本文關(guān)鍵詞： TFE3 慢病毒過(guò)表達(dá) Xp11.2易位性腎癌 ACHN細(xì)胞 mTOR pS6　出處：《南方醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景和目的:Xp11.2易位性腎癌是WHO新分類的一種腎癌,發(fā)病年齡輕、預(yù)后較差,主要特征為TFE3基因發(fā)生易位,導(dǎo)致其過(guò)表達(dá)。因目前尚無(wú)針對(duì)過(guò)表達(dá)TFE3建立的腎癌細(xì)胞學(xué)模型,故作用機(jī)制尚不明確,涉及的信號(hào)通路有待研究,其診斷及治療缺乏理論基礎(chǔ)。目的:1.篩選出適合構(gòu)建過(guò)表達(dá)TFE3慢病毒感染的腎癌細(xì)胞系,構(gòu)建并鑒定特異性慢病毒載體顆粒。2.建立慢病毒過(guò)表達(dá)TFE3穩(wěn)定感染的腎癌細(xì)胞系,研究過(guò)表達(dá)TFE3對(duì)其生物學(xué)影響。3.明確mTOR及pS6在Xp11.2易位性腎癌及過(guò)表達(dá)TFE3細(xì)胞系中的表達(dá),探討PI3K/AKT/mTOR通路在該類腫瘤中的作用。4.Affymetrix基因芯片檢測(cè)Xp11.2易位性腎癌相關(guān)的差異基因,篩選出更多精確的靶基因。方法:1.Real time PCR和Western blot方法檢測(cè)8種腎源性的細(xì)胞系中TFE3的表達(dá)水平,篩選一株進(jìn)行后續(xù)實(shí)驗(yàn),構(gòu)建特異性慢病毒過(guò)表達(dá)載體顆粒并鑒定其感染效率。2.構(gòu)建穩(wěn)定感染過(guò)表達(dá)TFE3的腎癌細(xì)胞系,分為過(guò)表達(dá)組(OE),陰性對(duì)照組(NC)和空載對(duì)照組(CON),并用Western blot進(jìn)行驗(yàn)證。3.MTT、平板克隆法檢測(cè)不同實(shí)驗(yàn)組的細(xì)胞增殖和克隆形成;利用流式細(xì)胞儀檢測(cè)其細(xì)胞周期。4.免疫組化方法檢測(cè)12例Xp11.2易位性腎癌和27例非Xp11.2易位性腎癌中mTOR和pS6的表達(dá)情況,同時(shí)用Western blot方法檢測(cè)不同細(xì)胞實(shí)驗(yàn)組中兩者的差異。5.Affymetrix基因芯片分析2例Xp11.2易位性腎癌患者癌組織與癌旁組織之間基因表達(dá)譜的差異。結(jié)果:1.Real-time PCR結(jié)果顯示TFE3基因在ACHN中表達(dá)豐度為中,Western blot結(jié)果顯示在ACHN中無(wú)目的條帶,因此選取ACHN細(xì)胞系作為靶細(xì)胞進(jìn)行慢病毒感染。成功構(gòu)建人TFE3特異性慢病毒過(guò)表達(dá)載體并獲得相應(yīng)高效率慢病毒。2.在實(shí)驗(yàn)組中,OE組病毒滴度=2×108TU/ml,MOI=10,Western blot結(jié)果顯示OE組中檢測(cè)到95KD的目的條帶,表明過(guò)表達(dá)TFE3穩(wěn)定感染ACHN細(xì)胞成功。OE組的細(xì)胞增殖水平和克隆數(shù)較NC組顯著升高(P0.05),說(shuō)明過(guò)表達(dá)TFE3能夠促進(jìn)ACHN細(xì)胞的增殖和克隆形成。細(xì)胞周期結(jié)果顯示,OE組較NC組的S期延長(zhǎng)(P0.05),說(shuō)明過(guò)表達(dá)TFE3能夠調(diào)控ACHN細(xì)胞的周期從而促進(jìn)細(xì)胞增殖。3.免疫組化法檢測(cè)石蠟標(biāo)本中的mTOR和pS6在Xp 11.2易位性腎癌較非Xp 11.2易位性腎癌中陽(yáng)性率顯著升高(P0.05),兩種蛋白在OE組中表達(dá)顯著增高(P0.05),說(shuō)明該腫瘤在組織學(xué)和細(xì)胞學(xué)層面都存在PI3K/AKT/mTOR通路的過(guò)度激活。4.Affymetrix基因芯片檢測(cè)Xp11.2易位性腎癌患者相關(guān)差異表達(dá)的基因。共篩選出差異表達(dá)基因1479個(gè),癌組織中上調(diào)基因790個(gè),下調(diào)基因689個(gè)。其中泛素化相關(guān)的RNF180基因下調(diào)5倍以上,提示該種腫瘤可能與泛素化有關(guān)。結(jié)論:1.過(guò)表達(dá)TFE3能夠促進(jìn)ACHN細(xì)胞的增殖,調(diào)控其細(xì)胞周期。2.在Xp 11.2易位性腎癌中存在PI3K/AKT/mTOR通路的過(guò)度激活。3.Xp11.2易位性腎癌可能與泛素化途徑相關(guān)。
[Abstract]:Background and objective: Xp11.2 translocated renal cell carcinoma (RCC) is a new type of renal cell carcinoma (RCC) classified by WHO. Its age is young and its prognosis is poor. The main feature is translocation of TFE3 gene, which results in its overexpression. There is no cytological model of RCC established for overexpression of TFE3 at present. Therefore, the mechanism of action is unclear, the signal pathway involved remains to be studied, and its diagnosis and treatment lack theoretical basis. Objective: 1. To screen and construct a cell line of renal cell carcinoma with overexpression of TFE3 lentivirus infection. To construct and identify the specific lentivirus vector particle. 2. To establish a cell line with stable infection of lentivirus overexpression TFE3, and to study the biological effects of overexpression of TFE3 on the cell line. 3. To clarify the expression of mTOR and pS6 in Xp11.2 translocated renal cell carcinoma and TFE3 cell line. To investigate the role of PI3K/AKT/mTOR pathway in this type of tumor. 4. Affymetrix gene chip was used to detect differentially expressed genes related to Xp11.2 translocation of renal cell carcinoma, and more accurate target genes were screened. Methods: 1. Real time PCR and Western blot were used to detect the expression of TFE3 in eight renal cell lines. One cell strain was screened for subsequent experiments to construct specific lentivirus overexpression vector particles and to identify its infection efficiency. 2. To construct a renal carcinoma cell line with stable overexpression of TFE3. Western blot was used to test the cell proliferation and clone formation in different experimental groups. The expression of mTOR and pS6 in 12 cases of Xp11.2 translocated renal cell carcinoma and 27 cases of non-#en1# translocated renal cell carcinoma were detected by flow cytometry. At the same time, Western blot method was used to detect the difference between the two cell groups. 5.Affymetrix gene chip was used to analyze the difference of gene expression profiles between cancer tissues and adjacent tissues of two patients with Xp11.2 translocated renal cell carcinoma. The expression abundance of ACHN was determined by Western blot. The results showed that there was no objective band in ACHN. Therefore, ACHN cell line was selected as the target cell for lentivirus infection. The human TFE3 specific lentivirus overexpression vector was successfully constructed and the corresponding high efficiency lentivirus was obtained. In the experimental group, the titer of OE group virus was 2 脳 10 8 TUU / ml moi 10 blot and the results showed that the human TFE3 specific lentivirus overexpression vector was found in the OE group. The target band of 95KD was detected, The results showed that the proliferation level and clone number of ACHN cells infected by overexpression of TFE3 were significantly higher than those of NC group, which indicated that overexpression of TFE3 could promote the proliferation and clone formation of ACHN cells. The cell cycle results showed that the cell cycle of OE group was higher than that of NC group. The S-phase prolongation (P0.05) of group S indicates that overexpression of TFE3 can regulate the cell cycle of ACHN cells and thus promote cell proliferation .3.Immunohistochemical method is used to detect the positive rate of mTOR and pS6 in Xp11.2 translocated renal cell carcinoma compared with non-Xp11.2 translocated renal cell carcinoma. The expression of two proteins in OE group was significantly higher than that in OE group, indicating that there was overexpression of PI3K/AKT/mTOR pathway in the tumor. 4. Affymetrix gene chip was used to detect the differentially expressed genes in patients with Xp11.2 translocation of renal cell carcinoma. A total of 1479 differentially expressed genes were screened. There were 790 up-regulated genes and 689 down-regulated genes in cancer tissues. The RNF180 gene associated with ubiquitization was down-regulated by more than 5 times, suggesting that the tumor might be related to ubiquitization. Conclusion: 1. Overexpression of TFE3 can promote the proliferation of ACHN cells. Regulation of cell cycle. 2. Overexpression of PI3K/AKT/mTOR pathway in Xp11.2 translocation RCC. 3.Xp11.2 translocation RCC may be associated with Ubiquitin pathway.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.11

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 沈勤;饒秋;余波;王璇;馬恒輝;周曉軍;;伴有TFE3擴(kuò)增的腎臟上皮樣血管平滑肌脂肪瘤[J];臨床與實(shí)驗(yàn)病理學(xué)雜志;2012年06期

2 薛彥詩(shī);甘衛(wèi)東;劉鐵石;張士偉;姚林方;連惠波;張古田;李笑弓;郭宏騫;;Xp11.2易位/TFE3基因融合相關(guān)性腎癌的臨床與病理分析[J];江蘇醫(yī)藥;2012年05期

3 梅榮;吳長(zhǎng)利;胡海龍;楊宇明;白玫;王仁杰;趙文魯;;Xp11.2易位/TFE3基因融合相關(guān)性腎癌的臨床病理特點(diǎn)(附1例報(bào)告)[J];山東醫(yī)藥;2014年01期

4 饒秋;周曉軍;吳波;馬恒輝;周航波;劉曉紅;陳潔宇;;Xp11.2易位/TFE3基因融合相關(guān)性腎癌的病理特征與臨床分析[J];中華病理學(xué)雜志;2007年04期

5 張棟;孫鵬;李鵬;;Xp11.2易位/TFE3基因融合相關(guān)性腎癌1例討論[J];泌尿外科雜志(電子版);2011年02期

6 顧聞?dòng)?楊斌;彭波;車建平;鄭軍華;;Xp11.2易位/TFE3基因融合相關(guān)性腎癌1例報(bào)告及文獻(xiàn)復(fù)習(xí)[J];臨床泌尿外科雜志;2012年06期

7 李琪;盧威;;成人Xp11.2易位/TFE3基因融合相關(guān)性腎癌1例[J];中國(guó)現(xiàn)代藥物應(yīng)用;2013年18期

8 邰艷紅;韋立新;石懷銀;;Xp11.2易位/TFE3基因融合相關(guān)性腎癌的臨床病理學(xué)特點(diǎn)[J];臨床與實(shí)驗(yàn)病理學(xué)雜志;2008年05期

9 鄒泓;李鋒;龐麗娟;胡文浩;李洪安;蔣金芳;梁偉華;孫振柱;王春;王嘉志;王諸成;;Xp11.2易位/TFE3基因融合相關(guān)性腎癌的臨床病理研究(附10例報(bào)道)[J];腫瘤;2009年06期

10 姜天嬌;楊青;段崇鋒;林東亮;;Xp11.2易位/TFE3基因融合相關(guān)性腎癌CT表現(xiàn)[J];醫(yī)學(xué)影像學(xué)雜志;2014年04期

相關(guān)會(huì)議論文 前2條

1 饒秋;周曉軍;吳波;馬恒輝;周航波;劉曉紅;陳潔宇;;Xp11.2易位/TFE3基因融合相關(guān)性腎癌11例報(bào)道及文獻(xiàn)復(fù)習(xí)[A];中華醫(yī)學(xué)會(huì)病理學(xué)分會(huì)2010年學(xué)術(shù)年會(huì)日程及論文匯編[C];2010年

2 甘衛(wèi)東;郭宏騫;戴玉田;連惠波;屈鋒;曾令奇;徐林鋒;;Xp11.2易位/TFE3基因融合相關(guān)性腎癌[A];第十五屆全國(guó)泌尿外科學(xué)術(shù)會(huì)議論文集[C];2008年

相關(guān)博士學(xué)位論文 前1條

1 方媛;過(guò)表達(dá)TFE3基因在Xp11.2易位性腎癌中的作用及其分子機(jī)制研究[D];南方醫(yī)科大學(xué);2017年

，

本文編號(hào)：1511298