摘要
目的:本研究拟用肝细胞生长因子(HGF)基因修饰内皮祖细胞(EPCs),以观察其对 EPCs 增殖、迁移、分泌一氧化氮(NO)及生血管能力的影响。方法:采用梯度离心法分离并培养大鼠骨髓 EPCs,荧光 DiI 标记乙酰化低密度脂蛋白(DiI-acLDL)摄取和异硫氰酸荧光素标记的荆豆凝集素(FITC-UEA-I)结合荧光双染法鉴定大鼠 EPCs。细胞分为 HGF 转染组、阴性对照组及空白对照组,HGF 组用脂质体介导法导入 pIRES2-EGFP-HGF 质粒,阴性对照组导入 pIRES2-EGFP 质粒,空白对照组不导入质粒,然后用荧光显微镜观察绿色荧光蛋白的合成及逆转录多聚酶链反应(RT-PCR)检测细胞 HGF mRNA 的表达来鉴定转染的 EPCs。用四唑盐(MTF)法、改良的 Bodyen 小室及硝酸盐还原法观察细胞增殖、移行及分泌 NO 能力;体外成管试验用于分析细胞生血管的能力。结果:原代细胞培养4天后发现可成梭形贴壁细胞,7天后细胞成集落状,原代细胞培养21大左右接近融合并呈典型的鹅卵石样排列,细胞 DiI-LDL 摄取试验阳性,FITC-UEA-I 染色阳性。转染18h 后 HGF 转染组及阴性对照组细胞绿色荧光蛋白检测呈阳性,转染率为40%,而空白组则呈阴性。RT-PCR 结果显示,HGF 转染组细胞内 mRNA 的表达显著高于对照组,而空白对照及阴性对照组 HGF mRNA 表达无显著性差异。HGF 转染组细胞增殖、移行能力及分泌 NO 量均显著高于对照组。在 ECMatrix 培养基上培养12h,HGF 转染组体外成管评分显著高于对照组。结论:HGF 基因修饰能够显著提高 EPCs 增殖、移行、分泌 NO 及生血管能力,改善 EPCs 的生物学功能。
Objective ：The aim of this study is to observe the effect of transfected HGF gene on the abilities of endotheliel progenitor cells （EPCs） to proliferate, migrate, secrete NO and angiogenesis. Methods ： Rat bone marrow-derived EPCs were separated by gradient centrifugation of Ficoll and cultured, and then identified by dual-staining ceils positive for both lectin and DiI-acLDL. EPCs were divide into three groups：transfected HGF, negative con- trol and blank control. In transfected HGF group, pIRES2-EGFP-HGF was transfected into EPCs via mediation by liposome, pIRES2-EGFP was transfected into EPCs in negative control group. No plasmid was transfected in blank control group. After transfection, the synthesis of EGFP was observed by fluorescence microscope, and the expression of HGF mRNA in EPCs was detected by RT-PCR. MTT and modified Bodyen chamber and nitriate recovery were used respectively to mesure the abilities of EPCs to proliferate, migrate and secrete NO. In vitro tube-formation analysis also was used to evaluate the angiogenic ability of EPCs. Results：The spindle ceils were found on the fourth day. On the seventh day, the cluster of ceils appeared, and the ceils looked like ＂cobblestone＂ on the 21st day. Double positive for DiI-acLDL uptake and lectin binding by direct fluorescent staining were observed in the ceils. 18h after transfection, EGFP was positive in transfected HGF and negative group, and the rate of trans- fected EPCs was up to 40%. However, EGFP was negative in blank control group. Results of RT-PCR indicated that the expressions of HGF mRNA were significantly higher in transfected HGF group than that in negative and blank control group, and there were no different between negative and blank control group. The abilities of EPCs to proliferate, migrate and secrete NO in transfected HGF group were superior to that in negative and blank control group. 12h after culured on ECMatrix,the values of in vitro tube-formation analysis in transfected HGF group were significantly higher than those in negative and blank control group. Conclusions：The EPCs modified by HGF gene could enhance the abilities of proliferate, migrate, secrete NO and angiogenesis, and hence improve some biologic property of ECPs.
出处
《中国循环杂志》
CSCD
北大核心
2007年第6期468-472,共5页
Chinese Circulation Journal
基金
国家自然科学基金(批准号:30570765
30700889)
重庆市自然科学基金(批准号:CSTC
2005bb5304)资助
关键词
内皮祖细胞
肝细胞生长因子
基因转染
大鼠
Endothelial progenitor cells
Hepatocyte growth factor
Gene transfer
Rat
