腺病毒介导HIF-1α修饰的脂肪源性干细胞异位成软骨的研究

A study on the ectopic cartilage formation of adipose-derived stem cells by adenovirus-mediated HIF-1αgene transfection

目的探讨重组腺病毒介导的低氧诱导因子-1α(HIF-1α)基因转染对脂肪源性干细胞(adipose-derivedstem cells,ASCs)异位成软骨的作用。方法构建能介导HIF-1α基因转染和表达的腺病毒载体(Ad-HIF-1α-GFP),转染体外培养的ASCs,RT-PCR检测其HIF-α基因的表达,同时检测转染后细胞Ⅱ型胶原的表达。随后将纤维蛋白胶分别与Ad-GFP-ASCs和Ad-HIF-1α-GFP-ASCs复合种植在裸鼠皮下,4周后行HE染色和甲苯胺蓝(Toluidine blue)染色进行观察。结果荧光显微镜观察证实Ad-HIF-1α-GFP-ASCs的转染效率可达到80%;RT-PCR检测结果显示Ad-GFP-ASCs组微弱表达HIF-1α,Ad-HIF-1α-GFP-ASCs组HIF-1α基因则高表达,且Ad-HIF-1α-GFP-ASCs组Ⅱ型胶原表达量明显高于Ad-GFP-ASCs组(P<0.05);形态学染色表明Ad-HIF-1α-GFP转染的ASCs主要为圆形,细胞伸展比率较低,但细胞数目较多,且细胞分泌较多的蛋白多糖。结论腺病毒介导的HIF-1α可诱导ASCs向软骨细胞分化。

Objective To investigate the effect of ectopic cartilage formation of adipose-derived stem cells （ASCs） by adenovirus-mediated HIF-1α gene transfection. Methods Adenoviral vector was constructed with full length human HIF-1α gene and Ascs was transfected in vitro. The expressions of HIF-1α gene and collagen Ⅱ gene were detected by RT-PCR in transfected group. Then Fibrin glue and those composites mixed with Ad-GFP-ASCs and Ad- HIF-1α-GFP-ASCs respectively were transplanted into those naked mice. Those samples were acquired after 4 weeks and were tested by H＆E and Toluidine blue staining. Results Confirmed by fluorescence microscope, the transfection efficiency of Ad-HIF-1α-GFP-ASCs was 80%. The expression of HIF-1α gene was positive in the Ad-HIF- 1α-GFP- ASCs group and negative in the Ad-GFP-ASCs group according to the RT-PCR test results, and the relative value of the expression of collagen II mRNA had significant difference between the two groups （P〈0.05）. Histology results suggested that in Ad-HIF- 1α-GFP group the transfected ASCs were round and a large amount of proteoglycan was around them. The percentage of stretch of cells was low, but a large number of cells existed. Conclusions Adenovirus-mediated HIF- 1α may induce the differentiation of adipose-derived stem cells into chondrocyte in vivo.