本文關(guān)鍵詞： 卵巢癌 適體 miRNA PTEN 嵌合體 甲基化 腫瘤干細(xì)胞 動(dòng)物模型　出處：《中南大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分Chi-29b嵌合體在卵巢上皮性癌細(xì)胞中的抗癌作用 目的：建立一種可以將miRNA-29b分子通過腫瘤組織特異性轉(zhuǎn)運(yùn)方式運(yùn)入腫瘤細(xì)胞,進(jìn)而影響抑癌基因PTEN的重新表達(dá),從而抑制卵巢癌生長的治療方法。 方法：構(gòu)建可以靶向結(jié)合卵巢癌細(xì)胞表面MUC1蛋白的適體和miR-29b構(gòu)成的嵌合體(Chi-29b)。Dicer酶體外消化Chi-29b,檢測Chi-29b能否釋放miR-29b。FACS法檢測Chi-29b的細(xì)胞內(nèi)化率。Western-blot法檢測OVCAR-3細(xì)胞中DNMT1、DNMT3A、DNMT3B、PTEN蛋白表達(dá)水平,甲基化技術(shù)檢測OVCAR-3細(xì)胞中PTEN啟動(dòng)子區(qū)的甲基化,RT-PCR檢測OVCAR-3細(xì)胞中PTEN mRNA表達(dá)水平,Hoechst33342法檢測細(xì)胞凋亡。 結(jié)果:Dicer酶能有效裂解Chi-29b嵌合體,并釋放miR-29b。Chi-29b嵌合體可以通過濃度依賴的方式特異性的轉(zhuǎn)運(yùn)到OVCAR-3細(xì)胞中；而且當(dāng)嵌合體Chi-29b濃度為250nM時(shí),達(dá)到細(xì)胞內(nèi)化的高峰拐點(diǎn)(81%),500nM和1000nM時(shí)的Chi-29b內(nèi)化數(shù)量更多。和對照組比較,Chi-29b嵌合體可以顯著下調(diào)OVCAR-3細(xì)胞中DNMT1, DNMT3A, DNMT3B的蛋白表達(dá)水平(P0.001),誘導(dǎo)PTEN基因啟動(dòng)子甲基化低表達(dá)(P0.01),上調(diào)PTEN mRNA和蛋白表達(dá)水平(P0.001和P0.01),并且可以顯著誘導(dǎo)OVCAR-3細(xì)胞凋亡(P0.001)。 結(jié)論：嵌合體Chi-29b能通過MUC1的靶向作用以濃度依賴方式進(jìn)入OVCAR-3細(xì)胞；OVCAR-3細(xì)胞中,Chi-29b通過甲基化機(jī)制上調(diào)PTEN基因和蛋白表達(dá),誘導(dǎo)細(xì)胞凋亡。 第二部分卵巢癌中Chi-29b嵌合體的抗化療耐藥效應(yīng)及機(jī)制 目的：確定在卵巢癌移植模型和化療藥物耐藥的卵巢癌腫瘤模型中MUC1核酸適體-miR-29b嵌合體的抗腫瘤作用,并進(jìn)一步探明相關(guān)的機(jī)制。 方法：構(gòu)建OVCAR-3-taxol細(xì)胞亞株,MTT法檢測其IC50值；將OVCAR-3-taxol細(xì)胞分別和1mg/ml紫杉醇、500nM的Chi-29b、1mg/ml紫杉醇加500nM的Chi-29b共孵育,MTT法檢測細(xì)胞增殖。裸鼠右后肢皮下注射OVCAR-3、OVCA-420、OVCAR-3-taxol細(xì)胞,建立相應(yīng)的異種卵巢癌移植動(dòng)物模型并分組；腹腔注射Chi-29b,評價(jià)腫瘤生長情況及其對ALDH1+細(xì)胞的影響,甲基化技術(shù)檢測移植瘤細(xì)胞中PTEN啟動(dòng)子的甲基化,RT-PCR法檢測PTENmRNA及ALDH1mRNA表達(dá)水平,Western-blot法檢測PTEN、MAPK4、 MAPK10、IGF1、AKT、Bax及血管動(dòng)蛋白的蛋白表達(dá)水平,Tunnel法檢測細(xì)胞凋亡水平。 結(jié)果：()VCAR-3-taxol細(xì)胞亞株的IC50值是5764±143μg/L；Chi-29b嵌合體能顯著抑制體外OVCAR-3-taxol細(xì)胞增殖(P0.001)。在OVCAR-3移植瘤中,和對照組比較,腹腔內(nèi)注射Chi-29b嵌合體能顯著抑制腫瘤生長(p0.001),顯著上調(diào)PTENmRNA表達(dá)(p0.001),顯著下調(diào)PTEN甲基化和MAPK4、IGF1蛋白的表達(dá)水平(p0.001)；然而在OVCA-420移植瘤中,Chi-29b通過下調(diào)MAPK4、MAPK10和IGF1蛋白表達(dá)而抑制腫瘤生長(p0.01),但不影響PTEN基因的表達(dá)。在OVCAR-3-taxol移植瘤中,和對照組比較,腹腔注射Chi-29b嵌合體能顯著抑制腫瘤生長和增加腫瘤細(xì)胞凋亡(p0.001),顯著上調(diào)PTENmRNA、PTEN和Bax蛋白表達(dá)水平(p0.001),顯著下調(diào)Akt、MAPK4、MAPK10和IGF1蛋白表達(dá)水平(p0.001)。OVCAR-3-taxol移植瘤中的ALDH1mRNA顯著高于OVCAR-3移植瘤中的表達(dá)水平(p0.001)；腹腔內(nèi)注射Chi-29b嵌合體能顯著降低這兩種移植瘤中的ALDH1mRNA表達(dá)水平(p0.001)和OVCAR-3-taxol移植瘤中的ALDH1+細(xì)胞數(shù)(p0.001)。 結(jié)論：Chi-29b在卵巢癌及卵巢癌耐藥的動(dòng)物模型中均能發(fā)揮有效的抗腫瘤作用；卵巢癌耐藥細(xì)胞的產(chǎn)生和ALDH1陽性細(xì)胞增加有關(guān)；Chi-29b通過抑制腫瘤干細(xì)胞活性發(fā)揮抗卵巢癌耐藥的作用；Chi-29b通過調(diào)節(jié)PTEN-Akt-Bax及MAPK和IGF信號途徑抑制卵巢癌的生長,并且可能和ALDH1有關(guān)。
[Abstract]:The anticancer effect of Chi-29b chimeras in epithelial ovarian cancer cells
Objective: to establish a therapeutic method that can transport miRNA-29b molecules into tumor cells through tumor tissue specific transport, thereby affecting the re expression of tumor suppressor gene PTEN, thereby inhibiting the growth of ovarian cancer.
Methods: construct chimera targeted binding aptamer and miR-29b ovarian cancer cell surface MUC1 protein A (Chi-29b).Dicer enzyme digestion in vitro Chi-29b, detection of Chi-29b can release miR-29b.FACS method to detect Chi-29b cell internalization rate of.Western-blot DNMT1 was detected in OVCAR-3 cells, DNMT3A, DNMT3B, PTEN protein expression, OVCAR-3 methylation detection cell PTEN in the promoter methylation level of PTEN, mRNA expression of OVCAR-3 cells was measured by RT-PCR, cell apoptosis was detected by Hoechst33342.
Results: Dicer enzyme can effectively cleaved Chi-29b chimeras, and the release of miR-29b.Chi-29b chimeras can be dependent on the concentration of specific transport into OVCAR-3 cells; and when the chimera Chi-29b concentration was 250nM, reached the peak of the cellular internalization of inflection point (81%), 500nM and 1000nM when the internalization of Chi-29b and control group in greater numbers. Comparison of Chi-29b chimeras can significantly reduce DNMT1, OVCAR-3 in DNMT3A cells, the expression level of DNMT3B protein (P0.001), inducible promoter methylation of PTEN gene low expression (P0.01), mRNA and upregulation of PTEN protein expression (P0.001 and P0.01), and significantly induced apoptosis in OVCAR-3 cells (P0.001).
Conclusion: chimeric Chi-29b can enter OVCAR-3 cells in a concentration dependent manner through the targeted action of MUC1. In OVCAR-3 cells, Chi-29b can upregulate PTEN gene and protein expression and induce cell apoptosis through methylation.
Anti chemotherapeutic resistance and mechanism of Chi-29b chimerism in second parts of ovarian cancer
Objective: to determine the antitumor effect of MUC1 aptamer -miR-29b chimeras in ovarian cancer transplantation models and chemotherapeutic drug resistant ovarian cancer models, and further explore the related mechanisms.
Methods: the OVCAR-3-taxol cell line, detect the IC50 value of MTT; OVCAR-3-taxol cells were 1mg/ml and paclitaxel, 500nM Chi-29b, 1mg/ml paclitaxel plus 500nM Chi-29b co incubation, cell proliferation was detected by MTT. The right hind nude mice subcutaneous injection of OVCAR-3, OVCA-420, OVCAR-3-taxol cells, to establish xenograft transplanted ovarian cancer animal model and the corresponding group; intraperitoneal injection of Chi-29b, the growth of tumor and its effects on ALDH1+ cells, the methylation of PTEN promoter methylation detection of tumor cells in the PTENmRNA and RT-PCR method to detect the expression level of ALDH1mRNA, detection of PTEN, Western-blot MAPK4, MAPK10, IGF1, AKT, Bax and angiomotin protein the expression, detect the apoptosis rate of Tunnel method.
Results: (IC50) VCAR-3-taxol cell line was 5764 + 143 g/L; Chi-29b chimera could significantly inhibit the proliferation of OVCAR-3-taxol cells in vitro (P0.001). In the OVCAR-3 transplantation tumor, compared with control group, intraperitoneal injection of Chi-29b chimeras can significantly inhibit tumor growth (p0.001), up regulate the expression of PTENmRNA (p0.001), significant downregulation of PTEN methylation and MAPK4 expression level of IGF1 protein (p0.001); however, in the OVCA-420 transplantation tumor in Chi-29b by downregulating MAPK4 expression of MAPK10 and IGF1 protein and inhibit tumor growth (P0.01), but did not affect the expression of PTEN gene. In the OVCAR-3-taxol transplantation tumor, compared with control group, intraperitoneal injection of Chi-29b chimera could significantly inhibit the growth of tumor cells and increase the apoptosis of tumor (p0.001), the significant increase in PTENmRNA, PTEN and Bax protein expression (p0.001), Akt MAPK4, MAPK10 was significantly down regulated, and the expression level of IGF1 protein (p0.001) ALDH1mRNA in.OVCAR-3-taxol xenograft was significantly higher than that in OVCAR-3 transplanted tumor (p0.001). Intraperitoneal injection of Chi-29b chimeras could significantly reduce ALDH1mRNA expression level (p0.001) and ALDH1+ cell number (p0.001) in two kinds of transplanted tumors.
Conclusion: Chi-29b can exert potent antitumor effects in animal models of ovarian cancer and ovarian cancer resistance; increase production and the positive cells of ALDH1 resistant ovarian cancer cells; Chi-29b by inhibiting the tumor stem cells play anti multidrug resistance in ovarian carcinoma; Chi-29b through inhibition of ovarian cancer and the regulation of PTEN-Akt-Bax and MAPK IGF signal the way of growth, and may be associated with ALDH1.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.31

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