【摘要】：研究背景:腎細(xì)胞癌簡稱腎癌,是泌尿系統(tǒng)最常見的惡性腫瘤之一,它的發(fā)病率很高,且近年來呈現(xiàn)出逐年增長的趨勢,因?yàn)閷ζ浒l(fā)生發(fā)展的分子機(jī)制的研究并不十分清楚,給它的早期診斷以及治療帶來了很多困擾。神經(jīng)元衍生的神經(jīng)營養(yǎng)因子(NDNF)基因,又名C4orf31。目前的研究發(fā)現(xiàn)其在神經(jīng)系統(tǒng)中分布廣泛,起著營養(yǎng)支持神經(jīng)元的作用,它還能維持內(nèi)皮細(xì)胞生存和血管形成,有利于血管的重建。我們課題組通過前期的轉(zhuǎn)錄組測序工作發(fā)現(xiàn)NDNF在腎癌中低表達(dá),但是,NDNF與腎癌發(fā)生發(fā)展的關(guān)系到底如何,目前尚無相關(guān)研究報道進(jìn)行說明,因此,本研究主要針對NDNF在腎癌中的表達(dá)以及一些生物學(xué)功能進(jìn)行了初步的研究。研究目的:1、檢測NDNF在腎癌組織及腎癌細(xì)胞系中的表達(dá);2、了解NDNF對腎癌細(xì)胞ACHN和786-0增殖、遷移以及侵襲能力的影響;3、檢測 NDNF 對腎癌細(xì)胞 ACHN 和 786-0 中 EMT 標(biāo)記物 E-cadherin、N-cadherin和Vimentin蛋白表達(dá)的影響,初步探索NDNF影響腎癌細(xì)胞轉(zhuǎn)移的分子機(jī)制。研究方法:首先,收集未經(jīng)放化療的69例臨床手術(shù)切除的腎癌組織及相應(yīng)的癌旁正常組織,采用RT-qPCR和Western blot檢測NDNF在腎癌組織及相應(yīng)的癌旁正常組織中的mRNA和蛋白表達(dá)水平;其次,采用相同的實(shí)驗(yàn)方法檢測NDNF在腎癌細(xì)胞系A(chǔ)CHN、OS-RC-2、786-0和769-P中的表達(dá)情況;然后,構(gòu)建NDNF穩(wěn)定表達(dá)的重組慢病毒表達(dá)細(xì)胞模型,采用CCK-8法和細(xì)胞平板克隆形成實(shí)驗(yàn)評估腎癌細(xì)胞的增殖能力;Transwell細(xì)胞遷移和侵襲實(shí)驗(yàn)檢測腎癌細(xì)胞的遷移和侵襲能力;最后,采用Western blot檢測EMT標(biāo)記物E-cadherin、N-cadherin和Vimentin蛋白在腎癌細(xì)胞中的表達(dá)水平,初步探索NDNF影響腎癌細(xì)胞遷移的分子機(jī)制。實(shí)驗(yàn)數(shù)據(jù)均采用SPSS22.0統(tǒng)計軟件進(jìn)行數(shù)據(jù)分析處理,所有實(shí)驗(yàn)都重復(fù)至少3次以上,實(shí)驗(yàn)結(jié)果數(shù)據(jù)用平均值±標(biāo)準(zhǔn)差(mean±S.D.)表示。實(shí)驗(yàn)結(jié)果的比較采用Independent sample t-test檢驗(yàn)。P0.05表示差異具有顯著的統(tǒng)計學(xué)意義。研究結(jié)果:1、NDNF在腎癌組織和腎癌細(xì)胞系中的mRNA和蛋白表達(dá)水平均下調(diào);2、腎癌細(xì)胞ACHN和786-0轉(zhuǎn)染過表達(dá)病毒后,NDNF的蛋白表達(dá)水平明顯高于相應(yīng)的陰性對照組細(xì)胞;3、NDNF抑制腎癌細(xì)胞增殖:CCK8細(xì)胞增殖試驗(yàn)中,腎癌細(xì)胞ACHN和786-0的NDNF過表達(dá)組的細(xì)胞生長曲線均明顯低于相應(yīng)的陰性對照組(P0.05)。細(xì)胞平板克隆形成實(shí)驗(yàn)中,ACHN細(xì)胞的NDNF過表達(dá)組細(xì)胞克隆數(shù)為89,陰性對照組為265,克隆形成率分別為9.8%、36.4% (P0.05) ; 786-0細(xì)胞的NDNF過表達(dá)組細(xì)胞克隆數(shù)為115,陰性對照組為158,克隆形成率分別為16.4%、22.7% (P0.05),即NDNF抑制了腎癌細(xì)胞ACHN和786-0的細(xì)胞增殖能力;4、NDNF抑制腎癌細(xì)胞遷移:NDNF過表達(dá)組的腎癌細(xì)胞穿過Transwell小室至其下側(cè)的細(xì)胞數(shù)量顯著少于陰性對照組的腎癌細(xì)胞。33%醋酸洗脫小室下側(cè)的細(xì)胞后測其平均吸光值,ACHN細(xì)胞株:0.55 (NDNF過表達(dá)組)、1.00 (陰性對照組)(P0.05) ; 786-0細(xì)胞株:0.68 (NDNF過表達(dá)組)、1.00 (陰性對照組)(P0.05)。即NDNF抑制了腎癌細(xì)胞ACHN和786-0的細(xì)胞遷移能力;5、NDNF抑制腎癌細(xì)胞侵襲:NDNF過表達(dá)組的腎癌細(xì)胞穿過Transwell小室至其下側(cè)的細(xì)胞數(shù)量顯著少于陰性對照組腎癌細(xì)胞。33%醋酸洗脫小室下側(cè)的細(xì)胞后測其平均吸光值,ACHN細(xì)胞株:0.47 (NDNF過表達(dá)組)、1.00 (陰性對照組)(P0.05) ; 786-0細(xì)胞株:0.71 (NDNF過表達(dá)組)、1.00 (陰性對照組)(P0.05)。即NDNF抑制了腎癌細(xì)胞ACHN和786-0的細(xì)胞侵襲能力;6、在腎癌細(xì)胞ACHN和786-0中,NDNF過表達(dá)組中的EMT的間質(zhì)表型標(biāo)記物N-cadherin和Vimentin蛋白的表達(dá)水平相較于陰性對照組均顯著下調(diào),但是上皮表型標(biāo)記物E-cadherin的蛋白表達(dá)水平顯著上調(diào)。研究結(jié)論:NDNF在腎癌組織及腎癌細(xì)胞系中的表達(dá)下調(diào);NDNF抑制腎癌細(xì)胞ACHN和786-0的增殖、遷移以及侵襲能力;NDNF下調(diào)腎癌細(xì)胞ACHN和786-0中N-cadherin和Vimentin蛋白的表達(dá)水平并上調(diào)E-cadherin蛋白的表達(dá)水平,這提示我們NDNF可能是通過EMT相關(guān)的通路來抑制腎癌細(xì)胞的遷移侵襲能力的。
[Abstract]:Background: renal cell carcinoma (RCC), called renal carcinoma, is one of the most common malignant tumors of the urinary system. Its incidence is very high, and in recent years it has been increasing year by year, because the molecular mechanism of its development is not very clear, which has brought a lot of trouble to its early diagnosis and treatment. The current study of the NDNF gene, also known as C4orf31., has found that it is widely distributed in the nervous system and plays a role in nourishing the neuron. It can also maintain endothelial cell survival and angiogenesis and facilitate the reconstruction of blood vessels. Our group has found that NDNF is low expression in renal cancer through the previous transcriptional sequencing work, but, NDNF and The relationship between the development of renal cancer and the development of renal cancer has not been reported. Therefore, this study is mainly aimed at the preliminary study of the expression of NDNF in renal carcinoma and some biological functions. 1, the expression of NDNF in renal carcinoma and renal cell carcinoma cell lines was detected. 2, NDNF was used to understand the ACHN and 786-0 of renal cell carcinoma cells. The effects of proliferation, migration and invasiveness; 3, to detect the effects of NDNF on the expression of E-cadherin, N-cadherin and Vimentin in renal cell carcinoma cells, ACHN and EMT, and to explore the molecular mechanism of NDNF affecting the metastasis of renal cell carcinoma. RT-qPCR and Western blot were used to detect the mRNA and protein expression levels of NDNF in the renal carcinoma tissue and the corresponding normal tissues. Secondly, the same experimental method was used to detect the expression of NDNF in the renal cell line ACHN, OS-RC-2786-0 and 769-P. Then, the recombinant lentivirus, which was expressed steadily in NDNF, was constructed. The cell model was expressed, the proliferation ability of renal cell carcinoma cells was evaluated by CCK-8 method and cell plate clone, and migration and invasion test of Transwell cells was used to detect the migration and invasion ability of renal cell carcinoma cells. Finally, the expression level of EMT marker E-cadherin, N-cadherin and Vimentin protein in renal cell carcinoma cells was detected by Western blot. The molecular mechanism of renal cell migration by NDNF was explored step by step. The experimental data were analyzed by SPSS22.0 software, all experiments were repeated more than 3 times, and the experimental data were expressed with mean standard deviation (mean + S.D.). The comparison of experimental results using Independent sample t-test test.P0.05 indicated that the difference was Significant statistical significance. 1, the mRNA and protein expression levels of NDNF in renal and renal carcinoma cells were all downregulated; 2, after ACHN and 786-0 transfected in renal cell carcinoma cells, the protein expression level of NDNF was significantly higher than that of the corresponding negative control group; 3, NDNF inhibited the proliferation of renal cell carcinoma cells: in CCK8 cell proliferation test, kidney The cell growth curves of the ACHN and 786-0 NDNF over expression groups were significantly lower than that in the corresponding negative control group (P0.05). In the cell plate clone formation experiment, the number of cell clones in the NDNF overexpression group of ACHN cells was 89, the negative control group was 265, the clone formation rate was 9.8%, 36.4% (P0.05), and the NDNF overexpression group of 786-0 cells was the cell gram. The number of augmentation was 115 and the negative control group was 158, and the clone formation rate was 16.4%, 22.7% (P0.05), that is, NDNF inhibited the proliferation of ACHN and 786-0 of renal cell carcinoma cells; 4, NDNF inhibited the migration of renal cell carcinoma cells: the number of cells passing through the NDNF overexpression group from the Transwell cell to its lower side was significantly less than that of the negative control group of renal cell carcinoma cell.33% The average absorbance of the cells in the lower side of the chamber was measured by acetic acid, ACHN cell lines: 0.55 (NDNF overexpressed group), 1 (negative control group) (P0.05); 786-0 cell lines: 0.68 (NDNF overexpression group), 1 (negative control group) (P0.05). That is, NDNF inhibited the cell migration ability of ACHN and 786-0 in renal cell carcinoma cells; 5, NDNF inhibited the invasion of renal cell carcinoma cells: NDNF over The number of cells passing through the Transwell cell to the lower side of the renal cell carcinoma cells in the expression group was significantly less than that in the negative control group of renal cancer cells.33% acetic acid eluting the lower side of the cell. The ACHN cell line: 0.47 (NDNF overexpression group), 1 (negative control group) (P0.05); 786-0 cell lines: 0.71 (NDNF overexpression group), 1 (negative pairs) (P0.05). That is, NDNF inhibited the cell invasiveness of ACHN and 786-0 of renal cell carcinoma cells; 6, in the ACHN and 786-0 of renal cell carcinoma cells, the expression level of the interstitial phenotypic markers N-cadherin and Vimentin protein in the NDNF overexpression group was significantly lower than that in the negative control group, but the protein expression level of the epithelial phenotype marker E-cadherin. The expression of NDNF in renal carcinoma and renal carcinoma cell lines is down regulated; NDNF inhibits the proliferation, migration and invasiveness of ACHN and 786-0 of renal cell carcinoma cells; NDNF downregulates the expression level of ACHN and N-cadherin and Vimentin protein in renal cell carcinoma cells and up regulation of E-cadherin protein expression, which suggests that NDNF may be The migration and invasion ability of renal cell carcinoma cells was inhibited by EMT related pathways.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.11

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