核心结合因子(Cbfa1／Runx2)重组腺病毒载体的构建、鉴定及在脂肪来源干细胞中的表达

Construction of Recombinant Adenovirus Cbfa1/Runx2 and Its Expression in Adipose-derived Stem Cells

目的:验证核心结合因子(Cbfa1/Runx2)的重组腺病毒载体可高效感染脂肪组织来源干细胞并表达蛋白。方法:分离、扩增脂肪组织来源干细胞并诱导其向骨细胞、脂肪细胞分化。将Cbfa1/Runx2cDNA的全长序列亚克隆到穿梭质粒pAdTrack上,在BJ5183细菌内和pAdEasy1同源重组,筛选阳性克隆,命名为Ad-Cb-fa1/Runx2。经酶切、PCR鉴定,线性化后以脂质体法转染293T细胞进行包装、扩增,利用报告基因GFP对病毒滴度和感染效率进行监测。通过RT-PCR、间接免疫荧光检测Ad-Cbfa1/Runx2感染脂肪组织来源干细胞后Cbfa1/Runx2基因的表达。结果:脂肪组织来源干细胞细胞数目两天增长一倍。在特定诱导条件下,可向成脂细胞、成骨细胞分化。酶切及PCR鉴定证实Cbfa1/Runx2基因重组腺病毒载体构建成功。Ad-Cbfa1/Runx2对脂肪组织来源干细胞的转染效率为81.39%。转染3天后,PCR、间接免疫荧光检测到脂肪组织来源干细胞中Cbfa1/Runx2蛋白的表达。结论:成功分离、培养了脂肪组织来源干细胞,构建了含有小鼠Cbfa1/Runx2基因的重组腺病毒载体,证明了Ad-Cbfa1/Runx可高效感染脂肪组织来源干细胞并表达Cbfa1/Runx2蛋白。

Objectives To construct the recombinant adenovirus that express core banding factor 1 （Cbfa1） and to isolate adipose-derived stem cells （ADSCs） for searching therapy genes and carrier cells for genes enhancing bone tissue engineering. Methods Adipose derived stem cells were harvested from inguinal groove of fourweek old male SD rats. Multi-differentiation of adipose derived stem ceils was induced by a classical method. Full-length Runx2 cDNA was digested and inserted into the pAdTrack vector, which was subsequently cotransformed into E. coli. BJ5183 with pAdEasy-1. The recombinant plasmid was identified by restriction enzyme digesting and PCR, and then the linearized recombinant plasmid was transfected into 293A cells using lipofectamine for adenovirus packaging. Recombinant adenovirus carrying the Cbfa1/Runx2 gene （called Ad-Cbfa1/Runx2） was purified by CsCI gradient ultracentrifugation. The viral titer was detected by GFP reporter gene. Then the Ad- Cbfa1/Runx2 was used to transduce adipose-derived stem cells. The Cbfa1/Runx2 expression was assessed by RT-PCR, immunofluorescence, and Western blot. Results Approximately 2 ×10^6 ADSCs were harvested. The doubling time of the cells was 48 hours, reaching saturation in 4.5 days. ADSCs could be induced to adipocyte and osteoblast. Restriction endonuclease digestion and RT- PCR confirmed that Cbfa1/Runx2 was cloned to the adenovirus vector successfully. The RNA and protein expressions of Cbfa1/Runx2 were detected after Ad-Cbfa1/Runx2 infected adipose-derived stem ceils by RT-PCR, immunofluorescence, and Western blot. Conclusions Adipose-derived stem cells can be isolated and infected by recombinant adenovirus Ad-Cbfa1/ Runx2 successfully. The Ad-Cbfa1/Runx2 can mediate the expression of Cbfa1/Runx2 in adipose-derived stem cells.