miR-34a对胃癌细胞增殖及5-FU药物敏感性的影响
发布时间：2018-10-31 19:45
【摘要】：目的在胃癌SGC7901细胞中,研究过表达miR-34a对肿瘤增殖及对化疗药物敏感性的影响,为胃癌的诊疗提供理论和实验依据。方法在前期的实验基础上,选择miR-34a基因序列,制备双链DNA oligo,应用基因重组技术克隆到pcDNA6.2-EGFP载体中DNA中,然后行测序鉴定。用Asc1和Pme1进行双酶切构建pLenti6.3-EGFP-miR-34a慢病毒载体。将构建好的载体混合包装质粒共转染293T细胞包装病毒,72h后离心去除细胞碎片收集病毒原液。将miR34a过表达慢病毒感染SGC7901细胞8h,使用5-FU药物处理24h,CCK-8检测miR-34a对SGC7901细胞增殖以及药物敏感性,以及对5-FU的协同药物作用。结果(1)双酶切证实所设计的miR-34a插入慢病毒载体是正确的,DNA测序验证插入的序列是准确的;应用293T细胞成功包装pLenti6.3-EGFP-miR-34a的重组慢病毒载体。(2)miR34a过表达慢病毒及阴性对照慢病毒感染SGC7901细胞后,使用CCK-8检测SGC7901细胞增殖情况,第1天、第2天、第3天细胞活力OD值分别为0.237、0.227,0.343、0.344,0.508、0.548,显示miR34a过表达后引起SGC7901细胞增殖减慢,但差异无统计学意义(P=0.9368)。(3)使用5-FU药物处理后,检测SGC7901细胞增殖情况,miR34a过表达慢病毒和阴性对照病毒细胞活力OD值分别为0.19、0.36,显示miR34a过表达后显著抑制了SGC7901细胞增值(P=0.0197)。结论成功构建miR-34a过表达慢病毒载体,并成功感染SGC7901细胞。miR34a过表达后引起SGC7901细胞增殖无明显变化,但miR34a过表达后显著提高了SGC7901细胞对5-FU的药物敏感性。
[Abstract]:Objective to study the effects of overexpression of miR-34a on tumor proliferation and sensitivity to chemotherapeutic agents in SGC7901 cells of gastric cancer and to provide theoretical and experimental evidence for the diagnosis and treatment of gastric cancer. Methods on the basis of previous experiments, the miR-34a gene sequence was selected and double-stranded DNA oligo, was cloned into pcDNA6.2-EGFP vector DNA by gene recombination technique, and then sequenced. PLenti6.3-EGFP-miR-34a lentivirus vector was constructed by double enzyme digestion of Asc1 and Pme1. The vector mixed packaging plasmid was co-transfected into 293T cell packaging virus. After 72 hours, the cell fragment collection virus solution was removed by centrifugation. MiR34a overexpression of lentivirus was infused into SGC7901 cells for 8 h, and CCK-8 was used to detect the proliferation and drug sensitivity of miR-34a to SGC7901 cells and the synergistic drug effect on 5-FU for 24 h after 5-FU treatment. Results (1) double enzyme digestion confirmed that the miR-34a inserted into the lentivirus vector was correct and the DNA sequence was accurate. The recombinant lentivirus vector of pLenti6.3-EGFP-miR-34a was successfully packaged by 293T cells. (2) after miR34a overexpression lentivirus and negative control lentivirus were infected with SGC7901 cells, the proliferation of SGC7901 cells was detected by CCK-8. On the 3rd day, the OD values of cell viability were 0.237 ~ 0.227 ~ 0.344 ~ 0.344 ~ 0.508 ~ 0.548, respectively. The results showed that the proliferation of SGC7901 cells was slowed down after over-expression of miR34a, but there was no significant difference (P _ (0.9368). (_ 3) after treatment with 5-FU. The proliferation of SGC7901 cells was detected, the OD values of lentivirus over-expressed by miR34a and those of negative control were 0.19 ~ 0.36, respectively, which showed that miR34a overexpression significantly inhibited the proliferation of SGC7901 cells (P0. 0197). Conclusion the lentivirus vector of miR-34a overexpression was successfully constructed and SGC7901 cells were successfully infected. MiR34a overexpression did not induce the proliferation of SGC7901 cells, but miR34a overexpression significantly increased the drug sensitivity of SGC7901 cells to 5-FU.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
本文编号：2303345
[Abstract]:Objective to study the effects of overexpression of miR-34a on tumor proliferation and sensitivity to chemotherapeutic agents in SGC7901 cells of gastric cancer and to provide theoretical and experimental evidence for the diagnosis and treatment of gastric cancer. Methods on the basis of previous experiments, the miR-34a gene sequence was selected and double-stranded DNA oligo, was cloned into pcDNA6.2-EGFP vector DNA by gene recombination technique, and then sequenced. PLenti6.3-EGFP-miR-34a lentivirus vector was constructed by double enzyme digestion of Asc1 and Pme1. The vector mixed packaging plasmid was co-transfected into 293T cell packaging virus. After 72 hours, the cell fragment collection virus solution was removed by centrifugation. MiR34a overexpression of lentivirus was infused into SGC7901 cells for 8 h, and CCK-8 was used to detect the proliferation and drug sensitivity of miR-34a to SGC7901 cells and the synergistic drug effect on 5-FU for 24 h after 5-FU treatment. Results (1) double enzyme digestion confirmed that the miR-34a inserted into the lentivirus vector was correct and the DNA sequence was accurate. The recombinant lentivirus vector of pLenti6.3-EGFP-miR-34a was successfully packaged by 293T cells. (2) after miR34a overexpression lentivirus and negative control lentivirus were infected with SGC7901 cells, the proliferation of SGC7901 cells was detected by CCK-8. On the 3rd day, the OD values of cell viability were 0.237 ~ 0.227 ~ 0.344 ~ 0.344 ~ 0.508 ~ 0.548, respectively. The results showed that the proliferation of SGC7901 cells was slowed down after over-expression of miR34a, but there was no significant difference (P _ (0.9368). (_ 3) after treatment with 5-FU. The proliferation of SGC7901 cells was detected, the OD values of lentivirus over-expressed by miR34a and those of negative control were 0.19 ~ 0.36, respectively, which showed that miR34a overexpression significantly inhibited the proliferation of SGC7901 cells (P0. 0197). Conclusion the lentivirus vector of miR-34a overexpression was successfully constructed and SGC7901 cells were successfully infected. MiR34a overexpression did not induce the proliferation of SGC7901 cells, but miR34a overexpression significantly increased the drug sensitivity of SGC7901 cells to 5-FU.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
【参考文献】
相关期刊论文 前1条
1 金懋林;;消化道恶性肿瘤化学治疗的进展[J];癌症进展;2007年01期
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