IL-6過表達(dá)對大腸癌細(xì)胞株凋亡的影響及其對5-FU敏感性的研究
發(fā)布時(shí)間：2018-11-04 20:56
【摘要】：目的:白介素6(IL-6)在大腸癌的發(fā)生、發(fā)展中產(chǎn)生了重要作用。因此,本實(shí)驗(yàn)通過電轉(zhuǎn)染白介素6基因來使大腸癌Lovo細(xì)胞中的白介素6過表達(dá),探討白介素6對大腸癌Lovo細(xì)胞增殖與凋亡的影響,用MTT法檢測白介素6基因過表達(dá)Lovo細(xì)胞株對5氟尿嘧啶(5-fluorouracil,5-Fu)的敏感性。方法:實(shí)驗(yàn)分為3組:正常Lovo細(xì)胞組、空載質(zhì)粒Lovo細(xì)胞組、過表達(dá)質(zhì)粒Lovo細(xì)胞組。1)建立白介素6基因過表達(dá)Lovo細(xì)胞株:通過采用分子克隆技術(shù)將白介素6基因插入表達(dá)載體pECFPN1的克隆位點(diǎn)之間,獲得重組質(zhì)粒IL-6-pECFP-N1,通過DNA測序鑒定陽性質(zhì)粒,以電穿孔轉(zhuǎn)染法將重組質(zhì)粒IL-6-pECFP-N1導(dǎo)入受體Lovo細(xì)胞中,獲取白介素6基因過表達(dá)Lovo細(xì)胞株。2)用酶聯(lián)免疫吸附測定法(ELISA)檢測白介素6蛋白的含量來判斷三組細(xì)胞白介素6的表達(dá)情況。3)轉(zhuǎn)導(dǎo)Lovo細(xì)胞株細(xì)胞凋亡檢測:用7-AAD/PE-Annexin V染色法流式細(xì)胞術(shù)檢測12h、36h、72h三個(gè)時(shí)間點(diǎn)各細(xì)胞株的凋亡或存活情況。4)MTT檢測不同濃度(20ug/ml、80ug/ml、160ug/ml、320ug/ml)的5-氟尿嘧啶處理各組細(xì)胞株后12h、36h、72h三個(gè)時(shí)間點(diǎn)的細(xì)胞增殖抑制情況。結(jié)果:1)測序結(jié)果顯示成功構(gòu)建了重組質(zhì)粒il-6-pecfp-n1。2)酶聯(lián)免疫吸附測定法(elisa)檢測白介素6蛋白的含量:白介素6蛋白在三組細(xì)胞中都有不同水平的表達(dá),在白介素6基因過表達(dá)lovo細(xì)胞株中表達(dá)水平最高,且含量明顯高于正常lovo細(xì)胞組、空載質(zhì)粒lovo細(xì)胞組(p0.05),而正常lovo細(xì)胞組與空載質(zhì)粒lovo細(xì)胞組白介素6的含量差別無統(tǒng)計(jì)學(xué)意義(p0.05)。3)7-aad/pe-annexinv染色法流式細(xì)胞術(shù)檢測結(jié)果表明白介素6基因過表達(dá)lovo細(xì)胞組細(xì)胞存活量明顯高于正常lovo細(xì)胞組細(xì)胞存活量與空載質(zhì)粒lovo細(xì)胞組細(xì)胞存活量,差異有統(tǒng)計(jì)學(xué)意義(p0.05)。而正常lovo細(xì)胞組細(xì)胞存活量與空載質(zhì)粒lovo細(xì)胞組細(xì)胞存活量差異無統(tǒng)計(jì)學(xué)意義(p0.05)。4)mtt檢測結(jié)果顯示5-氟尿嘧啶對各組細(xì)胞增殖均有抑制作用,抑制率呈時(shí)間-濃度依賴性(p0.05)。而三組間白介素6過表達(dá)lovo細(xì)胞組的增殖抑制率明顯高于正常lovo細(xì)胞組與空載質(zhì)粒lovo細(xì)胞組,差異具有統(tǒng)計(jì)學(xué)意義(p0.05),正常lovo細(xì)胞組與空載質(zhì)粒lovo細(xì)胞組的增殖抑制率無明顯差異(p0.05)。結(jié)論:1)成功構(gòu)建了重組質(zhì)粒il-6-pecfp-n1,通過電轉(zhuǎn)染重組質(zhì)粒成功獲得了白介素6基因過表達(dá)lovo細(xì)胞株。2)大腸癌lovo細(xì)胞轉(zhuǎn)染白介素6基因后,能夠使白介素6獲得相對高表達(dá)。3)白介素6基因過表達(dá)大腸癌lovo細(xì)胞株體外培養(yǎng)其凋亡率明顯受到抑制,結(jié)果顯示,白介素6對大腸癌lovo細(xì)胞的凋亡具有抑制作用。4)白介素6基因過表達(dá)大腸癌Lovo細(xì)胞株對5-氟尿嘧啶敏感,且隨時(shí)間及濃度的增加,增殖受到明顯抑制。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.34
本文編號：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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.34
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