【作者】 王铁君；

【导师】 杨海山；

【作者基本信息】 吉林大学 ， 放射医学， 2009， 博士

【摘要】 本研究以Chk-1和PARP-1为作用靶点,构建其RNAi表达载体,干扰细胞损伤修复机制,观察其辐射增敏作用,拟提高肿瘤治疗的敏感性,探索一种有效的肿瘤治疗途径。结果显示1.对经限制性内切酶酶切鉴定正确的Chk-1和PARP-1重组RNAi载体进行测序,结果与理论序列完全一致。然后以脂质体介导分别转染Lewis肺癌细胞株筛选出有效的Chk-1和PARP-1 RNA i载体,即pGPU6/GFP/Neo-Chk-1-536、pGPU6/GFP/Neo-PARP-1-1308。2. 2Gy照射+ Chk-1、PARP-1 siRNA组的吸光度值与2Gy照射组、Chk-1和PARP-1 siRNA组比较差异有显著意义(p<0.05)。RT-PCR和免疫组化检测结果显示转染细胞Chk-1、PARP-1表达水平降低。Chk-1和PARP-1 siRNA转染、2Gy照射可诱导细胞凋亡,与正常对照组比,差异显著(p<0.05),并且siRNA转染可增加2Gy照射引起的Lewis肺癌细胞凋亡(p<0.05)。3.pGPU6/GFP/Neo-Chk-1-536、pGPU6/GFP/Neo-PARP-1-1308联合照射组分别与各自联合照射组比,Lewis肺癌细胞的吸光度值有显著差异(p<0.05)。2Gy照射组+ pGPU6/GFP/Neo-Chk-1-536+ pGPU6/GFP/Neo-PARP-1-1308组对Lewis肺癌细胞的抑制率最高。表明Chk-1和PARP-1 RNAi对肿瘤有治疗作用,与放疗有协同作用;pGPU6/GFP/Neo-Chk-1-536和pGPU6/GFP/Neo-PARP-1-1308有协同作用。4.2Gy照射组与假照组和pGPU6/GFP/Neo-Chk-1-536组、pGPU6/GFP/Neo- PARP-1-1308组、pGPU6/GFP/Neo-Chk-1-536+ pGPU6/GFP/Neo- PARP-1-1308组比,肿瘤生长缓慢(p<0.05);联合Chk-1和PARP-1的siRNA治疗,与单纯照射组及单纯siRNA治疗组比,肿瘤生长缓慢(p<0.05)。pGPU6/GFP/ Neo-PARP-1-1308和pGPU6/GFP/Neo-Chk-1-536联合放疗,与各自分别联合放疗比,肿瘤明显生长缓慢(p<0.05)。表明pGPU6/GFP/Neo-PARP-1-1308、pGPU6/GFP/Neo-Chk-1-536对肿瘤与放疗有协同作用,pGPU6/GFP/Neo-Chk-1-536与pGPU6/GFP/Neo-PARP-1-1308有协同作用,pGPU6/GFP/Neo-Chk-1-536与pGPU6/GFP/Neo-PARP-1-1308联合治疗具有辐射增敏作用。5.镜下观察各组Lewis肺癌小鼠瘤组织的形态学变化。对照组癌细胞成片状分布,细胞生长旺盛,细胞核浓染,癌细胞几乎不见坏死。放射组癌组织内存在一些异常的肿瘤细胞,这些细胞呈小的巢状分布于正常的肿瘤细胞之间;细胞核、固缩、坏死;坏死的肿瘤细胞较对照组明显增多。Chk-1或PARP-1+放疗组癌组织存在大量异常的肿瘤细胞,这些细胞呈片状分布;细胞核染色较轻、固缩、坏死,部分细胞崩解;坏死的肿瘤细胞较其他组均明显增多。Chk-1和PARP-1联合放疗组癌组织细胞核固缩、坏死,坏死的肿瘤细胞较各自单独与放疗联合组均明显增多。6.激光共聚焦显微镜下观察发现,肿瘤局部照射后2天,Chk-1及PARP-1 siRNA表达载体即散在分布于小鼠肿瘤组织内。7.与2Gy照射组,pGPU6/GFP/Neo-Chk-1-536和pGPU6/GFP/Neo-PARP-1- 1308分别联合放疗可使肿瘤组织表达的Bcl2下调、Bax上调;pGPU6/GFP/Neo- Chk-1-536和pGPU6/GFP/Neo-PARP-1-1308联合放疗可明显下调肿瘤组织表达的Bcl2、上调Bax,表明siRNA与照射具有协同作用。8.与假照射组比,Chk-1和PARP-1的siRNA分别使肿瘤细胞的Chk-1和PARP-1 mRNA表达下调;2Gy照射后,应用siRNA治疗,与单纯照射组, Chk-1、PARP-1 mRNA表达量下降,但与未照射、单纯siRNA治疗组比下降不明显,表明siRNA与照射具有协同作用。pGPU6/GFP/Neo-Chk-1-536和pGPU6/GFP/Neo- PARP-1-1308有协同作用,联合治疗增加辐射敏感性。结论成功构建并筛选Chk-1、PARP-1的RNAi载体,针对Chk-1、PARP-1的RNAi可以阻断肿瘤细胞DNA损伤修复,引起细胞凋亡,从而提高放疗对肿瘤的抑制率,证实Chk-1、PARP-1 RNAi具有放疗增敏作用。更多还原

【Abstract】 In recent years, studies have shown that the specific gene blockers can inhibit the function of the key regulatory gene which play important in tumor cell DNA damage and repair process, can inhibit DNA repair, and enhance the effectiveness of their radiotherapy. Ionizing radiation can induce signal transduction pathway of DNA damage , activate checkpoint G1 / S and G2 / M, delay cell cycle progression, so take enough time for DNA damage repair. Currently Chk-1 and Chk2 are respectively believed that they are the key regulatory gene of the check points and play an important at G2 / M and G1 / S phase regulating downstream signal transduction pathway. The use of Chk-1 inhibitor UCN-01 could block Chk-1 function, eliminate the cell cycle arrest induced by drugs and radiation, increase cytotoxicity. But the existence of its side effects need further search and the corresponding experimental study to confirm, which limite their application in clinical tumor therapy.Poly（ADP-ribose）polymerase -1 （PARP-1） play an important role in DNA repair, cell death, proliferation and differentiation.The study shows that the blockers of PARP-1 can inhibit PARP-1-mediated DNA repair mechanism, improve radiotherapy and chemotherapy on tumor cell DNA damage by inhibiting PARP-1’s activity, thus may have a potential tumor therapeutic value. But there are many defects in blocker technology itself, which significantly limit their clinical application on tumor treatment.Small interference RNA（ siRNA） technology can better solve the problem brought about by the application of blockers of the above questions. It may be an effective therapeutic approaches that take Chk-1 and PARP-1 as target and interfere cell damage repair mechanism, improve the treatment of tumor sensitivity. This may provides a new way for radiotherapy for cancer gene therapy.ObjectiveThe RNAi expression vector of Chk-1, PARP-1 were designed、synthesised and identificated. They were studyed on the impact of tumor radiation therapy through the application in vitro and in vivo.They would be a new ways for treatment of cancer research.MethodsAccording to the log in GenBank Chk-1 No. NM₀07691, PARP-1 No. NM₀07415 and shRNA design principles, oligonucleotide used to construct RNAi vector wered design and synthesised, and the recombinant vector RNAi wered identificated through the application of restriction enzyme and sequencing messurement.They were transfected mouse Lewis lung cancer cell lines through Lipofectimine ? 2000 and observed transfection under fluorescence microscope after 48h,thus the Lewis lung cancer cells were re-collected respectively.The effective iRNA vector were chosen by RT-PCR technology.The effective siRNA expression vector were transfected to Lewis lung cancer cell lines. The radiation sensitizing effect of RNAi to Lewis lung cancer cell line were studyed. The inhibited rate of transfected cells were observed by MTT assay , cell apoptosis were observed by flow cytometry, expression of Chk-1 and PARP-1 were analysised by immunohistochemical.The animal models of tumor-bearing mice were built when C57BL / 6 inbred mice were inoculated 5×105 Lewis lung cancer cells into right hind limb subcutaneous. The tumor-bearing mice were irradiated 2Gy body by using deep X-ray treatment machine . RNAi （2 -5mg/kg body weight） were injected by the tail vein after irradiation 4h. Tumor size were observed, tumor-bearing mice survival and mortality were statisted, the expression of Chk-1 and PARP-1 were detected by RT-PCR and immunohistochemistry.ResultsThe correct recombinant PARP-1 RNAi vector by the restriction endonuclease digestion were sequencing analysised, the sequence alignment analysis were taken between sequence of measurements with the theoretical sequence .Results showed that the sequence is fully consistent with the theory. PARP-1 RNAi vector pGPU6/GFP/Neo-PARP-1-1308 were effectively screened when they transfected into Lewis lung cancer cell lines by Lipofectimine ? 2000.The absorbance values of siRNA transfection group and 2Gy irradiation was significant （p <0.05）than the control group, and that of 2Gy irradiation group + siRNA group was significant （ p <0.05） than 2Gy irradiation group and siRNA group respectively. there was no significant difference （P> 0.05） between missense sequence group and the normal control group. The same result took place between 2Gy irradiation group + missense sequence group and 2Gy irradiation group. The inhibited rate of Lewis lung cancer cells in 2Gy irradiation + siRNA group is the highest, 60.5 percent. RT-PCR test results also showed that Chk-1, PARP-1 mRNA expression level is lower in transfected cells .There was significant difference （p <0.05） on Lewis lung cancer cells apoptosis among siRNA transfection, 2Gy irradiation with the normal control group, , and siRNA transfection can increase the Lewis lung cancer cell apoptosis with 2Gy irradiation（p<0.05）. siRNA pGPU6/GFP/Neo-PARP-1-1308, pGPU6/GFP/Neo-Chk-1-536 make tumor smaller than sham irradiation group and the negative control group; 2Gy irradiation significantly reduced tumor volume small, slower growing than sham control group and negative control group.United siRNA treatment can decrease tumor volume than irradiation alone. United siRNA pGPU6/GFP/Neo-PARP-1-1308 and pGPU6/GFP/Neo-Chk-1-536 together radiotherapy significantly reduced tumor volume than radiotherapy alone ,but was not obviously reduced with the single united radiotherapy separately. That showed that the siRNA pGPU6/GFP/Neo-PARP-1-1308, pGPU6/GFP/Neo-Chk-1-536 have synergistic effect with radiation on the tumor therapy. but there was no synergy between siRNA pGPU6/GFP/Neo-Chk-1-536 and pGPU6/GFP/Neo-PARP-1-1308.siRNA pGPU6/GFP/Neo-Chk-1-536, pGPU6/GFP/Neo-PARP-1- 1308 could decrease tumor-bearing mice mortality than sham irradiation group, Tumor-bearing mice mortality decreased in 2Gy irradiation group than that in sham control group and negative control group,so is in united siRNA treatment than irradiation alone group.SiRNA pGPU6/GFP / Neo-Chk-1 -536 and pGPU6/GFP/Neo-PARP-1-1308 United radiotherapy significantly decreased mortality than radiotherapy alone and was not obviously reduced with the single united radiotherapy separately.That Showed that the siRNA pGPU6/GFP/Neo-Chk-1- 536, pGPU6/GFP/Neo-PARP-1-1308 have therapeutic effects on the tumor,and has synergistic effect with radiation therapy.Chk-1 expression of tumor cells was down-regulated with siRNA pGPU6/GFP/Neo-Chk-1-536 than sham irradiation group. Chk-1 mRNA expression decreased with the application of siRNA treatment after 2Gy irradiation than irradiation alone group, but decline was not obvious than non- irradiated group . PARP-1 expression of tumor cells was down-regulated with pGPU6/GFP/Neo- PARP-1-1308; PARP-1 mRNA expression decreased with the application of siRNA treatment after 2Gy irradiation than irradiation alone group, but the decline was not obvious than non-irradiated group. That indicated that siRNA has a synergy with irradiation .ConclusionThe RNAi vector of Chk-1, PARP-1 were successfully constructed and screened. The RNAi of Chk-1 and PARP-1 can enhance inhibition of Lewis lung cancer cells and the cell apoptosis induced radiotherapy. They has therapeutic effects on the tumor and has synergistic effect with radiation therapy.更多还原