CN104232574A - Method for in-vitro directional differentiation inducing of mesenchymal stem cell towards melanocyte - Google Patents
Method for in-vitro directional differentiation inducing of mesenchymal stem cell towards melanocyte Download PDFInfo
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Abstract
The invention relates to a method for in-vitro directional differentiation inducing of a mesenchymal stem cell towards melanocyte, particularly relates to a method for in-vitro directional differentiation inducing of the mesenchymal stem cell with an umbilical cord source towards the melanocyte, and belongs to the technical field of biology. The mesenchymal stem cell with the umbilical cord source is cultivated by adopting an explants method, and then the mesenchymal stem cell with the umbilical cord source is identified, so as to determine the characteristics of the mesenchymal stem cell; a culture medium is induced to carry out in-vitro inducing differentiation to form the melanocyte by using improved melanine; and finally the cultivated melanocyte is identified. An ES cell source and a marrow or adipose tissue-derived stromal cell source are limited or not easy to obtain while the stem cell can be easily obtained from the umbilical cord, the immunogenicity is very low, and an individual difference is not easily generated, so that the method is suitable for large-scale clinical application, and the induced melanocyte can be applied to epichrosis leucasmus therapy.
Description
Technical field
The present invention relates to a kind of method that derived mesenchymal stem cells in vitro is induced to melanocyte directed differentiation, what be specifically related to is the derived mesenchymal stem cells in vitro method of inducing to melanocyte directed differentiation in a kind of umbilical cord source, belongs to biological technical field.
Background technology
Vitiligo (vitiligo) is a kind of common local or whole body depigmentation dermatoses, this sick sickness rate is about 0.5-1%, relation is all had with inherited genetic factors, immune factor, oxidative stress is unbalance etc., the viewpoint of generally acknowledging at present is that the melanophore that composite factor causes is destroyed, thus the depigmentation caused, and hickie is formed.This sick methods for the treatment of is a lot, as medicine, and laser, operation etc., but curative effect is all imprecise, and even there is isomorphic effect in what have.
The method of cellular replacement therapy disease receives increasing concern in recent years, utilizes high proliferation and the multi-lineage potential of stem cell, makes it in patient body, be divided into target cell and the function substituting sick cell, thus improves and stop the development of the state of an illness.
The selection of seed cell is the key of cellular transplantation therapy disease, adopts the mescenchymal stem cell of embryonic stem cell (ES), derived from bone marrow and adipose-derived mescenchymal stem cell to be induced to differentiate into melanophore at present.The objective shortcoming of prior art: ES cell derived and marrow or fat mesenchymal stem cell limited source or not easily obtain, human umbilical cord mesenchymal stem cells (hUC-MSCs) compared with other mescenchymal stem cells of originating, primitiveness and self and multiplication capacity stronger; Immunogenicity is low, can not react by triggering immune; Draw materials conveniently, the high and pollution that is virus-free and tumour cell of purity, in view of the unrivaled advantage of mescenchymal stem cell in these other source, umbilical cord mesenchymal stem cells scientific research and clinical in have a wide range of applications.
At present, hUC-MSCs is from the acquisition of umbilical cord mainly from umbilical cord China Tong Shi glue, but it is separated and cultural method does not have the step of standard, and there is diverse ways different experiments room, mainly contains enzyme digestion and tissue explants adherent method.Enzyme digestion utilizes collagenase by the collegen filament degraded in umbilical cord, thus isolate cell.Enzyme digestion can obtain primary cell within a short period of time, but the method step is many, very easily produces chemical pollution and physical abuse to cell; The concentration of enzyme and the bad grasp of digestion time, and the collagenase used is expensive, is therefore not suitable for large-scale production.
Tissue explants adherent method mainly utilizes mescenchymal stem cell in fritter umbilical cord tissue to have stronger adhesive capacity to plastic culture bottle, progressively can move out in tissue block adherent culturing process, and endotheliocyte and hematopoietic lineage cells etc. are not adherent in tissue block, can change in liquid process in cultivation and be discarded gradually.The method step simple and fast, decreases the probability of cell physical abuse and chemical pollution, can keep the vigor of cell better; eliminate the use of enzyme simultaneously; decrease Financial cost, be also applicable to large-scale production, therefore planting block method has more advantage than enzyme digestion.And bibliographical information has no hUC-MSCs is induced to differentiate into melanophore.
Summary of the invention
The object of the invention is to overcome the defect existed in prior art, a kind of method that derived mesenchymal stem cells in vitro is induced to melanocyte directed differentiation is provided, the present invention plants by adopting the mescenchymal stem cell that block method cultivates umbilical cord source, adopt the melanocyte induced liquid after improvement, induce its vitro directed differentiation to be melanophore.
The method that derived mesenchymal stem cells in vitro of the present invention is induced to melanocyte directed differentiation, comprises the steps: the acquisition of umbilical cord tissue block; The cultivation of mescenchymal stem cell in primary umbilical cord source, the Secondary Culture of cell; The qualification of the mescenchymal stem cell in the umbilical cord source obtained; Differentiation Induction in vitro is melanophore, melanocytic cultivation and qualification.
(1) step of the acquisition of wherein said umbilical cord tissue block comprises:
A. after operating table obtains people umbilical cord, be immersed in 0.9% sterile saline, be asepticly transported to reality
Test room.
B. take out at super clean bench, containing 1% dual anti-(penicillin and Streptomycin sulphate) PBS buffer solution 3 times, with
Removing bloodstain and fully cleaning umbilical vein tube chamber, peel off arteriovenous, get umbilical cord reticular tissue, cut the tissue block into 4-5mm3 size.
(2) cultivation of mescenchymal stem cell, the subculture step of cell in the primary umbilical cord source described in comprise:
A. the DMEM/F12 (1:1) that tissue block adds 0.5-1mL in advance in 6 porocyte culture plate inner panels is inoculated
Substratum (containing 10% foetal calf serum (FBS)), puts culture plate and cultivates in cell culture incubator (37 DEG C, 5% volume CO2 saturated humidity).
B. change liquid first after cell cultures 24h, every 2-3d changes liquid once later, primary after cultivating 12-14 d
Cell grows and removes tissue block after covering with 60%.
C. the Secondary Culture of hUC-MSCs: put culture plate and cultivate in cell culture incubator, Conditions Temperature 37 DEG C,
5%CO2 saturated humidity, changes liquid after 24h first, later every 2-3d replaced medium, and microscope observing cell form is also taken pictures.
(3) hUC-MSCs obtained carries out immunophenotype respectively and becomes the detections such as fat skeletonization, to confirm its tool
There is the characteristic of mescenchymal stem cell.
(4) described Differentiation Induction in vitro is melanophore, and melanocytic culturing step comprises:
Be inoculated in 24 orifice plates by after hUC-MSCs passage, after using DMEM/12 substratum (containing 10%FBS) culturing cell to 70% to merge, change melanocyte inducing culture inducing culture, within 2 days, change liquid once, go down to posterity when cell reaches 60% fusion, continuous induction is cultivated 9 weeks.
The melanocyte induced liquid wherein improved, comprises 50ng/mL SCF, 20%MCDB201,4ng/mL bFGF, 100nM EDN3,50% L-Wnt3a supernatant, 20pM cholera toxin, 0.05 μM of dexamethasone, 50nM TPA, 20% low sugar DMEM, 1 × pancreas islet plain sheet selenium transferrin, 104M L-AA, 1 mg/mL linolic acid-bovine serum albumin.
(4) qualification by the external cell after melanocyte differen-tiation of hUC-MSCs, with determine differentiation-inducing after
To cell there is melanocytic characteristic.
technological merit of the present invention
(1) ES cell derived and marrow or fat mesenchymal stem cell limited source or not easily obtain, and umbilical cord
Easy acquisition, immunogenicity is extremely low, not easily produces individual difference, is applicable to mass-producing in enormous quantities and is applied to clinical;
(2) melanophore of inducing likely is applied to therapy of vitiligo.
Accompanying drawing explanation
fig. 1. plant block method and cultivate primary hUC-MSCs morphological observation figure; In figure, (a) is umbilical cord tissue block adherent culture diagram; (b) for primary hUC-MSCs grow after morphologic observation (x40); C () is primary cell colony flowing water shape growth conditions (x40); D () to cover with the 50%(x40 of substratum for P3 for cell).
Fig. 2. the form (oil red O stain ' 40) after human umbilical cord mesenchymal stem cells adipogenic induction; A () is control group; B () is induction group.
Fig. 3. the form (Alizarin red staining ' 40) after human umbilical cord mesenchymal stem cells osteogenic induction; A () is control group; B () is induction group.
Fig. 4. hUC-MSCs is to cellular form change diagram under light microscopic in melanophore Induction Process; A () is for before induction; B () is for induction is after 3 weeks; C () is for induction is after 5 weeks; D () is for induction is after 9 weeks.
Fig. 5. hUC-MSCs the relative expression to melanocyte genes involved in melanocyte differen-tiation process scheme (*:
p<0.05);
Fig. 6. melanophore anti-S-100, the HMB45 immunocytochemical stain result in hUC-MSCs source
(× 200); In figure, (a) is the expression of control group S-100, is negative; B () is the expression of induction group S-100, be positive; C () is the expression of control group HMB45, be negative; D () is the expression of induction group HMB45, be positive.
Fig. 7. anti-MITF, the SOX10 immunofluorescence figure (× 200) of melanophore in hUC-MSCs source; In figure, (a) is MITF immunofluorescence; B () is SOX10 immunofluorescence.
Embodiment
Below in conjunction with concrete embodiment, the present invention will be further described.
The method that the derived mesenchymal stem cells in vitro that umbilical cord is originated is induced to melanocyte directed differentiation, completes according to following steps:
(1) plant block method and cultivate primary hUC-MSCs(Secondary Culture and morphological observation)
A. after operating table obtains people umbilical cord, be immersed in 0.9% sterile saline, be asepticly transported to reality
Test room.
B. take out at super clean bench, containing 1% dual anti-(penicillin and Streptomycin sulphate) PBS buffer solution 3 times,
To remove bloodstain and fully to clean umbilical vein tube chamber, peel off arteriovenous, get umbilical cord reticular tissue, cut the tissue block into 4-5mm3 size.
C. inoculate tissue block in 6 porocyte culture plates, add in advance in plate 0.5-1mL DMEM/F12 (1:
1) substratum (containing 10%FBS), puts culture plate and cultivates in cell culture incubator (37 DEG C, 5% volume CO2 saturated humidity).
D. change liquid first after cell cultures 24h, every 2-3d changes liquid once later, primary after cultivating 12-14 d
Cell grows and removes tissue block after covering with 60%.
The morphological observation of primary hUC-MSCs Secondary Culture as shown in Figure 1.
(2) hUC-MSCs multi-lineage potential detects (scleroblast and stearoblast differentiation-inducing): detect and obtain
The cell obtained has stem cell properties.
A. hUC-MSCs is differentiation-inducing to adipocyte:
Be inoculated in 6 porocyte culture plates after 3rd generation hUC-MSCs trysinization, cell density is moderate, if adipogenic induction group 3 hole, and control group 3 hole.
Cell culture growth changes fat induced liquid into when merging to 80%: containing the IBMX of DMEM in high glucose 100 mg/L of 10%FBS, 1mM dexamethasone, 50mM indomethacin, 10mM Regular Insulin, control group adds regular growth nutrient solution.
Changed liquid once every 2-3 days between inductive phase, induction duration is 2 weeks, and induction terminates the change of rear Microscopic observation cellular form and fat drips formational situation and carries out the qualification of oil red staining, and basis of microscopic observation is also taken a picture, and result as shown in Figure 2.
B. hUC-MSCs is differentiation-inducing to scleroblast
Be inoculated in 6 porocyte culture plates after 3rd generation hUC-MSCs trysinization, cell density is moderate (with 10
5/ mL is advisable), be set as self-bone grafting group 3 hole, control group 3 hole.
Cell cultures adherent growth changes self-bone grafting liquid into after merging to 80%: containing 10%FBS DMEM in high glucose, 10mmol/Lb-phospho-glycerol, 200mmol/L xitix, 1.0mmol/L dexamethasone, control group adds regular growth nutrient solution.
The osteogenic induction time is 2 weeks, between inductive phase every 2-3 days, changes liquid once, and hystazarin red colouring qualification in 2 weeks, inverted phase contrast microscope is observed and taken a picture, and result as shown in Figure 3.
(3) hUC-MSCs is differentiation-inducing to melanophore
24 orifice plates are inoculated in after 3rd generation hUC-MSCs passage, after using DMEM/12 substratum (containing 10%FBS) culturing cell to 70% to merge, control group continues to cultivate, induction group changes melanocyte inducing culture: 50ng/mL SCF, 20%MCDB201, 4ng/mL bFGF, 100nM EDN3, 50% L-Wnt3a supernatant, 20pM cholera toxin, 0.05 μM of dexamethasone, 50nM TPA, 20% low sugar DMEM, 1 × pancreas islet plain sheet selenium transferrin, 104M L-AA, 1 mg/mL linolic acid-bovine serum albumin, within 2 days, change liquid once, cell goes down to posterity when reaching 60% fusion, continuous induction is cultivated 9 weeks.
(4) from following several respects prove differentiation-inducing after the cell that obtains there is melanocytic characteristic:
a. hUC-MSCs growth and morphologic observation between inductive phase
At hUC-MSCs in the differentiation-inducing process of melanophore, cellular form gradually became more elongated after 3 weeks
Fusiformis, kytoplasm enrich, transparency decline, after 5 weeks, part cell stretches gradually and occurs dual polarization and multipolarization, and after 9 weeks, attached cell form of diverse is common with multipolarization form, and similar melanocytic dendron shape structure, result is as shown in Figure 4.
B. RT-PCR detects related gene expression
HUC-MSCs, the induce to melanophore the 3rd, 5,7, after 9 weeks, compares with control group, induction group melanophore genes involved MITF, and the expression increase in time of TYRP1, SOX10, MC1RmRNA is significantly increased, (
p<0.05), result as shown in Figure 5
C. immunochemistry dyeing
Induction group cellular form is the melanoid cells Dendritic shape structure of dual polarization and multipolarization, after HMB45 and S-100 dyeing, cell space and dendron are dyed to brown, be positive, cellular control unit form is spindle shape, notable difference is had with induction group, S-100, HMB45 dyeing is negative, and result as shown in Figure 6.
D. immunofluorescence dyeing
Induction group cell through FITC mark two anti-hatch after, MITF, SOX10 all show green fluorescence, positive expression, and control group is then negative expresses, and result is as shown in Figure 7.
Claims (8)
1. a derived mesenchymal stem cells in vitro method of inducing to melanocyte directed differentiation, is characterized in that, bag
Draw together following steps: the acquisition of umbilical cord tissue block; The cultivation of mescenchymal stem cell in primary umbilical cord source, the Secondary Culture of cell; The qualification of the mescenchymal stem cell in the umbilical cord source obtained; Differentiation Induction in vitro is melanophore, melanocytic cultivation and qualification.
2. a kind of derived mesenchymal stem cells in vitro according to claim 1 is induced to melanocyte directed differentiation
Method, is characterized in that, described source for mesenchymal stem cells is in umbilical cord.
3. a kind of derived mesenchymal stem cells in vitro according to claim 1 is induced to melanocyte directed differentiation
Method, is characterized in that, the step of the acquisition of described umbilical cord tissue block comprises:
Operating table is immersed in 0.9% sterile saline after obtaining people umbilical cord, is asepticly transported to reality
Test room;
Take out at super clean bench, containing 1% dual anti-(penicillin and Streptomycin sulphate) PBS buffer solution 3 times, with
Removing bloodstain and fully cleaning umbilical vein tube chamber, peel off arteriovenous, get umbilical cord reticular tissue, cut the tissue block into 4-5mm3 size.
4. a kind of derived mesenchymal stem cells in vitro according to claim 1 is induced to melanocyte directed differentiation
Method, is characterized in that, the cultivation of mescenchymal stem cell, the subculture step of cell in described primary umbilical cord source comprise:
The DMEM/F12 that inoculation tissue block adds 0.5-1mL in advance in 6 porocyte culture plate inner panels cultivates
Base, puts culture plate and cultivates in cell culture incubator, and culture condition is 37 DEG C, 5% volume CO2 saturated humidity;
Change liquid first after cell cultures 24h, every 2-3d changes liquid once later, primary after cultivating 12-14 d
Cell grows and removes tissue block after covering with 60%;
The Secondary Culture of hUC-MSCs: put culture plate and cultivate in cell culture incubator, Conditions Temperature 37 DEG C,
5%CO2 saturated humidity, changes liquid after 24h first, later every 2-3d replaced medium, and microscope observing cell form is also taken pictures.
5. a kind of derived mesenchymal stem cells in vitro according to claim 1 is induced to melanocyte directed differentiation
Method, is characterized in that, described Differentiation Induction in vitro is melanophore, and melanocytic culturing step comprises:
Be inoculated in 24 orifice plates by after hUC-MSCs passage, after using DMEM/12 culture medium culturing cell to 70% to merge, change melanocyte inducing culture inducing culture, within 2 days, change liquid once, go down to posterity when cell reaches 60% fusion, continuous induction is cultivated 9 weeks.
6. a kind of derived mesenchymal stem cells in vitro according to claim 4 or 5 lures to melanocyte directed differentiation
The method led, is characterized in that, in described DMEM/F12 substratum, the ratio of DMEM and F12 is 1:1;
Containing 10% foetal calf serum in described DMEM/F12 substratum.
7. a kind of derived mesenchymal stem cells in vitro according to claim 5 lures to melanocyte directed differentiation
The method led, it is characterized in that, described melanocyte inducing culture based component comprises 50ng/mL SCF, 20%MCDB201,4ng/mL bFGF, 100nM EDN3,50% L-Wnt3a supernatant, 20pM cholera toxin, 0.05 μM of dexamethasone, 50nM TPA, 20% low sugar DMEM, 1 × pancreas islet plain sheet selenium transferrin, 104M L-AA, 1 mg/mL linolic acid-bovine serum albumin.
8. a kind of derived mesenchymal stem cells in vitro according to claim 1 method of inducing to melanocyte directed differentiation, it is characterized in that, the melanophore obtained is for leukodermic treatment.
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| CN106867962A (en) * | 2017-03-28 | 2017-06-20 | 周婧 | A kind of method that inducing neural ridge stem cell breaks up to chromatophore |
| CN106978386A (en) * | 2017-03-21 | 2017-07-25 | 安徽安龙基因医学检验所有限公司 | A kind of autologous fat mescenchymal stem cell directed differentiation is the method for melanocyte |
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| CN104673743A (en) * | 2015-02-13 | 2015-06-03 | 中国人民解放军第二军医大学 | Tissue block culture method for obtaining primary cell from animal tissue |
| CN105505859A (en) * | 2015-12-28 | 2016-04-20 | 江苏大学附属医院 | Method for inducing differentiation of fibroblasts into multifunctional stem cells |
| CN105505859B (en) * | 2015-12-28 | 2020-02-21 | 江苏大学附属医院 | Method for inducing and differentiating fibroblast into multifunctional stem cell |
| CN106978386A (en) * | 2017-03-21 | 2017-07-25 | 安徽安龙基因医学检验所有限公司 | A kind of autologous fat mescenchymal stem cell directed differentiation is the method for melanocyte |
| CN106867962A (en) * | 2017-03-28 | 2017-06-20 | 周婧 | A kind of method that inducing neural ridge stem cell breaks up to chromatophore |
| CN106867962B (en) * | 2017-03-28 | 2020-09-04 | 周婧 | Method for inducing neural crest stem cells to differentiate into pigment cells |
| CN109207420A (en) * | 2017-06-30 | 2019-01-15 | 株式会社爱茉莉太平洋 | The manufacturing method and artificial skin of artificial skin |
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| CN108373990A (en) * | 2018-03-19 | 2018-08-07 | 沈阳艾米奥生物工程技术研发中心有限公司 | People's amnioic epithelium stem cell is to melanocyte inductive differentiation medium and its method |
| CN108373990B (en) * | 2018-03-19 | 2021-08-24 | 沈阳艾米奥生物工程技术研发中心有限公司 | Culture medium for inducing differentiation from human amniotic epithelial stem cells to melanocytes and method thereof |
| JP2021514680A (en) * | 2018-03-29 | 2021-06-17 | エースソステム バイオストラテジーズ インコーポレイテッド | How to isolate stem cells from the human umbilical cord |
| JP7064254B2 (en) | 2018-03-29 | 2022-05-10 | エースソステム バイオストラテジーズ インコーポレイテッド | How to isolate stem cells from the human umbilical cord |
| CN111040984A (en) * | 2018-10-15 | 2020-04-21 | 美奇创新生物科技(北京)有限公司 | Method for forming skin fibroblasts by inducing differentiation of umbilical cord mesenchymal stem cells |
| CN112481194A (en) * | 2020-12-02 | 2021-03-12 | 深圳清华大学研究院 | Method for preparing cell suspension with melanocyte activity |
| CN112481194B (en) * | 2020-12-02 | 2023-03-21 | 深圳清华大学研究院 | Method for preparing cell suspension with melanocyte activity |
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Application publication date: 20141224 |