CN105087467A - Preparation method of kidney sertoli cells and special mediums thereof - Google Patents
Preparation method of kidney sertoli cells and special mediums thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of kidney sertoli cells and special mediums thereof, in particular provides a method of inducing mesenchymal stem cells into the kidney sertoli cells, and the method comprises the following steps: (1) inoculating the mesenchymal stem cells into a medium I; and (2) transferring the mesenchymal stem cells done in step (1) into a medium II so as to obtain the kidney sertoli cells, wherein the medium I is composed of a zooblast basic medium and solutes, and the solutes comprise 1.9 percent to 2.1 percent of fetal calf serum, 9.75ng/mL to 10.25ng/mL of activin A and 0.95*10 to 5mol/L to 1.05*10 to 5mol/L of tretinoin; and the medium II is composed of a zooblast basic medium and solutes, and the solutes comprise 1.9 percent to 2.1 percent of fetal calf serum, 9.75ng/mL to 10.25ng/mL of activin A, 0.95*10 to 7mol/L to 1.05*10 to 7mol/L of tretinoin and 19ng/ml to 21ng/ml of BMP7. An induction culture method of the invention is suitable for the mesenchymal stem cells of various sources, and is free of virus introduction, easy in operation and high in efficiency.
Description
Technical field
The present invention relates to by the method for mesenchyma stem cell differentiation induction cell, particularly the preparation method of Renal Podocytes and special culture media thereof.
Background technology
Kidney is one of most important organ of human body, its main Physiological Function is by generating urine to remove interior metabolism product and to enter body surplus substance and poisonous substance etc., retain moisture content and the adjustment of other useful matteies realization to body fluid, ionogen and acid base equilibrium through heavy absorptive function simultaneously, maintain the stable of organismic internal environment.
Renal Podocytes, i.e. renal capsule visceral layer epithelial cell are the important component maintaining glomerular filtration function normal configuration and function, and at maintenance glomerular permeability, antagonism capillary vessel cavity fluid hydrostaticpressure aspect plays a significant role.The many factors such as hypertension, diabetes, nephrotoxic drugs, primary glomerulonephritis, tumour can cause the damage of Renal Podocytes.Because podocyte is well differentiated thesocyte, once disappearance is renewable hardly, the progressive damage of podocyte is lost, cause the integrity of glomerular filtration membrane to be destroyed, form proteinuria, and albuminuretic appearance increases the weight of Podocytes in Renal Tissue further, the vicious cycle formed thus finally causes the generation of glomerular sclerosis, affect renal function, cause chronic renal disease (chronickidneydisease, CKD).Take various possible methods for the treatment of for these pathogenesis clinically, comprise and control blood pressure, blood sugar, blood fat, drug intervention inflammation-inhibiting, immunity moderation, the strict albumen that controls are taken in, are reduced proteinuria etc., delay the progress of kidney disease as much as possible.But because therapeutic response differs, some patients's state of an illness continuing advances, the Progressive symmetric erythrokeratodermia of renal function declines, and finally causes end stagerenaldisease (endstagerenaldisease, ESRD).
According to US Renal Data registration system (theUnitedStatesRenalDataSystem, USRDS) show, the incidence of CKD in adult is approximately 13.6%, and in recent years the sickness rate of CKD in continuous increase, end 2012, U.S. domestic ESRD patient reaches 636905 examples, and newly-increased patient 114813 example, wherein 88638 people die from ESRD.Hemodialysis, peritoneal dialysis and renal transplantation are still the main and the most effective treatment means of current CKD and ESKD, but dialysis can only partly replace the filtering function of kidney, renal transplantation to be still current ESRD patient gets well ideal methods for the treatment of.But transplant organ resource is seriously deficient, disease Man's Demands can not be met far away, overwhelming majority ESRD patient dies from chronic renal disease and related complication thereof owing to failing to accept renal transplantation in time, and chronic renal disease has become one of disease of serious threat human health.
Stem cell (stemcell) is a class hyperproliferation, has the cell of self and multi-lineage potential, and under certain condition, stem cell can be divided into several functions cell.Can be divided into according to source of human stem cell: embryonic stem cell (embryonicstemcells, ESCs), adult stem cell and induced multi-potent stem cells (inducedpluripotentstemcells, iPSs).Mescenchymal stem cell (mesenchymalstemcells, MSCs) be the adult stem cell that a class has immunoloregulation function and highly plastic, its wide material sources, draw materials conveniently, and have reduced immunogenicity, not by ethics restriction and the feature not becoming knurl danger, therefore being seed cell with MSCs, is Renal Podocytes by external evoked directed differentiation, repairs glomerular filtration function, improve the effect of renal function, there is higher clinical value.
Under suitable condition of in vitro culture, mescenchymal stem cell can be induced to differentiate into the multiple mature cells such as neurocyte, myocardial cell, insulin secreting cells, liver cell, in order to repair tissue and the organ of replacement function damage, the effective function improving corresponding damaged tissue.Recent study result also shows, the stem cell in multiple source has the potential to renal tissue cytodifferentiation, can substitute impaired kidney cell.At least 26 kinds of cells are comprised in ripe kidney, existing induction system, the kind of the induced dry-cell efficiency of breaking up to kidney cell and noble cells is all very limited in vitro, major part research report all concentrates on the differentiation-inducing of renal tubular cell, differentiation research for Renal Podocytes is also in desk study, and there is no report for derived mesenchymal stem cells in vitro to Renal Podocytes inducing culture system.Only there is pertinent literature to utilize induced multi-potent stem cells (ips) to induce and obtain Renal Podocytes, because iPSCs is induced by multiple adult cell reprogrammed to obtain, wide material sources, avoid the problem of ethnics Problem that embryonic stem cell faces and immunological rejection.But because current iPSCs transformation efficiency is low, reprogrammed mechanism indefinite and may cause the activation of oncogene in reprogrammed process, also limit the further application of iPSCs cell in clinical treatment.
Summary of the invention
The object of this invention is to provide a kind of preparation method and special culture media thereof of Renal Podocytes.
On the one hand, the invention provides mescenchymal stem cell induction is the method for Renal Podocytes, and it comprises: 1) be inoculated in by mescenchymal stem cell in substratum 1; And 2) by completing steps 1) mescenchymal stem cell transfer in substratum 2 to obtain Renal Podocytes, wherein said substratum 1 is made up of zooblast basic medium and solute, and described solute comprises: the foetal calf serum of 1.9%-2.1%, 9.75ng/mL-10.25ng/mLactivinA and 0.95 × 10
-5mol/L-1.05 × 10
-5mol/L vitamin A acid; Wherein said substratum 2 is made up of zooblast basic medium and solute, and described solute comprises: the foetal calf serum of 1.9%-2.1%, 9.75ng/mL-10.25ng/mLactivinA, 0.95 × 10
-7mol/L-1.05 × 10
-7mol/L vitamin A acid and 19ng/ml-21ng/mlBMP7.
Preferably, method of the present invention comprises the steps:
1) mescenchymal stem cell is inoculated in described substratum 1, cultivates 2-4 days; And
2) by completing steps 1) mescenchymal stem cell transfer in described substratum 2, cultivate and obtain Renal Podocytes in 5-7 days.
Preferably, in described step 1) in cultivate 3 days, and in described step 2) in cultivate 6 days.
Preferably, zooblast basic medium described in method of the present invention is DMEM/F12 substratum, and in substratum 1, solute comprises the foetal calf serum of 2%, the activinA of 10ng/mL and 10
-5the vitamin A acid of mol/L; And comprise the foetal calf serum of 2%, the activinA, 10 of 10ng/mL at substratum 2
-7the vitamin A acid of mol/L and the BMP7 of 20ng/ml.
Preferably, in described step 1 of the present invention) in, the starting point concentration of described mescenchymal stem cell in described substratum 1 can be 3 × 10
5individual/ml-6 × 10
5individual/ml, is preferably 5 × 10
5individual/ml; Preferably, the culture condition of culturing process can be: 37 DEG C, 5%CO2, relative air humidity be cultivate in the incubator of 95%.
Preferably, wherein said step 1) for add substratum 1 in 6 orifice plates, every hole 2ml, then inoculate the 3rd generation mescenchymal stem cell, make its concentration in the medium be 5 × 10
5individual/ml, 37 DEG C, 5%CO2, relative air humidity be cultivate 3 days in the incubator of 95%, suction in every 1.5 days is abandoned supernatant and adds the new substratum of 2ml 1; And described step 2) for inhaling the supernatant abandoned in 6 orifice plates of step 1, add substratum 2, every hole 2ml, 37 DEG C, 5%CO2, relative air humidity be cultivate 6 days in the incubator of 95%, suction in every 2 days is abandoned supernatant and adds the new substratum of 2ml.
Preferably, method feature of the present invention is that described mescenchymal stem cell is fat mesenchymal stem cell; Preferably, described fat mesenchymal stem cell is human mesenchymal stem cell; Preferably, described mescenchymal stem cell is third generation mescenchymal stem cell; Preferably, described mescenchymal stem cell is behaved fatty third generation mescenchymal stem cell; Preferably, described mescenchymal stem cell is cytokine CD29, CD44, CD105 and Flk-1 are the positive, and cytokine CD31, CD34, CD106 and HLA-DR are negative cell.
On the other hand, the present invention also provides a kind of test kit, and it comprises substratum 1 and substratum 2, and described substratum 1 and 2 is made up of zooblast basic medium and solute; In substratum 1, solute comprises: the foetal calf serum of 1.9%-2.1%, 9.75ng/mL-10.25ng/mLactivinA and 0.95 × 10
-5mol/L-1.05 × 10
-5mol/L vitamin A acid; And solute comprises in substratum 2: the foetal calf serum of 1.9%-2.1%, 9.75ng/mL-10.25ng/mLactivinA, 0.95 × 10
-7mol/L-1.05 × 10
-7mol/L vitamin A acid and 19ng/ml-21ng/mlBMP7.
In test kit of the present invention, wherein said zooblast basic medium is DMEM/F12 substratum, comprises the foetal calf serum of 2%, the activinA of 10ng/mL and 10 at substratum 1
-5the vitamin A acid of mol/L; And comprise the foetal calf serum of 2%, the activinA, 10 of 10ng/mL at substratum 2
-7the vitamin A acid of mol/L and the BMP7 of 20ng/ml.
On the other hand, the invention provides the Renal Podocytes utilizing method of the present invention to obtain, preferably Nephrin, Podocin and WT1 expression amount of described Renal Podocytes is higher than originated mescenchymal stem cell.
In another, the present invention also provides the Renal Podocytes that utilizes method of the present invention to obtain for the preparation of the purposes in treatment Renal Podocytes relative disease and the medicine at cellular transplantation therapy, and preferably described Renal Podocytes relative disease is minimal change glomerulonephritis, FSGS, membranous nephropathy, diabetic nephropathy.
Wherein, human activin A (activinA) of the present invention is purchased from PeproTech company of Britain, and catalog number is 120-14.Vitamin A acid available from Sigma, catalog number is R2625.Bone morphogenetic protein4 (bonemorphogeneticprotein7, BMP7) is purchased from PeproTech company, and catalog number is 120-03.
Vitamin A acid and activinA are inducible factor important in cell differentiation procedure, play an important role in the differentiation and development of various kinds of cell, in Induction Process, by adding different cytokines and combination of cytokines, its acting in conjunction is in stem cell, and induced dry-cell is to various cytodifferentiation.Wherein to cultivate disclosed before, although comprise these compositions, be not limited to these compositions, and working concentration and present patent application substratum working concentration and using method all inconsistent.In metanephros growth course, under BMP7 effect, metanephros mesenchymal cell contacts with ureteric bud and mutually assembles, and promotes the existence of metanephros mesenchymal cell.BMP7 also participates in the growth of podocyte simultaneously, is the important inducible factor of kidney development.
In described substratum, whole composition is all that induction is necessary, the vitamin A acid concentration that wherein inducing culture 1 uses is 100 times of substratum 2 vitamin A acid working concentration, first the vitamin A acid of high density and activinA combined induction 3 days is used, the mescenchymal stem cell in effective induced lipolysis source is to Renal Podocytes precursor cell differentiation, and the vitamin A acid of later stage lower concentration, and the epithelium that is situated between BMP7 and activinA combined action effective inducing cell generation in 6 days transforms and is divided into ripe Renal Podocytes further.
By method of the present invention, mescenchymal stem cell can obtain Renal Podocytes in vitro through two one-step inducing methods, and cell can express podocyte specific gene, and finally obtains Renal Podocytes.
Method for inducing and cultivating of the present invention, is suitable for various adipose-derived mescenchymal stem cell, and the virus-free importing of this method for inducing and cultivating, easy to operate, efficiency is high.Especially, the homogeneous Renal Podocytes of a large amount of differentiation and maturation, phenotype is obtained in the induction time that method of the present invention can be shorter in vitro, the podocyte obtained expresses slit diaphragm associated protein molecule, plays an important role for the structure and function maintaining renal glomerulus slit diaphragm.The Renal Podocytes that the inventive method obtains provides experimental basis and Clinical Evidence, for cellular transplantation therapy provides new approach for treating the podocyte relative diseases such as minimal change glomerulonephritis, FSGS, membranous nephropathy, diabetic nephropathy.
Accompanying drawing explanation
Fig. 1 is the form of human mesenchymal stem cell (seed cell of Renal Podocytes).
Fig. 2 is the immunophenotype detected result of human mesenchymal stem cell (seed cell of Renal Podocytes).
Fig. 3 derived mesenchymal stem cells in vitro of behaving breaks up the form of the Renal Podocytes obtained.
Fig. 4 derived mesenchymal stem cells in vitro of behaving breaks up the real-time quantitative PCR detected result of the Renal Podocytes obtained.
Fig. 5 A-Fig. 5 F is the immunocyte fluorescent dye detected result that people's derived mesenchymal stem cells in vitro breaks up the Renal Podocytes obtained.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
Penicillin, Streptomycin sulphate and trypsinase-EDTA are purchased from GIBCO company.Trizol purchased from American Invitrogen company, OligodT, M-MLV reversed transcriptive enzyme, Taq DNA polymerase, dNTP and RNA enzyme inhibitors are purchased from Japanese Takara company.
Goat anti human WT1 recombinant antibodies, goat anti-human Nephrin recombinant antibodies purchased from American R & D company.The anti-human Podocin polyclonal antibody of rabbit is purchased from Abcam company of Britain.The chicken anti-rabbit IgG antibody purchased from American SantaCruz company of the anti-sheep IgG antibody of AlexaFluor555 donkey, AlexaFluor555 donkey anti goat igg antibody purchased from American MolecularProbe company, isothiocyanic acid mark.
HBSS damping fluid: NaCl8g/L, KCl0.40g/L, NaHCO
30.35g/L, Na
2hPO
40.06g/L, KH
2pO
40.06g/L.
The preparation of embodiment 1, substratum
DMEM/F12 substratum (also known as DF12 substratum) is purchased from GIBCO company.Foetal calf serum (FBS) is purchased from GIBCO company.Human activin A (activinA) is purchased from PeproTech company of Britain, and catalog number is 120-14.Vitamin A acid available from Sigma, catalog number is R2625.Bone morphogenetic protein4 (bonemorphogeneticprotein7, BMP7) is purchased from PeproTech company, and catalog number is 120-03.
Substratum 1 is made up of DMEM/F12 substratum and solute; Described solute and the concentration in substratum 1 as follows: volumetric concentration is foetal calf serum, the 10ng/mLactivinA and 10 of 2%
-5mol/L vitamin A acid.
Substratum 2 is made up of DMEM/F12 substratum and solute; Described solute and the concentration in substratum 2 as follows: volumetric concentration is foetal calf serum, the 10ng/mLactivinA and 10 of 2%
-7mol/L vitamin A acid, 20ng/mlBMP7.
The inducing culture of embodiment 2, Renal Podocytes
One, preparation the 3rd generation mescenchymal stem cell
1, fatty tissue (source is people) after sterile collection adult lipsuction, in Biohazard Safety Equipment, cleans 2 times with the cell washing solution containing penicillin and Streptomycin sulphate, removes hemocyte and anaesthetic, the centrifugal 3min of 800rpm.
2, fatty tissue is gone in new 50ml centrifuge tube, add 0.05% collagenase P digestion tissue, in 37 DEG C of constant temperature culture oscillator concussion 30min.
3, postdigestive fatty tissue 100 eye mesh screen filters and collects filtrate, centrifugal 10 minutes of room temperature 1200rpm, collecting cell.
4, by the cell of step 3 37 DEG C, 5%CO2, relative air humidity be cultivate in the incubator of 95%, when cell reach 70%-80% converge time, 2 times are washed with D-Hank ' s liquid, with 1g/L trypsin Gibco company) conventional digestion, cell goes down to posterity according to 1:3, obtains 1st generation mesenchymal cell.
5, by 1st generation mesenchymal cell 37 DEG C, 5%CO2, relative air humidity be cultivate in the incubator of 95%, reach 70%-80% to converge, 2 times are washed with D-Hank ' s liquid, with 1g/L trypsin Gibco company) conventional digestion, cell goes down to posterity according to 1:3, obtains 2nd generation mesenchymal cell.
6, by 2nd generation mesenchymal cell 37 DEG C, 5%CO2, relative air humidity be cultivate in the incubator of 95%, reach 70%-80% to converge, 2 times are washed with D-Hank ' s liquid, then the trypsin Gibco company of 1g/L is used) conventional digestion, centrifugal 6 minutes of room temperature 1200rpm, collecting cell.
7, by the cell of step 6 37 DEG C, 5%CO2, relative air humidity be cultivate 4-6 hour in the incubator of 95%, reject substratum after cell attachment, washes 2 times with D-Hank ' s, be denoted as the 3rd generation mescenchymal stem cell.
Two, substratum combination is adopted to carry out inducing culture
1, the cultivation of first stage
(1) in 6 orifice plates, substratum 1 is added, every hole 2ml.
(2) inoculate in 6 orifice plates of step (1) the 3rd generation mescenchymal stem cell (make its concentration in the medium be 5 × 10
5individual/ml), 37 DEG C, 5%CO2, relative air humidity be cultivate 3 days (suction in every 1.5 days is abandoned supernatant and adds the new substratum of 2ml 1) in the incubator of 95%.
2, the cultivation of subordinate phase
Inhale the supernatant abandoned in 6 orifice plates of step 1, add substratum 2, every hole 2ml, 37 DEG C, 5%CO2, relative air humidity be cultivate 6 days (suction in every 2 days is abandoned supernatant and adds the new substratum of 2ml 2) in the incubator of 95%, centrifugal 5 minutes of room temperature 1200rpm, collecting cell (precipitation).
Three, the qualification of inducing effect
1, the 3rd generation mescenchymal stem cell values of immunophenotyping
Step one obtain the 3rd generation mescenchymal stem cell cellular form see Fig. 1.
By the phenotype of indirect immunofluorescence detection the 3rd generation mescenchymal stem cell, carry out flow cytometer detection after the mouse anti human CD29 marked with fluorescein isothiocyanate (FITC), CD34, CD44, CD105, CD106, HLA-DR and Flk-1 antibody (antibody is all purchased from BD company) mark, flow cytometer is ACCURIC6 (BectonDickinson).
The results are shown in Figure 2, X-coordinate represents cell fluorescence intensity, and ordinate zou represents cell count; P1 represents selected cell colony.Phenotypic results show, the 3rd generation mescenchymal stem cell CD29, CD44, CD105, Flk-1 be the positive, CD31, CD34, CD106 and HLA-DR molecule is feminine gender.
2, human mesenchymal stem cell is induced to differentiate into the qualification of Renal Podocytes
(1) identification of morphology
In substratum 1, cultivate cell after 3 days reach 80%-90% and converge, in culturing process, the metamorphosis of cell is as follows: fibroblast-like human mesenchymal stem cell is after the induction of 3 days, just having there is obvious change in cellular form, induces the 3rd day cell become large and become flat; In substratum 2, continue cultivation 6 days, cellular form changed further, at the 9th day of induction, visible cell became larger compared with the 3rd day, there is the structure of podocytic process sample in cell bifurcated, the podocyte of form conditions of similarity immortalization, and the cell cultivated 9 days in 1,2 substratum is shown in Fig. 3.
(2) Real-time PCR Analysis
Get in step 2 cultivate 6 days cell (by the 3rd generation mescenchymal stem cell in contrast), extract total serum IgE reverse transcription is cDNA, by the expression of real-time quantitative PCR qualification Renal Podocytes specificity marker gene Nephrin gene, Podocin gene and WT1 gene.The wherein albumen of Nephrin genes encoding, is positioned on the filtration crack film of podocyte, participates in forming glomerular filtration function; Podocin gene product protein localization, in renal glomerulus slit diaphragm district, plays an important role for the structure and function maintaining slit diaphragm as scaffolding protein.WT1 plays important regulating effect in rear kidney growth course, and at adult kidney, the albumen continuous expression of WT1 genes encoding is also limited on glomerular podocyte.
Adopt people β-actin as the contrast of each gene, primer used is as follows:
Primer for the Nephrin gene that increases is:
Upstream primer NephrinF:5 '-GAGGACCGAGTCAGGAACGA-3 ' (SEQIDNO:1);
Downstream primer NephrinR:5 '-TGACCGTGGAGCTCTGAGTGT-3 ' (SEQIDNO:2).
Primer for the Podocin gene that increases is:
Upstream primer PodocinF:5 '-CCTTTTCATGAGATCGTGACCAA-3 ' (SEQIDNO:3);
Downstream primer PodocinR:5 '-GCATTTTCCATTCGGTAGTAGCA-3 ' (SEQIDNO:4).
Primer for the WT1 gene that increases is:
Upstream primer WT1F:5 '-GTGACTTCAAGGACTGTGAACG-3 ' (SEQIDNO:5);
Downstream primer WT1R:5 '-CGGGAGAACTTTCGCTGACAA-3 ' (SEQIDNO:6).
β-actin primer for increasing is:
Upstream primer β-actinF:5 '-CTGGAACGGTGAAGGTGACA-3 ' (SEQIDNO:7);
Downstream primer β-actinR:5 '-AAGGGACTTCCTGTAACAATGCA-3 ' (SEQIDNO:8).
△ △ CT method is adopted to calculate the relative expression quantity of each gene.With the 3rd generation mescenchymal stem cell the expression amount of Nephrin gene (or Podocin, WT1 gene) for 1, calculate the relative expression quantity of each gene in the cell after adopting substratum 1 inducing culture.Adopt in the cell after substratum 1 inducing culture, the relative expression quantity of the relative expression quantity of Nephrin gene to be the relative expression quantity of 15.96, Podocin gene be 31.65, WT1 gene is 2.11.The results are shown in Figure 4.
(3) immunofluorescence dyeing qualification
By cultivate in step 29 days cell (by the 3rd generation mescenchymal stem cell in contrast) carry out immunofluorescence dyeing, detect the expression of podocyte mark Nephrin, Podocin and WT1.
The concrete grammar detecting Nephrin is as follows: cell is fixed with 80% ice ethanol, with the PBST buffer blind containing 1%BSA after PBS cleaning; Then sheep mouse-anti people Nephrin monoclonal antibody (working concentration is 1:50 dilution) is used to hatch 8 hours as 4 DEG C; After PBS buffer solution for cleaning, the more anti-sheep IgG of AlexaFluor555 (red fluorescence) donkey was as two anti-incubated at room 30 minutes; Nucleus (blue-fluorescence) is redyed with Hoechst33342 staining fluid (Sigma), at fluorescence microscopy Microscopic observation (OlympusDP72) with after PBS buffer solution for cleaning.3rd generation mescenchymal stem cell all do not show red fluorescence, be negative findings (Fig. 5 A).The cell more than 80% after substratum 1,2 inducing culture is adopted to show red fluorescence (Fig. 5 B).
Detect the concrete grammar of concrete grammar with Nephrin of Podocin, difference is only to adopt the anti-human Podocin monoclonal antibody of rabbit (working concentration is 1:50 dilution) as primary antibodie, to adopt the chicken anti-rabbit IgG of isothiocyanic acid mark (green fluorescence) to resist as two.3rd generation mescenchymal stem cell all do not show green fluorescence, be negative findings (Fig. 5 C).The cell more than 80% after substratum 1,2 inducing culture is adopted to show green fluorescence (Fig. 5 D).
Detect the concrete grammar of concrete grammar with Nephrin of WT1, difference is only to adopt Goat anti human WT1 monoclonal antibody (working concentration is 1:50 dilution) as primary antibodie, to adopt AlexaFluor555 (red fluorescence) donkey anti goat igg to resist as two.3rd generation mescenchymal stem cell all do not show red fluorescence, be negative findings (Fig. 5 E).The cell more than 80% after substratum 1,2 inducing culture is adopted to show red fluorescence (Fig. 5 F).
The results are shown in Figure 5A-F (Fig. 5 A, Fig. 5 C and Fig. 5 E be the 3rd generation mescenchymal stem cell, Fig. 5 B, Fig. 5 D and Fig. 5 F are the cell after adopting substratum 1,2 inducing culture).Result shows, after adopting substratum 1,2 inducing culture, most cells is the Nephrin positive (Fig. 5 B), Podocin (Fig. 5 D) positive and the WT1 positive (Fig. 5 F).
Above qualification result shows, adopts the cell after substratum inducing culture to be mainly Renal Podocytes (express the specificity marker gene of Renal Podocytes, and show the form of Renal Podocytes).
Claims (10)
1. be a method for Renal Podocytes by mescenchymal stem cell induction, it comprises:
1) mescenchymal stem cell is inoculated in substratum 1; And
2) by completing steps 1) mescenchymal stem cell transfer to obtain Renal Podocytes in substratum 2,
Wherein said substratum 1 is made up of zooblast basic medium and solute, and described solute comprises: the foetal calf serum of 1.9%-2.1%, 9.75ng/mL-10.25ng/mLactivinA and 0.95 × 10
-5mol/L-1.05 × 10
-5mol/L vitamin A acid;
Wherein said substratum 2 is made up of zooblast basic medium and solute, and described solute comprises: the foetal calf serum of 1.9%-2.1%, 9.75ng/mL-10.25ng/mLactivinA, 0.95 × 10
-7mol/L-1.05 × 10
-7mol/L vitamin A acid and 19ng/ml-21ng/mlBMP7.
2. method according to claim 1, is characterized in that in described step 1) in cultivate 2-4 days, and in described step 2) in cultivate 5-7 days, preferably in described step 1) in cultivate 3 days, and in described step 2) in cultivate 6 days.
3. method according to claim 1 and 2, wherein said zooblast basic medium is DMEM/F12 substratum,
The foetal calf serum of 2%, the activinA of 10ng/mL and 10 is comprised at substratum 1
-5the vitamin A acid of mol/L; And
The foetal calf serum of 2%, the activinA, 10 of 10ng/mL is comprised at substratum 2
-7the vitamin A acid of mol/L and the BMP7 of 20ng/ml.
4. the method according to claim 1-3, wherein in step 1) described in the concentration of mescenchymal stem cell be 3 × 10
5individual/ml-6 × 10
5individual/ml, is preferably 5 × 10
5individual/ml.
5. the method according to claim 1-4, is characterized in that described mescenchymal stem cell is fat mesenchymal stem cell; Preferably, described mescenchymal stem cell is human mesenchymal stem cell; Preferably, described mescenchymal stem cell is third generation mescenchymal stem cell; Preferably, described mescenchymal stem cell is behaved fatty third generation mescenchymal stem cell.
6. the method according to claim 1-5, is characterized in that the cytokine CD29 of described mescenchymal stem cell, CD44, CD105 and Flk-1 is the positive, cytokine CD31, CD34, CD106 and HLA-DR are negative cell.
7. a test kit, it comprises substratum 1 and substratum 2,
Wherein said substratum 1 is made up of zooblast basic medium and solute, and described solute comprises: the foetal calf serum of 1.9%-2.1%, 9.75ng/mL-10.25ng/mLactivinA and 0.95 × 10
-5mol/L-1.05 × 10
-5mol/L vitamin A acid;
Wherein said substratum 2 is made up of zooblast basic medium and solute, and described solute comprises: the foetal calf serum of 1.9%-2.1%, 9.75ng/mL-10.25ng/mLactivinA, 0.95 × 10
-7mol/L-1.05 × 10
-7mol/L vitamin A acid and 19ng/ml-21ng/mlBMP7.
8. test kit according to claim 7, wherein said zooblast basic medium is DMEM/F12 substratum,
Solute in described substratum 1 comprises the foetal calf serum of 2%, the activinA of 10ng/mL and 10
-5the vitamin A acid of mol/L; And
Solute in described substratum 2 comprises the foetal calf serum of 2%, the activinA, 10 of 10ng/mL
-7the vitamin A acid of mol/L and the BMP7 of 20ng/ml.
9. the Renal Podocytes that the method according to claim 1-6 obtains, preferably in described Renal Podocytes Nephrin, Podocin and WT1 expression amount higher than originated mescenchymal stem cell.
10. Renal Podocytes according to claim 9 for the preparation for the treatment of Renal Podocytes relative disease and the medicine at cellular transplantation therapy in purposes, preferably described Renal Podocytes relative disease is minimal change glomerulonephritis, FSGS, membranous nephropathy, diabetic nephropathy.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN107058214A (en) * | 2017-05-27 | 2017-08-18 | 广州润虹医药科技有限公司 | The culture medium and cultural method of induced multi-potent stem cell directed differentiation kidney cell |
| WO2019050350A3 (en) * | 2017-09-07 | 2019-04-25 | 차의과학대학교 산학협력단 | Stem cell-derived sertoli-like cell, preparation method therefor, and use thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834205A (en) * | 2015-12-07 | 2017-06-13 | 江苏齐氏生物科技有限公司 | A kind of Mouse Kidney sertoli cell is separated and cultural method |
| CN107058214A (en) * | 2017-05-27 | 2017-08-18 | 广州润虹医药科技有限公司 | The culture medium and cultural method of induced multi-potent stem cell directed differentiation kidney cell |
| CN107058214B (en) * | 2017-05-27 | 2020-05-08 | 广州润虹医药科技股份有限公司 | Culture medium and culture method for inducing pluripotent stem cells to directionally differentiate kidney cells |
| WO2019050350A3 (en) * | 2017-09-07 | 2019-04-25 | 차의과학대학교 산학협력단 | Stem cell-derived sertoli-like cell, preparation method therefor, and use thereof |
| WO2020253763A1 (en) * | 2019-06-18 | 2020-12-24 | 中国医学科学院基础医学研究所 | Heterogeneous stem cell population, preparation method therefor and use thereof |
| CN117871216A (en) * | 2024-03-12 | 2024-04-12 | 中日友好医院(中日友好临床医学研究所) | Three-dimensional visualization method for glomerular fissure membrane in paraffin specimen |
| CN117871216B (en) * | 2024-03-12 | 2024-06-04 | 中日友好医院(中日友好临床医学研究所) | Three-dimensional visualization method for glomerular fissure membrane in paraffin specimen |
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