfo
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- CN101224312B CN101224312B CN2007100366376A CN200710036637A CN101224312B CN 101224312 B CN101224312 B CN 101224312B CN 2007100366376 A CN2007100366376 A CN 2007100366376A CN 200710036637 A CN200710036637 A CN 200710036637A CN 101224312 B CN101224312 B CN 101224312B
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Abstract
The invention discloses a tissue-engineered androgen secretion tissue graft, comprising (1) pharmaceutically accepted biomaterial and (2) a testis body cell group, wherein, the testis body cell group comprises separated and purified Leydig cells (LC) and mixture of LC and other testis body cells. After the androgen secretion tissue disclosed by the invention is transplanted in the body, androgen required by various physiological activities can be effectively secreted. The invention also provides the specific manufacturing method and purposes of the androgen secretion tissue.
Description
Technical field
The present invention relates to medical science and biomedical engineering field, relate more specifically to the tissue-engineered androgen secretory tissue that the testis somatic cell makes up.
Background technology
Androgen is the masculinity endocrine hormone, mainly by the Leydig cell in the interstitial tissue of testis (Leydig cells, LC) synthetic and secretion, the metabolic process of multiple tissue and cell in the participation body.Absence of testis that a variety of causes causes or dysfunction all can cause the androgen level low, and male's different developmental phases physiology and psychology are caused material impact, finally cause patient's quality of life seriously to descend.In recent years, along with rhythm of life is accelerated, operating pressure increases, and the increasing of aging population, and the sickness rate of this type of disease has ascendant trend gradually.Regrettably, at the hypoandrogenism disease, clinical still do not have treat measure safely and effectively.At present, method commonly used is the supplemented with exogenous testosterone.Because the testosterone supplement therapy can't reach the physiological requirement that human body has rhythm and pace of moving things testosterone secretion, final regular meeting causes a series of severe complications such as erythrocytosis, hypertension, bone density be unusual.Also there is the scholar to attempt allogeneic testis transplantation and LC cell therapy.But the former can't extensive use because of problems such as transplant rejection, afunction and spermatogenic cell ethics; Latter Ze Yin LC source is limited, the forfeiture and to lack suitable growing carrier and specific tissue's microenvironment etc. former thereby limit its clinical expansion rapidly of testosterone secretion function.Tissue engineering technique provides new way for rebuilding the androgen secretory tissue.
Tissue engineering technique can will have the seed cell and the degradable biomaterial of function compound, formation has the cell-material composite of certain form and biological function, along with cell proliferation and material degradation, reconstructed tissue takes place, the final tissue with approximate normal morphology, histological structure and physiological function that forms is finished the physiological of histoorgan and is rebuild.Yet, related organization's engineering makes up studies show that of androgen secretory tissue both at home and abroad at present, though it is feasible technically that Method of Tissue Engineering makes up the androgen secretory tissue, the structure tissue transplantation interior back of body hormonal readiness is kept of short duration, can't reach therapeutic effect steady in a long-term.
As seen, this area presses for sets up a kind of new androgen secretory tissue constructing technology, makes the androgen secretory tissue of new structure can bring into play the androgen secretory function steadily in the long term in vivo.
Summary of the invention
The present invention aims to provide a kind of tissue-engineered androgen secretory tissue implant.
Another object of the present invention provides a kind of seed cell that makes up tissue-engineered androgen secretory tissue.
A further object of the present invention provides the preparation method of above-mentioned engineered tissue grafts and seed cell.
In a first aspect of the present invention, a kind of tissue-engineered androgen secretory tissue implant is provided, it comprises: (i) biomaterial; (ii) be inoculated in the seed cell of described biomaterial, described seed cell comprises: and Leydig cell (Leydig cells, LC) or contain the testis body cell mass of LC.
In another preference, comprise in the described testis body cell mass LC and Xie Ertuoli cell (Sertolicells, SC), its quantity ratio is 1: 0.01-30; Wherein LC and SC account for 50-100% of cell mass total amount.
In another preference, the quantity of LC and SC ratio is 1: 0.1-1.
In another preference, comprise also in the testis body cell mass that quantity is the myoid cell of 0-50%.
In another preference, described LC and SC account for 90-100% of cell mass total amount.
In another preference, described testis body cell mass is taken from mammal.
In another preference, described testis body cell mass is taken from mice, rat, rabbit, monkey, Canis familiaris L., cat, sheep, pig, cattle, people.
In a second aspect of the present invention, the preparation method of above-mentioned graft is provided, be about to (i) biomaterial; (ii) seed cell mixes and to obtain tissue-engineered androgen secretory tissue implant provided by the invention, and described seed cell comprises: LC or contain the testis body cell mass of LC.
In another preference, described biomaterial comprises (but being not limited to): (1) degradable biomaterial; (2) non-degradable biomaterial.
In another preference, described preparation method comprises step:
A. incite somebody to action (ii) seed cell, be inoculated on (i) biomaterial;
B. after treating that (ii) seed cell adheres on (i) biomaterial well, transplant in In vitro culture or the body, obtain tissue-engineered androgen secretory tissue implant as claimed in claim 1, described seed cell comprises: LC or contain the testis body cell mass of LC.
In another preference, described preparation method is: seed cell is inoculated on the degradable biomaterial, after treating that seed cell adheres on the Biodegradable material well, at once or be wrapped in degradable or non-degradable biomaterial core surface behind the In vitro culture certain hour, formation has the tissue grafts of certain form, transplants in In vitro culture or the body.
In another preference, the In vitro culture time is 0 hour-15 days, preferred 7-15 days, and more preferably 7 days.
In another preference, the In vitro culture of going ahead of the rest before transplanting in the body is transplanted in the body after 0 hour-15 days, and preferred In vitro culture is transplanting in the body after 2 hours-7 days, and more preferably In vitro culture is transplanted in the body after 2 hours-4 hours.
In another preference, transplant preferred allograft in the body, preferred more homogenic individual transplanting, more preferably autotransplantation.
In another preference, transplantation site includes, but is not limited in the body of described androgen secretory tissue graft: whole body is subcutaneous, muscular tissue is interior, in the intraperitoneal, omentum majus, in subcutaneous, the groin of interior, the tunica vaginalis of testis intracavity of kidney peplos, testis original position.
In a third aspect of the present invention, a kind of testis body cell mass is provided, it comprises LC and SC, and its quantity ratio is 1: 0.01-30; Wherein LC and SC account for 50-100% of cell mass total amount.
In a fourth aspect of the present invention, the preparation method of above-mentioned testis body cell mass is provided, it comprises step: LC and SC are mixed, and the quantity ratio during mixing is 1: 0.01-30.
In another preference, the quantity ratio when LC and SC mix in the described method is 1: 0.1-1.
In another preference, also comprise step in the described method: contain the myoid cell that quantity is total cellular score 0-50%.
In another preference, the method for the above-mentioned testis body cell mass of described preparation comprises step:
A. will obtain interstitial tissue of testis's inner cell by 100-600 order drainage screen with the testis tissue of composite collagen enzymic digestion, described compound adhesive protoenzyme contains type i collagen enzyme, II Collagen Type VI enzyme and neutral protease;
B. the interstitial tissue of testis's inner cell that obtains was cultivated in culture fluid 10 minutes-72 hours, got attached cell and obtain the somatic cell of common cultivation testis.
In another preference, step a is: obtain LC in the testis, purity is 90-100%, obtains SC in the testis simultaneously, and purity is 90-100%; Step b is: the ratio with LC and SC is 1: 0.01-30 mixes, and obtains the somatic cell of common cultivation testis, and the ratio of preferred LC and SC is 1: 0.1-1.
In a fifth aspect of the present invention, the purposes of above-mentioned testis body cell mass is provided, it can be used as the seed cell that makes up tissue-engineered androgen secretory tissue; Or be used to make up tissue-engineered androgen secretory tissue implant.
In another preference, described testis body cell mass can be used for diseases such as cellular transplantation therapy androgen hyposecretion.
In another preference, described tissue-engineered androgen secretory tissue implant can be used for treating testis paramophia, disappearance or androgen paracrisis disease.
In view of the above, the invention provides a kind of new androgen secretory tissue constructing technology, make the androgen secretory tissue of new structure can bring into play the androgen secretory function steadily in the long term in vivo.
Description of drawings
Fig. 1 has shown separation and purification and the qualification result of LC, wherein,
A represents the LC of purification; B represents 3 β-HSD chromogenic enzyme substrate result; C represents 3 β-HSD immunocytochemistry result; D represents 3 β-HSD immunofluorescence laser co-focusing testing result; E represents that fluidic cell detects the result of purifying cells 3 β-HSD; F represents blank.
Fig. 2 has shown that various somatic cells are cultivated and qualification result altogether in the testis, wherein,
A represents that the whole attached cell B of common cultivation are transmission electron microscope results; C represents Xie Ertuoli cell (Sertoli cell, SC) situation; D represents other attached cell except that LC; E represents the SC form of purification; F represents SC oil red coloration result.
Fig. 3 has shown androgen secretory tissue external structure and testing result, wherein,
A represents moulding PGA/PLA timbering material; B represents timbering material scanning electron microscope result; C represents that cell-material composite sees substantially; D represents the inverted phase contrast microscope observed result; E represents the scanning electron microscope result; F represents the result of Histological section.
Fig. 4 has shown transplantation model, construct in vitro and histology result in the body, wherein,
A represents to transplant animal model; Situation when B represents to transplant in the external structure composite body C and represents that body is implanted into 45 days; D represents to make up the section situation of organizing; E represents to make up tissue tissue and learns the section result; F: high power lens makes up the situation of organizing down.
Fig. 5 has shown castration and transplanting rat hormone in vivo level.
The specific embodiment
The inventor finds after deliberation, the external time-to-live and the androgen secretory function that can obviously prolong LC cultivated in LC and other testis somatic cell altogether, after the mixing testis body cell mass that will contain LC is inoculated into the timbering material of definite shape, through In vitro culture or be transplanted to the androgen secretory tissue that can form specific modality in the animal body, this is organized in the animal body can long-term surviving and significantly improve the plasma testosterone level of animal, and can carry out allograft and do not have rejection.
According to these results of study, the inventor proposes, the mixing testis body cell mass that contains LC is the elite seed cell that makes up the androgen secretory tissue, use the androgen secretory tissue endogenous generation enduringly androgen that this cell mixing makes up, and can accept the body physiological and regulate, thereby can be used for the treatment of relevant diseases such as hypoandrogenism and/or testis be damaged.The inventor carried out more extensive and the further investigation the basis on finished the present invention.
Seed cell
Seed cell provided by the invention comprises the LC that testis body cell mass or in-vitro separation purification obtain.
The testis body cell mass
Contain LC and SC in the testis body cell mass provided by the invention, both quantity ratios are 1: 0.01--30 preferably is 1: 0.1-1, more preferably is 1: 0.3-0.5.
Described LC can be that this area is commonly used, as the LC that obtains by the Percoll separation and purification.Preferably make with the following method and to obtain: just disappear with 0.25% composite collagen enzymic digestion testis tissue to testis tissue shape, organize loosely between convoluted seminiferous tubule, but convoluted seminiferous tubule stops digestion when not having obviously fracture continuously; Filter with Φ=33 μ m stainless steel filtering nets, filtrate is centrifugal; Carry out cell inoculation with precipitation, serum-free DMEM/F12 (1:1) culture fluid was cultivated 2 hours, and adherent cell collecting promptly gets LC.
Can contain stem cell and precursor in the testis body cell mass that is provided, wherein myoid cell has been proved to be the source of LC stem cell and precursor.
Can also contain extracellular matrix, cytokine or the cell of keeping or regulate LC quantity or function in the testis body cell mass that is provided.Such as but not limited to, stem cell factor (SCF), transforming growth factor (TGF), fibroblast growth factor (bFGF), epithelium growth factor (EGF), leukocyte inhibitory factor (LIF), insulin-like growth factor I (IGFI), islet cells, neuron.
LC
LC provided by the invention can be the LC that the method for this area routine obtains, as the LC that obtains by the Percoll separation and purification.Preferably make with the following method and to obtain: just disappear with 0.25% composite collagen enzymic digestion testis tissue to testis tissue shape, organize loosely between convoluted seminiferous tubule, but convoluted seminiferous tubule stops digestion when not having obviously fracture continuously; Filter with Φ 33 μ m stainless steel filtering nets, filtrate is centrifugal; Carry out cell inoculation with precipitation, serum-free DMEM/F12 (1:1) culture fluid was cultivated 2 hours, and adherent cell collecting promptly gets LC.
The preparation of testis body cell mass
The method of the above-mentioned testis body cell mass of acquisition provided by the invention is by the mixed of quantity than 1: 0.01-30 with LC and SC.
In this mixed system, can also allow to be mixed with the myoid cell that quantity is 0-50%.
A kind of preferred manufacturing procedure, be that testis is shredded, after 0.3% composite collagen enzymic digestion to whole organizational structuries disappearances are the mud shape, Φ 100 μ m strainer filterings, filtrate is centrifugal, get the sedimentation cell inoculated and cultured, serum-free DMEM/F12 (1:1) cultivates and collects whole attached cells after 24 hours, promptly gets and cultivates the testis somatic cell altogether.
A kind of more preferred manufacturing procedure is that separation and Culture obtains LC and SC respectively, and both purity are all at 90-100%, according to 1: the ratio of 0.01-30 is mixed, and obtains the somatic cell of common cultivation testis, and the ratio of a kind of preferred LC and SC is 1: 0.1-1.
Testis body cell mass purposes
Testis body cell mass provided by the invention is a kind of new seed cell, can be used for making up tissue-engineered androgen secretory tissue, and the latter is the complex of testis body cell mass and biomaterial; The cellular transplantation therapy that also can be used for hypoandrogenism disease patient.
The material that can be used for tissue-engineered androgen secretory tissue of the present invention is medically acceptable biomaterial, comprises (but being not limited to):
(1) degradable biomaterial comprises (but being not limited to):
(a) degradability synthesized polymer material, for example poly-'alpha '-hydroxy acids (as polylactic acid PLA, polyglycolic acid PGA, poly butyric PHB etc.), poly-anhydride (polyanhydrides), poly-phosphazo (polyphosphazenes), polyamino acid (polyamino acid), false polyamino acid (pesudo-polyamino acid), poe (polyorthoesters), polyester urethane (polyest erurethane), Merlon (polycarbonate), Polyethylene Glycol, poly(ethylene oxide), poly-P-Dioxane ketone (polydioxanone) etc.;
(b) natural degradable material, for example collagen (collagen), gelatin (gelatin), ammonia polyose of candy (glycosaminoglycan, GAGs), chitosan (chitosan), chitin (chitin), alginate, calcium alginate gel etc.; Various acellular matrixes;
(c) composite of the mixture of above-mentioned material or composite, especially macromolecular material and natural material.
(2) non-degradable biomaterial comprises (but being not limited to): Medpore, bioceramic, hydroxyapatite, silicone rubber, carbon fiber, terylene, real silk etc.
Graft
Graft of the present invention is meant the class androgen secretory tissue that the complex that contains testis body cell mass and biologic bracket material or this complex form through In vitro culture.The seed cell of cultured and amplified in vitro among the present invention is inoculated on the good degradable biological timbering material of biocompatibility can forms " seed cell-biomaterial " complex, with this complex through In vitro culture or be implanted in the body, degraded and absorbed gradually along with biomaterial, seed cell continues propagation justacrine substrate, finally substitutes biomaterial and forms corresponding androgen secretory tissue.Also can contain the non-degradable biomaterial in the graft among the present invention, mainly be and above-mentioned degradable biomaterial Application of composite,, keep the mode of appearance of transplanting as the kernel of graft.
Tissue-engineered androgen secretory tissue implant preparation method of the present invention is easy, a quantity of seeds cell is mixed getting final product with pharmaceutically acceptable biomaterial.
When Biodegradable material of the present invention is the syringeability material, adjust androgen secretory tissue cell concentration with this fluent material; When biomaterial of the present invention is the solidity material, adjust seed cell suspension concentration with culture fluid, mix with degradation material then.During mixing, the ratio of culture fluid and degradation material is not particularly limited, but is advisable with the cell suspension maximum that this material can adsorb, and inoculum density calculates according to the contained cell number of every ml cell suspension.Seed cell concentration in the tissue-engineered androgen secretory tissue of the present invention is about 1 * 10 usually
5-5 * 10
8/ ml or higher preferably is 5 * 10
6-5 * 10
7/ ml more preferably is 1 * 10
7-2 * 10
7/ ml.
The extracorporeal culturing method of graft of the present invention and culture fluid are to know in this area.A kind of preferable methods be with the method for preparing complex at 37 ℃ of DMEM culture fluid that add serum-free after cultivating 2h-4h, the next day change liquid.Suitable culture fluid comprises (but being not limited to): DMEM, preferred DMEM/F-12 (1:1) culture fluid.The serum content that adds in the culture fluid is 0-5 (v/v) %, and preferred 0-1% does not more preferably contain serum.
In addition, in tissue-engineered androgen secretory tissue implant of the present invention, also can add or compound other various cells, somatomedin, various transgene component, thereby promote that seed cell is divided into the androgen secretory cell, and keep the phenotype of androgen secretory cell and/or promote the growth of androgen secretory cell.Representational material comprises (but being not limited to): human chorionic gonadotropin (hCG), lutropin (LH), transforming growth factor-β 3, BMP-2, BMP-4, BMP-7, CDMP, platelet-derived growth factor-BB (PDGF-BB), insulin, insulin like growth factor (IGF)-1 and 2, alkaline fat stem cell factor (bFGF), keratinocyte growth factor (KGF), nerve growth factor (NGF) and hepatocyte growth factor (HGF) and epidermal growth factor (EGF) etc.
Tissue-engineered androgen secretory tissue with the inventive method forms is divided into two classes, and a class is the complex that the androgen secretory tissue makes up cell and the formation of syringeability biomaterial, and this complex can be injected directly into various transplantation sites in the body.Another kind of is that the androgen secretory tissue makes up cell and non-injectivity material forms complex, and this complex can directly be implanted to interior each transplantation site of body or be implanted to each transplantation site in the body again behind In vitro culture.
The graft preparation method
The preparation method of tissue-engineered androgen secretory tissue of the present invention is easy, and the described seed cell of some is mixed with pharmaceutically acceptable biomaterial, transplants to get final product in In vitro culture or body again.
The present invention makes up in the androgen secretory tissue process, and used biologic bracket material can be the degradable biological timbering material, also can be the mixture of degradable biological timbering material and non-degradable biologic bracket material.When timbering material is degradable biomaterial, the seed cell direct inoculation is formed complex to stock support; When timbering material is the mixture of degradable biomaterial and non-degradable biomaterial, earlier the non-degradable biomaterial is prepared into the kernel of definite shape, the external application degradable biomaterial forms the combined support material, again seed cell is inoculated into stock support and forms complex, also can be with cell-degradable biomaterial complex outer non-degradable biological support surface that spreads on prefabricated shape directly or behind the In vitro culture.Used seed cell preferably contains the LC of some and ratio and the common cultivation testis body cell mass of SC, and remains with LC stem cell and precursor as far as possible.The SC cell that is contained can also provide immune exemption effect for construct in vitro and transplanted tissue, survival after transplanting in the androgen secretory tissue allogeneic that is beneficial to make up and function performance; External lasting cultivation can form the homogeneous structure with certain form and histological structure after 7-15 days; Because the immunity exemption effect of SC and the LC stem cell and the precursor of reservation in the co-cultured cell, guarantee construct in vitro be organized in can be grown to serve as after 1-3 month have certain form, the tissue of histological structure and androgen secretory function; And the androgen secretory tissue of this structure can be kept the long period (at least 6 months) in the allogeneic individuality.
The present invention makes up the process of androgen secretory tissue, mainly comprises in In vitro culture and the body transplanting.Also can add mechanical stimulation in the In vitro culture process by mode centrifugal or vibration, or the mechanical environment by androgen secretory tissue in the bioreactor analogue body, tissue to external structure applies similar mechanical stimulation, promotes the maturation of androgen secretory tissue and the improvement of mechanical property.In addition, can also add in the In vitro culture process or compound other various cytokines, somatomedin, various transgene component, thereby promote that seed cell is divided into the androgen secretory cell, and keep the phenotype of androgen secretory cell and/or promote the growth of androgen secretory cell.
Use the method that the present invention determines, can successfully construct androgen secretory tissue with good organization's structure, biochemistry composition.This tissue is the continuous release testosterone in vivo, and accepts the physiological regulation of body, makes the interior androgen level of body remain on rational physiological level, for the damaged reconstruction of androgen secretory tissue provides solid experiment basis and technical foundation.
Major advantage of the present invention is:
1, with multiple somatic cell mixing in the testis or common the cultivation, each stage of development LC is had effects such as support, nutrition, function adjusting and immunoprotection, simultaneously, also kept stem cell and the precursor of LC as far as possible to greatest extent as seed cell.Therefore, the present invention's seed cell quantity and function of stable maintenance LC for long periods of making up the androgen secretory tissue.
2, can keep the androgen secretory function of certain level after the androgen secretory tissue graft of the present invention's structure implants for long periods; and, graft all can be survived and the performance function in homogenic individuality or in the allogeneic body because the SC that contains has effects such as immunoprotection.
3, the androgen secretory tissue graft that makes up of the present invention has certain mode of appearance, and can carry out prefabricatedly as required, therefore can carry out the perfect reconstruction of function and form simultaneously.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage ratio and umber by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Embodiment 1
The directly adherent culture method altogether of preparation testis body cell mass I-
To remove the rat testis of tunica albuginea with 0.25% collagenase NB4 (Serva, Germany) with tissue mass in the 1:1 ratio, 37 ℃, 100 times/minute concussions digest to the complete obiteration of testis tissue shape, be the mud shape, the PBS that adds 3 times of volumes stops enzymic digestion, with the aperture is the stainless steel filtering net filtration of 100um, 1500 left the heart 5 minutes, abandon supernatant, the gained cell inoculation is in culture dish, to contain the DMEM/F12 of 1% hyclone, 37 ℃, cultivate under 5%CO2 and the saturated humidity condition, change liquid after 24 hours first, continue original fluid and condition of culture and be cultured to 72 hours, the gained cell is the directly adherent testis body cell mass that obtains of cultivating altogether.
Inverted phase contrast microscope is observed the cell that mainly contains three kinds of representative configuration characteristics in visible this cell mass down, and LC, SC and a small amount of myoid cell [the quantity ratio is about 1:(1-5): (0.1-1), because of Mus difference in age, this ratio has certain difference].Cultivate after 24-48 hour as seen, LC is interconnected to the cell mass of sprawling of monolayer, be distributed with the island sample therebetween and assemble the SC cell of growth, the cell lamination is assembled, so that boundary is unclear, visible a large amount of stronger fat of refractivity drip distribution in the cell island, are mingled with the pipe week myoid cell of some between this two classes cell.
Embodiment 2
Preparation testis body cell mass II-LC and SC mixing method
(1) LC separation and Culture
To remove the rat testis of tunica albuginea with 0.25% collagenase NB4 (Serva, Germany) with tissue mass in the 1:1 ratio, 37 ℃, 100 times/minute concussions digest to the testis tissue shape and just disappear, organize loose between convoluted seminiferous tubule, but when convoluted seminiferous tubule does not have obviously fracture continuously, add the culture fluid or the PBS that double digestion suspension volume and stop digestion, filter with Φ=30 μ m stainless steel filtering nets, filtrate through 1500 rev/mins centrifugal 5 minutes, gained precipitates according to every gram testis tissue gained cell inoculation 20cm
2Density inoculate.37 ℃, 5%CO
2, 95%O
2Under the condition of 100% humidity, serum-free DMEM/F12 (1:1) culture fluid was cultivated 2 hours, reject supernatant and not adherent suspension cell.
(carry out LC and identify by enzyme-specific substrate reactions of 3 β-HSD) and immunocytochemistry reaction through 3 beta-hydroxysteroid dehydrogenases for the gained attached cell.See Fig. 1.
The result shows, the LC that obtains through the differential adherent method is based on two kinds of forms: polygon and spindle shape, visible a small amount of dikaryocyte.3 β-HSD specificity zymetology reaction, 3 β-HSD immunocytochemistry, laser co-focusing and flow cytometer are identified and are positive, and confirm that the gained cell is LC, drain cell instrument identification of cell positive rate〉95%.
(2) SC separation and Culture
To remove the rat testis of tunica albuginea with 0.25% collagenase NB4 (Serva, Germany) with tissue mass in the 1:1 ratio, 37 ℃, 100 times/minute concussions digest to the testis tissue shape and just disappear, organize loose between convoluted seminiferous tubule, but when convoluted seminiferous tubule does not have obviously fracture continuously, add the culture fluid or the PBS that double digestion suspension volume and stop digestion, filter with Φ=30 μ m stainless steel filtering nets, reject filtrate, collect convoluted seminiferous tubule tissue on the filter screen, remove once more with 0.25% collagenase NB4 (Serva, Germany) with tissue mass in the 1:1 ratio, 37 ℃, 100 times/minute concussions digest to the mud shape, and 1500 change 5 minutes centrifugal collecting precipitations, are inoculated in the culture dish, add the DMEM/F12 culture fluid that contains 5% hyclone, 37 ℃, cultivate under 5%CO2 and the saturated humidity condition, collect suspension cell after 2 hours, to another culture dish with the same terms High Density Cultivation (2 * 10
7/ cm
2), changing liquid first in 24 hours, adherent cell collecting was identified and purity analysis in 48 hours.
Through oil red dyeing and transmission electron microscope observing, the gained cell is SC, and purity is more than 90%.
The result shows that the SC cell of acquisition is typical epithelioid cell characteristics, and triangle or polygon, lamellar are connected to form the characteristics that " paving stone " sample is arranged.The SC that the testis tissue of Mus age less (＜15 days) obtains contains a large amount of fat and drips, the oil red stained positive, and transmission electron microscope can be examined and see the satellite corpusculum.
(3) LC mixes with SC
The LC that obtains in the said method and SC counting back are obtained the testis body cell mass by the mixed of 2: 1 or 1:2, can be directly used in the androgen secretory tissue and make up.
Embodiment 3
Make up tissue-engineered androgen secretory tissue I
Materials and methods
1. animal model preparation
(1) preparation castration mouse model: inner incision on the bilateral groin, cut off sheath, complete taking-up testis keeps epididymis, and the layering apposition suture is with integrity, unobstructed property and the closure of cavity of tunica vaginalis after the maintenance castration.
(2) determine to transplant animal model in the body: respectively at 1 week after the castration, 2 weeks, 3 weeks, in 4 weeks, in 6 weeks, blood is got in 8 weeks and 12 all heart punctures, detect level of serum testosterone, the monitoring testosterone begins to descend and the low-level critical point of keeping, and the time point of the selected low-level stable maintenance of testosterone determines to transplant in the body animal model as the Best Times of transplanting in the body with this.
2. seed cell preparation
(1) LC separation and Culture: the LC that obtains purification by embodiment 2 described methods.
(2) testis body cell mass separation and Culture: obtain to cultivate altogether the testis body cell mass by embodiment 1 described method.
3. biologic bracket material preparation
Degradable poly hydroxyacetic acid (PGA) 10mg, plasticity is diameter 8mm in advance, and the disk shape structure of thick 3mm drips a small amount of 0.1% polylactic acid (PLA) (dichloromethane is a solvent) solid shape, form the PGA/PLA biologic bracket material, standby through infiltration of 75% ethanol and ultraviolet radiation disinfection.
4. androgen secretory tissue external structure and functional evaluation:
(1) experiment grouping: 1. simple LC group; 2. cultivate the seed cell group altogether.
(2) the cell material complex makes up and In vitro culture: with two groups of cells respectively with same cell density (20 * 10
6Individual cell/ml) and total amount (6 * 10
6Individual cell /) drip on biomaterial scaffolds, cultivate under 37 ℃, 5%CO2 and the saturated humidity condition after 4 hours, add that serum-free DMEM/F12 (1:1) culture fluid is conventional to be cultivated, the next day change liquid.
(3) Biological Detection and functional evaluation: every day collecting cell material composite supernatant, detect its testosterone levels, observe the In vitro culture function and hold time.Draw materials during respectively at 1 week of In vitro culture and 2 weeks, histology makes up form, the material degradation situation of tissue, SABC identify each cell component ultrastructure in each cell component, electron microscopic observation complex, functional status and with the compatibility of stock support, determine transplant time in the body according to testing result.
5. make up and organize homogenic castration Mus to transplant and functional evaluation
(1) transplants in the body: be divided into different groups transplanting animal model in the homogenic Wister rat body, each treated animal is respectively through scrotal incision, cut off sheath, passivity is separated the exposure cavity of tunica vaginalis, the corresponding group cell-material composite of external structure is implanted, with the epididymis tissue encapsulation itself and otch are isolated, apposition suture sheath and each layer tissue, attention keeps the sealing and the integrity of sheath.
(2) lapse in histology, functional evaluation and the body: each group is observed forming process, material degradation process, histology's form and the vascularization situation of construct in vitro tissue respectively at transplanting 4 weeks of back, 6 weeks, 9 weeks, 12 weeks and drawing materials in 24 weeks.Blood is got in heart puncture, measures two groups with Access automatic immunity cell mass and transplants the Mus level of serum testosterone, and the result represents that with mean ± standard deviation the t that relatively goes in twos between group checks.
The result
1. animal model
(1) castration mouse model: survival rate is 100% after the Wister rat castration, the bilateral testes disappearance, and life habit does not have obvious change.
(2) determine to transplant animal model in the body: 1 week and 2 all level of serum testosterone and normal mice no difference of science of statistics after the castration, castration is after 3 weeks, level of serum testosterone begins to descend, in this phase, level of serum testosterone differs greatly between individuality, to the 4th week, testosterone levels descends obviously, and being maintained at reduced levels, in the 6th week, 8 weeks were compared no difference of science of statistics with 12 all level of serum testosterone and the 4th week.In view of the above, remove gesture 4 all animals as the animal model of transplanting in the body.
2.LC cultivate and evaluation: see Fig. 1.
The result shows, the LC that obtains through the differential adherent method is based on two kinds of forms: polygon and spindle shape, visible a small amount of dikaryocyte.3 β-HSD specificity zymetology reaction, 3 β-HSD immunocytochemistry, laser co-focusing and flow cytometer are identified and are positive, and confirm that the gained cell is LC, drain cell instrument identification of cell purity positive rate〉95%.
3. cultivate testis somatic cell morphology altogether: see Fig. 2.
The result shows, altogether various adherent somatic cells can symbiosis in the testis under the cultivation conditions, Electronic Speculum and oil red dyeing confirm that the Sertoli cell is the island sample and assembles growth, being rich in fat in the Sertoli cell drips, and the Sertoli iuntercellular forms desmosome and connects (shown in the arrow), and a large amount of LC and a small amount of fibroblast-like cell distribute between cell island week and island.
4. cell material complex biological characteristics: see Fig. 3.
The result shows, simple LC and cultivate the testis somatic cell altogether and biomaterial PGA/PLA has good bioaffinity, can begin to take shape in 2 weeks of In vitro culture and have certain form and histological structure, see cell well-grown on material under the inverted phase contrast microscope, the substrate secretion is vigorous; Scanning electron microscope is seen the mixing with cells growth and is had excellent biological compatibility with PGA; Histological section's visible tissue PGA that begins to take shape and do not degrade.
5. the androgen secretory tissue forms: see Fig. 4.
The result shows that two groups of cell material complex of gross examination of skeletal muscle in 2 months, all can be kept graft form and volume in transplanting animal body, and in the time of 3 months, simple LC group graft is little than the co-cultured cell group obviously.Form when co-cultured cell group graft still keeps transplanting, strong but pliable in texture, section sees that interior tissue forms well, the structure homogeneous, Histological section shows that transplanted tissue is made of fibrous septum and nest like cell group, sees the organization internal vascularization under the high power lens.
During to 6 months, simple LC transplantation group has been difficult to find transplanted tissue, and cultivation group graft form is kept well altogether, and volume slightly dwindles.
6. level of serum testosterone detects: see Fig. 5.
The result shows, two treated animal plasma testosterone level 4 weeks, 6 weeks, 9 weeks and all be significantly higher than simple castration Mus (P＜0.01) 12 weeks after transplanting, co-cultured cell material transplantation group testosterone raises to be maintained to and transplants 24 weeks of back.Transplant Mus for two groups and compare, co-cultured cell transplantation group plasma testosterone level is significantly higher than simple LC transplantation group (P＜0.01).
After 4 weeks of Wister rat castration, level of serum testosterone reduce to maintenance level (0.56 ± 0.054ng/ml, n=11); Plasma testosterone level rises beginning in the 6th week behind the simple LC of castration 4 above rat implantations of week, and to 9 to 12 weeks maintained certain level, after begin to fall after rise, to 24 week and castration Mus no difference of science of statistics; Co-cultured cell transplantation group testosterone levels is significantly increased when transplanting for 4 weeks, and the 24th week still maintained certain testosterone levels; 4th, 6,9 and 12 all two transplantation group plasma testosterone level have statistically-significant difference (P＜0.01).
Embodiment 4
Make up tissue-engineered androgen secretory tissue II
Method with reference to embodiment 3 makes up, and wherein applied seed cell comes from embodiment 2 prepared testis body cell masses.
The result is similar to Example 3.
Embodiment 5
Make up tissue-engineered androgen secretory tissue III
Seed cell and the external structure method used with embodiment 4 are identical, but the animal model of implanting is the allogeneic animal model.
The result is similar to Example 3, make up tissue can be in unusual fluctuation object of the same race the androgen of long-term surviving justacrine certain level.
Embodiment 6
Make up tissue-engineered androgen secretory tissue IV
Method with reference to embodiment 3 makes up, and different is that used timbering material is the PGA/Medpore compound rest that contains non-degradable material Medpore kernel.
The preparation method of compound rest: earlier with the prefabricated growth 1.3cm of Medpore, the testis form support of diameter 0.8cm forms compound rest with 15mg PGA fiber package around the Medpore kernel again as kernel.The seed cell direct inoculation is on the outer PGA fiber of compound rest.
The result is similar to Example 3, and newborn androgen secretory tissue and Medpore kernel have good interface and integrate.
Comparative Examples 1
Make up tissue-engineered androgen secretory tissue I '
LC that embodiment 2 is obtained and Sertoli cell are that 1: 0.005 mixed obtains the testis body cell mass by the quantity ratio.
Method with reference to embodiment 3 makes up and test then.
The result:
1. cultivate testis somatic cell morphology altogether: cultured cell contains the SC of minute quantity, and the cell island of formation is little and irregular, and the growth conditions of LC cell and embodiment 3 interior simple LC do not have remarkable morphology difference in the island.
2. cell material complex biological characteristics: the simple LC group in the similar embodiment 3.
3. engineering tissue forms: the simple LC group in the similar embodiment 3.
4. level of serum testosterone detects: the simple LC group in the similar embodiment 3.
Comparative Examples 2
Make up tissue-engineered androgen secretory tissue II '
LC that embodiment 1 is obtained and Sertoli cell are that 1: 50 mixed obtains the testis body cell mass by the quantity ratio.
Method with reference to embodiment 3 makes up and test then.
The result:
1. cultivate testis somatic cell morphology altogether: cultured cell contains a spot of LC, prolongs with incubation time, and cell quantity reduces relatively, can not form dominant growth, almost is difficult to see the cell of typical LC form to 1 week, is mainly SC in the cell mass.
2. cell material complex biological characteristics: cell and biomaterial adhere to good, but the androgen secretion level be starkly lower than the LC group and implement 3, implement 4 and implement the preparation of 5 methods respectively organize the androgen secretion level that complex produces, even be 0.
3. engineering tissue forms: similar embodiment 3, and can form homogeneous structure's structure of certain form and structure, and keep the long time, can vascularization, but tissue slice is observed morphology, forms cell and is mainly the SC that volume is big, bulk distributes.
4. level of serum testosterone detects: the plasma testosterone level of transplantation group castrated rats does not have obvious improvement, be starkly lower than simple LC group and implement 3, implement 4 and implement the preparation of 5 methods respectively organize the androgen secretion level that complex produces.
Discuss
For the above-mentioned reasons, the inventor thinks that the interior multiple somatic cell mixed transplantation of testis is hopeful to solve simultaneously the interior LC of shortage of graft stem cell, transplanting LC lacks several respects problems such as microenvironment and alloimmunity repulsion of growing.But because the equal adherent growth of intratesticular multiple somatic cell, therefore, just can obtain comprising multiple somatic cells such as the LC at different levels of LC stem cell and SC by adherent altogether cultivation, carrying out the androgen secretory tissue with these co-cultured cells as seed cell makes up, keep under the similar physiological status these intercellularly to connect each other and influence each other as far as possible, the physiological that can guarantee LC is upgraded and additional (because containing the LC stem cell), helping survival of LC longer-term and biological function again keeps, be expected to from cell quantity, many aspects such as function and kind satisfy the requirement of androgen secretory tissue structure to seed cell, make the cell composition and the function that make up tissue more approach physiological status.The more important thing is,, form the peculiar immunity of testis tissue and exempt microenvironment, be very beneficial for making up the immunity screen fraud after the tissue transplantation owing to wherein contain a large amount of SC and other multiple somatic cell, thus in allogeneic long-term surviving.
Result of the present invention shows, the cell mixing of LS and SC or cultivate the testis somatic cell altogether and can have excellent biological compatibility at external and degradable biomaterial, plasma testosterone level homogenic and allogeneic castration Mus can be obviously improved after cell-material composite implants, and original volume and form can be kept.The more important thing is in the visible graft of histology a large amount of vascularization are arranged, this and tissue engineering technique make up the interior forming process basically identical of body of other tissue, also are to make up the important assurance of organizing physiological regulation in long-term surviving and the acceptor.Therefore, the testis mixture cell of using cultivation altogether might construct the androgen secretory tissue with certain form, histological structure and testosterone secretion function fully as seed cell.
The above only is preferred embodiment of the present invention, be not in order to limit essence technology contents scope of the present invention, essence technology contents of the present invention is broadly to be defined in the claim scope of application, any technology entity or method that other people finish, if it is defined identical with the claim scope of application, or a kind of change of equivalence, all will be regarded as being covered by among this claim scope.
Claims (9)
1. a tissue-engineered androgen secretory tissue implant is characterized in that it comprises: (i) biomaterial; (ii) be inoculated in the seed cell of described biomaterial, described seed cell is the testis body cell mass that contains LC;
Comprise LC and Xie Ertuoli cell in the described testis body cell mass, its quantity ratio is 1: 0.01-30; Wherein LC and SC account for the 50-100% of cell mass total amount.
2. graft as claimed in claim 1 is characterized in that, the quantity ratio of LC and SC is 1: 0.1-1.
3. graft as claimed in claim 1 is characterized in that, comprises also in the testis body cell mass that quantity is the myoid cell of 0-50%.
4. the preparation method of graft as claimed in claim 1 is characterized in that, with (i) biomaterial; (ii) the seed cell mixing obtains tissue-engineered androgen secretory tissue implant as claimed in claim 1, and described seed cell is the testis body cell mass that contains LC;
Comprise LC and Xie Ertuoli cell in the described testis body cell mass, its quantity ratio is 1: 0.01-30; Wherein LC and SC account for the 50-100% of cell mass total amount.
5. a testis body cell mass is characterized in that, it comprises LC and SC, and its quantity ratio is 1: 0.01-30; Wherein LC and SC account for the 50-100% of cell mass total amount.
6. the preparation method of a testis body cell mass as claimed in claim 5 is characterized in that, it comprises step: LC and SC are mixed, and the quantity ratio during mixing is 1: 0.01-30.
7. preparation method as claimed in claim 6 is characterized in that, the quantity ratio during mixing is 1: 0.1-1.
8. preparation method as claimed in claim 6 is characterized in that it comprises step:
A. obtain LC in the testis, purity is 90-100%, obtains SC in the testis simultaneously, and purity is 90-100%;
B. the ratio with LC and SC is 1: 0.01-30 mixes, and obtains the somatic cell of common cultivation testis.
9. the purposes of a testis body cell mass as claimed in claim 5 is characterized in that, as the seed cell that makes up tissue-engineered androgen secretory tissue; Or be used to make up tissue-engineered androgen secretory tissue implant.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2362457Y (en) * | 1999-03-25 | 2000-02-09 | 天津市泌尿外科研究所 | Artificial testicle |
| JP2000139968A (en) * | 1998-11-13 | 2000-05-23 | Yasuto Takeuchi | Testicle replacement |
| US6620203B2 (en) * | 2000-04-28 | 2003-09-16 | Anthony Atala | Tissue engineered testicular prosthesis and use thereof |
| CN1513564A (en) * | 2003-08-20 | 2004-07-21 | 李庆昌 | Germ interstitial antisenility technology |
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2007
- 2007-01-19 CN CN2007100366376A patent/CN101224312B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000139968A (en) * | 1998-11-13 | 2000-05-23 | Yasuto Takeuchi | Testicle replacement |
| CN2362457Y (en) * | 1999-03-25 | 2000-02-09 | 天津市泌尿外科研究所 | Artificial testicle |
| US6620203B2 (en) * | 2000-04-28 | 2003-09-16 | Anthony Atala | Tissue engineered testicular prosthesis and use thereof |
| CN1513564A (en) * | 2003-08-20 | 2004-07-21 | 李庆昌 | Germ interstitial antisenility technology |
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