[成骨细胞条件培养基对骨髓间充质干细胞分化的影响]

Zhou Kunpeng, Chen Xiaohe, Chang Li, Li Xiuqun, Li Xunhu, Zhang Jue, Deng Li

Division of Stem cell and Tissue Engineering, State key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu Sichuan, 610041, P.R. China.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009 Feb;23(2):145-50.

OBJECTIVE

To study the effect of rat osteoblast conditioned culture medium on the BMSCs differentiation of allogeneic rat and to find a new approach to provide seed cells for bone tissue engineering.

METHODS

BMSCs and osteoblasts were harvested from 10 healthy one-week-old SD rats (male and female, weighing 20-30 g) by adherent method and enzyme digestion method respectively. Cell identification was conducted. Osteoblast conditioned culture medium was prepared by mixing supernatant of osteoblasts at passage 1-5 with complete medium (1:1). Then, BMSCs at passage 2 were co-cultured with osteoblast conditioned culture medium (inducement group) and complete medium (control group), respectively. The morphological changes of co-cultured BMSCs were observed by inverted phase contrast microscope, the growth condition of BMSCs was detected by MTT method, the expressions of ALP, Col I and osteocalcin (OCN) in the co-cultured BMSCs were tested by immunohistochemistry staining, and the expressions of Col I and OCN mRNA were detected by RT-PCR.

RESULTS

In the inducement group, BMSCs grew bigger, changing from long fusiform to flat and polygon with protuberance 7 days after co-culture; the presence of cell colony-like growth was observed 9 days after co-culture. Cell growth curve demonstrated that the counts of BMSCs was increased with time, there were more cells in the control group than that of the inducement group, and there was a significant difference in cell counts between the control and the inducement group 4-7 days after co-culture (P < 0.05). For the inducement group, ALP staining was positive 12 days after co-culture, the calcium nodules were appeared 18 days after co-culture, Col I and OCN were positive 21 days after co-culture, and the expressions of Col I and OCN mRNA were detected by RT-PCR 21 days after co-culture.

CONCLUSION

Rat osteoblast conditioned culture medium can significantly induce the differentiation of allogeneic rats' BMSCs towards osteoblasts.

目的

研究大鼠成骨细胞条件培养基对同种异体大鼠骨髓间充质干细胞（BMSCs）分化的影响，寻找为骨组织工程提供种子细胞的新途径。

方法

分别采用贴壁法和酶消化法从10只健康的1周龄SD大鼠（雌雄不限，体重20 - 30 g）中获取BMSCs和成骨细胞，并进行细胞鉴定。将第1 - 5代成骨细胞的上清液与完全培养基按1:1混合制备成骨细胞条件培养基。然后，将第2代BMSCs分别与成骨细胞条件培养基（诱导组）和完全培养基（对照组）共培养。通过倒置相差显微镜观察共培养的BMSCs的形态变化，采用MTT法检测BMSCs的生长状况，通过免疫组织化学染色检测共培养的BMSCs中碱性磷酸酶（ALP）、I型胶原（Col I）和骨钙素（OCN）的表达，通过逆转录-聚合酶链反应（RT-PCR）检测Col I和OCN mRNA的表达。

结果

诱导组中，共培养7天后BMSCs体积增大，由长梭形变为扁平多角形且有突起；共培养9天后观察到细胞呈集落样生长。细胞生长曲线显示，BMSCs数量随时间增加，对照组细胞数量多于诱导组，共培养4 - 7天后对照组与诱导组细胞数量差异有统计学意义（P < 0.05）。诱导组共培养12天后ALP染色呈阳性，共培养18天后出现钙结节，共培养21天后Col I和OCN呈阳性，共培养21天后通过RT-PCR检测到Col I和OCN mRNA的表达。