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目的构建含有miR-363-3p的慢病毒表达载体,获得能够稳定表达miR-363-3p的DU145细胞株并初步进行细胞形态学实验。方法利用PCR扩增miR-363-3p片段,插入慢病毒载体p CDH-CMV-MCS-EF1-tRFP中构建重组慢病毒质粒p CDH-miR-363-3p。将慢病毒包装三质粒表达系统的重组慢病毒质粒p CDH-miR-363-3p,包装质粒ps PAX以及包膜质粒p MD2.G共转染人胚肾上皮细胞(HEK293T)细胞中,同时以慢病毒空载体p CDH-vector作为阴性对照。收集含有病毒颗粒细胞的病毒上清液,测定Lentivirus-miR-363-3p和Lentivirus-vector的病毒滴度。再用重组慢病毒Lentivirus-miR-363-3p及阴性对照Lentivirus-vector以相同感染复数(MOI)的病毒量分别感染DU145细胞,72 h后,在荧光显微镜下通过观察红色荧光蛋白(RFP)的表达情况观察被感染细胞数。采用反转录实时定量PCR(RT-q PCR)方法检测两组细胞系中miR-363-3p表达情况。将获得的两组细胞进行平板划痕实验,初步观察其细胞生长行为的差异。结果 PCR成功扩增出miR-363-3p片段,插入慢病毒载体p CDH-CMV-MCSEF1-tRFP后,利用限制性内切酶酶切鉴定和核酸序列测定证实重组慢病毒质粒p CDH-miR-363-3p构建成功。通过慢病毒包装三质粒表达系统获得表达miR-363-3p的重组慢病毒,测得滴度约为1.5×107efu/ml。以相同MOI的重组慢病毒Lentivirus-miR-363-3p以及Lentivirus-vector分别感染DU145细胞72 h后,在荧光显微镜下能够明显观察到红色荧光,且RT-q PCR检测结果显示,感染miR-363-3p DU145细胞中miR-363-3p表达水平显著高于感染miR-vector DU145细胞,差异有统计学意义(P<0.01)。平板划痕实验结果显示划痕后24 h时两组细胞生长具有明显差异(P<0.01),但48 h时两组生长的细胞均已完全覆盖划痕。结论成功构建了含miR-363-3p的慢病毒表达载体,获得的重组慢病毒能够有效地感染DU145细胞,为下一步实验奠定了坚实的基础。初步的细胞形态学实验-平板划痕显示划痕后24 h两组细胞生长存在差异。
Objective To construct a lentiviral vector containing miR-363-3p and obtain DU145 cell line which can stably express miR-363-3p and to conduct preliminary cell morphology experiments. Methods The miR-363-3p fragment was amplified by PCR and inserted into lentiviral vector pCDH-CMV-MCS-EF1-tRFP to construct recombinant lentiviral plasmid pCDH-miR-363-3p. The recombinant lentiviral plasmid pCDH-miR-363-3p, packaging plasmid ps PAX and envelope plasmid p MD2.G were co-transfected into human embryonic kidney epithelial cells (HEK293T) cells at the same time Lentiviral empty vector pCDH-vector served as a negative control. Virus supernatant containing virus granulosa cells was collected and the virus titer of Lentivirus-miR-363-3p and Lentivirus-vector was determined. DU145 cells were infected with the recombinant lentivirus Lentivirus-miR-363-3p and the negative control Lentivirus-vector at the same multiplicity of infection (MOI), respectively. After 72 hours, the cells were infected with red fluorescent protein (RFP) The expression was observed infected cells. Reverse transcription-based real-time PCR (RT-q PCR) was used to detect miR-363-3p expression in both cell lines. The two groups of cells obtained plate scratch test, preliminary observation of the differences in cell growth behavior. Results The fragment of miR-363-3p was successfully amplified by PCR. After insertion of the lentiviral vector pCDH-CMV-MCSEF1-tRFP, restriction endonuclease digestion and nucleic acid sequence analysis confirmed that the recombinant lentiviral plasmid pCDH-miR- 363-3p build successfully. Recombinant lentivirus expressing miR-363-3p was obtained by lentivirus-packaging three plasmid expression system and the titer was about 1.5 × 107efu / ml. The DU145 cells were infected with recombinant lentivirus Lentivirus-miR-363-3p and Lentivirus-vector at the same MOI for 72 h, respectively. Fluorescence microscopy showed that red fluorescence was observed. The results of RT-q PCR showed that miR-363 The expression level of miR-363-3p in miR-3p DU145 cells was significantly higher than that in miR-vector DU145 cells (P <0.01). The results of plate scratch test showed that there was a significant difference in cell growth between the two groups at 24 h after scratching (P <0.01), but the cells grown in both groups had completely covered the scratch at 48 h. Conclusion The lentiviral vector containing miR-363-3p was successfully constructed. The recombinant lentivirus obtained could effectively infect DU145 cells, which laid a solid foundation for further experiments. Preliminary cell morphological experiments - flat scratches showed scratch 24 h after the two groups of cell growth differences.