骨髓间充质干细胞培养、冻存及体外诱导神经干细胞分化的研究
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摘要
哺乳动物的骨髓中含大量成体干细胞,而且取材方便,成为成体干细胞研究焦点。骨髓间充质干细胞(Marrow mesenchymal stem cells,MSCs)是继造血干细胞(Hematopoietic stem cell,HSCs)之后近年来研究较多的一种骨髓源多能干细胞。MSCs不仅具有强大的增殖分化能力,易于在体外大量繁殖,诱导分化,而且具有向胞外分泌多种细胞因子的特性,参与细胞微环境的构建。体内移植具有低免疫原性,在器官移植、自身免疫性疾病及替代治疗中有广阔的应用前景。
本文以SD(sprague-dawley)大鼠为实验材料,首先探讨了MSCs的体外分离、扩增的方法。取SD大鼠双侧股骨、胫骨,采用全骨髓法贴壁培养MSCs,以梯度密度接种骨髓细胞。研究结果显示,以0.5～1×107个/mL接种骨髓细胞可以有效缩短原代培养的时间,传代细胞生长状态稳定,P2、P4、P6代生长曲线基本相同。免疫荧光法鉴定表面抗原CD29、CD44阳性表达,不表达造血细胞系表面抗原CD34+。免疫荧光双标鉴定P3代细胞的纯度,其CD29阳性率为97.96±0.35%,CD44阳性表达率为98.14±0.19%。上述结果充分证明本方法适于在体外快速获得大量具有较高生物学活性的高纯度MSCs。
在此基础上,对MSCs低温冻存进行了研究。以MSCs基本培养基、胎牛血清、DMSO为主要成分,设计不同配比的冻存保护液。以纯化的MSCs(P3),1×106个/mL密度,-80℃冻存7 d后复苏,台盼蓝据染法检测复苏率,结果显示以80%DMEM+10%FBS+10%DMSO作为冻存保护液,复苏率为61.00±3.61%,显著优于其余各组。复苏后的细胞能够在体外增殖,并正常传代。依此方法冻存P4代MSCs,于冻存1、3、6个月后复苏,复苏率分别为61.67±4.41%、61.67±3.33%和65.00±11.54%,差异不显著(P>0.05),复苏后细胞增殖、传代正常。对复苏后传代培养的MSCs行免疫荧光鉴定,其表面抗原CD29与CD44阳性表达,说明冻存后的MSCs仍可保持原有的生物学活性。
此外,本实验还对MSCs旁分泌作用对NSCs分化的影响进行了研究。以出生24 h的SD大鼠乳鼠作为供体,分离、培养海马源性NSCs,并诱导其分化;以免疫荧光法检测NSCs表面抗原Nestin,神经元表面抗原NSE及星形胶质细胞表面抗原GFAP。研究结果表明,以无血清培养的方法联合应用碱性成纤维细胞生长因子和表皮生长因可以使体外培养的NSCs表现出良好的持续增殖能力,并且能够稳定地传代扩增,且传代后的NSCs表达Nestin阳性。在去除培养液中的生长因子并添加10% FBS后,NSCs能够分化成为NSE阳性表达的神经元和GFAP阳性表达的星形胶质细胞。在建立NSCs培养方法的基础之上,以MSCs条件培养液诱导NSCs分化,并以上述添加FBS的培养液作为对照,7 d后行免疫荧光双标鉴定,发现与对照组相比,MSCs培养液对NSCs向神经元和星形胶质细胞分化具有促进作用。
There exists a large number of adult stem cells in the marrow of mammals. It is convenient to get the experimental materials from mammals. Therefore, the marrow of mammals became the central issue of researches on adult stem cells. The marrow mesenchymal stem cells(MSCs) is one kind of bone marrow-derived multipotent stem cells, and it was studied more in recent years following after researches on hematopoietic stem cells. The MSCs has strong ability of multiplication and differentiation and it is easy to multiply and induced differentiation in vitro. Moreover, it has the characteristic of secreting varied cell facters to the extracellular space, and it is involved in the restructure of cell micro-environment. They have low immunogenicity when transplanted in vivo. Therefore, there are broad application prospects for MSCs in organ transplantation, autoimmune diseases and replacement therapy.
In this article, SD(sprague-dawley) rats were used as MSCs donors. We investigated the method of isolation and multiplication at first. After getting the bilateral femurs and shinbones of SD rats, we cultured the bone marrow cells by adherent method, and then inoculated marrow cells by the method of density gradient centrifugation. The results showed that it could shorten the time of primary culture effectively with the density of 0.5～1×107cells/mL. The growth of passage cells was stable and the growth curves of P2, P4 and P6 were similar to each other. With immunofluorescence method, it was identified that the surface antigen CD29 and CD44 positive expressed while surface antigen CD34 of hematopoietic cell lines did not express. Double immunofluorescence techniques were used to identify the purity of the P3 cells, The respective positive rates of CD29 and CD44 were 97.96±0.35%and 98.14±0.19%. The above results completely demonstrated that this method was appropriate to gain a large quantity of high purified MSCs in vitro which have high biological activity.
The cryopreservation of MSCs were studied based on the results above. Using LG-DMEM medium, fetal calf serum and DMSO as the main materials, we designed freezing protection solution that had different mixture ratios. P3 MSCs were stored under the condition of 1×106cells/mL density and -80℃for 7 days and then revived. The thawed rate was detected with trypan blue staining method. The results showed that the thawed rate of group F10(80%DMEM+10%FBS+10%DMSO)was 61.00±3.61%, which was notedly better than the other groups. The thawed MSCs could multiply in vitro and pass on from generation to generation normally. P4 MSCs were stored by this method, and revived after one, three, six months. The thawed rates were 61.67±4.41%, 61.67±3.33%, 65.00±11.54%, here was no significant difference among them (P>0.05). The ultiplication and subcultured of MSCs was normal after thawed. The immunofluorescence identification of MSCs subcultured after thawed showed that the surface antigen CD29 and CD44 positive expressed. It illustrated that the MSCs after cryopreservation still had biological activities.
In addition, we did research on the influence of the paracrine interactions of MSCs on the differentiation of NSCs. The neonatal SD rats within 24h were used as NSCs donors, We isolated and cultured the hippocampus-derived NSCs, and then induced them to differentiate. NSCs surface antigen Nestin, neuron surface antigen NSE and fibrous astrocyte surface antigen GFAP were detected with the immunofluorescence method. The results indicated that the serum-free culture combined with basic fibroblast growth factor and epidermal growth factor could make NSCs showing good ability of continuing proliferation, passing from generation to generation stably, and the NSCs after passage were Nestin positive expressed. After removing the growth factor and adding 10%FBS, NSCs could differentiate into neuron that NSE positive expressed and astrocyte that GFPA positive expressed. Subsequently, NSCs were induced to differentiate in the MSCs culture solution, and using the above medium which added FBS 10% as a contrast. 7 days later, by means of immunofluorescence techniques, it was identified that the MSCs culture solution had stimulative effect on the differentiation of NSCs into neuron and astrocyte compared with the controls.
本文以SD(sprague-dawley)大鼠为实验材料,首先探讨了MSCs的体外分离、扩增的方法。取SD大鼠双侧股骨、胫骨,采用全骨髓法贴壁培养MSCs,以梯度密度接种骨髓细胞。研究结果显示,以0.5～1×107个/mL接种骨髓细胞可以有效缩短原代培养的时间,传代细胞生长状态稳定,P2、P4、P6代生长曲线基本相同。免疫荧光法鉴定表面抗原CD29、CD44阳性表达,不表达造血细胞系表面抗原CD34+。免疫荧光双标鉴定P3代细胞的纯度,其CD29阳性率为97.96±0.35%,CD44阳性表达率为98.14±0.19%。上述结果充分证明本方法适于在体外快速获得大量具有较高生物学活性的高纯度MSCs。
在此基础上,对MSCs低温冻存进行了研究。以MSCs基本培养基、胎牛血清、DMSO为主要成分,设计不同配比的冻存保护液。以纯化的MSCs(P3),1×106个/mL密度,-80℃冻存7 d后复苏,台盼蓝据染法检测复苏率,结果显示以80%DMEM+10%FBS+10%DMSO作为冻存保护液,复苏率为61.00±3.61%,显著优于其余各组。复苏后的细胞能够在体外增殖,并正常传代。依此方法冻存P4代MSCs,于冻存1、3、6个月后复苏,复苏率分别为61.67±4.41%、61.67±3.33%和65.00±11.54%,差异不显著(P>0.05),复苏后细胞增殖、传代正常。对复苏后传代培养的MSCs行免疫荧光鉴定,其表面抗原CD29与CD44阳性表达,说明冻存后的MSCs仍可保持原有的生物学活性。
此外,本实验还对MSCs旁分泌作用对NSCs分化的影响进行了研究。以出生24 h的SD大鼠乳鼠作为供体,分离、培养海马源性NSCs,并诱导其分化;以免疫荧光法检测NSCs表面抗原Nestin,神经元表面抗原NSE及星形胶质细胞表面抗原GFAP。研究结果表明,以无血清培养的方法联合应用碱性成纤维细胞生长因子和表皮生长因可以使体外培养的NSCs表现出良好的持续增殖能力,并且能够稳定地传代扩增,且传代后的NSCs表达Nestin阳性。在去除培养液中的生长因子并添加10% FBS后,NSCs能够分化成为NSE阳性表达的神经元和GFAP阳性表达的星形胶质细胞。在建立NSCs培养方法的基础之上,以MSCs条件培养液诱导NSCs分化,并以上述添加FBS的培养液作为对照,7 d后行免疫荧光双标鉴定,发现与对照组相比,MSCs培养液对NSCs向神经元和星形胶质细胞分化具有促进作用。
There exists a large number of adult stem cells in the marrow of mammals. It is convenient to get the experimental materials from mammals. Therefore, the marrow of mammals became the central issue of researches on adult stem cells. The marrow mesenchymal stem cells(MSCs) is one kind of bone marrow-derived multipotent stem cells, and it was studied more in recent years following after researches on hematopoietic stem cells. The MSCs has strong ability of multiplication and differentiation and it is easy to multiply and induced differentiation in vitro. Moreover, it has the characteristic of secreting varied cell facters to the extracellular space, and it is involved in the restructure of cell micro-environment. They have low immunogenicity when transplanted in vivo. Therefore, there are broad application prospects for MSCs in organ transplantation, autoimmune diseases and replacement therapy.
In this article, SD(sprague-dawley) rats were used as MSCs donors. We investigated the method of isolation and multiplication at first. After getting the bilateral femurs and shinbones of SD rats, we cultured the bone marrow cells by adherent method, and then inoculated marrow cells by the method of density gradient centrifugation. The results showed that it could shorten the time of primary culture effectively with the density of 0.5～1×107cells/mL. The growth of passage cells was stable and the growth curves of P2, P4 and P6 were similar to each other. With immunofluorescence method, it was identified that the surface antigen CD29 and CD44 positive expressed while surface antigen CD34 of hematopoietic cell lines did not express. Double immunofluorescence techniques were used to identify the purity of the P3 cells, The respective positive rates of CD29 and CD44 were 97.96±0.35%and 98.14±0.19%. The above results completely demonstrated that this method was appropriate to gain a large quantity of high purified MSCs in vitro which have high biological activity.
The cryopreservation of MSCs were studied based on the results above. Using LG-DMEM medium, fetal calf serum and DMSO as the main materials, we designed freezing protection solution that had different mixture ratios. P3 MSCs were stored under the condition of 1×106cells/mL density and -80℃for 7 days and then revived. The thawed rate was detected with trypan blue staining method. The results showed that the thawed rate of group F10(80%DMEM+10%FBS+10%DMSO)was 61.00±3.61%, which was notedly better than the other groups. The thawed MSCs could multiply in vitro and pass on from generation to generation normally. P4 MSCs were stored by this method, and revived after one, three, six months. The thawed rates were 61.67±4.41%, 61.67±3.33%, 65.00±11.54%, here was no significant difference among them (P>0.05). The ultiplication and subcultured of MSCs was normal after thawed. The immunofluorescence identification of MSCs subcultured after thawed showed that the surface antigen CD29 and CD44 positive expressed. It illustrated that the MSCs after cryopreservation still had biological activities.
In addition, we did research on the influence of the paracrine interactions of MSCs on the differentiation of NSCs. The neonatal SD rats within 24h were used as NSCs donors, We isolated and cultured the hippocampus-derived NSCs, and then induced them to differentiate. NSCs surface antigen Nestin, neuron surface antigen NSE and fibrous astrocyte surface antigen GFAP were detected with the immunofluorescence method. The results indicated that the serum-free culture combined with basic fibroblast growth factor and epidermal growth factor could make NSCs showing good ability of continuing proliferation, passing from generation to generation stably, and the NSCs after passage were Nestin positive expressed. After removing the growth factor and adding 10%FBS, NSCs could differentiate into neuron that NSE positive expressed and astrocyte that GFPA positive expressed. Subsequently, NSCs were induced to differentiate in the MSCs culture solution, and using the above medium which added FBS 10% as a contrast. 7 days later, by means of immunofluorescence techniques, it was identified that the MSCs culture solution had stimulative effect on the differentiation of NSCs into neuron and astrocyte compared with the controls.
引文
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