【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(lái)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(duì)大腸癌Lovo細(xì)胞增殖與凋亡的影響,用MTT法檢測(cè)白介素6基因過表達(dá)Lovo細(xì)胞株對(duì)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(cè)序鑒定陽(yáng)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(cè)定法(ELISA)檢測(cè)白介素6蛋白的含量來(lái)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(cè):用7-AAD/PE-Annexin V染色法流式細(xì)胞術(shù)檢測(cè)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(cè)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(cè)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(cè)定法(elisa)檢測(cè)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(wú)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(cè)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(wú)統(tǒng)計(jì)學(xué)意義(p0.05)。4)mtt檢測(cè)結(jié)果顯示5-氟尿嘧啶對(duì)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(wú)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(duì)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(duì)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(duì)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(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.34

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 ;大腸癌發(fā)病率升高[J];中國(guó)腫瘤臨床與康復(fù);2016年02期

2 李夢(mèng)瑤;張留偉;段芳芳;劉志科;馬子晴;吉振鵬;陳大方;;大腸癌發(fā)病風(fēng)險(xiǎn)評(píng)估方法[J];中國(guó)慢性病預(yù)防與控制;2016年02期

3 廖德貴;王珊;黃世章;賴妙玲;黃海杰;;IL-6在不同病理類型的結(jié)腸癌組織中的表達(dá)水平及意義[J];臨床醫(yī)學(xué)工程;2015年07期

4 吳強(qiáng);莊坤;袁慧鈞;李慧軍;;姜黃素與5氟尿嘧啶聯(lián)合應(yīng)用抗鼻咽癌的實(shí)驗(yàn)研究[J];哈爾濱醫(yī)科大學(xué)學(xué)報(bào);2014年05期

5 張強(qiáng);金曉燕;;晚期胃腸道惡性腫瘤患者惡液質(zhì)形成與腫瘤壞死因子-α和白介素-6濃度的關(guān)系[J];中國(guó)現(xiàn)代醫(yī)生;2014年06期

6 馮秋娟;柯長(zhǎng)濤;李妙丹;李穎欣;程小廣;曾今誠(chéng);;白細(xì)胞介素6對(duì)結(jié)腸癌Lovo細(xì)胞細(xì)胞周期的影響[J];國(guó)際檢驗(yàn)醫(yī)學(xué)雜志;2012年23期

7 彭向陽(yáng);彭偉雄;黃振華;黃思輝;邱振雄;何加揮;;IL-6和MMP-9在胃癌組織中的表達(dá)及其相關(guān)性[J];中國(guó)熱帶醫(yī)學(xué);2012年08期

8 盧燦榮;張士武;張勇;衛(wèi)勃;;胰腺癌CEA,CA199,IL-6和MCP-1在血清中的表達(dá)及臨床意義[J];科技導(dǎo)報(bào);2012年14期

9 江陳;常家聰;;大腸癌的治療方法研究進(jìn)展[J];安徽醫(yī)藥;2012年02期

10 李群珍;鄧紅;;二十二碳六烯酸增強(qiáng)5氟尿嘧啶對(duì)人肝癌細(xì)胞的細(xì)胞毒活性[J];現(xiàn)代消化及介入診療;2011年03期

，

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