端粒酶反转录酶基因介导的人骨髓基质干细胞永生化研究

Telomerase reverse transcriptase gene mediated immortalization of human bone marrow stromal cells

目的建立永生化人骨髓基质干细胞系,为组织工程提供种子细胞库及标准化细胞系。方法取单克隆培养的人骨髓基质干细胞,采用脂质体转染法将人端粒酶反转录酶(hTERT)基因导入细胞,G418筛选后获得阳性克隆,体外连续扩增培养,RT-PCR检测转染细胞内hTERT基因的整合与表达。取转染后第100代细胞,用成骨诱导条件培养基(普通培养基加地塞米松、β-甘油磷酸、维生素C)、软骨诱导条件培养基(普通培养基加地塞米松、TGF-β、维生素C)、心肌细胞诱导条件培养基(普通培养基加5-氮胞苷)分别向成骨、软骨、心肌细胞诱导培养。于培养后第7、14、21、28天,采用免疫细胞化学染色观察成骨细胞诱导Ⅰ型胶原、骨钙素的表达;采用茜素红染色观察钙结节形成;采用免疫细胞化学染色观察软骨细胞诱导Ⅱ型胶原的表达,同时进行甲苯胺蓝染色;采用免疫细胞化学染色观察心肌细胞诱导横纹肌肌动蛋白的表达,分析细胞系向成骨、软骨及心肌细胞分化的能力。结果 hTERT稳定转染入人骨髓基质干细胞,转化细胞端粒酶表达阳性。转化细胞在体外长期连续培养下生长良好,已传至136代。取第100代细胞进行诱导分化,结果显示其能向成骨、软骨及心肌细胞分化。结论外源性hTERT的导入可致人骨髓基质干细胞永生化,并维持成体干细胞的多向分化特性,可为组织工程提供种子细胞库并为组织工程种子细胞研究提供标准化细胞。

Objective To generate an immortalized human bone marrow stromal cell line and provide seed cells and standard cell line for tissue engineering research. Methods The primary human bone marrow stromal ceils （hMSCs） were monoclonally cultured and transfected with human telomerase reverse transcriptase （hTERT） gene by liposome-mediated transfection, the positive clones obtained after G418 selection were then cultured continuously in vitro, and the gene integration and expression of hTERT was detected by RT- PCR. The 100th passage cells were induced into osteoblasts, chondrocytes and myocytes by cultivation with osteoblast-conditioned medium （standard medium plus dexamethasone, glycerol-phosphoric acid-β and vitamin C）, chondrocyte-conditioned medium （standard medium plus TGF- β, dexamethasone and vitamin C） and myocyte-conditioned medium （standard medium plus 5-azacitidine）, respectively. The differentiation potency of transfected cells was analyzed at the 7th, 14th, 21st and 28th day. The expression of collogen I and osteocalcin were observed by immunocytochemical staining and the formation of calcium nodes were observed by alizarin red staining in osteoblasts; the expression of collogen II was observed by immumocytochemical staining and the secretion of mucopolysaccharides was observed by toluidine blue staining in chondrocytes; and the expression of actin was observed by immumocytochemical staining in cadioeytes. Results The hTERT gene was transfected into hMSCs stably, and the telomerase activity was positive in transfected cells. The transfected cells have now undergone more than 136 passages and continue to proliferate vigorously. The results of inducing differentiation showed that the transfected could differentiated into osteoblasts, chondrocytes and myocytes in vitro. Conclusion The transfection of exogenous hTERT gene may immortalize hMSCs and preserve the multipotentiality, and provide seed cells and standard cell line for tissue engineering research.