hPDGF-A／hBD2双基因修饰骨髓源间充质干细胞外源基因体外与体内的表达

Exogenous Gene Expression In Vitro and In Vivo in Bone Marrow Mesenchymal Stem Cells Modified by hPDGF-A and hBD_2

本研究观察腺病毒介导hPDGF-A/hBD2双基因转染大鼠骨髓间充质干细胞(BM-MSC)体外以及移植后体内局部的外源基因表达情况。将已成功构建和包装的hPDGF-A/hBD2双基因共表达腺病毒体外感染原代分离培养的BM-MSC后,采用免疫荧光细胞化学法检测目的基因蛋白的表达。收集经重组腺病毒感染的BM-MSC的无血清培养上清,采用划痕试验检测培养上清中分泌的hPDGF-A的促迁移效应。将重组腺病毒感染的BM-MSC局部点状注射至大鼠放射创伤复合伤创面,在各时相点进行冰冻切片并观察创面移植的BMSC的分布,同时采用免疫组织化学的方法观察外源基因的表达。结果表明:在体外重组腺病毒感染后的大鼠BM-MSC表达报告基因EGFP。免疫荧光细胞化学检测显示双基因修饰的BM-MSC表达hPDGF-A和hBD2。划痕实验证实双基因修饰BMSC培养上清组在8、12、24和30小时的愈合面积百分比显著高于对照组(p<0.05)。荧光显微镜观察大鼠创面移植的外源性基因修饰的BM-MSC显示,至少在移植后2周之内BM-MSC可以较高水平地表达以EGFP为指示的外源基因。创面免疫组织化学染色表明,外源基因从第3天开始表达,第7天达到高峰,第21天仍有少量表达。结论:hPDGF-A/hBD2双基因修饰BM-MSC在体外、体内均可使目的基因得到有效表达,成为hPDGF-A/hBD2双基因修饰BMSC促进难愈创面愈合的基础。

The objective of this study was to investigate the expression of exogenous hPDGF-A and hBD2 in genemodified bone marrow mesenchymal stem cells （BM-MSCs） in vitro and in vivo. Recombinant adenovirus vector expressing hPDGF-A/hBD2 genes was constructed and packaged into virion. Primary isolated and cultured BM-MSCs were transfected by using hPDGF-A hBD2, then the expressions of exogenous hPDGF-A/hBD2 were detected by immunocytochemical staining in vitro. The conditioned medium （serum-free cultured supernatant of BM-MSCs transfected with recombinant adenovirus ） collected from gene-modified BM-MSCs was applied to scratch wound on monolayer cells of multipotential cell line 10T1/2 in order to confirm the stimulative effect of hPDGF-A on cell migration. Geneodified BM-MSCs were topically transplanted on wound of rats with radiation and skin excision combined injury. The distribution of BM-MSCs and expression of hPDGF-A/hBD2 on the wound was observed by fluorescent microscopy and immunohistochemical staining respectively. The results indicated that the rat BM-MSCs transfected with recomlinant adenovirus could express the EGFP in vitro. The immunflnorescent cytochemistry assay showed that the gene-modified BM-MSCs expressed the hPDGF-A and hBD2. The scratch test confimed that the percentage of healing area of wound in cultured supernatant group of gene-modified BM-MSCs was significanly higher than that in control group on 8, 12, 24 and 48 hours （p 〈 0.05 ）. The fluorescence microscopy of exogenous gene-mod-ified BM-MSCs transplanted on wound revealed that the gene-modified BM-MSCs could higher express exogenous genes of EGFP at least within 2 weeks. The immunohistochemistry staining of wound indicated that the expression of exogenous genes began from day 3, reached to peak on day 7, and still visible on day 21 even though the expression became weak because of the possible dilution of the exogenous genes during cell division. It is concluded that efficient expression of exogenous hPDGF-A/hBD2 in gene-modified BM-MSCs are demonstrated both in vitro and in vivo, which suggests that the molecular mechanism underlying chronic wound-heating accelerated by the strategy combining cell therapy with gene therapy.