体外下调NY-SAR-35对胶质瘤细胞生物学行为的影响
发布时间：2018-08-25 13:41
【摘要】：目的:研究NY-SAR-35对胶质瘤细胞恶性生物学行为的影响。方法:(1)用qRT-PCR筛选出高表达NY-SAR-35的胶质瘤细胞;(2)用带有荧光的特异性干扰NY-SAR-35表达的序列转染胶质瘤细胞,对转染条件进行优化;(3)CCK-8法检测干扰NY-SAR-35对胶质瘤细胞生长能力的影响;(4)流式细胞仪检测干扰NY-SAR-35对胶质瘤细胞周期和凋亡的影响;(5)Western blot检测细胞周期蛋白A(Cyclin A)、细胞周期蛋白激酶2(CDK2)、凋亡相关蛋白caspase-3和caspase-9的表达水平变化;(6)通过Matrigel粘附实验检测胶质瘤细胞黏附能力变化;(7)划痕修复实验、Transwell迁移实验检测胶质瘤细胞迁移能力;(8)Transwell Matrigel侵袭实验检测胶质瘤细胞侵袭能力、Wester blot检测上皮细胞钙粘蛋白(E-cadherin)和N-钙粘蛋白(N-cadherin)表达水平变化。结果:(1)NY-SAR-35的蛋白表达可见于细胞质和细胞核中,但主要在细胞核中;选取A172和U251两株胶质瘤细胞株作为实验对象;(2)经qRT-PCR检测,筛选出干扰效率最高的两组干扰序列作为实验组;(3)CCK-8法检测发现,下调NY-SAR-35表达后,两株胶质瘤细胞增殖能力较对照组显著下降,差异具有统计学意义(P0.01);(4)流式细胞仪检测细胞周期,显示胶质瘤细胞NY-SAR-35表达下调后,S期细胞增加,G2/M期细胞减少(P小于0.01),G0/G1期细胞所占百分比无统计学差异(P0.05);Western blot结果显示NY-SAR-35下调后Cyclin A的表达增加,CDK2减少,与对照组比较差异具有统计学意义(P0.01);(5)流式细胞仪检测细胞凋亡,结果显示NY-SAR-35干扰后胶质瘤细胞的凋亡增加,与对照组比较差异具有统计学意义(P0.01);Western blot结果显示NY-SAR-35下调后caspase-3的表达增加,与对照组比较其差异具有统计学意义(P0.01),caspase-9的表达无变化,与对照组比较差异不具有统计学意义(P0.05);(6)粘附实验结果显示在干扰NY-SAR-35后,两株胶质瘤细胞的黏附能力均下降,与对照组比较其差异具有统计学意义(P0.01);(7)在划痕修复实验中,当干扰NY-SAR-35 12小时时,两株胶质瘤细胞的修复率无统计学意义(P0.05);当24小时时,实验组的修复率明显低于对照组,其差异具有统计学意义(P0.01);同时,Transwell迁移实验显示下调NY-SAR-35可以导致穿膜细胞数量减少,与对照组比较差异具有统计学意义(P0.01);(8)Transwell Matrigel侵袭实验显示干扰NY-SAR-35后,细胞的穿膜数量明显降低(P0.01);Western blot检测干扰后E-cadherin的表达量增加,N-cadherin的表达量降低,与对照组比较差异具有统计学意义(P0.01)。结论:下调NY-SAR-35可减弱胶质瘤细胞的恶性生物学行为;提示阻断NY-SAR-35的表达具有用于胶质瘤多途径治疗的潜在临床价值。
[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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41
本文编号：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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41
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