cxcr4过表达hucblate epcs移植后肢缺血组织血管新生的实验研究.docx

优秀毕业论文 精品参考文献资料 对照组缺血后肢肌内直接注射500 对照组缺血后肢肌内直接注射500 u 1EGM-2。28天后，脱颈法处死动物， 立即取缺血后肢腓肠肌，放入10倍体积的4％多聚甲醛固定。 5．HE染色检测缺血后肢肌肉间、肌肉旁微小血管情况；免疫组织化学染色 检测肌肉间、肌肉旁新生血管CD31阳性内皮细胞的表达情况，并计算 CD31阳性新生微血管数量。 结果： 1．rAAV。一CXCR4感染组HUCB—late EPCs中CXCR4蛋白的表达高于rAAV。-GFP 感染组和对照组。 2．3组均在基质胶上成功形成管腔样结构，单位面积基质胶形成的管腔面 积分别为0．531±0．023ram2、0．5H±0．026ram2、0．523±0．021mm2。， rAAV。一CXCR4、rAAV。一GFP感染对late EPCs的体外成血管能力无影响 (乃0．05)。 3．与空白组比较对照组和实验组CD3]+表达的新生微血管明显增多，实验 组新生微血管表达数量较对照组多(P(O．05)，分别为：肌肉间(2．32 ±0．90、5．18±0．86、8．39±0．91)；肌肉旁(5．32±0．93、8．12±O．95、 12．45±1．01)。 关键词：趋化因子受体一4(CXCR4)，后肢缺血模型，血管新生 结论： 1．9种不同血清型rAAV对人脐带血Late EPCs的转导效率存在明显差异， 其中rAAV。的转导效率最高、rAAV：次之、rAAV。最低。 2．CXCR4过表达可以促进人脐带血]ate EPCs在SD大鼠后肢缺血组织新生 微血管的形成。 万方数据 AbstractPart Abstract Part I乃evaluate the transduction efficiency of human umbilical cord．derived late EPCs with nine recombinant adeno—associated virus serotypes Obj ective To evaluate the transduction efficiency of(HUCB-late EPCs)with nine rAAVserotypes，and apply the rAAV serotype that has highest transduction efficiency as CXCR4 gene vector． Methods 1．rAAV vector production system relies on the polyetherimide(PEI)一modified triple plasmid transfection method(transfection of 293 cells with pAAV-RCl．9，pHelper, and pAAV-CMV-GFP)．Three days after HEK 293 cell transfection in the plate format，nuclei were harvested and disrupted，and the virus was then separated， purified，followed by SDS—PAGE and titration by quantitative PCR using primers specific for the GFP wansgene．According to the experimental results，select the highest transfection efficiency of recombinant adeno—associated virus serotype as CXCR4 gene vectoL to obtain rAAV-CXCR4 2．Human umbilical cord blood mononuclear cells(MNCs)were isolated by density—gradient cemrifugation from human umbilical cord blood，and induced to endothelial progenitor cells(EPCs)．After cultured for 4 weeks，late EPCs were obtained． 万方数据 3． 3．Ten thousand late EPCs per well were plated in a 24一well plate with 500山of media．Immediately after the platin