携带肝细胞生长因子基因的减毒沙门菌促血管形成的实验研究  

Stimulatory effect of hepatocyte growth factor gene on angiogenesis mediated by attenuated Salmonella

作　　者：哈小琴[1] 郭馨云[1] 葛秀洁[1] 杨迎桂[1] 朱晓红[1] 张萍[1] 杨淑娟[1] 薛荣利[1] 张媛媛[1] 肖娜娜[1] 

机构地区：[1]兰州军区兰州总医院检验中心/甘肃省干细胞与基因药物重点实验室,730050

出　　处：《中国医药生物技术》2015年第6期522-527,共6页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(81273568);甘肃省杰出青年基金(2011GS03841);解放军总后勤部后勤科研项目(CLZ11J09)

摘　　要：目的探讨携带肝细胞生长因子(HGF)基因的减毒沙门菌体外促血管形成的效应。方法构建携带HGF基因真核表达载体的减毒沙门菌菌株(TPH),体外转染人脐静脉内皮细胞(HUVEC)后观察目的基因表达及其增殖活性;评价TPH的细胞表达上清对鸡胚绒膜尿囊膜血管生成效应的刺激效应。结果构建的TPH菌株在体外可有效转染HUVEC并表达有活性的HGF蛋白,6×10~5个HUVEC细胞可表达160~190 ng的HGF蛋白,和对照组相比,TPH转染组细胞表达上清可明显促进HUVEC增殖,而且具有剂量-效应关系;TPH转染组细胞表达上清也刺激鸡胚绒膜尿囊膜小血管生成,其血管数量(133.0±11.5)/cm^2明显大于转染TP的HUVEC细胞上清组(83.3±5.5)/cm^2和对照组(62.7±7.1)/cm^2(P<0.05)。结论携带HGF基因的减毒沙门菌可有效转染HUVEC并促进细胞增殖,刺激小血管新生,可能在组织创面愈合中起重要作用,在胃溃疡治疗中有潜在的应用价值。Objective To investigate the stimulatory effect of TPH, a recombinant attenuatedSalmonella strain carrying hepatocyte growth factor （HGF） gene eukaryotic expression vector, on angiogenesisin vitro. Methods A stable strain （TPH）, recombinant attenuatedSalmonella carrying HGF gene eukaryotic expression vector, was constructed. The TPH was transfected into human umbilical vein endothelial cells （HUVEC）. And the express level of HGF interest protein was assessed by ELISA. Effects of the expression product after TPH transfection on HUVEC proliferation and angiogenesis were assayed by MTT and chicken embryos chorionic allantois membrane （CAM） stimulating activity, respectively. Results The TPH could be effectively transfected into HUVEC in vitro and highly express HGF interest protein. The expression concentration of HGF protein is （160 - 190）ng/ml by 6 ＆#215; 105 cells. The expression supernatant after TPH transfection could significantly stimulate the proliferation of HUVEC, with a dose-effect relation, compared with the supernatant （same volume as HGF expression supernatant） from TP-transfected and control groups. The expression of HGF supernatant after TPH transfection also could significantly promote the angiogenesis of chorioallantoic membrane （CAM）. The numbers of blood vessel in expression supernatant from TP-transfected group （83.3 ＆#177; 5.5）/cm2 and PBS group （62.7 ＆#177; 7.1）/cm2 were significant fewer than that in HGF expression product from TPH-transfected group （133.0 ＆#177; 11.5）/cm2 （P＆lt; 0.05）. Conclusion TPH can effectively transfect HUVEC and promote the cell proliferation, stimulate angiogenesis, may play an important role in wound healing in organization, has potential application value in gastric ulcer treatment.

关 键 词：肝细胞生长因子 基因转移技术 血管生成诱导剂 转化 细菌 减毒沙门菌 

分 类 号：R378.22[医药卫生—病原生物学]