發(fā)布時間：2018-01-08 20:08

  本文關(guān)鍵詞：基于腫瘤干細(xì)胞miRNA調(diào)控研究片仔癀抑制肝癌細(xì)胞生長的作用機(jī)制　出處：《福建中醫(yī)藥大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 片仔癀 肝癌 干細(xì)胞 miRNA 生長

【摘要】：目的:研究片仔癀干預(yù)對肝癌細(xì)胞生長的影響及調(diào)控機(jī)制;建立肝癌干細(xì)胞富集體系,篩選肝癌干細(xì)胞差異表達(dá)miRNA;從肝癌干細(xì)胞miRNA調(diào)控進(jìn)一步明確片仔癀對肝癌干細(xì)胞生長的干預(yù)作用和調(diào)控機(jī)制。方法:1、培養(yǎng)肝癌HepG2和BEL-7402細(xì)胞,并給予不同濃度的片仔癀(0-0.75 mg/mL)干預(yù),通過倒置顯微鏡觀察細(xì)胞形態(tài)和密度,采用MTT實驗檢測細(xì)胞活力,應(yīng)用集落形成實驗檢測細(xì)胞存活能力,通過PI染色和流式細(xì)胞儀檢測細(xì)胞周期改變,通過Hoechst染色觀察細(xì)胞凋亡情況,采用Western-blot檢測片仔癀干預(yù)對肝癌細(xì)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(xì)胞培養(yǎng)基懸浮培養(yǎng)條件下培養(yǎng)HepG2細(xì)胞,采用倒置顯微鏡觀察克隆球和親本細(xì)胞生長情況,通過RT-PCR檢測OCT4表達(dá)和流式細(xì)胞儀檢測CD133、CD90陽性細(xì)胞比例,進(jìn)一步采用miRNA芯片篩選克隆球和親本細(xì)胞miRNA差異表達(dá),并通過Q-PCR實驗對差異表達(dá)進(jìn)行驗證。3、采用無血清腫瘤干細(xì)胞培養(yǎng)基懸浮培養(yǎng)HepG2細(xì)胞富集肝癌干細(xì)胞,并給予片仔癀干預(yù),通過臺盼藍(lán)染色計數(shù)檢測細(xì)胞生長情況,通過PI染色和流式細(xì)胞儀檢測細(xì)胞周期改變,通過Hoechst染色觀察細(xì)胞凋亡情況;采用高內(nèi)涵觀察細(xì)胞克隆球形成能力改變,通過皮下移植瘤模型觀察細(xì)胞體內(nèi)致瘤能力變化;采用Western-blot檢測Bax、Bcl-2、CyclinD1、CDK4、SOX2、OCT4、p21 的表達(dá),通過 Q-PCR、Western-blot 檢測miR-483-5p及其靶基因CDKN1A(p21)的表達(dá)。結(jié)果:1、不同濃度片仔癀干預(yù)HepG2和BEL-7402細(xì)胞后,顯著抑制肝癌細(xì)胞生長和降低細(xì)胞活力,明顯抑制細(xì)胞存活能力和阻滯細(xì)胞周期從G0/G1到S期的轉(zhuǎn)換的過程,顯著誘導(dǎo)細(xì)胞凋亡;顯著降低Bcl-2/Bax比例,下調(diào)細(xì)胞周期調(diào)控蛋白CyclinD1、CDK4的表達(dá);明顯下調(diào)肝癌干細(xì)胞標(biāo)志物OCT4和SOX2的表達(dá)。2、采用無血清腫瘤干細(xì)胞培養(yǎng)基懸浮培養(yǎng)HepG2細(xì)胞,成功獲得HepG2克隆球細(xì)胞,且所獲得的克隆球細(xì)胞高表達(dá)干性基因OCT4,干細(xì)胞標(biāo)記物CD133和CD90雙陽性細(xì)胞比例明顯升高,證實了無血清腫瘤干細(xì)胞培養(yǎng)基懸浮培養(yǎng)具有富集肝癌干細(xì)胞的作用;miRNA芯片檢測結(jié)果證實與親本細(xì)胞相比,HegG2克隆球中有234個miRNA表達(dá)顯著上調(diào),有218個miRNA表達(dá)顯著下調(diào)(cut off1.5倍,P0.05),其中miR-483-5p、miR-582-5p在HegG2克隆球中的表達(dá)顯著上調(diào)(P0.05),Q-PCR檢測進(jìn)一步驗證了二者在HepG2克隆球表達(dá)上調(diào)的結(jié)果。3、片仔癀干預(yù)對HepG2克隆球細(xì)胞具有抑制生長和降低細(xì)胞活力的作用,具有抑制細(xì)胞從G0/G1期到S期轉(zhuǎn)換的過程和誘導(dǎo)細(xì)胞凋亡的作用;能夠顯著抑制細(xì)胞體外克隆球形成能力和體內(nèi)致瘤能力及生長的作用;片仔癀干預(yù)對HepG2克隆球細(xì)胞具有顯著降低Bcl-2/Bax比例,下調(diào)細(xì)胞周期調(diào)控蛋白CyclinD1、CDK4的表達(dá);明顯下調(diào)肝癌干細(xì)胞標(biāo)志物OCT4和SOX2的表達(dá);此外可顯著下調(diào)miR-483-5p的表達(dá),同時上調(diào)其靶基因CDKN1A/p21在mRNA和蛋白水平的表達(dá)。結(jié)論:片仔癀通過調(diào)控細(xì)胞增殖凋亡相關(guān)調(diào)控因子Bcl-2/Bax比例、CyclinD1和CDK4的表達(dá)具有顯著抑制肝癌細(xì)胞生長的作用,且具有下調(diào)干性基因SOX2和OCT4表達(dá)的作用;成功復(fù)制無血清腫瘤干細(xì)胞培養(yǎng)基懸浮培養(yǎng)富集肝癌干細(xì)胞體系,證實miR-483-5p、miR-582-5p等多個miRNA在肝癌干細(xì)胞中具有顯著調(diào)控效應(yīng);片仔癀干預(yù)顯著抑制肝癌干細(xì)胞生長和體內(nèi)外致瘤能力,且通過調(diào)控細(xì)胞增殖、凋亡相關(guān)蛋白Bcl-2/Bax比例、CyclinD1、CDK4和miR-483-5p及其靶基因CDKN1A(p21)表達(dá)進(jìn)而抑制肝癌干細(xì)胞生長和誘導(dǎo)細(xì)胞凋亡可能是其抑制肝癌細(xì)胞生長的重要機(jī)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(xué)位授予單位】：福建中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.7

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