HIF-1α促进BMSCs成骨及成血管作用的体内外研究

The study on HIF-1α gene in promoting the formation of bone and blood vessel by BMSCs in vitro and in vivo

目的:运用基因工程技术,检测HIF-1α基因诱导骨髓间充质干细胞(BMSCs)在体内、外向成血管和成骨方向分化的作用。方法:(1)HIF-1α基因突变后,应用Lentivirus构建Lenti-LacZ、Lenti-WT、Lenti-MT。(2)分别用Lenti-LacZ、Lenti-WT及Lenti-MT转染BMSCs,检测①细胞转染效率;②目的基因在mRNA及蛋白水平的表达;③目的基因在BMSCs细胞内的定位。(3)BMSCs成功转染目的基因后,分别在特定时间点提取总RNA和蛋白,通过RT-PCR和Western印迹检测目的基因对BMSCs成血管和成骨因子表达的调控作用。(4)行碱性磷酸酶(alkalinephosphatase,ALP)和钙结节的表达检测。(5)检测生物支架材料-明胶海绵(GS)的结构、形态及细胞附着情况。(6)建立F344大鼠颅骨双侧直径5 mm的标准骨缺损模型,分别将细胞复合支架材料植入骨缺损区。术后8周取材,行Microfil灌注,观察血管形成,通过大体标本观察、X线及Micro-CT检查、组织学和形态学检测等观察骨修复情况,采用SPSS10.0软件包对结果进行单因素方差分析,评价修复效果。结果:当感染复数(MOI)=15时,BMSCs的转染效率最高。免疫荧光检测表明,目的基因在BMSCs细胞核内。体外常氧条件下,HIF-1α能够显著上调BMSCs的成骨和成血管因子的表达,且ALP和钙结节结果表明目的基因可诱导BMSCs骨向分化。体内实验结果显示,8周时,Lenti-WT组和Lenti-MT组对骨缺损的修复作用明显强于Lenti-LacZ组,而Lenti-MT组又优于Lenti-WT组。结论:HIF-1α基因可以显著提高BMSCs成血管和成骨活性。

PURPOSE： Tissue engineering technique was used to explore the function of HIF-1α gene in promoting thedifferentiation of BMSCs into blood vessels and bone both in vitro and in vivo. METHODS： （1） Mutation of HIF-lct gene, and construction of Lenti-WT, Lenti-MT and Lenti-LacZ. （2） BMSCs were treated with Lenti-LacZ, Lenti-WT and Lenti- MT. Then the cells were detected by ①morphology and transduction efficiency; ②expression of target gene at the level of mRNA and protein; ③detecting intracellular location. （3） After transduction, RNA and protein were extracted, the expression of osteogenic and angiogenic factor were detected by RT-PCR and Western-blot. （4） Alkaline phosphatase and alizarin red was used to detect alkaline phosphatase （ALP） and calcium nodes. （5）Observation the surface of scaffold material-gelatin sponge （GS）. （6） Establishment of bilateral calvarial bone defect （5mm in diameter） in F344 rat model. The scaffolds containing BMSCs were implanted to repair bone defects. After 8 weeks newly formed blood vessels and bone formation were observed by Microfil perfusion, gross specimen observation, X-ray, Micro-CT, histological, morphological observation. Statistical analysis was conducted using ANOVA with SPSS10.0 software package. RESULTS： When MOI was 15, the transduction efficiency could reach optimization. The results of immunofluorescence showed that HIF-Iot gene was located in cell nucleus, expression of target gene was detected at both mRNA and protein level. After gene transduction, expression of osteogenic and angiogenic factor at the mRNA and protein levels were significantly increased. Results of ALP and ARS showed that the target gene could induce the osteogenic differentiation of BMSCs treated by HIF-lct. In vivo study showed different effect in repairing skull defects in rats among Lenti-LacZ and other groups： After 8 weeks, bone repair in Lenti-MT and Lenti-WT groups was better than that in Lenti-LacZ group, and the best result was found in Lenti-WT group. CONCLUSIONS： HIF-lct could promote BMSCs to forme blood vessels and bone. Supported by National Natural Science Foundation of China （81100788）, Key Project of Chinese Ministry of Education （212080）, China Postdoctoral Science Foundation （2012M510114）, Grants of Scientific Research of BSKY （XJ201109） and Young Top-notch Talent Support Scheme from Anhui Medical University.