IL-6过表达对大肠癌细胞株凋亡的影响及其对5-FU敏感性的研究
发布时间：2018-11-04 20:56
【摘要】：目的:白介素6(IL-6)在大肠癌的发生、发展中产生了重要作用。因此,本实验通过电转染白介素6基因来使大肠癌Lovo细胞中的白介素6过表达,探讨白介素6对大肠癌Lovo细胞增殖与凋亡的影响,用MTT法检测白介素6基因过表达Lovo细胞株对5氟尿嘧啶(5-fluorouracil,5-Fu)的敏感性。方法:实验分为3组:正常Lovo细胞组、空载质粒Lovo细胞组、过表达质粒Lovo细胞组。1)建立白介素6基因过表达Lovo细胞株:通过采用分子克隆技术将白介素6基因插入表达载体pECFPN1的克隆位点之间,获得重组质粒IL-6-pECFP-N1,通过DNA测序鉴定阳性质粒,以电穿孔转染法将重组质粒IL-6-pECFP-N1导入受体Lovo细胞中,获取白介素6基因过表达Lovo细胞株。2)用酶联免疫吸附测定法(ELISA)检测白介素6蛋白的含量来判断三组细胞白介素6的表达情况。3)转导Lovo细胞株细胞凋亡检测:用7-AAD/PE-Annexin V染色法流式细胞术检测12h、36h、72h三个时间点各细胞株的凋亡或存活情况。4)MTT检测不同浓度(20ug/ml、80ug/ml、160ug/ml、320ug/ml)的5-氟尿嘧啶处理各组细胞株后12h、36h、72h三个时间点的细胞增殖抑制情况。结果:1)测序结果显示成功构建了重组质粒il-6-pecfp-n1。2)酶联免疫吸附测定法(elisa)检测白介素6蛋白的含量:白介素6蛋白在三组细胞中都有不同水平的表达,在白介素6基因过表达lovo细胞株中表达水平最高,且含量明显高于正常lovo细胞组、空载质粒lovo细胞组(p0.05),而正常lovo细胞组与空载质粒lovo细胞组白介素6的含量差别无统计学意义(p0.05)。3)7-aad/pe-annexinv染色法流式细胞术检测结果表明白介素6基因过表达lovo细胞组细胞存活量明显高于正常lovo细胞组细胞存活量与空载质粒lovo细胞组细胞存活量,差异有统计学意义(p0.05)。而正常lovo细胞组细胞存活量与空载质粒lovo细胞组细胞存活量差异无统计学意义(p0.05)。4)mtt检测结果显示5-氟尿嘧啶对各组细胞增殖均有抑制作用,抑制率呈时间-浓度依赖性(p0.05)。而三组间白介素6过表达lovo细胞组的增殖抑制率明显高于正常lovo细胞组与空载质粒lovo细胞组,差异具有统计学意义(p0.05),正常lovo细胞组与空载质粒lovo细胞组的增殖抑制率无明显差异(p0.05)。结论:1)成功构建了重组质粒il-6-pecfp-n1,通过电转染重组质粒成功获得了白介素6基因过表达lovo细胞株。2)大肠癌lovo细胞转染白介素6基因后,能够使白介素6获得相对高表达。3)白介素6基因过表达大肠癌lovo细胞株体外培养其凋亡率明显受到抑制,结果显示,白介素6对大肠癌lovo细胞的凋亡具有抑制作用。4)白介素6基因过表达大肠癌Lovo细胞株对5-氟尿嘧啶敏感,且随时间及浓度的增加,增殖受到明显抑制。
[Abstract]:Objective: interleukin 6 (IL-6) plays an important role in the occurrence and development of colorectal cancer. Therefore, the effect of interleukin 6 on the proliferation and apoptosis of colorectal cancer Lovo cells was investigated by electrotransfection of interleukin 6 gene to induce the overexpression of interleukin 6 in Lovo cells of colorectal cancer. The sensitivity of IL 6 gene overexpression Lovo cell line to 5 fluorouracil 5 Fu was detected by MTT assay. Methods: the experiment was divided into three groups: normal Lovo cell group, empty plasmid Lovo cell group, and control group. The over expression plasmid Lovo cell line was established. The recombinant plasmid IL-6-pECFP-N1, was obtained by inserting the IL 6 gene into the clone site of the expression vector pECFPN1 by molecular cloning technique. The positive plasmid was identified by DNA sequencing, and the recombinant plasmid IL-6-pECFP-N1 was transfected into the receptor Lovo cells by electroporation transfection. Interleukin-6 gene overexpression in Lovo cell line. 2) Detection of interleukin 6 protein content by enzyme linked immunosorbent assay (ELISA) to determine the expression of interleukin 6 in three groups of cells. 3) transduction of Lovo cell line Death detection: 12 h by flow cytometry with 7-AAD/PE-Annexin V staining, Apoptosis or survival of each cell line at three time points for 72 h after 36 h. 4) MTT was used to detect 5-fluorouracil (5-fluorouracil) at different concentrations (20ug / ml 80ugr / ml) for 12 h and 36 h after treatment with 5-fluorouracil. Inhibition of cell proliferation at three time points at 72 h. Results: 1) sequencing results showed that recombinant plasmid il-6-pecfp-n1.2 was successfully constructed to detect the content of interleukin 6 protein by (elisa): the expression of interleukin 6 protein was different in the three groups. The expression level of IL-6 gene overexpression lovo cell line was the highest, and the content was significantly higher than that of normal lovo cell line. The expression level of IL-6 gene over-expressed lovo cell line was significantly higher than that of normal lovo cell line (p0.05). However, there was no significant difference in the content of interleukin 6 between normal lovo cell group and blank plasmid lovo cell group (p0.05). 3) the results of flow cytometry with 7-aad/pe-annexinv staining showed that interleukin 6 gene was overexpressed in lovo. The cell survival in the cell group was significantly higher than that in the normal lovo cell group and the no-loaded plasmid lovo cell group. The difference was statistically significant (p0.05). However, there was no significant difference in cell survival between normal lovo cell group and no-loaded plasmid lovo cell group (p0.05). The results of mtt showed that 5-fluorouracil could inhibit the proliferation of the cells in each group. The inhibition rate was time-concentration dependent (p0.05). The inhibition rate of lovo cells proliferation in the three groups was significantly higher than that in the normal lovo cells group and the blank plasmid lovo cell group (p0.05). There was no significant difference in the inhibition rate of proliferation between the normal lovo cell group and the blank plasmid lovo cell group (p0.05). Conclusion: 1) Recombinant plasmid il-6-pecfp-n1, was successfully constructed and overexpressed lovo cell line was obtained by electrotransfection of IL 6 gene. 2) IL 6 gene was transfected into lovo cells of colorectal cancer. The overexpression of interleukin 6 gene in lovo cell line of colorectal cancer was significantly inhibited in vitro, and the results showed that the apoptosis rate of the cell line was significantly inhibited. Interleukin 6 can inhibit apoptosis of colorectal cancer lovo cells. 4) over expression of interleukin 6 gene is sensitive to 5-fluorouracil in colorectal cancer Lovo cell line, and the proliferation is inhibited with the increase of time and concentration.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.34
本文编号：2311113
[Abstract]:Objective: interleukin 6 (IL-6) plays an important role in the occurrence and development of colorectal cancer. Therefore, the effect of interleukin 6 on the proliferation and apoptosis of colorectal cancer Lovo cells was investigated by electrotransfection of interleukin 6 gene to induce the overexpression of interleukin 6 in Lovo cells of colorectal cancer. The sensitivity of IL 6 gene overexpression Lovo cell line to 5 fluorouracil 5 Fu was detected by MTT assay. Methods: the experiment was divided into three groups: normal Lovo cell group, empty plasmid Lovo cell group, and control group. The over expression plasmid Lovo cell line was established. The recombinant plasmid IL-6-pECFP-N1, was obtained by inserting the IL 6 gene into the clone site of the expression vector pECFPN1 by molecular cloning technique. The positive plasmid was identified by DNA sequencing, and the recombinant plasmid IL-6-pECFP-N1 was transfected into the receptor Lovo cells by electroporation transfection. Interleukin-6 gene overexpression in Lovo cell line. 2) Detection of interleukin 6 protein content by enzyme linked immunosorbent assay (ELISA) to determine the expression of interleukin 6 in three groups of cells. 3) transduction of Lovo cell line Death detection: 12 h by flow cytometry with 7-AAD/PE-Annexin V staining, Apoptosis or survival of each cell line at three time points for 72 h after 36 h. 4) MTT was used to detect 5-fluorouracil (5-fluorouracil) at different concentrations (20ug / ml 80ugr / ml) for 12 h and 36 h after treatment with 5-fluorouracil. Inhibition of cell proliferation at three time points at 72 h. Results: 1) sequencing results showed that recombinant plasmid il-6-pecfp-n1.2 was successfully constructed to detect the content of interleukin 6 protein by (elisa): the expression of interleukin 6 protein was different in the three groups. The expression level of IL-6 gene overexpression lovo cell line was the highest, and the content was significantly higher than that of normal lovo cell line. The expression level of IL-6 gene over-expressed lovo cell line was significantly higher than that of normal lovo cell line (p0.05). However, there was no significant difference in the content of interleukin 6 between normal lovo cell group and blank plasmid lovo cell group (p0.05). 3) the results of flow cytometry with 7-aad/pe-annexinv staining showed that interleukin 6 gene was overexpressed in lovo. The cell survival in the cell group was significantly higher than that in the normal lovo cell group and the no-loaded plasmid lovo cell group. The difference was statistically significant (p0.05). However, there was no significant difference in cell survival between normal lovo cell group and no-loaded plasmid lovo cell group (p0.05). The results of mtt showed that 5-fluorouracil could inhibit the proliferation of the cells in each group. The inhibition rate was time-concentration dependent (p0.05). The inhibition rate of lovo cells proliferation in the three groups was significantly higher than that in the normal lovo cells group and the blank plasmid lovo cell group (p0.05). There was no significant difference in the inhibition rate of proliferation between the normal lovo cell group and the blank plasmid lovo cell group (p0.05). Conclusion: 1) Recombinant plasmid il-6-pecfp-n1, was successfully constructed and overexpressed lovo cell line was obtained by electrotransfection of IL 6 gene. 2) IL 6 gene was transfected into lovo cells of colorectal cancer. The overexpression of interleukin 6 gene in lovo cell line of colorectal cancer was significantly inhibited in vitro, and the results showed that the apoptosis rate of the cell line was significantly inhibited. Interleukin 6 can inhibit apoptosis of colorectal cancer lovo cells. 4) over expression of interleukin 6 gene is sensitive to 5-fluorouracil in colorectal cancer Lovo cell line, and the proliferation is inhibited with the increase of time and concentration.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.34
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