miR-34a對(duì)胃癌細(xì)胞增殖及5-FU藥物敏感性的影響
發(fā)布時(shí)間：2018-10-31 19:45
【摘要】：目的在胃癌SGC7901細(xì)胞中,研究過表達(dá)miR-34a對(duì)腫瘤增殖及對(duì)化療藥物敏感性的影響,為胃癌的診療提供理論和實(shí)驗(yàn)依據(jù)。方法在前期的實(shí)驗(yàn)基礎(chǔ)上,選擇miR-34a基因序列,制備雙鏈DNA oligo,應(yīng)用基因重組技術(shù)克隆到pcDNA6.2-EGFP載體中DNA中,然后行測序鑒定。用Asc1和Pme1進(jìn)行雙酶切構(gòu)建pLenti6.3-EGFP-miR-34a慢病毒載體。將構(gòu)建好的載體混合包裝質(zhì)粒共轉(zhuǎn)染293T細(xì)胞包裝病毒,72h后離心去除細(xì)胞碎片收集病毒原液。將miR34a過表達(dá)慢病毒感染SGC7901細(xì)胞8h,使用5-FU藥物處理24h,CCK-8檢測miR-34a對(duì)SGC7901細(xì)胞增殖以及藥物敏感性,以及對(duì)5-FU的協(xié)同藥物作用。結(jié)果(1)雙酶切證實(shí)所設(shè)計(jì)的miR-34a插入慢病毒載體是正確的,DNA測序驗(yàn)證插入的序列是準(zhǔn)確的;應(yīng)用293T細(xì)胞成功包裝pLenti6.3-EGFP-miR-34a的重組慢病毒載體。(2)miR34a過表達(dá)慢病毒及陰性對(duì)照慢病毒感染SGC7901細(xì)胞后,使用CCK-8檢測SGC7901細(xì)胞增殖情況,第1天、第2天、第3天細(xì)胞活力OD值分別為0.237、0.227,0.343、0.344,0.508、0.548,顯示miR34a過表達(dá)后引起SGC7901細(xì)胞增殖減慢,但差異無統(tǒng)計(jì)學(xué)意義(P=0.9368)。(3)使用5-FU藥物處理后,檢測SGC7901細(xì)胞增殖情況,miR34a過表達(dá)慢病毒和陰性對(duì)照病毒細(xì)胞活力OD值分別為0.19、0.36,顯示miR34a過表達(dá)后顯著抑制了SGC7901細(xì)胞增值(P=0.0197)。結(jié)論成功構(gòu)建miR-34a過表達(dá)慢病毒載體,并成功感染SGC7901細(xì)胞。miR34a過表達(dá)后引起SGC7901細(xì)胞增殖無明顯變化,但miR34a過表達(dá)后顯著提高了SGC7901細(xì)胞對(duì)5-FU的藥物敏感性。
[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.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2
本文編號(hào)：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.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 金懋林;;消化道惡性腫瘤化學(xué)治療的進(jìn)展[J];癌癥進(jìn)展;2007年01期
，本文編號(hào)：2303345
本文鏈接：http://sikaile.net/yixuelunwen/zlx/2303345.html
最近更新
教材專著
