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目的:研究人类肿瘤相关基因CHD5基因miR-shRNA慢病毒对结直肠癌Lovo细胞的影响。方法:利用软件设计对CHD5基因干扰有效的序列,合成靶序列,退火形成双链DNA,酶切后与载体相连接。将重组慢病毒载体pPRIME-TET-GFP-CHD5与慢病毒包装质粒pCMV-VSV-G,pRSV-Rev,pMDLg-pRRE共转染293FT细胞,将包装重组慢病毒感染人类结直肠癌Lovo细胞。通过荧光定量PCR和Western blot验证CHD5基因在细胞中的表达情况,应用MTT检测CHD5低表达对Lovo细胞增殖的影响。结果:成功构建pPRIME-TET-GFP-CHD5重组质粒,经酶切及序列测定正确,包装的病毒滴度为3.1×106TU/ml。用制备的病毒上清感染Lovo细胞后,荧光定量PCR和Western blot分析结果显示该慢病毒可分别在转录和蛋白质水平上抑制Lovo细胞CHD5基因的表达,并使得Lovo细胞增殖失控。结论:成功构建CHD5慢病毒表达载体,表达的慢病毒可有效的感染Lovo细胞,提高Lovo细胞的增殖能力。
Objective: To study the effect of human tumor-associated gene CHD5 gene miR-shRNA lentivirus on colorectal cancer Lovo cells. Methods: Using software to design a sequence which is effective to interfere with CHD5 gene, the target sequence was synthesized and double-stranded DNA was annealed to form a double-stranded DNA. After digested, the target vector was ligated with the vector. 293FT cells were co-transfected with the recombinant lentiviral vector pPRIME-TET-GFP-CHD5 and the lentiviral packaging plasmid pCMV-VSV-G, pRSV-Rev and pMDLg-pRRE and the recombinant lentivirus was packaged to infect human colorectal cancer Lovo cells. The expression of CHD5 gene was verified by real-time PCR and Western blot. The effect of CHD5 low expression on the proliferation of Lovo cells was detected by MTT assay. Results: The recombinant plasmid pPRIME-TET-GFP-CHD5 was successfully constructed. After digestion and sequencing, the virus titer was 3.1 × 10 6 TU / ml. Fluorescence quantitative PCR and Western blot analysis showed that the lentivirus could inhibit the expression of CHD5 gene in Lovo cells at the transcriptional and protein levels respectively and the proliferation of Lovo cells was uncontrolled after Lovo cells were infected with the prepared virus supernatant. Conclusion: The CHD5 lentiviral vector was successfully constructed. The expressed lentivirus could effectively infect Lovo cells and enhance the proliferation of Lovo cells.