鼠重组腺病毒介导的骨形成蛋白-9转染大鼠牙囊干细胞的实验研究

Dental follicle stem cells transduced with recombinant bone morphogenetic protein-9 adenovirus promote osteogenesis in rats

目的:探讨鼠重组腺病毒介导的骨形成蛋白9基因转染大鼠牙囊干细胞的可行性及其转染后的成骨作用,以获得可用于牙周骨组织再生工程的基因修饰的种子细胞。方法:取大鼠下颌骨,解剖显微镜下体外分离培养纯化鉴定牙囊干细胞,腺病毒介导的骨形成蛋白9基因转染第三代牙囊干细胞,并设立空白对照组,绿色荧光病毒组(GFP组)。通过观察细胞形态及生长曲线变化,荧光显微镜及RT-PCR检测转染后骨形成蛋白9基因mRNA的表达,碱性磷酸酶及钙茜素红染色测定转染后牙囊干细胞的成骨活性。结果:与未转染对照组比较,转染组细胞转染2周后形成钙化结节,停滞期延长,数量轻度下降,倍增时间延长。牙囊干细胞转染骨形成蛋白9基因后12 h后即有荧光表达,转染3,6,9,12 d后骨形成蛋白9mRNA均呈阳性表达且逐渐增强,未转染对照组呈阴性。转染组碱性磷酸酶活性随转染时间的延长呈升高趋势,ALP染色及茜素红钙结节染色为阳性:未转染对照组碱性磷酸酶染色及钙茜素红染色均呈弱阳性表达,转染组显著高于未转染组。结论:鼠重组腺病毒介导的RBMP-9基因可以成功地转染大鼠牙囊干细胞,转染后牙囊干细胞高表达骨形成蛋白9,且具有明显的成骨作用。

Objectives： To investigate the possibility of dental follicle stem cells （DFSCs）transfected by adenovirus-mediated bone morphngenetic protein- 9 in rats and the osteogenesis following transfection for obtaining the cells of bone regenertation in periodontal tissue engineering. Methods： DFSCs were obtained from mandibular bone of rat, isolated and purified in vitro. DFSCs incubated at passage 3 were transfeetod with adenoviros carrying bone morphogenetic protein - 9 （multiphcity of infection = 100） in the transfection group. A blank control group and Green Fluorescent Protein group{ GFP） were set. Changes in cell morphology and growth curve were measured. Bone morphogenetic protein - 9 mRNA expression was detected by using fluorescence microscope and RT - PCR following transfection. Osteogenic activity was determined by alkaline phosphatase and calcium-alizarin red staining. Results： Calcified nodule formed at 2 weeks following transfection. No significant difference was detected in cell morphology or calcified nodule in the blank control group. Compared with the blank control group, cell stationary phase prolonged, number was slightly decreased, and doubling time prolonged in the transfection group. Fluorescence expression of bone morphogenetie protein- 9 was found at 12 hours following transfection. Bone merphngenetic protein - 9 mRNA expression showed positive reaction and became increased at 3, 6, 9, 12 days following transfection, but negative reaction in the blank control group. The activity of alkaline phosphatase had an increased tendency with prolonged transfection time. Calcium - alizarin red staining showed calcified nodule. Cells in the blank control group were weakly positive for alkaline phosphatase and calcium- alizarin red staining. Conclusions： Adenovirus carrying bone morphogenetic protein - 9 can successfully transfect rat DFSCs, Following transfection, DFSCs highly express human bone morphogenetic protein- 9, and have evidence osteogenesis.