本文選題：脈絡(luò)膜黑色素瘤 + 缺氧誘導(dǎo)因子-1α��； 參考：《青島大學(xué)》2014年碩士論文

【摘要】：目的：以特異性小干擾RNA (siRNA)沉默缺氧培養(yǎng)的人眼脈絡(luò)膜黑色瘤細(xì)胞(OCM-1)中缺氧誘導(dǎo)因子-1α (HIF-1α)基因,觀察其對(duì)細(xì)胞骨架分子波形蛋白(Vimentin)表達(dá)的影響,以初步探討HIF-1α對(duì)人眼脈絡(luò)膜黑色瘤上皮-間質(zhì)轉(zhuǎn)化(EMT)的調(diào)控作用。 方法：在正常培養(yǎng)基中加入100μmo1/L的氯化鈷(CoCl2)以模擬腫瘤內(nèi)部缺氧微環(huán)境,構(gòu)建靶向HIF-1α的siRNA干擾載體,同時(shí)設(shè)立β-actin陽性對(duì)照、無義寡核苷酸陰性對(duì)照及空載脂質(zhì)體對(duì)照組,以LipofectamineTM2000為載體介導(dǎo)siRNA轉(zhuǎn)染缺氧培養(yǎng)的OCM-1細(xì)胞,以RT-PCR和Western blot方法檢測(cè)缺氧培養(yǎng)前后及轉(zhuǎn)染前后HIF-1α Vimentin mRNA和蛋白的表達(dá)情況。 結(jié)果:OCM-1細(xì)胞經(jīng)缺氧培養(yǎng)后可由上皮細(xì)胞樣形態(tài)轉(zhuǎn)變成間質(zhì)細(xì)胞樣形態(tài)。與常氧組相比,單純?nèi)毖踅MHIF-1α mRNA表達(dá)無明顯差異(P0.05),蛋白表達(dá)顯著升高(P0.01),而Vimentin mRNA和蛋白的表達(dá)均顯著升高(P0.01)。缺氧培養(yǎng)的各組之間相比,陽性對(duì)照組β-actin mRNA表達(dá)明顯下降(P0.01),說明轉(zhuǎn)染成功；HIF-1a干擾組HIF-1α和Vimentin mRNA和蛋白表達(dá)均明顯下降(P0.01)；無義寡核苷酸陰性對(duì)照組及空載脂質(zhì)體對(duì)照組各檢測(cè)因素均無明顯差別(P0.05)。 結(jié)論： 1.缺氧培養(yǎng)可使OCM-1細(xì)胞由上皮細(xì)胞樣形態(tài)轉(zhuǎn)變成間質(zhì)細(xì)胞樣形態(tài),提示缺氧微環(huán)境可促使OCM-1細(xì)胞發(fā)生EMT。 2.缺氧可在蛋白水平上調(diào)OCM-1細(xì)胞中HIF-1α的表達(dá),提示缺氧對(duì)HIF-1α的調(diào)控主要發(fā)生在轉(zhuǎn)錄后水平。 3.HIF-1α可通過轉(zhuǎn)錄激活Vimentin,使其表達(dá)上調(diào),提示HIF-1α可能參與調(diào)控OCM-1細(xì)胞的EMT,進(jìn)而影響其侵襲、轉(zhuǎn)移能力。 4.可成功利用LipofectamineTM2000為載體將siRNA轉(zhuǎn)入OCM-1細(xì)胞內(nèi)。 5.HIF-lα-siRNA轉(zhuǎn)染OCM-1細(xì)胞可成功下調(diào)HIF-1α及Vimentin的表達(dá),進(jìn)一步說明缺氧條件下Vimentin的表達(dá)、EMT的發(fā)生受HIF-1α的調(diào)控；從分子水平抑制HIF-1α的表達(dá),可能抑制腫瘤侵襲、轉(zhuǎn)移,從而可為腫瘤治療提供新方向。
[Abstract]:Aim: to study the effect of hypoxia inducible factor 1 偽 (HIF-1 偽) gene on cytoskeleton vimentin (vimentin) expression in cultured human eye choroidal melanoma cells (OCM-1) by silencing hypoxia with specific small interfering RNA (siRNA). To investigate the effect of HIF-1 偽 on epithelial-interstitial transformation (EMT) of human choroidal melanoma. Methods: to construct siRNA interference vector targeting HIF-1 偽 by adding 100 渭 mol / L Cobalt chloride (CoCl2) into normal medium to simulate the anoxic microenvironment of tumor, and to set up 尾 -actin positive control, negative control group and no-load liposome control group. The expression of HIF-1 偽 Vimentin mRNA and protein was detected by RT-PCR and Western blot before and after hypoxia culture. Results the cells of OCM-1 could be transformed from epithelial cells to interstitial cells by hypoxia. Compared with normoxic group, the expression of HIF-1 偽 mRNA in hypoxia group was not significantly different (P0.05), but the expression of Vimentin mRNA and protein was significantly increased (P0.01). The expression of 尾 -actin mRNA in positive control group was significantly decreased (P0.01), which indicated that the expression of HIF-1 偽 and Vimentin mRNA and protein in HIF-1a interference group decreased significantly (P0.01). There was no significant difference between negative control group and no-load liposome control group (P0.05). Conclusion: 1. Hypoxia can up-regulate the expression of HIF-1 偽 in OCM-1 cells at the protein level, suggesting that the regulation of HIF-1 偽 by hypoxia mainly occurs at the post-transcriptional level. 3. HIF-1 偽 can activate Vimentinthrough transcription and up-regulate the expression of HIF-1 偽. These results suggest that HIF-1 偽 may be involved in the regulation of EMTs of OCM-1 cells, thus affecting their invasion and metastasis ability. 4. The expression of HIF-1 偽 and Vimentin in OCM-1 cells was successfully down-regulated by transfection of siRNA with LipofectamineTM2000. 5. HIF-l 偽 -siRNA transfected OCM-1 cells could down-regulate the expression of HIF-1 偽 and Vimentin, which further indicated that the expression of Vimentin was regulated by HIF-1 偽 and inhibited the expression of HIF-1 偽 at molecular level. It may inhibit tumor invasion and metastasis, thus providing a new direction for tumor therapy.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 趙娟;周占宇;王爽;苑伏香;盧發(fā)艷;;siRNA特異性沉默HIF-1α對(duì)缺氧培養(yǎng)的OCM-1細(xì)胞中Vimentin表達(dá)的影響[J];國(guó)際眼科雜志;2014年03期

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本文編號(hào)：2078124