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为了构建携带增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)基因的慢病毒载体,观察GDNF基因大鼠骨髓间充质干细胞(rat mesenchymal stem cells,rMSC)的表达,本研究采用RT-PCR(reverse transcription-PCR)方法从P0大鼠小脑组织中扩增出GDNF基因编码区636 bp的片段,通过限制性内切酶酶切、T4DNA连接酶连接,将GDNF插入慢病毒转移载体PNL-IRES2-EGFP,构建PNL-GDNF-IRES2-EGFP。在脂质体介导下将构建成功的慢病毒三质粒系统共转染人胚肾细胞系(293T)包装生产慢病毒,测定病毒滴度。感染rMSCs,荧光显微镜下观察EGFP的表达、转导效率,RT-PCR、Western blot方法分别检测GDNF mRNA和蛋白的表达情况。结果显示:GDNF的基因序列经测序后与GeneBank报道的序列完全一致,重组慢病毒载体质粒PNL-GDNF-IRES2-EGFP经双酶切鉴定正确;三质粒共转染293T细胞后荧光激发可见大量绿色荧光,收集、浓缩病毒后测定其滴度为5.3×107pfu/ml;感染rMSCs结果显示:GDNF-rMSCs组5 d转导效率为93.3%±3.17%,传代培养4周,下降到81.1%±3.59%,差异具有统计学意义(P<0.01)。RT-PCR(real time-PCR)、Western blot显示GDNF成功在rMSCs中表达。本结果表明,我们已经成功构建带有EGFP、GDNF基因的慢病毒载体,并获得GDNF-rMSCs基因工程细胞,但提高该工程细胞外源基因的稳定表达技术仍需进一步探讨。
To construct a lentiviral vector carrying enhanced green fluorescent protein (EGFP) and glial cell line-derived neurotrophic factor (GDNF) gene, we observed the expression of GDNF gene in rat bone marrow mesenchyme In this study, a 636-bp fragment of GDNF gene was amplified from the rat cerebellum by means of RT-PCR (reverse transcription-PCR). The restriction endonuclease Digested with T4 DNA ligase and inserted into GDNF lentiviral vector PNL-IRES2-EGFP to construct PNL-GDNF-IRES2-EGFP. The constructed lentivirus plasmid system was co-transfected into human embryonic kidney cell line (293T) under the mediation of liposome to produce lentivirus, and the virus titer was determined. The cells were infected with rMSCs. The expression of EGFP and transduction efficiency were observed by fluorescence microscopy. The expression of GDNF mRNA and protein were detected by RT-PCR and Western blot respectively. The results showed that the sequence of GDNF gene was identical with the sequence reported by GeneBank. The recombinant lentiviral plasmid PNL-GDNF-IRES2-EGFP was identified by double enzyme digestion. Three plasmids co-transfected 293T cells showed a large number of green The results showed that the transduction efficiency of GDNF-rMSCs was 93.3% ± 3.17% after 5 days of culture, down to 81.1% ± 3.59 after subculture for 4 weeks %, The difference was statistically significant (P <0.01). RT-PCR (real time-PCR), Western blot showed that GDNF was successfully expressed in rMSCs. The results show that we have successfully constructed lentiviral vectors with EGFP and GDNF genes and obtained GDNF-rMSCs genetically engineered cells. However, it is still necessary to further explore the improvement of the stable expression of the exogenous genes in this engineering cell.