發(fā)布時(shí)間：2018-04-28 09:03

  本文選題：肝癌細(xì)胞 + 全反式維甲酸　； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:體外探討全反式維甲酸(all-trans-retinoic acid,ATRA)是否通過(guò)調(diào)控micro RNA 200家族(mi R200s)逆轉(zhuǎn)肝癌細(xì)胞的上皮細(xì)胞間充質(zhì)轉(zhuǎn)化(epithelial mesenchymal transition,EMT),從而抑制腫瘤細(xì)胞的增殖遷徙侵襲,促進(jìn)其成熟分化,并尋找相關(guān)下游靶基因,探討可能的分子生物學(xué)機(jī)制,為ATRA用于肝癌的臨床治療提供新的思路。方法:以小鼠肝癌細(xì)胞Hepa1-6為研究對(duì)象,首先體外給予0、0.1、1.0和10.0μmol/L不同終濃度的ATRA處理,臺(tái)盤(pán)藍(lán)拒染實(shí)驗(yàn)計(jì)數(shù)細(xì)胞,繪制細(xì)胞生長(zhǎng)曲線(xiàn),克隆形成實(shí)驗(yàn)、結(jié)晶紫染色檢測(cè)細(xì)胞增殖及克隆形成能力;Hoechst檢測(cè)細(xì)胞凋亡,劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞的平行遷徙能力,Transwell實(shí)驗(yàn)檢測(cè)縱向遷徙及侵襲能力。熒光定量PCR(real-time PCR)法檢測(cè)肝前體細(xì)胞標(biāo)志甲胎蛋白(alpha fetoprotein,AFP)及成熟肝細(xì)胞標(biāo)志白蛋白(albumin,ALB)和細(xì)胞角蛋白18(cytokeratin 18,CK18),酪氨酸轉(zhuǎn)氨酶(tyrosine aminotransferase,TAT),載脂蛋白B(Apo lipoprotein B,Apo B)的m RNA水平表達(dá);Western Blot及免疫熒光檢測(cè)AFP、ALB、CK18的蛋白水平表達(dá)。吲哚菁綠(Indocyanine green,ICG)攝取釋放實(shí)驗(yàn)及過(guò)碘酸希夫(Periodic acid-Schiff,PAS)染色檢測(cè)細(xì)胞的代謝解毒及糖原合成功能。Real-time PCR檢測(cè)上皮標(biāo)志蛋白-上皮細(xì)胞鈣粘蛋白(epithelia cadherin,E-cadherin),以及間質(zhì)標(biāo)志蛋白:神經(jīng)型鈣粘蛋白(nerve cadherin,N-cadherin)、鋅指蛋白轉(zhuǎn)錄因子snail、波形蛋白Vimentin和上皮-間質(zhì)轉(zhuǎn)化因子twist的m RNA表達(dá)。初步篩選出對(duì)Hepa1-6細(xì)胞作用最佳的ATRA濃度。其次,體外用最佳濃度的ATRA處理Hepa1-6細(xì)胞,real-time PCR檢測(cè)mi R200s的表達(dá)情況,篩選出表達(dá)變化具有顯著差異的mi R200成員mi R200a-3p、mi R200c-3p、mi R141-3p。采用抑制mi RNA200功能的三種特異性mi RNA antagomir處理Hepa1-6細(xì)胞,再用最佳濃度的ATRA繼續(xù)培養(yǎng)細(xì)胞,分為:a.空白對(duì)照組;b.ATRA組;c.mi RNA antagomir Negative Control(NC)+ATRA組;d.mi R-200s antagomir+ATRA共三組。MTT分析法檢測(cè)細(xì)胞增殖情況,克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞克隆形成,Annexin V-FITC/PI雙染法流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡,劃痕實(shí)驗(yàn)檢測(cè)細(xì)胞的平行遷徙能力,Transwell實(shí)驗(yàn)檢測(cè)縱向遷徙能力及侵襲能力,Real-time PCR檢測(cè)成熟肝細(xì)胞標(biāo)志蛋白ALB、CK18的表達(dá),ICG及PAS染色檢測(cè)細(xì)胞的成熟分化作用。轉(zhuǎn)錄因子芯片檢測(cè)ATRA處理前后345種轉(zhuǎn)錄因子活性,獲得差異因子,生物信息學(xué)分析可能受mi R-200分子調(diào)控的差異因子靶基因,并進(jìn)行real-time PCR法及western blot驗(yàn)證。結(jié)果:第一部分:0.1、1.0和10.0μmol/L ATRA處理后Hepa1-6細(xì)胞的增殖、遷徙、侵襲能力較control組明顯下降(p0.05),凋亡率增加,ICG攝取及PAS染色陽(yáng)性細(xì)胞數(shù)顯著增多(p0.05),上皮標(biāo)志蛋白E-cadherin,CK18及成熟肝細(xì)胞標(biāo)志ALB、CK18、TAT、Apo B的表達(dá)明顯上調(diào),肝腫瘤細(xì)胞標(biāo)志AFP下降,間質(zhì)標(biāo)志蛋白N-cadherin,Vimentin、Snail、Twist的表達(dá)明顯下調(diào)(p0.05),這些調(diào)節(jié)作用呈現(xiàn)出對(duì)ATRA的劑量依賴(lài)性,10.0μmol/L ATRA組的作用最強(qiáng)。第二部分:1)Real-time PCR結(jié)果顯示10.0μmol/LATRA誘導(dǎo)后Hepa1-6細(xì)胞中mi RNA200a-3p、200c-3p、141-3p的m RNA表達(dá)明顯上調(diào)(p0.05),而其他7個(gè)mi R200家族分子的表達(dá)與對(duì)照組相比無(wú)統(tǒng)計(jì)學(xué)差異(p0.05)。2)ATRA處理后Hepa1-6細(xì)胞的增殖、凋亡、遷移、侵襲以及成熟分化作用較對(duì)照組出現(xiàn)與第一部分相同的作用趨勢(shì)。NC+ATRA組與ATRA誘導(dǎo)組Hepa1-6細(xì)胞的增殖、凋亡率、遷移、侵襲能力、成熟肝細(xì)胞標(biāo)志表達(dá)及分化能力均無(wú)差異(p0.05)。而與NC+ATRA組相比,mi R-200a/200c/141-3p antagomir+ATRA三組Hepa1-6細(xì)胞的增殖率、劃痕愈合率及侵襲細(xì)胞均增多(p0.05),其中mi R-200a/200c-3p antagomir+ATRA兩組增多更明顯,甚至達(dá)到control組水平,但三組克隆形成率均較NC+ATRA組無(wú)差異;同時(shí)三組凋亡率均較NC+ATRA組均明顯下降(p0.05)。只有mi R-200a/200c-3p antagomir+ATRA兩組與NC+ATRA組相比,transwell縱向遷移能力增強(qiáng),ALB、CK18的m RNA表達(dá)下調(diào),PAS及ICG陽(yáng)性細(xì)胞數(shù)減少(p0.05),mi R-141-3p antagomir不影響ATRA對(duì)肝癌細(xì)胞的縱向遷移和促進(jìn)分化成熟作用。3)轉(zhuǎn)錄因子芯片結(jié)果顯示ATRA處理后有8個(gè)轉(zhuǎn)錄因子活性上調(diào),35個(gè)轉(zhuǎn)錄因子活性下調(diào),生物信息學(xué)結(jié)果分析mi R-200a/200c/141-3p與klf12、zeb1、Pou4f1、Ets1、Jun基因相關(guān),相對(duì)應(yīng)于AP2、AREB1、Brn-3、Ets1/PEA3及Fra-1/JUN轉(zhuǎn)錄因子,而其他7個(gè)mi R-200亞型與ATRA調(diào)控的43個(gè)差異因子均不相關(guān)。ATRA誘導(dǎo)后轉(zhuǎn)錄因子Jun、Fra-1、Pouf1、Etv4、zeb1的m RNA表達(dá)顯著下調(diào),klf12表達(dá)上調(diào)(p0.05)。并且mi R-200a/200c/141-3p antagomira特異性抑制了mi R-200a/200c/141-3p的生物學(xué)作用后,三組較mi RNA Negative control組Jun、Fra-1、Pouf1、Etv4、zeb1的蛋白表達(dá)顯著上調(diào)(p0.05),Klf12的蛋白表達(dá)未見(jiàn)明顯改變。結(jié)論:ATRA呈濃度依賴(lài)性逆轉(zhuǎn)小鼠肝癌細(xì)胞Hepa1-6的上皮間質(zhì)轉(zhuǎn)化,抑制細(xì)胞的增殖、遷移及侵襲能力,促進(jìn)細(xì)胞凋亡及成熟分化,并具有成熟肝細(xì)胞的合成代謝功能。ATRA可能通過(guò)調(diào)控mi R-200a-3p、mi R-200c-3p、mi R-141-3p逆轉(zhuǎn)肝癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化,其中mi R-200a-3p、mi R-200c-3p、mi R-141-3p均參與介導(dǎo)ATRA抑制肝癌細(xì)胞增殖、平行遷移、侵襲的作用及促凋亡作用;同時(shí)mi R-200a-3p、mi R-200c-3p還介導(dǎo)了ATRA抑制肝癌細(xì)胞縱向遷移及促進(jìn)分化成熟的作用,并且mi R-200s調(diào)控肝癌EMT的發(fā)生可能與Jun、Fra-1、Pouf1、Etv4、zeb1相關(guān)。
[Abstract]:Objective: To investigate in vitro whether all-trans-retinoic acid (ATRA) can reverse the epithelial mesenchymal transition (epithelial mesenchymal transition, EMT) by regulating the micro RNA 200 family (MI R200s), thus inhibiting the proliferation and invasion of tumor cells, promoting its maturation and differentiation, and searching for the related downstream targets. To explore the possible molecular biological mechanism to provide new ideas for the clinical treatment of liver cancer by ATRA. Methods: the mouse hepatoma cell Hepa1-6 was used as the research object. First, the ATRA treatment of the different final concentrations of 0,0.1,1.0 and 10 mu mol/L was given in vitro, the cell blue stain experiment was used to count the fine cell, the cell growth curve was plotted, the cloning experiment was made and the crystallization was crystallized. Cell proliferation and clone formation ability were detected by purple staining. Hoechst was used to detect cell apoptosis, scratch test to detect the parallel migration ability of cells. Transwell test was used to detect vertical migration and invasion ability. Fluorescence quantitative PCR (real-time PCR) method was used to detect alpha fetoprotein (alpha fetoprotein, AFP) and mature hepatocyte marker albumin (real-time PCR) method. Albumin, ALB) and cytokeratin 18 (cytokeratin 18, CK18), tyrosine aminotransferase (tyrosine aminotransferase, TAT), apolipoprotein B (Apo lipoprotein B, Apo) level expression. Periodic acid-Schiff (PAS) staining was used to detect the metabolic detoxification of cells and the function of glycogen synthesis by.Real-time PCR to detect epithelial marker protein, epithelia cadherin, E-cadherin, and interstitial marker protein: neuronal cadherin (nerve cadherin, N-cadherin), zinc finger protein transcription factor Snail, vimentin M RNA expression of tin and epithelial mesenchymal transition factor twist. The optimum concentration of ATRA for Hepa1-6 cells was screened. Secondly, Hepa1-6 cells were treated with the best concentration of ATRA in vitro. Real-time PCR was used to detect the expression of MI R200s. Hepa1-6 cells were treated with three specific mi RNA antagomir, which inhibit the function of MI RNA200, and then the cells were continued to be cultured with the best concentration of ATRA, divided into a. blank control group, b.ATRA group and c.mi RNA antagomir group. Cell clones were formed, cell apoptosis was detected by Annexin V-FITC/PI double staining flow cytometry, parallel migration ability was detected by scratch test, vertical migration ability and invasion ability were detected by Transwell test, Real-time PCR was used to detect the mature hepatocyte marker protein ALB, CK18 surface expression, ICG and PAS staining to detect the maturation and differentiation of cells. The activity of 345 transcription factors before and after ATRA treatment was detected by the recording factor chip, and the difference factor was obtained. The bioinformatics analysis could be regulated by the MI R-200 molecule, and the real-time PCR method and Western blot were used to verify. The results were as follows: the first part: the proliferation, migration, and invasion energy of Hepa1-6 cells after the treatment of 0.1,1.0 and 10 micron ATRA. Compared with control group (P0.05), the apoptosis rate increased, the number of ICG uptake and PAS staining positive cells increased significantly (P0.05), the epithelial marker protein E-cadherin, CK18 and mature hepatocyte marker ALB, CK18, TAT, Apo B, the expression of liver tumor cell markers decreased. P0.05, which showed a dose dependence on ATRA, the 10 micron mol/L ATRA group had the strongest effect. The second part: 1) Real-time PCR results showed mi RNA200a-3p in Hepa1-6 cells induced by 10 mu mol/LATRA, 200c-3p, and the expression and control of the other 7 family members. Compared with the control group, the proliferation, apoptosis, migration, invasion and mature differentiation of Hepa1-6 cells were similar to those of the control group after ATRA treatment. The proliferation, apoptosis, migration, invasiveness, the expression and differentiation of mature hepatocyte markers were no more than that of the control group. The proliferation, apoptosis, migration, invasion and maturation of Hepa1-6 cells were compared with those of the control group after ATRA treatment. The difference (P0.05). Compared with the NC+ATRA group, the proliferation rate of Hepa1-6 cells in the MI R-200a/200c/141-3p antagomir+ATRA three groups, the scar healing rate and the invasive cells increased (P0.05), and the increase of MI R-200a/200c-3p antagomir+ATRA two group was more obvious, even reached the control group, but the three groups had no difference compared with those of the NC+ATRA group. The apoptosis rate of the three groups were all significantly lower than those in the NC+ATRA group (P0.05). Only the MI R-200a/200c-3p antagomir+ATRA two groups were compared with the NC+ATRA group, and the vertical migration ability of Transwell was enhanced, the m RNA expression of ALB, CK18 decreased and the number of PAS and positive cells decreased. The transcriptional factor.3) transcriptional factor chip results showed that 8 transcriptional factors were up-regulated after ATRA treatment, and the activity of 35 transcriptional factors was down. The bioinformatics results showed that MI R-200a/200c/141-3p was associated with klf12, ZEB1, Pou4f1, Ets1, and Jun genes, and corresponded to AP2, AREB1, Brn-3, and transcription factors, and the other 7 The 43 difference factors regulated by ATRA are not related to.ATRA induced transcription factor Jun, Fra-1, Pouf1, Etv4, the RNA expression of M is down significantly, and klf12 expression is up regulated (P0.05). The protein expression of TV4, ZEB1 increased significantly (P0.05), and the expression of Klf12 protein was not obviously changed. Conclusion: ATRA has a concentration dependent reversal of the epithelial transformation of Hepa1-6 in mouse hepatoma cells, inhibiting cell proliferation, migration and invasion, promoting cell apoptosis and maturation, and having the metabolic function.ATRA of mature liver cells. By regulating mi R-200a-3p, MI R-200c-3p and MI R-141-3p to reverse the epithelial mesenchymal transition of hepatoma cells, MI R-200a-3p, MI R-200c-3p and MI R-141-3p are involved in inhibiting the proliferation, parallel migration, invasion and apoptosis of hepatoma cells. And promote the role of differentiation and maturation, and MI R-200s regulation of liver cancer EMT may be related to Jun, Fra-1, Pouf1, Etv4, ZEB1.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.7

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 鄭巖松,林永X,呂新生,石錚;γ-干擾素對(duì)肝癌細(xì)胞株Hep-G2 Fas,Bcl-2表達(dá)的影響[J];中國(guó)普通外科雜志;2001年02期

2 周晶,沈志祥;過(guò)氧化物酶體增殖物激活受體與肝癌[J];腫瘤防治研究;2004年11期

3 段紀(jì)成;楊家和;劉凱;吳孟超;;單用絲裂霉素或表柔比星對(duì)肝癌細(xì)胞株的抑制作用[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2007年04期

4 楊盛力;彭靜;陳孝平;;中藥逆轉(zhuǎn)肝癌多藥耐藥的研究進(jìn)展[J];肝膽外科雜志;2008年06期

5 魏莉;李?lèi)?ài)民;羅榮城;;肝癌多藥耐藥及逆轉(zhuǎn)的研究進(jìn)展[J];中國(guó)藥房;2009年07期

6 王智;;硒-甲基硒代半胱氨酸誘導(dǎo)肝癌SMMC-7721細(xì)胞株的凋亡及機(jī)制探討[J];江蘇醫(yī)藥;2012年11期

7 李培坤;耿小平;;肝癌耐藥的基因?qū)W研究進(jìn)展[J];中華腫瘤防治雜志;2012年12期

8 龔民族;;大白鼠肝癌細(xì)胞株FSK 7901,FSK 7902的建立和特點(diǎn)[J];解剖學(xué)報(bào);1981年03期

9 曹韻貞,湯釗猷;人體肝癌細(xì)胞株研究進(jìn)展[J];腫瘤;1985年03期

10 ;視黃酸逆轉(zhuǎn)入肝癌細(xì)胞株的研究[J];上海醫(yī)科大學(xué)學(xué)報(bào);1987年05期

相關(guān)會(huì)議論文 前10條

1 魏緒勇;王建國(guó);馮曉文;王雁;曹?chē)?guó)強(qiáng);陳海;徐驍;鄭樹(shù)森;;肝癌組織中氨基�；�-1表達(dá)及作用[A];2012中國(guó)器官移植大會(huì)論文匯編[C];2012年

2 魏緒勇;王建國(guó);馮曉文;王雁;曹?chē)?guó)強(qiáng);陳海;徐驍;鄭樹(shù)森;;肝癌組織中氨基酰化酶-1表達(dá)及作用[A];2012年浙江省外科學(xué)學(xué)術(shù)年會(huì)論文集[C];2012年

3 唐瑞峰;馬艷鵬;吳建飛;孫建;張文華;黃亮;;白細(xì)胞介素2和白細(xì)胞介素6對(duì)肝癌細(xì)胞表達(dá)血管內(nèi)皮生長(zhǎng)因子B的調(diào)節(jié)[A];第6屆全國(guó)疑難及重癥肝病大會(huì)論文集[C];2011年

4 唐瑞峰;孫超;馬艷鵬;吳建飛;孫建;張文華;;白細(xì)胞介素2和白細(xì)胞介素6對(duì)肝癌細(xì)胞表達(dá)血管內(nèi)皮生長(zhǎng)因子D的調(diào)節(jié)[A];第6屆全國(guó)疑難及重癥肝病大會(huì)論文集[C];2011年

5 任軍;;肝癌的生物治療[A];第三屆中國(guó)腫瘤學(xué)術(shù)大會(huì)教育論文集[C];2004年

6 陳啟楨;林雅慧;;樟芝菌種于中藥草固態(tài)發(fā)酵萃取液對(duì)抗人類(lèi)肝癌動(dòng)物試驗(yàn)功效研究[A];海峽兩岸第十屆菌物學(xué)暨第三屆食藥用菌學(xué)術(shù)研討會(huì)論文摘要集[C];2011年

7 楊建林;樊曉暉;;一株新發(fā)現(xiàn)的新城疫病毒高效殺傷肝癌細(xì)胞及其作用機(jī)制研究[A];2009醫(yī)學(xué)前沿論壇暨第十一屆全國(guó)腫瘤藥理與化療學(xué)術(shù)會(huì)議論文集[C];2009年

8 孫文潔;廖正凱;周福祥;張紅艷;黃成虎;熊杰;周云峰;;放射線(xiàn)對(duì)肝癌細(xì)胞中端粒酶啟動(dòng)子活性的影響[A];2007第六屆全國(guó)放射腫瘤學(xué)學(xué)術(shù)年會(huì)論文集[C];2007年

9 王魯;湯釗猷;薛瓊;孫惠川;陳軍;高冬梅;趙燕;陳潔;;β干擾素抑制肝癌腫瘤生長(zhǎng)和復(fù)發(fā)轉(zhuǎn)移的實(shí)驗(yàn)研究[A];2000全國(guó)腫瘤學(xué)術(shù)大會(huì)論文集[C];2000年

10 葉依霞;周永健;李瑜元;張宗勝;;廣州市人群非酒精性脂肪性肝病的自然病程及危險(xiǎn)因素[A];中華醫(yī)學(xué)會(huì)第十六次全國(guó)病毒性肝炎及肝病學(xué)術(shù)會(huì)議論文匯編[C];2013年

相關(guān)重要報(bào)紙文章 前8條

1 李運(yùn)紅邋胡顏;科學(xué)家從壁虎中分離出抗肝癌成分[N];新華每日電訊;2007年

2 張中橋;AFP可作為治療肝癌的新靶標(biāo)[N];中國(guó)醫(yī)藥報(bào);2006年

3 印高樂(lè);臺(tái)灣研究發(fā)現(xiàn)黃體酯酮可降低肝癌復(fù)發(fā)率[N];醫(yī)藥經(jīng)濟(jì)報(bào);2004年

4 李運(yùn)紅邋胡顏;我國(guó)科學(xué)家首次從壁虎中分離出抗腫瘤活性成分[N];醫(yī)藥經(jīng)濟(jì)報(bào);2007年

5 記者 陳建強(qiáng)邋通訊員 李運(yùn)紅 胡顏;國(guó)內(nèi)首次從壁虎中分離出抗腫瘤活性成分[N];光明日?qǐng)?bào);2007年

6 李運(yùn)紅邋胡顏;國(guó)內(nèi)首次從壁虎中分離出抗腫瘤活性成分[N];科技日?qǐng)?bào);2007年

7 劉道安邋運(yùn)紅 胡顏;我國(guó)學(xué)者從壁虎中分離出抗腫瘤活性成分[N];中國(guó)醫(yī)藥報(bào);2007年

8 劉道安邋通訊員 運(yùn)紅 胡顏;壁虎中分離出抗腫瘤活性成分[N];健康報(bào);2007年

相關(guān)博士學(xué)位論文 前10條

1 張余琴;MiR-20a激活PTEN/PI3K/AKT通路誘導(dǎo)肝癌輻射抵抗[D];南方醫(yī)科大學(xué);2015年

2 葉真龍;Argonaute-2蛋白在肝癌血管生成和擬態(tài)中的功能研究[D];第二軍醫(yī)大學(xué);2015年

3 劉s，

本文編號(hào)：1814625