體外下調(diào)NY-SAR-35對(duì)膠質(zhì)瘤細(xì)胞生物學(xué)行為的影響
發(fā)布時(shí)間：2018-08-25 13:41
【摘要】：目的:研究NY-SAR-35對(duì)膠質(zhì)瘤細(xì)胞惡性生物學(xué)行為的影響。方法:(1)用qRT-PCR篩選出高表達(dá)NY-SAR-35的膠質(zhì)瘤細(xì)胞;(2)用帶有熒光的特異性干擾NY-SAR-35表達(dá)的序列轉(zhuǎn)染膠質(zhì)瘤細(xì)胞,對(duì)轉(zhuǎn)染條件進(jìn)行優(yōu)化;(3)CCK-8法檢測(cè)干擾NY-SAR-35對(duì)膠質(zhì)瘤細(xì)胞生長(zhǎng)能力的影響;(4)流式細(xì)胞儀檢測(cè)干擾NY-SAR-35對(duì)膠質(zhì)瘤細(xì)胞周期和凋亡的影響;(5)Western blot檢測(cè)細(xì)胞周期蛋白A(Cyclin A)、細(xì)胞周期蛋白激酶2(CDK2)、凋亡相關(guān)蛋白caspase-3和caspase-9的表達(dá)水平變化;(6)通過(guò)Matrigel粘附實(shí)驗(yàn)檢測(cè)膠質(zhì)瘤細(xì)胞黏附能力變化;(7)劃痕修復(fù)實(shí)驗(yàn)、Transwell遷移實(shí)驗(yàn)檢測(cè)膠質(zhì)瘤細(xì)胞遷移能力;(8)Transwell Matrigel侵襲實(shí)驗(yàn)檢測(cè)膠質(zhì)瘤細(xì)胞侵襲能力、Wester blot檢測(cè)上皮細(xì)胞鈣粘蛋白(E-cadherin)和N-鈣粘蛋白(N-cadherin)表達(dá)水平變化。結(jié)果:(1)NY-SAR-35的蛋白表達(dá)可見于細(xì)胞質(zhì)和細(xì)胞核中,但主要在細(xì)胞核中;選取A172和U251兩株膠質(zhì)瘤細(xì)胞株作為實(shí)驗(yàn)對(duì)象;(2)經(jīng)qRT-PCR檢測(cè),篩選出干擾效率最高的兩組干擾序列作為實(shí)驗(yàn)組;(3)CCK-8法檢測(cè)發(fā)現(xiàn),下調(diào)NY-SAR-35表達(dá)后,兩株膠質(zhì)瘤細(xì)胞增殖能力較對(duì)照組顯著下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.01);(4)流式細(xì)胞儀檢測(cè)細(xì)胞周期,顯示膠質(zhì)瘤細(xì)胞NY-SAR-35表達(dá)下調(diào)后,S期細(xì)胞增加,G2/M期細(xì)胞減少(P小于0.01),G0/G1期細(xì)胞所占百分比無(wú)統(tǒng)計(jì)學(xué)差異(P0.05);Western blot結(jié)果顯示NY-SAR-35下調(diào)后Cyclin A的表達(dá)增加,CDK2減少,與對(duì)照組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.01);(5)流式細(xì)胞儀檢測(cè)細(xì)胞凋亡,結(jié)果顯示NY-SAR-35干擾后膠質(zhì)瘤細(xì)胞的凋亡增加,與對(duì)照組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.01);Western blot結(jié)果顯示NY-SAR-35下調(diào)后caspase-3的表達(dá)增加,與對(duì)照組比較其差異具有統(tǒng)計(jì)學(xué)意義(P0.01),caspase-9的表達(dá)無(wú)變化,與對(duì)照組比較差異不具有統(tǒng)計(jì)學(xué)意義(P0.05);(6)粘附實(shí)驗(yàn)結(jié)果顯示在干擾NY-SAR-35后,兩株膠質(zhì)瘤細(xì)胞的黏附能力均下降,與對(duì)照組比較其差異具有統(tǒng)計(jì)學(xué)意義(P0.01);(7)在劃痕修復(fù)實(shí)驗(yàn)中,當(dāng)干擾NY-SAR-35 12小時(shí)時(shí),兩株膠質(zhì)瘤細(xì)胞的修復(fù)率無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);當(dāng)24小時(shí)時(shí),實(shí)驗(yàn)組的修復(fù)率明顯低于對(duì)照組,其差異具有統(tǒng)計(jì)學(xué)意義(P0.01);同時(shí),Transwell遷移實(shí)驗(yàn)顯示下調(diào)NY-SAR-35可以導(dǎo)致穿膜細(xì)胞數(shù)量減少,與對(duì)照組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.01);(8)Transwell Matrigel侵襲實(shí)驗(yàn)顯示干擾NY-SAR-35后,細(xì)胞的穿膜數(shù)量明顯降低(P0.01);Western blot檢測(cè)干擾后E-cadherin的表達(dá)量增加,N-cadherin的表達(dá)量降低,與對(duì)照組比較差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論:下調(diào)NY-SAR-35可減弱膠質(zhì)瘤細(xì)胞的惡性生物學(xué)行為;提示阻斷NY-SAR-35的表達(dá)具有用于膠質(zhì)瘤多途徑治療的潛在臨床價(jià)值。
[Abstract]:Objective: to study the effect of NY-SAR-35 on malignant biological behavior of glioma cells. Methods: (1) glioma cells with high expression of NY-SAR-35 were screened by qRT-PCR, and (2) glioma cells were transfected with fluorescent specific interfering NY-SAR-35 sequence. The transfection conditions were optimized; (3) CCK-8 assay was used to detect the effect of interfering NY-SAR-35 on glioma cell growth; (4) flow cytometry was used to detect the effect of interfering NY-SAR-35 on glioma cell cycle and apoptosis; (5) cyclin A (Cyclin A), cell cycle was detected by) Western blot. Protein kinase 2 (CDK2), the expression of apoptosis-related proteins caspase-3 and caspase-9; (6) the adhesion ability of glioma cells was detected by Matrigel adhesion assay; (7) the scratch repair assay was used to detect the migration ability of glioma cells; (8) the invasion of) Transwell Matrigel was detected by transwell migration assay. The expression of cadherin (E-cadherin) and N-cadherin (N-cadherin) in epithelial cells was detected by Wester blot. Results: (1) the protein expression of NY-SAR-35 was found in cytoplasm and nucleus, but mainly in the nucleus. Two glioma cell lines, A172 and U251, were selected as experimental objects. (2) qRT-PCR was used to detect the expression of A172 and U251 glioma cells. The two sets of interference sequences with the highest interference efficiency were selected as the experimental group. (3) after down-regulation of NY-SAR-35 expression, the proliferative ability of the two glioma cells was significantly lower than that of the control group, and the difference was statistically significant (P0.01); (4) flow cytometry was used to detect the cell cycle. There was no significant difference in the percentage of G _ 0 / G _ 1 phase cells in G _ 2 / M phase (P < 0.01) after the down-regulation of NY-SAR-35 expression in glioma cells. The results of Western blot showed that the expression of Cyclin A increased and CDK2 decreased after NY-SAR-35 down-regulation. Compared with the control group, the difference was statistically significant (P0.01); (5) flow cytometry was used to detect the apoptosis of glioma cells. The results showed that the apoptosis of glioma cells increased after NY-SAR-35 interference. Compared with the control group, the results of Western blot showed that the expression of caspase-3 increased after down-regulation of NY-SAR-35, but there was no change in the expression of caspase-9 after down-regulation of NY-SAR-35. There was no significant difference compared with the control group (P0.05); (6). The results of adhesion test showed that the adhesion ability of the two glioma cells decreased after interfering with NY-SAR-35, and the difference was statistically significant (P0.01); (7) in the scratch repair experiment compared with the control group. When interfering with NY-SAR-35 for 12 hours, the repair rate of the two glioma cells was not statistically significant (P0.05), but at 24 hours, the repair rate of the experimental group was significantly lower than that of the control group. At the same time, the down-regulation of NY-SAR-35 resulted in a decrease in the number of transmembrane cells compared with the control group (P0.01); (8) Transwell Matrigel invasion test showed that after interfering with NY-SAR-35. The number of transmembrane was significantly decreased (P0.01) and the expression of N-cadherin increased after the interference of E-cadherin by Western blot (P0.01), which was significantly different from that of the control group (P0.01). Conclusion: down-regulation of NY-SAR-35 can attenuate the malignant biological behavior of glioma cells, suggesting that blocking the expression of NY-SAR-35 has potential clinical value for multipathway therapy of glioma.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41
本文編號(hào)：2203045
[Abstract]:Objective: to study the effect of NY-SAR-35 on malignant biological behavior of glioma cells. Methods: (1) glioma cells with high expression of NY-SAR-35 were screened by qRT-PCR, and (2) glioma cells were transfected with fluorescent specific interfering NY-SAR-35 sequence. The transfection conditions were optimized; (3) CCK-8 assay was used to detect the effect of interfering NY-SAR-35 on glioma cell growth; (4) flow cytometry was used to detect the effect of interfering NY-SAR-35 on glioma cell cycle and apoptosis; (5) cyclin A (Cyclin A), cell cycle was detected by) Western blot. Protein kinase 2 (CDK2), the expression of apoptosis-related proteins caspase-3 and caspase-9; (6) the adhesion ability of glioma cells was detected by Matrigel adhesion assay; (7) the scratch repair assay was used to detect the migration ability of glioma cells; (8) the invasion of) Transwell Matrigel was detected by transwell migration assay. The expression of cadherin (E-cadherin) and N-cadherin (N-cadherin) in epithelial cells was detected by Wester blot. Results: (1) the protein expression of NY-SAR-35 was found in cytoplasm and nucleus, but mainly in the nucleus. Two glioma cell lines, A172 and U251, were selected as experimental objects. (2) qRT-PCR was used to detect the expression of A172 and U251 glioma cells. The two sets of interference sequences with the highest interference efficiency were selected as the experimental group. (3) after down-regulation of NY-SAR-35 expression, the proliferative ability of the two glioma cells was significantly lower than that of the control group, and the difference was statistically significant (P0.01); (4) flow cytometry was used to detect the cell cycle. There was no significant difference in the percentage of G _ 0 / G _ 1 phase cells in G _ 2 / M phase (P < 0.01) after the down-regulation of NY-SAR-35 expression in glioma cells. The results of Western blot showed that the expression of Cyclin A increased and CDK2 decreased after NY-SAR-35 down-regulation. Compared with the control group, the difference was statistically significant (P0.01); (5) flow cytometry was used to detect the apoptosis of glioma cells. The results showed that the apoptosis of glioma cells increased after NY-SAR-35 interference. Compared with the control group, the results of Western blot showed that the expression of caspase-3 increased after down-regulation of NY-SAR-35, but there was no change in the expression of caspase-9 after down-regulation of NY-SAR-35. There was no significant difference compared with the control group (P0.05); (6). The results of adhesion test showed that the adhesion ability of the two glioma cells decreased after interfering with NY-SAR-35, and the difference was statistically significant (P0.01); (7) in the scratch repair experiment compared with the control group. When interfering with NY-SAR-35 for 12 hours, the repair rate of the two glioma cells was not statistically significant (P0.05), but at 24 hours, the repair rate of the experimental group was significantly lower than that of the control group. At the same time, the down-regulation of NY-SAR-35 resulted in a decrease in the number of transmembrane cells compared with the control group (P0.01); (8) Transwell Matrigel invasion test showed that after interfering with NY-SAR-35. The number of transmembrane was significantly decreased (P0.01) and the expression of N-cadherin increased after the interference of E-cadherin by Western blot (P0.01), which was significantly different from that of the control group (P0.01). Conclusion: down-regulation of NY-SAR-35 can attenuate the malignant biological behavior of glioma cells, suggesting that blocking the expression of NY-SAR-35 has potential clinical value for multipathway therapy of glioma.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.41
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
1 李敏;林俊;;細(xì)胞凋亡途徑及其機(jī)制[J];國(guó)際婦產(chǎn)科學(xué)雜志;2014年02期
2 羅彬;云翔;范蓉;林永達(dá);張慶梅;何少健;陳芳;肖紹文;謝小薰;;頂體素結(jié)合蛋白和脆性X智障基因1鄰近蛋白在大腸癌中的表達(dá)及意義[J];廣東醫(yī)學(xué);2014年07期
3 安立峰,董震;RNA干擾——腫瘤研究的新工具[J];中華腫瘤雜志;2005年07期
4 范蓉,肖紹文,羅昱,廖興勝,張小靜,許濤,謝小薰;癌-睪丸抗原基因NY-SAR-35在人腦膜瘤中的表達(dá)[J];中華神經(jīng)外科雜志;2005年02期
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