成人自体血清体外扩增培养骨髓间充质干细胞的生物学特性分析

Biological characteristics of adult human marrow mesenchymal stem cells cultured in autoserum

目的:为避免种子细胞培养过程中应用异体或异种血清的排斥及伦理问题,制备成人自体血清体外扩增培养骨髓间充质干细胞,观察其增殖及生长特征。方法:实验于2005-01/2006-01在北华大学医学院细胞生物实验室完成。①细胞来源:选取北华大学医学院附属医院收治的行骨髓干细胞保健治疗的成年志愿者3名,25～30岁,对本实验均知情同意。②实验方法:空腹无菌采集志愿者外周静脉血40mL,4℃静置4h,37℃静置4h,500g离心30min,吸取上清,即自体血清。从骨髓血中提取间充质干细胞,利用DMEM/F12培养基添加自体血清进行黏附培养和扩增。原代记为P1,按1∶3进行传代接种,第2代记为P2,以此类推。每次传代培养的细胞接种密度严格控制与原代培养接种密度相同的水平。③实验评估:倒置光学显微镜下观察骨髓间充质干细胞生长和形态变化,计数和MTT法观察原代和传代培养的增殖及生长特征,免疫组化法分析骨髓间充质干细胞纯度。结果:①原代及传代培养的成人骨髓间充质干细胞的光镜观察:原代培养24h后可见贴壁细胞,部分细胞有伪足伸展,第3天换液时有散在克隆生长,第5天呈克隆式增殖,10～12d可达90%细胞融合,基本铺满孔底面,呈骨髓间充质干细胞特征性的旋涡状生长,排列有方向性,旋涡中心细胞呈多层分布,细胞界限不清,细胞形态多呈梭形。传代细胞的生长较原代快,4h内即发现有贴壁细胞,24h内完全贴壁并伸展,第6～8天即可达到80%以上融合。②成人骨髓间充质干细胞的生长曲线分析:原代接种后2～6d为生长潜伏期,第7天开始形成大小不一的多个细胞克隆,至第9天细胞克隆进一步扩大,细胞数目呈指数级递增,为对数增殖期;第10～12天细胞生长进入平台期,原代培养结束。传至5代内的细胞生长旺盛,生长特性基本与第1代一致。③成人骨髓间充质干细胞免疫组化鉴定结果:P0、P1、P3、P5代细胞CD34染色呈阴性,而CD105染色阳性细胞在95%以上。结论:以自体血清体外扩增培养的骨髓间充质干细胞纯度高、增殖活性强,且至少在5代内生长旺盛并维持其生物特性。

AIM： To avoid the heterogenous or heterologous serum rejection and ethic problems in seed cell culture, the marrow mesenchymal stem cells （MSCs） from bone marrow were cultured in vitro, and the regeneration and growth characters of cultured MSCs were observed. METHODS： The experiment was performed in the laboratory of cytobiology of Beihua University Medical College from January 2005 to January 2006.①Three adult volunteers aged 25-30 years, taking health protection with bone marrow stem cell were selected from the affiliated hospital of Beihua University. They were informed all about this experiment.②40 mL peripheral venous blood was harvested from the volunteers under sterile condition, put at 4℃ for 4 hours, then at 37℃ for 4 hours, and centrifuged for 30 minutes （500 g） to take the serum （autoserum）. MSCs were extracted from the bone marrow blood and cultured and proliferated in vitro using DMEM/F12 with autoserum. P1 represented the primary culture. Then the cells were cultured through the passages with the ratio 1：3. Every time the cell density of serial subcultivation was controlled to the same as the primary culture.③The growth and morphologic change of MSCs were observed under inverted light microscope. The cell proliferation and growth of primary culture and serial subcultivation were observed by cell counting and MTT methods. The purity of cells was analyzed with immunohistochemistry. RESULTS： ①After 24 hours of primary culture, there was adherence cell and part of them had pseudopodia stretching. There were scattered cloning on the 3^rd day during medium exchange. On day 5, clone proliferation was observed. After 10 to 12 days, 90% cell fused, and nearly covered the undersurface of the hole. MSCs presented distinctive vortex growth and arranged regularly. In the center of vortex, the cell was multi-layer distributed with obscure boundary and fusiform shape. Serial subcultivation cell grew faster than the primary culture cells. Adherent cells were found within 4 hours and fully adhered and spread within 24 hours. On days 6 and 8, above 80% cells were fused.②Two to six days after primary culture was the growth latency; many clones formed with different sizes since the 7^th day, and on the 9th day, the cell clones became lager. The number of cells was increased by degrees of index number, this period was called logarithm proliferation period. From the 10^th to 12^th days, cell growth went into platform period, and primary culture was finished. Within 5 passages, the characters of the cell were the same to the first passage.③lmmunohistochemical results showed that the cells of P0, P1, P3, and P5 were negative for CD34, but cells above 95% were positive for CD105.CONCLUSION： High purity and active proliferation MSCs are cultured by autoserum, which grow prosperously and maintain its biological characters for at least 5 passages.