Ad-Rb94联合放疗对恶性肿瘤细胞生长的抑制作用
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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HepG_2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1.6×10~8pfu/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HepG_2停留在G_2/M期的细胞增加,G_0/G_1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G_2/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。
Objective
Gene 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 NH_2-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.
Methods
The 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.
Results
First 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 G_2/M phases that was treated Ad-Rb94,radiotherapy and combination of Ad-Rb94 and radiotherapy increased,while the number of cells of G_0/G_1 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%).
Conclusions
First 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 G_2/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.
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HepG_2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1.6×10~8pfu/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HepG_2停留在G_2/M期的细胞增加,G_0/G_1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G_2/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。
Objective
Gene 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 NH_2-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.
Methods
The 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.
Results
First 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 G_2/M phases that was treated Ad-Rb94,radiotherapy and combination of Ad-Rb94 and radiotherapy increased,while the number of cells of G_0/G_1 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%).
Conclusions
First 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 G_2/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.
引文
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[1]Keall PJ,lammering G,Lin PS,el al.Tumor control probability predictions for genetic radiotherapy[J].Int J Radial Oncol Biol Phys,2003,57(1):255-263.
[2]Teh BS,Aquilar-Cordova E,Vlachaki MT,et al.Combining radiotherapy with gene therapy(from the bench to the bedside):A novel treatment strategy for prostate cancer[J].Oncologist,2002,7(5):458-466.
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[4]Garnett CT,Palena C,Chakraborty M,el al.Sublethal irradiation of human tumor cells modulates phenotype resulting in enhanced killing by cytotoxic T lymphocytes[J].Cancer Res,2004,64(21):7985-7994.
[5]Liao YP,Wang CC,Butterfield LH,et al.Ionizing radiation affects human MART-1 melanoma antigen processing and presentation by dendriticcells[J].J Immunol,2004,173(4):2462-2469.
[6]Tsai CH,I-long JH,Hsieh KF,et al.Tetracycline-regulated intratumoral expression of interleukin-3 enhances the efficacy of radiation therapy for murine prostate cancer[J].Cancer Gene Therapy,2006,13(12):1082-1092.
[7]Fujita T,Timme TL,Tabata,K,et al.Cooperative effects of adenoviral vector-mediated interleukin 12 gene therapy with radiotherapy in a preclinical model of metastatic prostate cancer[J].Gene Therapy,2007,14(3):227-236.
[8]Gupta VK,Park JO,Jaskowiak NT,et al.Combined gene therapy and ionizing radiation is a novel approach to treat human esophageal adenocarcinoma[J].Ann Surg Oncol,2002,9(5):500-504.
[9]唐瑶云,赵素萍,徐婧,等.FCU/5-氟胞嘧啶自杀基因前体药物系统联合放射治疗鼻咽癌的实验研究[J].中华病理学杂志,2006,35(8):483-487.
[10]Chen JK,Hu L J,Wang DF,et al.Cytosine deaminase/5-fluorocytosine exposure induces bystander and radiosensitization effects in hypoxic glioblastoma cells in vitro[J].Int J Radiat Oncol,2007,67(5):1538-1547.
[11]陈道桢,刘璐,唐秋莎,等.HSV-TK/GCV自杀基因系统联合γ射线治疗宫 颈癌的实验研究[J].中华放射医学与防护杂志,2006,26(1):39-42.
[12]陈传本,潘建基,徐鹭英.重组人p53腺病毒注射液结合放射治疗鼻咽癌Ⅱ期临床试验观察[J].中华医学杂志,2003,83(23):2033-2035.
[13]张珊文,肖绍文,刘长清,等.头颈鳞癌基因治疗结合放射治疗的临床研究[J].中华肿瘤杂志,2005,27(7):426-428.
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[17]Zhang X,Multani AS,Zhou JH,et al.Adenoviral-mediated retinoblastoma 94produces rapid telomere erosion,chromosomal crisis,and caspase-dependent apoptosis in bladder cancer and immortalized human urothelial cells but not in normal urothelial cells[J].Cancer Res,2003,63(4):760-765.
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