【作者】 张耀文；

【导师】 曹永珍；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2009， 硕士

【摘要】 目的基因治疗是当前肿瘤生物治疗的研究热点,也是以后肿瘤治疗的发展趋势,放疗是目前肿瘤治疗的主要方法,放疗和基因治疗两者之间存在着相互增强的作用。增强的机理是射线照射后增加基因转染的效率和（或）促进基因的表达,另外基因转染后可增加肿瘤细胞对射线照射的敏感性。人Rb基因（视网膜母细胞瘤基因）是最早克隆出来的人类肿瘤抑制基因,Rb94基因是Rb基因的一部分,野生型pRb110蛋白N端缺失112个氨基酸残基即为pRb94蛋白。有研究表明Rb94基因有比Rb基因更好的抑瘤效应。本实验目的是通过含有Rb94基因的重组腺病毒载体（Ad-Rb94）转染体外培养的人肝癌细胞系HepG₂及人宫颈癌细胞系Hela,观察Rb94基因的抑瘤效应,然后联合X射线照射,研究两者的协同抑瘤作用,为临床恶性肿瘤的基因治疗和放射治疗的联合应用提供实验依据。方法构建好的含有Rb94基因的腺病毒表达载体,经PacI进行酶切后,在脂质体的作用下转染293A细胞进行扩增,制备大量的腺病毒颗粒。用PCR法鉴定重组Ad-Rb94载体中Rb94基因的存在,测定病毒颗粒滴度。选择肝癌和宫颈癌两株细胞进行实验,实验分组设计为空白对照组、Ad-lacZ对照组、Ad-Rb94组、放疗组和Ad-Rb94联合放疗组。用MTT法和流式细胞术观察Rb94基因联合放疗对细胞生长及细胞周期、凋亡的影响。结果1.构建的重组腺病毒表达载体经PCR鉴定含有Rb94目的基因;2.腺病毒滴度较高,为1.6×10⁸pfu/ml,达到体外和体内实验的要求;3.空白对照组与Ad-lacZ组比较差异无统计学意义（p＞0.05）;4.肝癌HepG2细胞生长曲线:Ad-Rb94基因组、放疗组和Ad-Rb94基因联合放疗组HepG2细胞的生长均受到抑制,尤其在转染后96h细胞存活数量最低,与Ad-lacZ组和空白对照组比较差异均有显著性（P＜0.05）。联合治疗组HepG2细胞生长最慢,Ad-Rb94与放疗联合应用后96h,对HepG2细胞生长的抑制率达55.63%±8.97%,显著高于单用Ad-Rb94组（31.61%±3.25%,p＜0.05）和放疗组（20.06%±4.33%,p＜0.05）;5.宫颈癌Hela细胞生长曲线:Ad-Rb94基因组、放疗组和Ad-Rb94基因联合放疗组Hela细胞的生长均受到抑制,尤其在转染后96h细胞存活数量最低,与Ad-lacZ组和空白对照组比较差异均有显著性（P＜0.05）。联合治疗组Heta细胞生长最慢,Ad-Rb94与放疗联合应用后96h,对Hela细胞生长的抑制率达51.84%±3.84%,显著高于单用Ad-Rb94组（37.40%±5.65%,p＜0.05）和放疗组（35.42%±2.47%,p＜0.05）;6.肝癌HepG2细胞周期和凋亡情况:流式细胞术检测结果显示,Ad-Rb94组、放疗组及Ad-Rb94联合放疗组HepG₂停留在G₂/M期的细胞增加,G₀/G₁期和S期细胞数量减少,凋亡细胞数量增加。Ad-Rb94联合放疗组HepG2细胞凋亡率27.1%,显著高于单用Ad-Rb94组（18.2%）和放疗组（11-3%）:结论1.所构建的重组腺病毒表达载体Ad-Rb94扩增后,建立了可获得高滴度含Rb94基因腺病毒（Ad-Rb94）库,经PCR验证含有Rb94目的基因,表明扩增成功;2.重组腺病毒Ad-Rb94能抑制肝癌细胞和宫颈癌细胞的生长,通过与Ad-lacZ对照比较,表明对肿瘤细胞的抑制作用来自转导的Rb94基因,而不是腺病毒载体本身;3.Ad-Rb94组、放疗组及Ad-Rb94联合放疗组对HepG2和Hela细胞的生长抑制作用呈时间依赖性,联合治疗组具有协同作用;Ad-Rb94组、放疗组及Ad-Rb94联合放疗组阻滞肝癌HepG2细胞周期于G₂/M期,促进细胞凋亡,联合治疗组具有协同作用,为今后用于临床恶性肿瘤的治疗提供了一个新的治疗方案。更多还原

【Abstract】 ObjectiveGene therapy is the hot point of the tumor biological treatment at present,and also the trend of the tumor therapy in the future.Radiotherapy has been the major method of the cancer treatment,and the effect of inhibiting tumor is enhanced each other between gene therapy and radiotherapy.The mechanism may be:radiation improves the efficiency of gene transfection and（or） increases the expression of protein,on the other hand the transfection of the gene improves the sensitivity of the radiation.The retinoblastoma gene（Rb） is the earliest isolated human suppressor gene,Rb94 gene is the part of Rb gene.Rb94 protein lacks 112 amino acid residues at NH₂-terminal of Rb protein.Studies indicate that the function of the Rb94 gene is better than that of the full length Rb gene in the inhibiting tumor effect.The aim of this study is to check the inhibiting effect of tumor cells growth by transfecting human hepatoma cell lines HepG2 and human cervix cancer Hela cells with adenovirus-mediated expression vector of Rb94 gene（Ad-Rb94）.We study the combined effect of Ad-Rb94 with X-radiation to provide the experimental data for clinical therapy of the cancer gene therapy combined with radiotherapy.MethodsThe recombinant adenovirus vector was restricted with PacI,then transfected 293A cells by liposomes to amplify the recombinant adenovirus express vector, finally we got a lot of the recombinant adenovirus Rb94 particles.Results showed that Rb94 gene was confirmed in Ad-Rb94 vector by PCR.We choosed human hepatoma cell lines HepG2 and human cervix cancer Hela cells.The experiment groups were designed as blank control、Ad-lacZ、Ad-Rb94、radiotherapy and combination of Ad-Rb94 and radiotherapy.The cell growth inhibition was assessed by MTT,and cell cycle and apoptosis were detected by flow cytometry.ResultsFirst Results showed that expected Rb94 gene was confirmed in Ad-Rb94 vector by PCR.Second The titer of adenovirus was 1.6×108 pfu/ml,which reached the standard of experiment.Third There was no statistical significance compared with Ad-lacZ group and blank control.Fourth The growth curve of HepG2 cells:The growth of HepG2 cells that was treated with Ad-Rb94,radiotherapy and combination of Ad-Rb94 and radiotherapy was slower than that of both Ad-lacZ and blank control significantly（p＜0.05）, especially at 96h after transfection.The growth inhibition rate of HepG2 cells of combined Ad-Rb94 with radiotherapy group at 96h after transfection was 55.63%±8.97%,which was significantly higher than that of Ad-Rb94 group（31.61%±3.25%,p＜0.05） and radiotherapy group（20.06%±4.33%,p＜0.05）.Fifth The growth curve of Hela cells:The growth of Hela cells that was treated with Ad-Rb94,radiotherapy and combination of Ad-Rb94 and radiotherapy was slower than that of both Ad-lacZ and blank control significantly（p＜0.05）,especially at 96h after transfection.The growth inhibition rate of Hela cells of HepG2 cells of combined Ad-Rb94 with radiotherapy group at 96h after transfection was 51.84%±3.84%,which was significantly higher than that of Ad-Rb94 group（37.40%±5.65%,p＜0.05） and radiotherapy group（35.42%±2.47%,p＜0.05）.Sixth The cell cycle and apoptosis of HepG2 cells:The number of cells of G₂/M phases that was treated Ad-Rb94,radiotherapy and combination of Ad-Rb94 and radiotherapy increased,while the number of cells of G₀/G₁ and S phases decreased; cells of apoptosis increased.The apoptotic rate of HepG2 cells of combined Ad-Rb94 with radiotherapy group was 27.1%,which was significantly higher than of Ad-Rb94 group（18.2%） and radiotherapy group（11.3%）.ConclusionsFirst We got high titer adenovirus particles containing Rb94 gene by amplifying recombinant adenovirus express vector of Rb94 gene,and results showed that expected Rb94 gene was confirmed by PCR.It indicated that amplification was successful. Second Recominant adenovirus mediated retinoblastoma 94 gene can inhibit the growth of human hepatoma cells and human cervix cancer cells.Compared with Ad-lacZ control group,the inhibition of Ad-Rb94 arised from Rb94 gene,rather than adenovirus.Third There was a time-dependent inhibition of cell proliferation in Ad-Rb94、radioterapy and combined treatment groups on HepG2 and Hela.Ad-Rb94 gene therapy combined with radiotherapy showed synergism of the inhibition. Ad-Rb94,radiotherapy and combined administration of Ad-Rb94 and radiotherapy arrested HepG2 cells in the G₂/M phase,and increased levels of apoptosis.Ad-Rb94 gene therapy combined with radiotherapy showed synergism of the inhibition.And synergism of combined group could provide a new treatment for clinical tumor therapy.更多还原