人骨髓基质干细胞体外诱导和鉴定

Induction of human mesenchymal stem cells in vitro and related identification

目的建立人自体骨髓基质干细胞(human menchymal stem cells,hMSCs)体外分离、鉴定体系,建立经诱导表达成骨、软骨、脂肪细胞表型的体外培养体系,探讨作为骨组织工程种子细胞的可能性,为组织工程技术应用打下基础。方法抽取患者骨髓5ml,以密度梯度法分离hMSCs,使用流式细胞仪鉴定细胞表型;应用免疫组化和分子生物学技术,对hMSCs进行成骨、软骨、脂肪细胞的诱导培养和表型鉴定。结果第2代hMSCs阳性细胞表达率CD105(78±6)%,CD166(43±7)%,CD29(69±12)%;hMSCs成骨诱导培养第3代平均扩增(163.4±13.4)倍,形成钙结节,骨细胞转录因子、骨钙素、骨桥蛋白和I型胶原免疫荧光阳性,RT-PCR证实有I型胶原、骨钙素、骨桥蛋白和骨结合素mRNA表达,对照组阴性;成软骨细胞诱导培养后II型胶原、SOX9(SRY-type HMGbox9,是在哺乳动物性别决定和软骨生成中起着关键调控作用的一个基因)。免疫荧光阳性,RT-PCR证实II型胶原、软骨聚集蛋白聚糖mRNA表达,对照组阴性;成脂肪细胞诱导培养后,油红-O染色阳性,RT-PCR证实PPAR2 mRNA的表达,对照组阴性。结论通过密度梯度法可获得较高纯度hMSCs,经体外培养能保持多项分化潜能,体外诱导培养第3代仍可表达各种细胞表型,能够满足骨组织工程临床应用对种子细胞质和量的需要。

OBJECTIVE To establish the isolation and identification system of human mesenchymal stem cells （hMSCs） in vitro, and then to establish a stable cultured system in which the hMSCs can be induced to osteoblasts, chondroblasts and lipoblasts in vitro. To study the possibility if hMSCs can be used as the seed cells in bone tissue engineering.This study would lay a foundation for the clinical application of tissue engineering. METHODS 5ml bone marrow of the patient was aspirated from lilac crest. The human MSCs were isolated using Percoll gradient centrifugation. The hMSCs were cultured in DMEM medium in vitro, FACS （fluorescence axtivated cell sorter） was used to identify the phenotype of hMSCs. The hMSCs were cultured in vitro, and induced to osteoblasts, chondrocytes and lipocytes. The changes of phenotype were tested by mmunohistochemisty and molecular biology technique. RESULTS The hMSCs were isolated by Percoll gradient centrifugation from patient＇s bone marrow. The expression rate of MSC markers including CD105, CD166, CD29 were （78±6） %, （43±7） %, （69±12） % respectively. The phenotype of osteoblasts induced from hMSCs was verified by the mineralized nodes formation when cultured in vitro and Col, and OCN expression showed by immunohistochemistry and RT-PCR. The phenotype of chondrocytes induced from hMSCs was verified by type, collagen, SOX9 expression with immunohistochemical staining technique and type, collagen, aggrecan mRNA expression with RT-PCR. The lipocytes＇ phenotype was verified by positive result of oil red O staining and PPAR 2mRNA expression by RT-PCR. CONCLUSION The highly pure hMSCs can be harvested by means of density gradient centrifugation, and the hMSCs have multi-potentiality of cell differentiation via induced culture in vitro. The third passage of hMSCs can be induced to express osteoplast phenotype and meet the qualitative and quantitative demands of seed cells when bone tissue engineering was used in clinical practice.