本文選題：表皮生長(zhǎng)因子受體Ⅲ型突變體 + 趨化因子受體�。� 參考：《武漢大學(xué)》2014年博士論文

【摘要】：目的：探討表皮生長(zhǎng)因子受體Ⅲ型突變體(epidermal growth factor receptor variant Ⅲ, EGFRvⅢ)和趨化因子受體CXCR4在人腦成膠質(zhì)母細(xì)胞瘤中的表達(dá)及其對(duì)成膠質(zhì)母細(xì)胞瘤A172細(xì)胞增殖、侵襲性遷移的影響和相互關(guān)系。方法：應(yīng)用免疫組織化學(xué)染色法檢測(cè)EGFRvⅢ和趨化因子受體CXCR4在60例人腦成膠質(zhì)細(xì)胞瘤及其瘤旁2cm正常腦組織中的表達(dá)水平。建立穩(wěn)定高表達(dá)EGFRvⅢ的成膠質(zhì)母細(xì)胞瘤A172細(xì)胞株,應(yīng)用細(xì)胞計(jì)數(shù)法、MTT法、細(xì)胞克隆形成實(shí)驗(yàn)以及裸鼠成瘤實(shí)驗(yàn)檢測(cè)EGFRvⅢ對(duì)成膠質(zhì)細(xì)胞瘤A172細(xì)胞增殖的影響,應(yīng)用細(xì)胞劃痕實(shí)驗(yàn)和transwell小室侵襲實(shí)驗(yàn)檢驗(yàn)EGFRvⅢ對(duì)成膠質(zhì)母細(xì)胞瘤A172細(xì)胞侵襲遷移的作用,再檢測(cè)高表達(dá)EGFRvⅢ后的遷移相關(guān)受體蛋白CXCR4的表達(dá)情況,進(jìn)一步探討EGFRvⅢ促進(jìn)A172細(xì)胞侵襲遷移的可能分子機(jī)制,應(yīng)用si RNA技術(shù)抑制CXCR4基因表達(dá)后,檢測(cè)高表達(dá)EGFRvⅢ的成膠質(zhì)母細(xì)胞瘤A172細(xì)胞的增殖、侵襲遷移能力的變化情況。結(jié)果：EGFRvⅢ和CXCR4在成膠質(zhì)母細(xì)胞瘤組織中的陽(yáng)性表達(dá)率明顯高于其瘤旁2cm正常腦組織(PEGFRvⅢ0.01; PCXCR40.01)；應(yīng)用RT-PCR和蛋白印記技術(shù)證實(shí)EGFRvⅢ高表達(dá)的細(xì)胞系A(chǔ)172細(xì)胞成功建立；與轉(zhuǎn)染空載體對(duì)照組比較,高表達(dá)EGFRvⅢ的A172細(xì)胞增殖能力明顯增加；通過(guò)連續(xù)6天的細(xì)胞計(jì)數(shù)及繪制細(xì)胞動(dòng)態(tài)生長(zhǎng)曲線(xiàn)發(fā)現(xiàn),細(xì)胞培養(yǎng)第5天,A172-EGFRvⅢ細(xì)胞數(shù)目[(9.1±1.1)×105]約為A172-Control細(xì)胞數(shù)目[(3.5±0.2)×105]的3倍(P0.05)；對(duì)照組和高表達(dá)EGFRvIII組的細(xì)胞克隆數(shù)分別為(50±10)個(gè)和(140±25)個(gè),差異有統(tǒng)計(jì)學(xué)意義(P0.01)；MTT實(shí)驗(yàn)檢測(cè)時(shí),在細(xì)胞培養(yǎng)至第3天,穩(wěn)定高表達(dá)EGFRvIII的A172-EGFRvⅢ細(xì)胞增殖率約為轉(zhuǎn)染空載體對(duì)照組A172-Control細(xì)胞的2倍(P0.05)；高表達(dá)EGFRvⅢ組的裸鼠皮下移植瘤的體積和質(zhì)量均高于對(duì)照組(P體積0.01；P質(zhì)量0.01)。在細(xì)胞劃痕實(shí)驗(yàn)中,對(duì)照組和高表達(dá)EGFRvⅢ組的A172細(xì)胞遷移距離分別為(265±75)um和(454±85)um,高表達(dá)EGFRvⅢ組約為對(duì)照組的1.7倍,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。在transwell小室侵襲實(shí)驗(yàn)中,對(duì)照組和高表達(dá)EGFRvⅢ組A172細(xì)胞的穿膜數(shù)目分別為(8.0±3.1)個(gè)/視野和(18.0±2.5)個(gè)/視野,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。通過(guò)RT-PCR和蛋白印記技術(shù)發(fā)現(xiàn),高表達(dá)EGFRvⅢ可明顯促進(jìn)趨化因子受體CXCR4的表達(dá)；進(jìn)一步的功能實(shí)驗(yàn)發(fā)現(xiàn),應(yīng)用si RNA技術(shù)抑制趨化因子受體CXCR4的表達(dá)后,繪制細(xì)胞動(dòng)態(tài)生長(zhǎng)曲線(xiàn)圖時(shí),在細(xì)胞培養(yǎng)的第5天,A172-EGFRvⅢ-CXCR4siRNA細(xì)胞數(shù)目[(3.2±1.2)×105]約為A172-EGFRvⅢ-Scrambled siRNA細(xì)胞數(shù)目[(11.8±0.2)×105]的1/4(P0.05)；細(xì)胞克隆形成實(shí)驗(yàn)結(jié)果,A172-EGFRvⅢ-CXCR4siRNA細(xì)胞克隆形成數(shù)目約(40±10)個(gè),而A172-EGFRvⅢ-Scrambled siRNA細(xì)胞克隆數(shù)目高達(dá)(98±23)個(gè),二者差異有統(tǒng)計(jì)學(xué)意義(P0.01); BrdU摻入實(shí)驗(yàn)結(jié)果顯示：A172-EGFRvⅢ-Scrambled siRNA細(xì)胞BrdU陽(yáng)性率為30%, A172-EGFRvⅢ-CXCR4siRNA細(xì)胞的BrdU陽(yáng)性率下降為18%,二者間差異有統(tǒng)計(jì)學(xué)意義(P0.05)。細(xì)胞劃痕實(shí)驗(yàn)結(jié)果：高表達(dá)EGFRvIII組的A172細(xì)胞遷移距離為(495±75)um, RNA干擾抑制CXCR4表達(dá)后細(xì)胞遷移距離為(260±67)um,細(xì)胞遷移距離降低約50%,差異具有統(tǒng)計(jì)學(xué)意義(P0.05), transwell小室侵襲實(shí)驗(yàn)結(jié)果：高表達(dá)EGFRvⅢ組A172細(xì)胞的穿膜數(shù)目為(18.5±2.8)個(gè)/視野,RNA干擾抑制CXCR4表達(dá)后細(xì)胞穿膜數(shù)目為(10.1±2.6)個(gè)/視野,二組差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論：EGFRvⅢ和CXCR4在人腦成膠質(zhì)細(xì)胞瘤組織中高表達(dá),并且可促進(jìn)成膠質(zhì)母細(xì)胞瘤A172細(xì)胞的增殖、侵襲遷移,通過(guò)上調(diào)趨化因子受體CXCR4是EGFRvⅢ促進(jìn)膠質(zhì)母細(xì)胞瘤A172細(xì)胞的增值、侵襲性遷移的重要途徑之一。
[Abstract]:Objective: To investigate the expression of epidermal growth factor receptor type III mutant (epidermal growth factor receptor variant III, EGFRv III) and chemokine receptor CXCR4 in human glioblastoma and its effect on the proliferation and invasive migration of glioblastoma A172 cells and their relationship. The expression level of EGFRv III and chemokine receptor CXCR4 in 60 human glioblastoma and 2cm normal brain tissue near the tumor was detected by staining. The A172 cell line of glioblastoma stable with high expression of EGFRv III was established. The cell count method, MTT method, cell clone formation, and nude mouse tumorigenesis test were used to detect the formation of EGFRv III. The effect of A172 cell proliferation in stromal tumor cells, the effect of EGFRv III on the invasion and migration of glioblastoma A172 cells and the expression of the migration related receptor protein CXCR4 after the high expression of EGFRv III were detected by the cell scratch test and the Transwell chamber invasion test, and the possibility of EGFRv III to promote the invasion and migration of A172 cells was further explored. Molecular mechanism, using Si RNA technique to suppress CXCR4 gene expression, to detect the proliferation of glioblastoma A172 cells with high expression of EGFRv III and the change of invasion and migration. Results: the positive expression rate of EGFRv III and CXCR4 in glioblastoma tissues was significantly higher than that of the normal brain tissue adjacent to the tumor of 2cm (PEGFRv III 0.01; PCXCR40.0). 1): using RT-PCR and protein imprinting technique, the cell line A172 cells with high expression of EGFRv III were successfully established, and the proliferation ability of A172 cells with high expression of EGFRv III was obviously increased compared with that of the empty vector control group, and the cell culture for fifth days and A172-EGFRv III cells by the continuous 6 days cell count and the plotting of the cell dynamic growth curve. The number [(9.1 + 1.1) * 105]] was about the number of A172-Control cells (3.5 + 0.2) x 105] (P0.05), and the number of cell clones in the control group and the high expression EGFRvIII group were (50 + 10) and (140 + 25) respectively, and the difference was statistically significant (P0.01). The MTT test was used to stabilize the A172-EGFRv III cells expressing EGFRvIII in the cell culture to third days. The proliferation rate was about 2 times (P0.05) of the A172-Control cells in the transfected empty vector control group, and the volume and quality of the subcutaneous transplanted tumor of the high expression EGFRv III group were higher than those of the control group (P volume 0.01, P mass 0.01). The migration distance of the control group and the high expression EGFRv III group was (265 + 75) um and (454 + 85) um respectively in the cell scratch test. The high expression of EGFRv III was about 1.7 times that of the control group, and the difference was statistically significant (P0.05). In the Transwell chamber invasion experiment, the number of A172 cells in the control group and the high expression EGFRv III group A172 cells were (8 + 3.1) / visual field and (18 + 2.5) / visual field, and the difference was statistically significant (P0.05). The results were found by RT-PCR and protein imprinting technique. High expression of EGFRv III can significantly promote the expression of chemokine receptor CXCR4; further functional experiments have found that the number of A172-EGFRv III -CXCR4siRNA cells (3.2 + 1.2) * 105] is about A172-EGFRv III -Scra (3.2 + 1.2) * 105] at the fifth day of cell culture after the application of Si RNA technology to inhibit the expression of chemokine receptor CXCR4. Mbled siRNA cell number [(11.8 + 0.2) x 105] 1/4 (P0.05); cell clone formation experimental results, A172-EGFRv III -CXCR4siRNA cell clone formation number (40 + 10), and A172-EGFRv III -Scrambled siRNA cell clone number is up (98 + 23), two differences have statistical significance (P0.01); BrdU incorporation experiment results show: Dialectical The positive rate of BrdU in -Scrambled siRNA cells was 30%, the positive rate of BrdU in A172-EGFRv III -CXCR4siRNA cells decreased to 18%, and the difference between the two groups was statistically significant (P0.05). The results of the cell scratch test were (495 + 75) um for the high expression of EGFRvIII group, and the cell migration distance of RNA dry disturbance rejection was (260 + 67), and the cell migration distance was (260 + 67). The cell migration distance was reduced by about 50%, the difference was statistically significant (P0.05), and the results of Transwell chamber invasion experiment: the number of A172 cells in the high expression EGFRv III group was (18.5 + 2.8) / visual field, and the number of cell penetrating membrane was (10.1 + 2.6) / visual field after RNA interference inhibition CXCR4 expression. The difference was statistically significant (P0.05). Conclusion: EGFRv III and CXCR4 are highly expressed in human glioblastoma tissue, and can promote the proliferation of glioblastoma A172 cells and invasion and migration. The up regulation of chemokine receptor CXCR4 is one of the important ways to promote the proliferation of A172 cells in glioblastoma and the invasive migration of glioblastoma cells.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R739.41

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 路偉;付玲娣;楊鈞;錢(qián)濤;;趨化因子CXCL12及其受體CXCR4在人腦星形細(xì)胞腫瘤中的表達(dá)及意義[J];腦與神經(jīng)疾病雜志;2010年03期

2 王東林;劉毅;高寶蓮;;腦膠質(zhì)瘤60例CXCR_4與EGFR的表達(dá)及意義[J];陜西醫(yī)學(xué)雜志;2008年09期

3 董倫;浦佩玉;王虎;王廣秀;康春生;焦德讓;;EGFR、p53與Ki-67在國(guó)人膠質(zhì)瘤中表達(dá)研究[J];中華神經(jīng)外科雜志;2006年07期

4 楊世昕,卞修武,蔣雪峰,陳劍鴻,王清良,王吉民;腦膠質(zhì)瘤趨化因子受體CXCR4表達(dá)的臨床意義[J];中國(guó)微侵襲神經(jīng)外科雜志;2005年08期

，

本文編號(hào)：1806925