外源性骨髓间质干细胞尾静脉注射对去势大鼠成骨微环境作用的研究

目的 研究骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSCs)经尾静脉注射对去势大鼠成骨微环境作用后骨质疏松症(osteoporosis,OP)模型大鼠体内BMSCs增殖及成骨、成脂向分化能力的改变.方法 选用同一批次体重相近的10周龄健康雌性SD大鼠行双侧卵巢切除术,3个月后即建立OP动物模型.同样方法选用8周龄健康雌性SD绿色荧光大鼠作为注射用BMSCs来源.实验分为BMSCs注射组与对照组.将1×106个GFP标记BMSCs通过尾静脉注射入BMSCs注射组大鼠.对照组大鼠注入同等体积的PBS缓冲液.注射1周后,分离、培养、纯化两组大鼠骨髓BMSCs,体外培养传代至3～4代后用于实验.MTT法测定不同培养天数的BMSCs生长曲线；脂滴油红0染色法和茜素红染色法检测两组大鼠BMSCs的成骨、成脂能力；RT-PCR法检测大鼠BMSCs成骨相关蛋白Runx2、骨钙素、骨桥蛋白及成脂相关蛋白过氧化物酶体增殖物激活受体y和脂蛋白酯酶mRNA的表达.结果 与对照组大鼠BMSCs相比,BMSCs注射组大鼠BMSCs生长曲线升高变化明显；成脂向诱导后,脂滴数量明显减少；成骨向诱导后,钙化结节明显增多.RT-PCR结果显示,相比对照组,BMSCs注射组大鼠Runx2、骨钙素和骨桥蛋白mRNA表达上调,而过氧化物酶体增殖物激活受体γ和脂蛋白酯酶mRNA表达下调.结论 通过外源性BMSCs尾静脉注射,可以增强OP模型大鼠BMSCs的增殖及成骨分化能力,降低其成脂分化能力,为OP的干细胞治疗提供可能.

Effect of tail vein injection of exogenous BMSCs on osteogenic microenvironment in ovariectomized rats

Objective To investigate the effect of tail vein injection of exogenous bone marrow mesenchymal stem cells (BMSCs) on proliferative, osteogenic and adipogenic potential of BMSCs in ovariectomized osteoporotic rats. Methods The animal model of osteoporosis (OP) was established 3 months after ovariectomy (bilateral ovarian resection)in healthy 10-week-old female SD rats with the same batch and weight. The healthy 8-week-old SD GFP rats were selected in the same way as the BMSCs source. The experiment was divided into BMSCs group and control group. The BMSCs (1×10⁶) marked with GFP were injected into BMSCs group rats by tail vein injection. One week after injection, BMSCs were isolated, cultured and purified in vitro to the 3rd, 4th passage in all groups. The MTT method was used to detect the growth curves of BMSCs. The oil red O dyeing and alizarin red staining (ARS) were used to compare the adipogenic and osteogenic potential between the two kinds of BMSCs, respectively. The RT-PCR was used to detect the expression of osteogenesis-related genes (Runx2, osteocalcin and osteopontin) and adipogenesis-related genes (PPARγ and LPL) in BMSCs. Results Compared with the control group, the growth curve of BMSCs in BMSCs group rose significantly; the calcified nodule increased significantly after osteogenic induction; the lipid droplet decreased significantly after adipogenic induction. RT-PCR results showed that the Runx2, osteocalcin and osteopontin mRNA expression increased, while the peroxisome PPARγ and LPL mRNA expression decreased in BMSCs group. Conclution By tail vein injection of exogenous BMSCs, the proliferative and osteogenic potential of BMSCs in ovariectomized osteoporotic rats is enhanced, while the adipogenic potential is reduced, which provides a possibility of stem cell therapy for osteoporosis.