【作者】 石华；

【导师】 宋方洲；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2007， 博士

【摘要】 子宫颈癌（cervical cancer, CC）是危害广大妇女健康最常见的恶性肿瘤之一,我国每年新发病例有13.5万,近年来发现,年轻妇女中宫颈癌的发病率呈明显上升的趋势,发病以每年2%³%的速度增长。宫颈癌越来越引起人们广泛的关注。近年来,随着医学分子生物学理论和技术的发展以及对宫颈癌发病机制认识的不断深入,尤其是DNA重组技术和基因转移技术逐步成熟,研究者们提出了利用基因转移技术向体内导入目的基因,从而对宫颈癌发病的分子环节进行干预的防治策略,为宫颈癌的治疗开辟了新途径。本研究拟以人白介素24（hIL-24）和HPV E6 siRNA作为分子工具,采用真核表达载体为基因导入工具对人宫颈癌中具有代表性的CaSki细胞株进行基因治疗的实验研究,主要探讨外源性hIL-24的表达对治疗宫颈癌CaSki细胞所发挥的作用及其内在机制,以及联合RNAi技术是否具有协同效应。以期为宫颈癌基因治疗的进一步深入研究奠定必要的实验基础。研究方法:1. hIL-24基因的克隆鉴定,构建真核表达载体及表达鉴定。培养CaSki细胞,提取其RNA作为模板,采用RT-PCR方法扩增hIL-24基因全部编码序列,将其克隆至真核表达载体pcDNA3.1（+）上进行测序分析。将测序正确的重组质粒转染入体外培养的CaSki细胞中进行表达。Western blot鉴定是否成功表达hIL-24蛋白。2. hIL-24基因诱导人宫颈癌CaSki细胞凋亡的实验。①以脂质体包裹重组真核表达质粒pcDNA3.1（+）-hIL-24转染CaSki细胞。②利用RT-PCR技术检测处理后CaSki细胞中HPV E6癌基因的mRNA水平变化。③Western blot分析处理后CaSki细胞中抑癌蛋白P53水平的变化。④处理后的CaSki细胞凋亡检测:透射电镜观察,流式细胞仪分析,TUNEL实验,hochest 33342荧光染色。3.细胞体外迁移、侵袭实验。①采用细胞体外迁移、侵袭实验检测pcDNA3.1（+）-hIL-24处理后CaSki细胞迁移、侵袭能力的改变。②Western blot检测与细胞迁移、侵袭能力有关的E-cadherin, MMP-2和p38 MAPK蛋白水平。4. HPV-16 E6 siRNA与hIL-24基因联合诱导人宫颈癌CaSki细胞的凋亡。①携带HPV-16 E6 siRNA与hIL-24基因的质粒载体分别以单独或联合的方式转染人宫颈癌CaSki细胞,处理后利用RT-PCR技术检测细胞中HPV E6癌基因的mRNA水平变化。②Western blot分析细胞中抑癌蛋白P53水平的变化。③流式细胞仪分析细胞凋亡情况。5.动物实验。①筛选稳定转染pcDNA3.1（+）-hIL-24质粒的CaSki细胞株,注射裸鼠,观察细胞在裸鼠皮下的成瘤情况,定期测量肿瘤的体积大小,处死裸鼠,称瘤重,计算肿瘤抑制率。②建立荷瘤裸鼠模型,于瘤体内注射脂质体包裹的pcDNA3.1（+）-hIL-24质粒,定期测量肿瘤的体积大小,处死裸鼠,称瘤重,计算肿瘤抑制率。观察肿瘤组织的病理改变。实验结果:1.①分别以人宫颈癌CaSki和Hela细胞总RNA为模板,经RT-PCR,琼脂糖凝胶电泳发现只有CaSki细胞扩增出一条特异性条带,长度大约为650 bp,与预期大小一致。而Hela细胞未扩增出特异性条带。序列分析结果显示,与GenBank公布的编号为NM₀06850的序列进行比对后发现有两处碱基的点突变,即223 bp处（G→A）伴随着氨基酸的改变（Ala→Thr）和370 bp处（T→C）伴随着氨基酸的改变（Tyr→His）。②成功构建人白介素24真核表达载体pcDNA3.1（+）-hlL-24。③蛋白印迹实验发现, CaSki细胞的培养上清和细胞裂解液中都不能检测到IL-24蛋白,而转染pcDNA3.1（+）-hlL-24真核表达质粒的CaSki细胞的培养上清和细胞裂解液中都能检测到IL-24蛋白。2.转染pcDNA3.1（+）-hIL-24的CaSki细胞中HPV E6癌基因的mRNA水平明显下降,抑癌蛋白P53水平明显增加,细胞凋亡率明显升高。3.①迁移和侵袭实验中转染pcDNA3.1（+）-hIL-24的CaSki细胞穿过Transwell小室膜的细胞数明显减少。②Western blot结果显示转染pcDNA3.1（+）-hIL-24的CaSki细胞中MMP-2水平下降, p38 MAPK和E-cadherin水平升高。4.共转染pcDNA3.1（+）-hIL-24和pGensil-CH1组与单独转染组CaSki细胞相比,HPV E6癌基因的mRNA水平明显下降,抑癌蛋白P53水平明显增加,细胞凋亡率明显升高。5.裸鼠成瘤实验发现,稳定转染pcDNA3.1（+）-hIL-24后的CaSki细胞成瘤能力明显降低;对荷瘤裸鼠瘤内注射pcDNA3.1（+）-hIL-24质粒进行基因治疗后,肿瘤生长受到抑制,肿瘤的体积与重量明显低于对照组。病理检查发现,处理组肿瘤生长不佳,细胞分裂相少见,组织内有较多坏死区域。结论:1.蛋白印迹实验发现, CaSki细胞的培养上清和细胞裂解液中都不能检测到IL-24蛋白,说明可能存在转录后调控,使IL-24 mRNA不能被翻译成蛋白质。而转染pcDNA3.1（+）-hlL-24真核表达质粒的CaSki细胞的培养上清和细胞裂解液中都能检测到IL-24蛋白,说明构建的pcDNA3.1（+）-hlL-24真核表达质粒能在CaSki细胞内成功表达分泌型的IL-24蛋白,且表达的蛋白具有免疫原性。2.实验组CaSki细胞中HPV E6癌基因的mRNA水平明显下降,抑癌蛋白P53水平显著增加,促进宫颈癌CaSki细胞凋亡。以上结果表明以真核表达载体介导的hIL-24基因在体外能显著促进宫颈癌CaSki细胞的凋亡。3.转染pcDNA3.1（+）-hIL-24的CaSki细胞迁移和侵袭能力均下降,与MMP-2水平下降, E-cadherin水平升高相关。4.用HPV-16 E6 siRNA与hIL-24基因联合诱导人宫颈癌CaSki细胞的凋亡。本实验RT-PCR结果表明两者两者单独使用时均能抑制E6癌基因的表达,联合时则抑制效应增强;Western blot结果显示两者单独使用时,通过抑制E6癌基因的表达或其他一些途径,均能使P53蛋白水平得到恢复,联合时则效应增强;流式结果显示两者联合使用时具有协同效应,能显著提高肿瘤细胞凋亡率。5.稳定转染pcDNA3.1（+）-hIL-24后的CaSki细胞成瘤能力明显降低;重组质粒pcDNA3.1（+）-hIL-24对荷瘤裸鼠有一定治疗作用。更多还原

【Abstract】 Cervical cancer remains as one of the biggest killers of women worldwide. Despite significant achievements in the treatment of cervical cancer, it is still a deadly disease that readily metastasize in the earlier period, hence newer therapeutical modalities are needed.Advanced in our understanding of the molecular biology of tumor cells have led to the development of new trestment strategies, including gene therapy. The molecular basis of cancer is now known to involve activation of dominant oncogenes and inactivation of tumor suppressor genes, these genetic events may represent novel targets for cancer therapy.IL-24 was identified through subtraction hybridization from a human melanoma cell line by P.B.Fisher in 1995. Up to now, a large body of data has accumulated to demonstrate that IL-24 results in growth suppression and induction of apoptosis in a broad range of cancer cells. RNA interference (RNAi) is a post-transcriptional regulatory manner of gene expression, it is a conservative mode in organism evolution, and it has the effect of counteract invading of virus and maintenance stablity of genome. RNAi could knockdown the expression of gene in mammalians, which provide a new approach for gene therapy of tumor. It was indicated that specifically inhibiting over-expression of oncogene, cancer related genes or mutant gene by RNAi and retained them in silence status could achieve the result of anticancer effect.In this study, we investigated the effect of hIL-24 gene inducing the apoptosis and reducing metastasis.of human cervical cancer CaSki cells and its possible mechanism, evaluated the effect of hIL-24 gene associated with HPV-16 E6 siRNA on inducing apoptosis of CaSki cells. And we observed the effect of gene therapy on the CaSki xenograft tumor in nude mice with hIL-24 expression recombinant plasmids, which providing experimental evidence for the treatment of cervical cancer by gene therapy. We develop this study according to following procedure:1. To clone hIL-24 gene and construct its eukaryotic expression vector, then transfect it into CaSki cells to express hIL-24 protein. METHODS: hIL-24 gene was acquired by RT-PCR from the total RNA of CaSki cell and cloned into plasmid pcDNA3.1(+) to generate the eukaryotic expression vector named pcDNA3.1(+)-hIL-24. pcDNA3.1(+)-hIL-24 was transfected into CaSki cells to express hIL-24 protein.RESULTS: PCR and sequencing revealed that hIL-24 gene was successfully cloned into plasmid pcDNA3.1(+) and its eukaryotic expression vector was constructed. The vector was able to express hIL-24 protein after transfected into CaSki cells.CONCLUSION: The hIL-24 eukaryotic expression vector was successfully constructed.2. To investigate hIL-24 gene inducing the apoptosis of human cervical cancer CaSki cells and its possible mechanism. METHODS: CaSki cells were transfected with hIL-24 eukaryotic expression vector by lipofectamine2000. The effect of hIL-24 on the transcription of HPV E6 gene was analyzed by RT-PCR. The effect of hIL-24 on P53 protein was analyzed by Western Blot. Apoptosis of CaSki cells induced by hIL-24 gene was analyzed by FACS. RESULTS: Transcription level of HPV E6 gene decreased significantly, the amount of P53 protein increased significantly and the apoptosis rate of CaSki cells increased significantly after hIL-24 transfection. CONCLUSION: HIL-24 gene can inhibit the transcription of HPV E6 gene, recover the activity of P53 protein, then induce the apoptosis of CaSki cells.3. To evaluate the effect of the ectopic production of MDA-7/IL-24 on cell migration and invasion by human cervical cancer cells.METHODS: We investgated the ability of metastasis of CaSki cells treated with hIL-24 by Cell migration assay and Cell invasion assay .We analysed the level of proteins associated with these processes by Western blot. RESULTS: Cervical cancer cells (CaSki) treated in vitro with hIL-24 migrated and invaded less than cells treated with PBS or vector control. MDA-7/IL-24 inhibited migration and invasion by down-regulating the production of matrix metalloproteinase-2 (MMP-2) and by up- regulating the production of E-cadherin relative to PBS and vector control. CONCLUSION: These results show that MDA-7/IL-24 inhibits invasion and migration of cervical cancer cells by down- or up-regulating proteins associated with these processes, resulting in reduced metastasis. Thus, Ad-mda7 should be considered a therapeutic agent that can inhibit primary tumor growth and prevent metastasis.4. To evaluate the effect of HPV-16 E6 siRNA associated with hIL-24 gene on inducing apoptosis of CaSki cells.Methods: Transfecting CaSki cells with hIL-24 gene eukaryotic expression vector and HPV-16 E6 siRNA vector separately and associately. The transcription of HPV E6 gene was analyzed by RT-PCR, P53 protein was analyzed by Western blot, apoptosis of CaSki cells was analyzed by FACS. Results: After transfecting, transcription level of HPV E6 gene decreased in all the three group of cells, the co-transfection group was significant; the amount of P53 protein increased in all the three group of cells, the co-transfection group was significant; and the rate of apoptosis of CaSki cells increased in all the three group of cells, the co-transfection group was significant.Conclusion: Both HPV-16 E6 siRNA and hIL-24 gene can inhibit the transcription of HPV E6 gene, recover the activity of P53 protein, then induce the apoptosis of CaSki cells. HPV-16 E6 siRNA associated with hIL-24 gene can significantly induce apoptosis of CaSki cells with positive cooperativity.5. The inhibition effect of hIL-24 on CaSki cell xenograft tumor in nude mice.Methods: Obtain the cell line expression recombinant plasmids by G418 screening. The cell lines were innoculated on the back of Balb/c nude mice subcutaneouly, and the growth of tumors were observed; the mice were sacrificed and tumor were decoherenced and weighed, the inhibition ratio of tumor growth was calculated. CaSki cell was innoculated into nude mice subcutaneouusly and the CaSki xenograft tumor model was constructed, the recombinant plasmids were injectioned into the tumor admixed with liposome, the volume and weight of tumor were measured. The pathological change was observed by HE stain.Results: the cell lines expressing recombinant plasmids were obtained. The oncogenicity of CaSki cell transfected by pcDNA3.1(+)-hIL-24 was lower obviously; After treated by recombinant plasmid, the growth of tumor was inhibited, the weight and volume of the pcDNA3.1(+)-hIL-24 treated group were lower than that of control group(P<0.05). There were many necrosis zone in tumor tissue treated by pcDNA3.1(+)-hIL-24, and there were necrosis cell and apoptosis body could be found in the CaSki xenograft tumour.Conclusion: the ectopic production of MDA-7/IL-24 gene could degrade the oncogenicity of CaSki cell; the treatment of plasmid expressing hIL-24 could inhibite growth of CaSki xenograft tumor.更多还原