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目的探讨外源性人VEGF165基因在HaCaT细胞中表达的可行性及其对体外培养的猪毛囊的影响。方法通过脂质体将与增强型绿色荧光蛋白(EGFP)基因共表达的血管内皮细胞生长因子(VEGF)真核表达载体PIRES2-EGFP-VEGF165瞬时转染HaCaT细胞,应用激光共聚焦显微镜观察EGFP 在HaCaT细胞内的表达,同时利用ELISA法检测VEGF在培养细胞被转染的上清液中的表达。进一步将该上清液加至体外培养的猪毛囊中,显微镜下测量毛囊的平均生长长度,并观察毛囊的形态学变化。结果成功地将VEGF真核表达载体PIRES2-EGFP-VEGF165瞬时转染了HaCaT细胞,以激光共聚焦显微镜观察可见细胞内EGFP的表达,同时用ELISA法证实了上清液中VEGF呈高水平表达,并且该上清液可以明显促进体外培养的猪毛囊生长,延缓其进入退行期。结论应用脂质体能够成功地将外源性人VEGF165基因转染HaCaT细胞,并进行有效表达,其表达的VEGF在体外具有促进猪毛囊生长的生物学话性。
Objective To investigate the feasibility of exogenous VEGF165 gene expression in HaCaT cells and its effect on pig hair follicles cultured in vitro. Methods HaCaT cells were transiently transfected with PIRES2-EGFP-VEGF165, a co-expression vector of enhanced green fluorescent protein (EGFP) gene, and the expression of EGFP was detected by laser confocal microscopy HaCaT cells, and the expression of VEGF in supernatant of cultured cells was detected by ELISA. Further, the supernatant was added to the pig hair follicles cultured in vitro, the average growth length of the hair follicles was measured under a microscope, and the morphological changes of the hair follicles were observed. Results The VEGF eukaryotic expression vector PIRES2-EGFP-VEGF165 was transiently transfected into HaCaT cells. The expression of EGFP was observed by laser confocal microscopy. The VEGF expression in the supernatant was confirmed by ELISA. And the supernatant can significantly promote the growth of pig hair follicles cultured in vitro, to delay its entry into the degenerative phase. Conclusion Liposomes can successfully transfect exogenous human VEGF165 gene into HaCaT cells and express it efficiently. The expressed VEGF has the biological activity to promote the growth of porcine hair follicles in vitro.