發(fā)布時間：2018-01-08 20:08

  本文關鍵詞：基于腫瘤干細胞miRNA調控研究片仔癀抑制肝癌細胞生長的作用機制　出處：《福建中醫(yī)藥大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 片仔癀 肝癌 干細胞 miRNA 生長

【摘要】：目的:研究片仔癀干預對肝癌細胞生長的影響及調控機制;建立肝癌干細胞富集體系,篩選肝癌干細胞差異表達miRNA;從肝癌干細胞miRNA調控進一步明確片仔癀對肝癌干細胞生長的干預作用和調控機制。方法:1、培養(yǎng)肝癌HepG2和BEL-7402細胞,并給予不同濃度的片仔癀(0-0.75 mg/mL)干預,通過倒置顯微鏡觀察細胞形態(tài)和密度,采用MTT實驗檢測細胞活力,應用集落形成實驗檢測細胞存活能力,通過PI染色和流式細胞儀檢測細胞周期改變,通過Hoechst染色觀察細胞凋亡情況,采用Western-blot檢測片仔癀干預對肝癌細胞Bax、Bcl-2、CyclinD1、CDK4、OCT4 和 SOX2 的影響。2、分別在完全培養(yǎng)基常規(guī)培養(yǎng)和無血清腫瘤干細胞培養(yǎng)基懸浮培養(yǎng)條件下培養(yǎng)HepG2細胞,采用倒置顯微鏡觀察克隆球和親本細胞生長情況,通過RT-PCR檢測OCT4表達和流式細胞儀檢測CD133、CD90陽性細胞比例,進一步采用miRNA芯片篩選克隆球和親本細胞miRNA差異表達,并通過Q-PCR實驗對差異表達進行驗證。3、采用無血清腫瘤干細胞培養(yǎng)基懸浮培養(yǎng)HepG2細胞富集肝癌干細胞,并給予片仔癀干預,通過臺盼藍染色計數(shù)檢測細胞生長情況,通過PI染色和流式細胞儀檢測細胞周期改變,通過Hoechst染色觀察細胞凋亡情況;采用高內涵觀察細胞克隆球形成能力改變,通過皮下移植瘤模型觀察細胞體內致瘤能力變化;采用Western-blot檢測Bax、Bcl-2、CyclinD1、CDK4、SOX2、OCT4、p21 的表達,通過 Q-PCR、Western-blot 檢測miR-483-5p及其靶基因CDKN1A(p21)的表達。結果:1、不同濃度片仔癀干預HepG2和BEL-7402細胞后,顯著抑制肝癌細胞生長和降低細胞活力,明顯抑制細胞存活能力和阻滯細胞周期從G0/G1到S期的轉換的過程,顯著誘導細胞凋亡;顯著降低Bcl-2/Bax比例,下調細胞周期調控蛋白CyclinD1、CDK4的表達;明顯下調肝癌干細胞標志物OCT4和SOX2的表達。2、采用無血清腫瘤干細胞培養(yǎng)基懸浮培養(yǎng)HepG2細胞,成功獲得HepG2克隆球細胞,且所獲得的克隆球細胞高表達干性基因OCT4,干細胞標記物CD133和CD90雙陽性細胞比例明顯升高,證實了無血清腫瘤干細胞培養(yǎng)基懸浮培養(yǎng)具有富集肝癌干細胞的作用;miRNA芯片檢測結果證實與親本細胞相比,HegG2克隆球中有234個miRNA表達顯著上調,有218個miRNA表達顯著下調(cut off1.5倍,P0.05),其中miR-483-5p、miR-582-5p在HegG2克隆球中的表達顯著上調(P0.05),Q-PCR檢測進一步驗證了二者在HepG2克隆球表達上調的結果。3、片仔癀干預對HepG2克隆球細胞具有抑制生長和降低細胞活力的作用,具有抑制細胞從G0/G1期到S期轉換的過程和誘導細胞凋亡的作用;能夠顯著抑制細胞體外克隆球形成能力和體內致瘤能力及生長的作用;片仔癀干預對HepG2克隆球細胞具有顯著降低Bcl-2/Bax比例,下調細胞周期調控蛋白CyclinD1、CDK4的表達;明顯下調肝癌干細胞標志物OCT4和SOX2的表達;此外可顯著下調miR-483-5p的表達,同時上調其靶基因CDKN1A/p21在mRNA和蛋白水平的表達。結論:片仔癀通過調控細胞增殖凋亡相關調控因子Bcl-2/Bax比例、CyclinD1和CDK4的表達具有顯著抑制肝癌細胞生長的作用,且具有下調干性基因SOX2和OCT4表達的作用;成功復制無血清腫瘤干細胞培養(yǎng)基懸浮培養(yǎng)富集肝癌干細胞體系,證實miR-483-5p、miR-582-5p等多個miRNA在肝癌干細胞中具有顯著調控效應;片仔癀干預顯著抑制肝癌干細胞生長和體內外致瘤能力,且通過調控細胞增殖、凋亡相關蛋白Bcl-2/Bax比例、CyclinD1、CDK4和miR-483-5p及其靶基因CDKN1A(p21)表達進而抑制肝癌干細胞生長和誘導細胞凋亡可能是其抑制肝癌細胞生長的重要機制之一。
[Abstract]:Objective: To study pientzehuang intervention on the growth of liver cancer cells and regulation mechanism; establish system of enrichment of liver cancer stem cells, the expression of miRNA in liver cancer stem cells were screened from liver cancer stem cells; miRNA regulation to further clarify the Pianzihuang on hepatocarcinoma stem cells the intervention and control mechanism. Methods: 1. Cultured HepG2 and BEL-7402 cells, and with different concentrations of pientzehuang (0-0.75 mg/mL) intervention by inverted microscope. Cell morphology and density were observed, the cell viability by MTT assay and colony formation assay, using cell viability, by PI staining and flow cytometry cell cycle change and apoptosis was observed by Hoechst staining, detected by Western-blot intervention on pientzehuang Bax cells, Bcl-2, CyclinD1, CDK4, OCT4 and SOX2.2, respectively, in complete medium and serum-free cultured tumor stem Cells cultured HepG2 cells under the condition of medium, observe the clones and parental cell growth by inverted microscope, and flow cytometry to detect the expression of OCT4 CD133 by RT-PCR, the proportion of CD90 positive cells, further using miRNA chip and the expression of miRNA cell clones screening differences, and verified by Q-PCR.3 experiments on the difference the expression of cell culture medium in suspension culture of HepG2 cell enrichment of liver stem cells in serum tumor stem, and give pientzehuang intervention by trypan blue staining to detect the growth of cells, by PI staining and flow cytometry cell cycle change and apoptosis was observed by Hoechst staining; the high content observed cell clone ball formation the ability to change, tumorigenicity changes through subcutaneous transplanted tumor cells in vivo models were detected by Western-blot; Bax, Bcl-2, CyclinD1, CD K4, SOX2, OCT4, p21 expression by Q-PCR, detection of CDKN1A miR-483-5p and its target gene Western-blot (p21) expression. Results: 1 different concentrations of HepG2 and BEL-7402 cells after the intervention of pianzaihuang, significantly inhibited the growth and cell viability of hepatoma cells, inhibit the conversion process of cell survival and cell cycle arrest from G0/G1 to the S phase, significantly induced cell apoptosis; significantly reduced the proportion of Bcl-2/Bax, downregulation of cell cycle regulatory protein CyclinD1, CDK4 expression was down regulated expression of.2; liver cancer stem cell marker OCT4 and SOX2, the cell culture medium in suspension culture of HepG2 cell tumor stem cells, successful acquisition of HepG2, high expression of dry OCT4 gene cloning and cells obtained by double positive cells were stem cell marker CD133 and CD90 increased significantly, confirming no serum tumor stem cell culture has rich nutrient medium In the role of cancer stem cells; miRNA chip test results confirmed that compared with the parent cells, 234 miRNA upregulated the expression of HegG2 in 218 clones, miRNA expression was significantly reduced (cut off1.5 times, P0.05), where miR-483-5p, miR-582-5p expression was significantly up-regulated in HegG2 clones in (P0.05), further Q-PCR test to verify the two results in the upregulation of the expression of.3 HepG2 clones, pientzehuang intervention has reduced cell viability and growth inhibition effect on HepG2 cells, inhibit the cells from G0/G1 phase to S phase transition process and induces cell apoptosis; can inhibit the in vitro clone forming ability and tumorigenicity in vivo and the growth of the intervention significantly reduced the proportion of Bcl-2/Bax; pientzehuang with HepG2 cells, downregulation of cell cycle regulatory protein CyclinD1, CDK4 expression was significantly down regulated in liver cancer stem cells; The expression of OCT4 and SOX2; in addition significantly downregulated the expression of miR-483-5p and its target gene CDKN1A/p21 expression upregulation in mRNA and protein level. Conclusion: pientzehuang through the regulation of cell proliferation and apoptosis related regulation factors Bcl-2/Bax ratio significantly inhibit the growth of hepatocellular carcinoma cells the expression of CyclinD1 and CDK4, and down regulate the expression of stemness genes SOX2 and OCT4; successful replication without cell culture medium suspension culture enrich liver cancer stem cell system, confirmed that miR-483-5p serum tumor stem, miR-582-5p and other miRNA has significant effects on liver cancer stem cells; inhibit cell growth and pientzehuang intervention in liver cancer stem outside the tumorigenic ability, and through the regulation of cell proliferation, apoptosis protein ratio of Bcl-2/Bax, CyclinD1, CDK4 and miR-483-5p and its target gene CDKN1A (p21) expression and inhibit the growth and induce liver cancer stem cells Apoptosis may be one of the important mechanisms to inhibit the growth of hepatoma cells.

【學位授予單位】：福建中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R735.7

【二級參考文獻】

相關博士學位論文 前2條

1 仝利民;日本護理保險制度及其對上海的啟示[D];華東師范大學;2008年

2 謝建華;中國老齡產業(yè)發(fā)展的理論與政策問題研究[D];中國社會科學院研究生院;2003年

相關碩士學位論文 前10條

1 賈琳;護理保險供需分析及我國護理保險制度的建構[D];沈陽師范大學;2012年

2 馮麒婷;國外長期照護保險計劃比較分析[D];中國社會科學院研究生院;2012年

3 張鑫;我國長期護理保險可行性研究[D];首都經濟貿易大學;2012年

4 徐斌秀;福利多元主義視角下中國老年社會長期護理保險制度的建構[D];南京大學;2011年

5 劉靜;中國人口老齡化背景下長期護理保險研究[D];遼寧大學;2011年

6 徐雯怡;論我國長期護理保險體制的構建[D];華東政法大學;2010年

7 張星;人口老齡化背景下我國老年長期護理保險的研究[D];吉林財經大學;2010年

8 張斌;我國長期護理保險發(fā)展研究[D];蘇州大學;2008年

9 夏秀梅;我國開展商業(yè)性長期護理保險研究[D];鄭州大學;2006年

10 鄒小菲;長期護理保險的全球發(fā)展趨勢及對中國的展望[D];青島大學;2005年

，

本文編號：1398485