脑源性神经营养因子基因修饰人骨髓间充质干细胞有限细胞系的建立

Establishment of the definite cell line of brain-derived neurotrophic factor gene-modified human bone marrow mesenchymal stem cells

目的:建立脑源性神经营养因子基因修饰的人骨髓间充质干细胞系。方法:实验于2004-02/2005-06在解放军军事医学科学院干细胞研究中心完成。人类骨髓细胞来源于解放军三○七医院骨髓移植治疗的6名健康成年骨髓供者,男3名,女3名,年龄25～45岁。常规分离培养人骨髓间充质干细胞,流式细胞术检测骨髓间充质干细胞表面分子CD34、CD44、CD45,采用重组逆转录病毒载体将脑源性神经营养因子基因转入人骨髓间充质干细胞。检测脑源性神经营养因子基因修饰的骨髓间充质干细胞的增殖特性,细胞内脑源性神经营养因子的蛋白表达,细胞培养液中脑源性神经营养因子含量,以及PCR检测脑源性神经营养因子-骨髓间充质干细胞中外源性脑源性神经营养因子基因DNA。结果:①人工分离培养出的骨髓间充质干细胞,形态、大小一致,呈长梭型或长多边形,从P0至P20基本保持一致,P2代骨髓间充质干细胞表达基质细胞表面标志CD44,不表达造血系细胞表面标志CD34、CD45。②反转录病毒感染骨髓间充质干细胞的感染效率约为10%,修饰后形成的脑源性神经营养因子-骨髓间充质干细胞在形态上始终保持一致,与骨髓间充质干细胞几乎完全一样,生长速率与未经基因修饰的骨髓间充质干细胞基本相同,培养10代后细胞停止分裂,免疫细胞化学染色显示脑源性神经营养因子-骨髓间充质干细胞的脑源性神经营养因子阳性率超过98%,培养72h后培养基中脑源性神经营养因子的含量较之骨髓间充质干细胞提高约20000倍。③PCR对脑源性神经营养因子-骨髓间充质干细胞基因组DNA进行扩增,得到外源性脑源性神经营养因子条带。结论:利用反转录病毒载体进行基因修饰得到了稳定的脑源性神经营养因子基因修饰的人骨髓间充质干细胞有限细胞系,为基因治疗提供了一种以多潜能成体干细胞为载体的种子细胞。

AIM： To establish brain-derived neurotrophic factor （BDNF） gene-modified human bone marrow mesenchymal stem （BDNF-MSCs） cell line. METHODS： The experiment was conducted in the Research Center for Stem Cell, Beijing Academy of Military Medical Sciences between February 2004 and June 2005. Human bone marrow mesenchymal stem cells （MSCs） were provided by 6 healthy adult marrow donors including 3 males and 3 females aged 25-45 years treated by marrow transplantation in the 307 Hospital of Chinese PLA. MSCs were isolated and incubated by routine way, and the surface antibodies of CD34, CD44 and CD45 were detected by flow cytometry. The BDNF genes were transfected to human MSCs with recombinant retrovirus vector containing human BDNF gene. The proliferation and BDNF protein expression were detected, and exogenous BDNF genes in BDNF-MSCs were detected by PCR. RESULTS： ①The artificial MSCs possessed unanimous morphous and size with long spindle or long polygon from passage 0 to passage 20, positive expression of CD44 and negative expression of CD34 and CD45 were found in MSCs of passage 2. ②The infection rate of retrovirus infecting MSCs was about 10%, there were no difference between BDNF- MSCs and MSCs in morphology and proliferation rate, but BDNF-MSCs could not proliferate after passage 10. Immunocytochemical staining suggested the positive rate BDNF exceeded 98% in BDNF-MSCs, content of BDNF in medium was increased 20 000 times in BDNF-MSCs than that in MSCs. ③Exogenous BDNF gene belt was discovered in BDNF-MSCs after amplification by PCR. CONCLUSION： Stable BDNF gene-modified human bone marrow mesenchymal stem cell line is established by retrovirus vectors, which provide a kind of seed cell with multiple potential adult stem cells as carrier.