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1、上海交通大学硕士学位论文RNA干扰PNAS-2后U937细胞凋亡及细胞周期变化的研究姓名：肖菲申请学位级别：硕士专业：内科血液学指导教师：陈芳源20070401Caspases9 均无变化，推测抑制 PNAS-2 表达后凋亡的上调并不通过经典的内源性和外源性凋亡通路。 第二部分 RNA 干扰 PNAS-2 后 U937 细胞的细胞周期变化分析 实验一 利用流式细胞仪和基因芯片分析 RNA 干扰 PNAS-2 后 U937 细胞的细胞周期变化 第二部分 RNA 干扰 PNAS-2 后 U937 细胞的细胞周期变化分析 实验一 利用流式细胞仪和基因芯片分析 RNA 干扰 PNAS-2 后 U937
2、 细胞的细胞周期变化 目的 采用流式细胞仪和基因芯片技术比较 RNA 干扰法封闭 U937 细胞中PNAS-2 基因后与细胞周期有关的变化情况， 从而了解 PNAS-2 在细胞周期通路中的可能发挥的作用。 方法 急性单核细胞白血病细胞株 U937 分别转染已构建的靶向封闭 PNAS-2的干扰组质粒和对照质粒组组质粒，使用流式细胞仪分析干扰组和对照组细胞周期的变化。采用 affymetrix u133A 2.0 genechip 及技术比较两组细胞 间基因表达的差异。 结果 流式细胞仪检测见干扰组细胞出现 G2/M 期的阻滞,差异有统计学意义；基因芯片筛选结果提示共有 1651 条出现上调，14
3、04 条出现下调。其中比值2 倍的共有 336 条，上调 114 条，下调 222 条。根据功能分类初步筛选出细胞周期相关的基因共 20 条，上调 12 条，下调 8 条。 结论 抑制 PNAS-2 表达后细胞出现 G2/M 期的阻滞可导致凋亡的上升；细胞周期素 cyclinD、 cyclinE、转录因子 E2F 的上调可以使细胞 G1 期到 S 期加速变化；cyclin A 表达上调，cyclin B 表达下调可能是细胞 G2/M 期阻滞的原因，ATM 基因可调控 DNA 损伤修复，它的下调也可能参与了细胞周期的阻滞过程。 实验二 利用蛋白质芯片技术检测 RNA 干扰 PNAS-2 后 U9
4、37 细胞的细胞周期相关蛋白质表达的变化 实验二 利用蛋白质芯片技术检测 RNA 干扰 PNAS-2 后 U937 细胞的细胞周期相关蛋白质表达的变化 目的 采用蛋白质芯片技术比较RNA干扰法封闭U937细胞后PNAS-2蛋白质水平后与细胞周期有关的变化情况，从而了解 PNAS-2 在细胞周期通路中的可能发挥的作用。 方法 急性单核细胞白血病细胞株 U937 分别转染已构建的靶向封闭 PNAS-2的干扰组质粒和对照组质粒。采用蛋白质芯片及技术比较干扰组和对照组细胞间蛋白质水平表达的差异。 结果 蛋白质芯片筛选结果提示共检测有 720 条蛋白质。有 266 条出现 1.5倍以上的上调变化（37
5、%），有 103 条出现 1.5 倍以上的下调变化（14%）。其中与细胞周期有关的蛋白质变化共有 29 条，上调 25 条，下调 4 条。细胞周期素蛋白 CyclinE、CyclinA 上调，ATM 蛋白、P53 蛋白上调，泛素化通路相关蛋白均上调。 结论 蛋白质芯片结果提示抑制PNAS-2基因后G2/M期阻滞与Cyclin A上调有关；同时，p53蛋白的上调在此变化中也起了非常重要的作用；泛素化调控通路参与其中，但具体定位仍需进一步研究。 关键词 关键词 基因芯片，蛋白质芯片，RNA 干扰，PNAS-2，细胞凋亡，细胞周期 STUDY OF APOPTOSIS AND CELL CYCLE
6、CHANGES AFTER SUPPRESS PNAS-2 EXPRESSION BY RNA INTERFERENCE IN U937 CELL LINE ABSTRACT Part one: Apply Gene chip to study of apoptosis relate gene changes after suppress PNAS-2 expression by RNA interference in u937 cell line Objective: To study the roll of PNAS-2 in apoptosis pathway, we apply gen
7、echip to study apoptosis relate gene changes after suppress PNAS-2 gene expression by RNA interference in U937 leukemia cell line. Methods: After transfect interference and control recombinant plasmids into U937 cells, G418 combined with GFP+ cell subpopulation sorting by FCM selecting to purify tan
8、sfected cells. Apply both semiquantitative PCR and Realtime PCR to evaluate the RNAi efficiency. Use affymetrix u133A 2.0 genechip to study gene changes between interference and control transfected group. Select 2 up-regulated and 6 down-regulated genes and run RT-PCR to confirm the result of genech
9、ip. Results: Confocal microscopy comfirm we have got almost pure tranfected U937 cells, both semiquantitative PCR and Realtime PCR confirm the inhibit rate are over 70%. The result of microarray indicate 1651 genes are up-regulated and 1404 downregulated in PNAS-2-shRNA transfected group compare wit
10、h control, among them 22 apoptosis relate genes change rates are above 2 times including 11up-regulated and 12 downregulated. We also confirm the result is reliable by applying RT-PCR. Conclusion: We have confirm that inhibition of PNAS-2 can induce apoptosis previously. In this study, we presume th
11、e mechanism may concerned with over-expression of c-jun and activition of JNK pathway, also may concerned with the down-regulation of VEGF gene. Part two: Study cell cycle relate changes after suppress PNAS-2 expression by RNA interference in u937 cell line Experiment one: Apply flow cytometer and G
12、ene chip to study cell cycle relate changes after suppress PNAS-2 expression by RNA interference in u937 cell line Objective: To study the roll of PNAS-2 in cell cycle pathway, we apply flow cytometer and genechip to study cell cycle relate changes after suppress PNAS-2 gene expression by RNA interf
13、erence in U937 leukemia cell line. Methods: After transfect interference and control recombinant plasmids into U937 cells, G418 combined with GFP+ cell subpopulation sorting by FCM selecting to purify tansfected cells. flow cytometer observe the cell cycle changes. Use affymetrix u133A 2.0 genechip
14、to study gene changes between interference and control transfected group. Results: The result of flow cytometer showed the cells arrest appeared in G2/M period ；The result of microarray indicate 1651 genes are up-regulated and 1404 downregulated in interference group compare with control, among them
15、 20 cell cycle relate genes change rates are above 1.5 times including 12up-regulated and 8 downregulated. Conclusion: Inhibition of PNAS-2 can induce a G2/M phase arrest,which can improve the apoptosis.The up-regulation of cyclin D、cyclin E and E2F can accelerat G1 to S phases progression. the G2/M
16、 phase arrest maybe due to the arise of cyclin A and the down of cyclin B。 ATM can repair the DNA damage, the decrease maybe involve in the cell cycle. Experiment two: Apply Protein microchips to study cell cycle relate changes after suppress PNAS-2 expression by RNA interference in u937 cell line Objective: To study the roll of PNAS-2 in cell cycle pathway, we apply microarray chip to study cell cycle relate changes after suppress P
