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目的构建八聚体结合转录因子4(Oct4)腺病毒真核表达载体pAV.Ex1d.-CMV>mOCT4/IRES/eGFP,探讨Oct4腺病毒载体对小鼠心肌再生的作用。方法利用Gateway技术构建pAV.Ex1d.-CMV>mOCT4/IRES/eGFP载体,腺病毒包装后,用微量进样器直接注射到小鼠心肌,利用RT-PCR、免疫荧光分别检测心肌组织中Oct4的表达,HE染色检测Oct4表达后心肌组织是否正常。构建小鼠心梗模型,HE染色鉴定,在梗死区周围直接注射Oct4腺病毒载体,4周后Western blot法检测心肌梗死区肌钙蛋白的表达。结果免疫荧光法检测到外源性Oct4在心肌细胞核中表达呈红色荧光,RT-PCR在Oct4腺病毒组中可检测到Oct4的表达,而空病毒组与对照组则检测不到;HE染色结果表明Oct4在活体心肌表达后心肌组织形态正常,心肌梗死区周围注射Oct4腺病毒载体4周后肌钙蛋白在Oct4组的表达量与对照组、空病毒组比较有明显差异(P<0.5)。结论外源Oct4可在活体心肌中表达,能促进梗死区心肌的再生。
Objective To construct the OctA adenovirus eukaryotic expression vector pAV.Ex1d.-CMV> mOCT4 / IRES / eGFP to study the effect of Oct4 adenovirus vector on myocardial regeneration in mice. Methods The vector pAV.Ex1d.-CMV> mOCT4 / IRES / eGFP was constructed by Gateway technique. After being packaged, the adenoviruses were directly injected into the myocardium of mice by microinjector. The expression of Oct4 in myocardium was detected by RT-PCR and immunofluorescence. The expression of Oct4 protein was detected by HE staining. The myocardial infarction model was constructed and identified by HE staining. Oct4 adenovirus vector was injected directly into the infarct area. Western blot was used to detect the expression of troponin 4 weeks after myocardial infarction. Results The expression of exogenous Oct4 in the nucleus of cardiomyocytes was detected by immunofluorescence. The expression of Oct4 was detected by RT-PCR in Oct4 adenovirus group, but not in the empty virus group and the control group. The result of HE staining The results showed that the expression of Oct4 in normal myocardium was normal. The expression of troponin in Oct4 group after injection of Oct4 adenovirus vector around myocardial infarction area was significantly lower than that in control group and empty virus group (P <0.5). Conclusion Exogenous Oct4 can be expressed in live myocardium and can promote myocardial regeneration in infarct zone.