【作者】 龚平生；

【导师】 罗贵民；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 survivin是新近发现的凋亡抑制蛋白(inhibitor of apoptosis proteins,IAP)家族的新成员,特异性表达于人的胚胎组织以及多数人类肿瘤细胞。在几乎所有常见肿瘤中,survivin都有表达。Survivin具有抑制凋亡、参与细胞周期调控、促进血管生成等生物学作用,与肿瘤细胞对辐射及化疗药物的耐受性增强有关,是肿瘤靶向治疗的重要基因之一。本研究旨在探讨survivin蛋白在X射线诱导HeLa细胞凋亡及染色体不稳定性(CIN)过程中所发挥的生物学作用。采用流式细胞术(FCM)检测X射线照射HeLa细胞后survivin蛋白表达、细胞凋亡和CIN的变化规律。结果显示,survivin在HeLa细胞中表达异常高,并且具有较高的转录水平;单纯X射线照射不能诱导HeLa细胞发生凋亡;受照细胞中发生CIN的细胞数随受照剂量的增高而显著增加,并且在接受4.0Gy照射后随着培养时间延长,发生CIN的细胞也随之增加。由于survivin具有组织表达选择性,使它成为RNAi技术应用的理想靶向基因。根据RNAi原理,利用pENTR-basic载体能够转录产生小的发卡状siRNA的特点,本研究成功构建了以survivin基因为靶点的pENTR-anti-SVV载体,在mRNA水平干扰survivin蛋白表达,以期对survivin蛋白在X射线诱导HeLa细胞凋亡及X射线诱导CIN过程中所产生的生物学影响进行研究。结果表明,干扰survivin蛋白表达后,电离辐射可以显著诱导细胞凋亡的产生,同时,发生CIN的细胞数也比单纯照射组显著增多。以上结果说明干扰survivin蛋白表达与辐射相结合可以加强辐射诱导的细胞凋亡及CIN的发生,本研究将为增强肿瘤辐射敏感性、提高放射治疗疗效提供理论依据。更多还原

【Abstract】 Survivin is a new member of the inhibitior of apoptosis protein (IAP) family. It possesses specific biological effects, such as, depressing apoptosis, regulating cell cycle and progressing angiogenesis. The expression of survivin is always on a high level in tumor tissues and can be detected at the early stage of tumorigenesis. The overexpression of survivin results in the inhibition of apoptosis, the progression of tumor angiogenesis and the invasion of tumor towards other tissues. And, survivin appears to be involved in the resistance of tumor tissues towards radiotherapy and chemical drugs. Accordingly, survivin is becoming a helpful potential target for tumor treatment. In this project, RNAi is used to interfer the expression of survivin, in order to investigate its biological effect and to explore the biological mechanism of survivin on apoptosis and chromosome instability in HeLa cells induced by X-rays. Results of the study will be helpful to elucidate the influence of RNAi on survivin radiobiological effect.1. Survivin expression in cells from different tissuesThe expression of survivin was detected by FCM in six sorts of human carcinoma from different tissues, such as, HeLa, HCT-8, SKOV-3, K562, SGC-7901, B16 and in one sort of cell from human normal hepatogenic tissue, HL-7702. Results showed that the expression of survivin was on the high level in six sorts of tumor cells, respectively, under the standard culture conditions (P<0.05~P<0.001). However, the expression of survivin could not be detected in HL-7702 cells (P>0.05).2. Effects of ionizing radiation on the expression of survivin and its biological effect2.1 Effects of ionizing radiation on the expression of survivinFCM was applied to detect the change of survivin expression in HeLa cells at 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that the expression of survivin in 6.0 Gy groups increased significantly, compared with sham-irradiated groups (P>0.05).FCM was applied to detect the change of survivin expression in HeLa cells during 2~72 h exposed to 4.0 Gy X-rays. Results showed that the expression of survivin was on a higher level at 4 h, 8 h, 48 h and 72 h, and they were significant difference with the control groups (P<0.01~ P<0.001).2.2 Effects of ionizing radiation on mRNA level of survivinRT-PCR assay was employed for the measurement of mRNA in HeLa cells at 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that survivin mRNA tended to increase in 1.0~6.0 Gy groups compared with the control groups.RT-PCR assay was employed for the measurement of mRNA in HeLa cells during 2~72 h exposed to 4.0 Gy X-rays. Results showed that survivin mRNA increased at 8 h, 12 h and 24 h after 4.0 Gy irradiated.2.3 Effects of ionizing radiation on apoptosis of HeLa cellsFCM was applied to detect the change of apoptosis in HeLa cells at 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that the number of apoptosis in 2.0 Gy groups declined significantly, compared with the sham-irradiated groups (P<0.05). However, the number of apoptosis had no significant difference between the irradiation groups with other doses and the sham-irradiated groups (P>0.05). It indicated that 0.5~6.0 Gy X-rays could not induced apparently the increase of apoptosis in HeLa cells.FCM was applied to detect the change of the number of apoptosis in HeLa cells during 2~72 h exposed to 4.0 Gy X-rays. Results showed that it was no significant difference between the irradiation groups and the control groups at different observed time (P>0.05).2.4 Effects of ionizing radiation on the distribution of cell cycle in HeLa cellsFCM was applied to analyze the distribution of cell cycle in HeLa cells at 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that the percentage in G2+M phase was increased significantly in HeLa cells exposed to 2.0 Gy, 4.0 Gy and 6.0 Gy, which was significant different with the sham-irradiated groups (P<0.001, respectively).FCM was applied to analyze the distribution of cell cycle in HeLa cells during 2~72 h exposed to 4.0 Gy X-rays. Results showed that the percentage in G2+M phase rose from12 h to 72 h. The increase was sharper than that of the control groups (P<0.05~ P<0.001).2.5 Effects of ionizing radiation on the number of chromosome of HeLa cellsFCM was applied to detected the changes of the number of chromosome in HeLa cells at 24 h exposed to X-rays with the doses of 0.5~6.0 Gy. Results showed that the percentage of tetraploid cells was increased significantly in HeLa cells exposed to 2.0 Gy, 4.0 Gy and 6.0 Gy, compared with the sham-irradiated groups (P<0.01~P<0.001), and the percentage of octoploid cells in these groups was also more than those of the sham-irradiated groups (P<0.05~P<0.01).FCM was applied to detected the number of chromosome in HeLa cells during 2~72 h exposed to 4.0 Gy X-rays. Results showed that the percentage of tetraploid cells remained the increase in HeLa cells during 2 h~48 h. They were significant difference with the control groups (P<0.05~ P<0.001). And, the percentage of octoploid cells was also significantly increased at 4 h, 24 h, 48 h and 72 h (P<0.01~P<0.001).3. Effects of shRNA on the expression of survivin and the biological effects3.1 Construction of the RNAi vectorSucceed to construct the RNAi vector, pENTR-anti-SVV, which was the recombinant plasmid including an insert fragment of shRNA. To identify the sequence veracity of shRNA, the plasmid was treated by sequencing process. Results indicated that the sequence of shRNA was consistent with what was designed.3.2 The efficiency of transfection in HeLa cells transfected with shRNAUnder the same transfected conditions, FCM was used to examine the intensity of green fluorescence in HeLa cells transfected with pcDNA3.1-GFP which acted as the control vector, in order to elevate the efficiency of transfection of pENTR-anti-SVV indirectly. It was showed that the efficiency was up to 64.64% at 48 h.3.3 Effects of shRNA on mRNA level of survivin in HeLa cells FCM was applied to measure the change of survivin on mRNA level in HeLa cells at 24 h after 4.0 Gy X-irradiation or/and transfected with pENTR-anti-SVV for 48 h. Results showed that mRNA was depressed apparently in the groups of transfected with pENTR-anti-SVV, while there was special product in the other control groups, such as, the untreated groups, irradiated groups, irradiated groups transfected with pENTR-basic.3.4 Effects of shRNA on the expression of survivin in HeLa cellsFCM was applied to detect the time course of changes of survivin expression in HeLa cells transfected with pENTR-anti-SVV for 48 h and exposed to 4.0 Gy X-irradiation. Results showed that the treated groups transfected with pENTR-anti-SVV had the lower level on the expression of survivin at 12 h, 24 h and 48 h, compared with the control groups transfected with pENTR-basic (P<0.001, respectively).3.5 Effects of shRNA on apoptosis in HeLa cellsFCM was applied to detect the percentage of apoptosis in HeLa cells transfected with pENTR-anti-SVV for 48 h and exposed to 4.0 Gy X-irradiation. Results showed that the treated groups transfected with pENTR-anti-SVV showed an apparently increase in apoptosis spanning 12 h to 24 h (P<0.05 and P<0.001, respectively). It suggested that the interference of survivin expression could induce the incident of apoptosis in HeLa cells.3.6 Effects of shRNA on the distribution of cell cycle in HeLa cellsFCM was applied to analyze the distribution of cell cycle in HeLa cells transfected with pENTR-anti-SVV for 48 h and exposed to 4.0 Gy X-irradiation. Results showed that the percentage in G2+M phase of the treated groups transfected with pENTR-anti-SVV were increased at 12 h and 48 h, which was significant different with the the control groups transfected with pENTR-basic (P<0.05 and P<0.01, respectively).3.7 Effects of shRNA on the number of the chromosome in HeLa cellsFCM was applied to detect the change of the number of chromosome in HeLa cells transfected with pENTR-anti-SVV for 48 h and exposed to 4.0 Gy X-irradiation. Results showed that the percentage of tetraploid cells was increased in the treated groups transfected with pENTR-anti-SVV at 24 h and 24 h. They were significant difference with the control groups transfected with pENTR-basic (P<0.01 and P<0.05, respectively). And, the percentage of octoploid cells in the treated groups was also significantly increased at 12 h, 24 h and 48 h (P<0.01~P<0.001).Results of the project will provide the possible evidence for the further research on survivin biological effects.更多还原