體外下調(diào)NY-SAR-35對膠質(zhì)瘤細胞生物學(xué)行為的影響
發(fā)布時間：2018-08-25 13:41
【摘要】：目的:研究NY-SAR-35對膠質(zhì)瘤細胞惡性生物學(xué)行為的影響。方法:(1)用qRT-PCR篩選出高表達NY-SAR-35的膠質(zhì)瘤細胞;(2)用帶有熒光的特異性干擾NY-SAR-35表達的序列轉(zhuǎn)染膠質(zhì)瘤細胞,對轉(zhuǎn)染條件進行優(yōu)化;(3)CCK-8法檢測干擾NY-SAR-35對膠質(zhì)瘤細胞生長能力的影響;(4)流式細胞儀檢測干擾NY-SAR-35對膠質(zhì)瘤細胞周期和凋亡的影響;(5)Western blot檢測細胞周期蛋白A(Cyclin A)、細胞周期蛋白激酶2(CDK2)、凋亡相關(guān)蛋白caspase-3和caspase-9的表達水平變化;(6)通過Matrigel粘附實驗檢測膠質(zhì)瘤細胞黏附能力變化;(7)劃痕修復(fù)實驗、Transwell遷移實驗檢測膠質(zhì)瘤細胞遷移能力;(8)Transwell Matrigel侵襲實驗檢測膠質(zhì)瘤細胞侵襲能力、Wester blot檢測上皮細胞鈣粘蛋白(E-cadherin)和N-鈣粘蛋白(N-cadherin)表達水平變化。結(jié)果:(1)NY-SAR-35的蛋白表達可見于細胞質(zhì)和細胞核中,但主要在細胞核中;選取A172和U251兩株膠質(zhì)瘤細胞株作為實驗對象;(2)經(jīng)qRT-PCR檢測,篩選出干擾效率最高的兩組干擾序列作為實驗組;(3)CCK-8法檢測發(fā)現(xiàn),下調(diào)NY-SAR-35表達后,兩株膠質(zhì)瘤細胞增殖能力較對照組顯著下降,差異具有統(tǒng)計學(xué)意義(P0.01);(4)流式細胞儀檢測細胞周期,顯示膠質(zhì)瘤細胞NY-SAR-35表達下調(diào)后,S期細胞增加,G2/M期細胞減少(P小于0.01),G0/G1期細胞所占百分比無統(tǒng)計學(xué)差異(P0.05);Western blot結(jié)果顯示NY-SAR-35下調(diào)后Cyclin A的表達增加,CDK2減少,與對照組比較差異具有統(tǒng)計學(xué)意義(P0.01);(5)流式細胞儀檢測細胞凋亡,結(jié)果顯示NY-SAR-35干擾后膠質(zhì)瘤細胞的凋亡增加,與對照組比較差異具有統(tǒng)計學(xué)意義(P0.01);Western blot結(jié)果顯示NY-SAR-35下調(diào)后caspase-3的表達增加,與對照組比較其差異具有統(tǒng)計學(xué)意義(P0.01),caspase-9的表達無變化,與對照組比較差異不具有統(tǒng)計學(xué)意義(P0.05);(6)粘附實驗結(jié)果顯示在干擾NY-SAR-35后,兩株膠質(zhì)瘤細胞的黏附能力均下降,與對照組比較其差異具有統(tǒng)計學(xué)意義(P0.01);(7)在劃痕修復(fù)實驗中,當(dāng)干擾NY-SAR-35 12小時時,兩株膠質(zhì)瘤細胞的修復(fù)率無統(tǒng)計學(xué)意義(P0.05);當(dāng)24小時時,實驗組的修復(fù)率明顯低于對照組,其差異具有統(tǒng)計學(xué)意義(P0.01);同時,Transwell遷移實驗顯示下調(diào)NY-SAR-35可以導(dǎo)致穿膜細胞數(shù)量減少,與對照組比較差異具有統(tǒng)計學(xué)意義(P0.01);(8)Transwell Matrigel侵襲實驗顯示干擾NY-SAR-35后,細胞的穿膜數(shù)量明顯降低(P0.01);Western blot檢測干擾后E-cadherin的表達量增加,N-cadherin的表達量降低,與對照組比較差異具有統(tǒng)計學(xué)意義(P0.01)。結(jié)論:下調(diào)NY-SAR-35可減弱膠質(zhì)瘤細胞的惡性生物學(xué)行為;提示阻斷NY-SAR-35的表達具有用于膠質(zhì)瘤多途徑治療的潛在臨床價值。
[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)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.41
本文編號：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)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.41
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