【摘要】：目的利用RNA干擾（RNAi）技術(shù)沉默GPR137基因在多種惡性膠質(zhì)瘤細(xì)胞中的表達(dá)，，闡明GPR137對(duì)惡性膠質(zhì)瘤細(xì)胞增殖、克隆形成能力等方面的影響。 方法利用免疫組化技術(shù)對(duì)29例不同級(jí)別人膠質(zhì)瘤組織中GPR137的表達(dá)及與病理分級(jí)相關(guān)性進(jìn)行分析。利用半定量反轉(zhuǎn)錄－聚合酶鏈反應(yīng)（semi-quantitative RT-PCR）檢測(cè)GPR137基因在五種惡性膠質(zhì)瘤細(xì)胞（U251,U-87MG, U-118MG, A172,U373）中的表達(dá)。構(gòu)建針對(duì)GPR137基因的短發(fā)卡(shRNA)慢病毒表達(dá)載體，包裝成病毒顆粒并感染人膠質(zhì)瘤U251和A172細(xì)胞，采用實(shí)時(shí)定量聚合酶鏈反應(yīng)和Western印跡法驗(yàn)證GPR137基因的mRNA和蛋白的表達(dá)，確定其抑制效率。采用噻唑藍(lán)(MTT)比色法和克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞增殖和克隆形成能力情況，應(yīng)用流式細(xì)胞分析法檢測(cè)細(xì)胞周期分布情況。觀察沉默GPR137基因?qū)251和A172細(xì)胞增殖、克隆形成和周期分布的影響。 結(jié)果不同級(jí)別人膠質(zhì)瘤組織中GPR137表達(dá)均異常上調(diào)，且與膠質(zhì)瘤臨床病理分級(jí)呈正相關(guān)。通過(guò)半定量反轉(zhuǎn)錄－聚合酶鏈反應(yīng)證實(shí)，GPR137基因在五種膠質(zhì)瘤細(xì)胞中均顯著表達(dá)。實(shí)時(shí)定量聚合酶鏈反應(yīng)和Western印跡法顯示，GPR137-shRNA能有效抑制U251和A172細(xì)胞中GPR137基因的mRNA和蛋白的表達(dá)。MTT和克隆形成實(shí)驗(yàn)顯示，感染后的膠質(zhì)瘤細(xì)胞的增殖和克隆形成能力明顯受到抑制。流式細(xì)胞分析法顯示，感染后的膠質(zhì)瘤細(xì)胞周期停滯在S期。上述差異均具有統(tǒng)計(jì)學(xué)意義P＜0.05㖞。 討論以往的文獻(xiàn)已經(jīng)證實(shí)信號(hào)轉(zhuǎn)導(dǎo)通路和相關(guān)的膜蛋白，特別是G蛋白偶聯(lián)受體(GPCRs)家族在膠質(zhì)瘤的形成中發(fā)揮著重要的作用。腫瘤細(xì)胞通過(guò)G蛋白偶聯(lián)受體得以存活、增殖、侵犯周?chē)M織、并躲避免疫機(jī)制。人體內(nèi)已知的800多種G蛋白偶聯(lián)受體大部分已經(jīng)明確其結(jié)構(gòu)和功能，并成為藥物作用靶標(biāo)。然而，目前仍有100多種被稱(chēng)為孤兒受體的G蛋白偶聯(lián)受體，其結(jié)構(gòu)和作用仍然不清楚，GPR137正是其中一種孤兒受體。GPR137位于人第11號(hào)染色體的R56區(qū)，在中樞神經(jīng)系統(tǒng)內(nèi)廣泛表達(dá)。已經(jīng)有文獻(xiàn)報(bào)道GPR137基因參與體內(nèi)一些腫瘤的形成過(guò)程，如肝癌、乳腺癌、前列腺癌等。但在腦膠質(zhì)瘤中的作用國(guó)內(nèi)外尚未有報(bào)道。本研究中，GPR137在多種腦膠質(zhì)瘤細(xì)胞中高表達(dá)。敲減GPR137后，U251和A172細(xì)胞增殖、克隆形成受到顯著抑制，而且細(xì)胞周期停滯在S期。說(shuō)明GPR137在膠質(zhì)瘤的增殖，克隆形成等方面發(fā)揮著重要的作用。然而相關(guān)的分子機(jī)制有待后續(xù)研究證實(shí)。綜上所述，GPR137基因在體外對(duì)膠質(zhì)瘤細(xì)胞的增殖、克隆形成具有重要影響。同時(shí)，為GPR137成為膠質(zhì)瘤靶向治療基因提供了理論依據(jù)。
[Abstract]:Objective to silence the expression of GPR137 gene in various malignant glioma cells by RNA interference (RNAi), and to elucidate the effect of GPR137 on the proliferation and cloning ability of malignant glioma cells. Methods Immunohistochemical technique was used to analyze the expression of GPR137 in 29 cases of human gliomas of different grades and its correlation with pathological grade. Semi-quantitative reverse transcription-polymerase chain reaction (semi-quantitative RT-PCR) was used to detect the expression of GPR137 gene in five kinds of malignant glioma cells (U251, UX87MG, U18MG, A172, U373). A short hairpin (shRNA) lentivirus expression vector targeting GPR137 gene was constructed and packaged into virus particles and infected human glioma U251 and A172 cells. The expression of mRNA and protein of GPR137 gene was verified by real-time quantitative polymerase chain reaction and Western printing, and its inhibitory efficiency was determined. The cell proliferation and clone formation ability were detected by thiazolyl blue (MTT) colorimetric assay and clone formation assay, and the cell cycle distribution was detected by flow cytometry. To observe the effect of silencing GPR137 gene on proliferation, clone formation and cycle distribution of U251 and A172 cells. Results the expression of GPR137 in different grades of human gliomas was abnormally up-regulated, which was positively correlated with the clinicopathological grade of gliomas. The expression of GPR137 gene in five kinds of glioma cells was confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Real-time quantitative polymerase chain reaction and Western staining showed that GPR137-shRNA could effectively inhibit the expression of GPR137 gene mRNA and protein in U251 and A172 cells. GPR137-shRNA and clone formation assay showed that the proliferation and clone formation ability of infected glioma cells were significantly inhibited. Flow cytometry showed that the infected glioma cell cycle stagnated in S phase. The above differences were statistically significant (P < 0.05). It has been confirmed that signal transduction pathway and related membrane proteins, especially G protein-coupled receptor (GPCRs) family, play an important role in the formation of glioma. Tumor cells survive, proliferate, invade surrounding tissues and avoid immune mechanisms through G-protein-coupled receptors. Most of the more than 800 kinds of G protein coupling receptors known in human body have been identified in their structure and function, and have become the target of drug action. However, there are still more than 100 kinds of G protein-coupled receptors called orphan receptors, and their structure and function are still unclear. GPR137 is one of the orphan receptors. GPR137 is located in the R56 region of human chromosome 11 and is widely expressed in the central nervous system. It has been reported that GPR137 gene is involved in the formation of some tumors in vivo, such as liver cancer, breast cancer, prostate cancer and so on. However, the role of glioma has not been reported at home and abroad. In this study, GPR137 was highly expressed in a variety of glioma cells. After knockout of GPR137, U251 and A172 cells proliferated and clone formation was significantly inhibited, and the cell cycle stagnated in S phase. It is suggested that GPR137 plays an important role in the proliferation and clone formation of gliomas. However, the related molecular mechanisms need to be confirmed by follow-up studies. In conclusion, GPR137 gene plays an important role in the proliferation and cloning of glioma cells in vitro. At the same time, it provides a theoretical basis for GPR137 to become a targeted therapeutic gene for glioma.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R739.41

【共引文獻(xiàn)】

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