基因重组野生型p53腺相关病毒治疗膀胱癌的实验研究
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摘要
膀胱移行细胞癌是我国泌尿生殖系最常见的恶性肿瘤。膀胱肿瘤术后易复发为其主要特征,并且复发肿瘤往往恶性度逐渐增高。治疗膀胱肿瘤以手术治疗和膀胱灌注药物治疗为主,但目前仍未找到有效控制膀胱肿瘤进展和复发的方法。所以人类在一直致力于探索新的有效的治疗方法。基因治疗经过近20多年的历程正逐步成熟,已逐渐被接受为继手术、化疗、放疗等常规治疗方法之后极有希望的新的疾病治疗方法。
由于p53基因在调节细胞周期和生长、诱导细胞凋亡的过程中发挥着重要作用,在膀胱肿瘤中p53突变率超过50%,因此利用p53基因治疗肿瘤成为研究的热点。基因导入系统是肿瘤基因治疗的核心技术,如何将目的基因导入机体细胞或肿瘤细胞并获得安全有效的表达是这一研究的重点。目前研究和应用最多的是腺病毒载体和逆转录病毒载体。但是各种病毒载体各有其优缺点,如逆转录病毒在宿主细胞内的随机整合,可以导致原癌基因的激活或功能基因插入失活的潜在危险。虽然腺病毒载体的研究也取得了进展,但也存在着一些问题如:毒副反应、有些腺病毒会引起急性感染、宿主体内存在病毒特异性免疫反应,腺病毒载体在宿主体内表达时间短,需要重复给药而加剧机体的免疫排斥反应等。因此人们正在寻找新的方法和载体使目的基因能够高效率地作用于肿瘤细胞。
腺相关病毒载体具有逆转录病毒和腺病毒的许多优点,并且与其它几种病毒相比,腺相关病毒载体具有安全性好、无致病性、免疫原性低、物理性质稳定、感染细胞谱广,可介导外源基因长期稳定表达等优点,被视为最有前途的基因治疗载体之一,成为目前基因治疗载体研究的热点。目前腺相关病毒载体基因治疗研究已在代谢性疾病和遗传性疾病中广泛开展,但其在膀胱肿瘤基因治疗中的研究尚少。本实验利用腺相关病毒包装平台—rAAV-Helper free system,分别构建携带人野生型p53基因和增强绿色荧光蛋白报告基因
Bladder transitional cell carcinoma (BTCC) is one of the most common urological malignant diseases in China. After operation BTCC easily recurs and the recurrence cancer often increases its progression. Operation and intravesical drug instillation are the major treatments. However there is no satisfying method to control its progression and recurrence. Therefore human being is still exploring new effective treatments of BTCC. For more than twenty years research gene therapy has gradually become mature and are accepting as extremely promising approach to treating cancer after regular treatment such as operation, chemotherapy and radiotherapy.p53 is the most frequently involved gene in cancer and its product P53 has a fundamental role in the control of cell proliferation and apoptosis. There are more than 50% bladder cancers possessing mutation of p53 gene. Therefore p53 gene therapy has become hot spot of research. Gene introduction system is the core technique of gene therapy. The key point of this research is how to introduce the interested gene into organism cell and tumor cell in order to express effective and safety. Most common viral vectors in gene therapy are recombinant retroviruses and adenoviruses. However, the vectors can be associated with serious adverse events due to the biologic behavior of the vector system. Genes carried by retroviral vectors are randomly integrated into the genome of the cell. This potentially leads to insertional mutagenesis. Adenoviral vectors are highly immunogenic, possibly causing a systemic inflammatory response syndrome leading to multiple organ failure. Therefore, new method and vector need to be developed so that interested gene could affect tumor cell efficiently.Adeno-associated virus (AAV) offers many desirable features of retroviruses
and adenovirus without some of their potential drawbacks for application in gene therapy. The recombinant AAV vectors can transducer both dividing and non-dividing cells in vitro and in vivo. Efficient and long-term transduction in vivo and the lack of both cytotoxicity and cellular immune responses are the hallmark features of AAV-mediated gene transfer. AAV was regarded as one of the extreme vectors in gene therapy in future. Although AAV vectors have been extensively used in gene therapy for genetic and metabolic diseases, few studies have used this vector system for bladder cancer gene therapy. This study used the package platform of AAV: rAAV-Helper free system to construct rAAV-wt-p53 vector and rAAV-EGFP vector and observed the rAAV-wt-p53 therapeutic effect and synergistic action with the cisplatin on bladder cancer in vitro and in vivo.Parti The package of rAAV-wt-p53 vector drugObjective: To construct AAV vector plasmids with gene engineering and molecular biological, packing rAAV-wt-p53 and rAAV-EGFP with three plasmids co-transfection techniques.Methods: 1. BamH I and Xba I fragment containing the full-length cDNA for human wild-type p53 was isolated from a plasmid pcDNAp53. The plasmid pCMV-MCS was also double digested by the same enzymes and ligated with wild-type p53 cDNA to construct interim plasmid pCMV-wt-p53. The structure of plasmid pCMV-wt-p53 was verified by restriction enzyme digested, PCR and sequencing. After verifying the plasmid pCMV-wt-p53, continue to next step.2. To avoid the lost of ITR which could decrease the yield of viral vector, the pCMV-wt-p53 was digested by Not I and the open read frame containing wt-p53 cDNA was cloned into ITR contained plasmid pAAV-MCS which was also digested by Not I . The plasmid was verified by restriction enzyme digested, PCR
and sequencing. The sequence of the sense primer and anti-sense used to PCR verified were part of L-ITR and R-ITR of pAAV-wt-p53 plasmid, the product of PCR was 3.5kb. 140bp ITR part of pAAV-wt-p53 plasmid could cut out by double digested using Not I and Pst I . Primer used to sequence the pAAV-wt-p53 plasmid was the same of sequencing primer of pCMV-wt-p53.3.The process of construction pAAV-EGFP plasmid was similar to that of pAAV-wt-p53. The EGFP sequence was derived from polymerase chain reaction amplification of plasmid pEGFP-Nl using a pair of primers. The sense primer was leaded-in Sal I site and the anti-sense primer was leaded-in Bgl II site. The product of PCR was 720bp EGFP sequence, which has Sal I and Bgl II site in each terminal. The EGFP sequence was double digested by Sal I and Bgl II .The plasmid pCMV-MCS was also double digested by the same enzymes and ligated with EGFP to construct plasmid pCMV-EGFP, which was verified by PCR and sequence. The plasmid pCMV-EGFP and pAAV-MCS were digested by Not I , and the open read frame containing EGFP was cloned into ITR containing pAAV-MCS fragment to construct plasmid pAAV-EGFP. The plasmid was verified by restriction enzyme digested, PCR and sequencing.4. HEK293 cells were co-transfected by electroporation using pAAV-wt-p53(or pAAV-EGFP), pRC and pHelper to package rAAV-wt-p53 and rAAV-EGFP. the cells were under Chloroform-NaCl sediment-Chloroform extract to dissociate and purify rAAV. Viral particle of purified rAAV were assayed by AVSachTM ELISA. Use SDS-PAGE Coomassie brilliant blue staining to observe capsid protein of rAAV. rAAV-wt-p53 was also identified with electronic microscope.Results: In this study we used the technique of gene engineering and molecular biology to construct pCMV-wt-p53 and pCMV-EGFP plasmid, then we cloned the open read frame containing interested gene into pAAV-MCS to
construct pAAV-wt-p53 and pAAV-EGFP. Thus we successfully avoid the lost of ITR in pAAV plasmid, which could decrease the yield of viral vector. All constructed plasmid was verified by PCR and sequencing. HEK293 cells was co-transfected by electroporation using three plasmids. We obtain high quality rAAV-wt-p53 and rAAV-EGFP after dissociating and purifying. The viral particles of rAAV were 2x1011. Under SDS-PAGE and Coomassie brilliant blue staining we could find three notable viral capsid protin strap of AAV. We observed the viral particle under electron microscope and found that rAAV were located uniformly, the diameters were about 20-25 nm, and the figure were polyhedron. The successful packaging of rAAV establishes the foundation for later experiment using this vector in vivo and in vitro.Part 2Study on rAAV-wt-p53 drug gene therapy of bladder cancer in vitro Objective: bladder cancer cell transfected with wild-type p53 mediated by rAAV in vitro to observe the effect on apoptosis and growth of cell, and analyze the synergistic effect combination with cisplatin on bladder cancer cell.Methods: 1. Bladder cancer cell 5637 is the cell line contains mutant p53 gene. In order to confirm this gene mutation, The part of p53 cDNA contains mutant site was amplified by RT-PCR and then sequenced the PCR product to confirm p53 mutation.2. 5637 cells were transected with rAAV-EGFP under different dose and observed cell under fluorescence microscope 96 hours later.3. 5637 cells were transfected with rAAV-wt-p53 or rAAV-EGFP, used un-transfected 5637 cells as control group, 96 hours later 5637 cells protein were extracted and western blot was performed to detect the proteins from different cell groups.
4. 5637 cells were transfected with rAAV-wt-p53 or rAAV-EGFP, 72 hours later the apoptosis rate was determined by flow cytometry with CellQuest software.5. 5637 cells were seeded in 96-well tissue culture plates. The cells were infected with either rAAV-wt-p53 or rAAV-TGFP or the same volume PBS and incubated. After infecting trypisnized counted every three well cells of different groups on the 1st, 2nd, 3rd, 4th, 5th days respectively. Drew the growth curve of the cells.6. 5637 cells were seeded in 96-well tissue culture plates. Added different dose viral particle of rAAV-wt-p53 and different dose of cisplatin alone or combined with another. After 72 hours the cells were assessed by MTT and calculated the IC50 of cisplatin and rAAV-wt-p53 alone and combined with another, then calculated the synergic index with the Isobole equation to analyze the synergistic effect of these two drugs.Results: 1. RNA was successful extracted from 5637 cells and cDNA was obtained by RNA reversed transcription. The product of PCR with primers of p53 was sequenced and found the codon 280 has changed from AGA to ACA. This confirms the mutation of p53 in 5637 cells.2.The 5637 cells were found to express green fluorescence protein under fluorescence microscope after infection with rAAV-EGFP. The percentage of emission green fluorescent cell were increased alone with increase dose of rAAV-EGFP.3. p53 protein of 5637 cells were detected by western blot. Untreated cells showed expression of p53 protein. Following transduction with rAAV-wt-p53 we observed an increased concentration of p53 protein in 5637 cells, whereas the concentration of p53 protein in 5637 cell after transduction with rAAV-EGFP was the same with the untreated 5637 cell.
4. The result of apoptosis rate of 5637 cells treated with rAAV-wt-p53 or rAAV-EGFP indicated that: The rate of apoptotic cells of cells treated with rAAV-wt-p53 was increased compared with cells treated with rAAV-EGFP or untreated cells.5. The influence of rAAV-wt-p53 on 5637 cell growth was assessed. rAAV-wt-p53 could inhibit the growth of 5637 cell and presented dose-effect relationship. But the rAAV-EGFP has no obviously inhibitory effect on cell growth. The drug synergism between cisplatin and rAAV-wt-p53 was analyzed, the synergic index D is less than 1. These two drugs have coordinate repression on cell growth.Part 3The study of rAAV-wt-p53 gene therapy of bladder cancer of nude mice in vivoObjective: To construct bladder cancer animal model using nude mice and study the therapeutical effect of rAAV-wt-p53 and cisplatin on bladder cancer model in vivo to observe the effect of anti-tumor and assess the interaction between two therapies.Methods: l.Cell suspension of 5637 cells were injected into the subcutaneously of right scapular region of nude mice, The number of injected cells were 5><106. The nude mice were raised under SPF condition and observed the xenograft tumor growth.2. Bearing tumor nude mice were randomly divided into 4 groups: normal group, cisplatin treatment group, rAAV-wt-p53 treatment group and cisplatin combined with rAAV-wt-p53 treatment group. 4 weeks later of treatment, the nude mice were killed by dislocate vertebrae cevicales. The xenografts tumors were weighted and fixed for HE stain. The liver tissue and nephridial tissue were
also fixed for HE stain. The drug interaction between cisplatin and rAAV-wt-p53 was analyzed by Jing-Zheng formula: Q=Eab/(Ea+Eb-Ea>
由于p53基因在调节细胞周期和生长、诱导细胞凋亡的过程中发挥着重要作用,在膀胱肿瘤中p53突变率超过50%,因此利用p53基因治疗肿瘤成为研究的热点。基因导入系统是肿瘤基因治疗的核心技术,如何将目的基因导入机体细胞或肿瘤细胞并获得安全有效的表达是这一研究的重点。目前研究和应用最多的是腺病毒载体和逆转录病毒载体。但是各种病毒载体各有其优缺点,如逆转录病毒在宿主细胞内的随机整合,可以导致原癌基因的激活或功能基因插入失活的潜在危险。虽然腺病毒载体的研究也取得了进展,但也存在着一些问题如:毒副反应、有些腺病毒会引起急性感染、宿主体内存在病毒特异性免疫反应,腺病毒载体在宿主体内表达时间短,需要重复给药而加剧机体的免疫排斥反应等。因此人们正在寻找新的方法和载体使目的基因能够高效率地作用于肿瘤细胞。
腺相关病毒载体具有逆转录病毒和腺病毒的许多优点,并且与其它几种病毒相比,腺相关病毒载体具有安全性好、无致病性、免疫原性低、物理性质稳定、感染细胞谱广,可介导外源基因长期稳定表达等优点,被视为最有前途的基因治疗载体之一,成为目前基因治疗载体研究的热点。目前腺相关病毒载体基因治疗研究已在代谢性疾病和遗传性疾病中广泛开展,但其在膀胱肿瘤基因治疗中的研究尚少。本实验利用腺相关病毒包装平台—rAAV-Helper free system,分别构建携带人野生型p53基因和增强绿色荧光蛋白报告基因
Bladder transitional cell carcinoma (BTCC) is one of the most common urological malignant diseases in China. After operation BTCC easily recurs and the recurrence cancer often increases its progression. Operation and intravesical drug instillation are the major treatments. However there is no satisfying method to control its progression and recurrence. Therefore human being is still exploring new effective treatments of BTCC. For more than twenty years research gene therapy has gradually become mature and are accepting as extremely promising approach to treating cancer after regular treatment such as operation, chemotherapy and radiotherapy.p53 is the most frequently involved gene in cancer and its product P53 has a fundamental role in the control of cell proliferation and apoptosis. There are more than 50% bladder cancers possessing mutation of p53 gene. Therefore p53 gene therapy has become hot spot of research. Gene introduction system is the core technique of gene therapy. The key point of this research is how to introduce the interested gene into organism cell and tumor cell in order to express effective and safety. Most common viral vectors in gene therapy are recombinant retroviruses and adenoviruses. However, the vectors can be associated with serious adverse events due to the biologic behavior of the vector system. Genes carried by retroviral vectors are randomly integrated into the genome of the cell. This potentially leads to insertional mutagenesis. Adenoviral vectors are highly immunogenic, possibly causing a systemic inflammatory response syndrome leading to multiple organ failure. Therefore, new method and vector need to be developed so that interested gene could affect tumor cell efficiently.Adeno-associated virus (AAV) offers many desirable features of retroviruses
and adenovirus without some of their potential drawbacks for application in gene therapy. The recombinant AAV vectors can transducer both dividing and non-dividing cells in vitro and in vivo. Efficient and long-term transduction in vivo and the lack of both cytotoxicity and cellular immune responses are the hallmark features of AAV-mediated gene transfer. AAV was regarded as one of the extreme vectors in gene therapy in future. Although AAV vectors have been extensively used in gene therapy for genetic and metabolic diseases, few studies have used this vector system for bladder cancer gene therapy. This study used the package platform of AAV: rAAV-Helper free system to construct rAAV-wt-p53 vector and rAAV-EGFP vector and observed the rAAV-wt-p53 therapeutic effect and synergistic action with the cisplatin on bladder cancer in vitro and in vivo.Parti The package of rAAV-wt-p53 vector drugObjective: To construct AAV vector plasmids with gene engineering and molecular biological, packing rAAV-wt-p53 and rAAV-EGFP with three plasmids co-transfection techniques.Methods: 1. BamH I and Xba I fragment containing the full-length cDNA for human wild-type p53 was isolated from a plasmid pcDNAp53. The plasmid pCMV-MCS was also double digested by the same enzymes and ligated with wild-type p53 cDNA to construct interim plasmid pCMV-wt-p53. The structure of plasmid pCMV-wt-p53 was verified by restriction enzyme digested, PCR and sequencing. After verifying the plasmid pCMV-wt-p53, continue to next step.2. To avoid the lost of ITR which could decrease the yield of viral vector, the pCMV-wt-p53 was digested by Not I and the open read frame containing wt-p53 cDNA was cloned into ITR contained plasmid pAAV-MCS which was also digested by Not I . The plasmid was verified by restriction enzyme digested, PCR
and sequencing. The sequence of the sense primer and anti-sense used to PCR verified were part of L-ITR and R-ITR of pAAV-wt-p53 plasmid, the product of PCR was 3.5kb. 140bp ITR part of pAAV-wt-p53 plasmid could cut out by double digested using Not I and Pst I . Primer used to sequence the pAAV-wt-p53 plasmid was the same of sequencing primer of pCMV-wt-p53.3.The process of construction pAAV-EGFP plasmid was similar to that of pAAV-wt-p53. The EGFP sequence was derived from polymerase chain reaction amplification of plasmid pEGFP-Nl using a pair of primers. The sense primer was leaded-in Sal I site and the anti-sense primer was leaded-in Bgl II site. The product of PCR was 720bp EGFP sequence, which has Sal I and Bgl II site in each terminal. The EGFP sequence was double digested by Sal I and Bgl II .The plasmid pCMV-MCS was also double digested by the same enzymes and ligated with EGFP to construct plasmid pCMV-EGFP, which was verified by PCR and sequence. The plasmid pCMV-EGFP and pAAV-MCS were digested by Not I , and the open read frame containing EGFP was cloned into ITR containing pAAV-MCS fragment to construct plasmid pAAV-EGFP. The plasmid was verified by restriction enzyme digested, PCR and sequencing.4. HEK293 cells were co-transfected by electroporation using pAAV-wt-p53(or pAAV-EGFP), pRC and pHelper to package rAAV-wt-p53 and rAAV-EGFP. the cells were under Chloroform-NaCl sediment-Chloroform extract to dissociate and purify rAAV. Viral particle of purified rAAV were assayed by AVSachTM ELISA. Use SDS-PAGE Coomassie brilliant blue staining to observe capsid protein of rAAV. rAAV-wt-p53 was also identified with electronic microscope.Results: In this study we used the technique of gene engineering and molecular biology to construct pCMV-wt-p53 and pCMV-EGFP plasmid, then we cloned the open read frame containing interested gene into pAAV-MCS to
construct pAAV-wt-p53 and pAAV-EGFP. Thus we successfully avoid the lost of ITR in pAAV plasmid, which could decrease the yield of viral vector. All constructed plasmid was verified by PCR and sequencing. HEK293 cells was co-transfected by electroporation using three plasmids. We obtain high quality rAAV-wt-p53 and rAAV-EGFP after dissociating and purifying. The viral particles of rAAV were 2x1011. Under SDS-PAGE and Coomassie brilliant blue staining we could find three notable viral capsid protin strap of AAV. We observed the viral particle under electron microscope and found that rAAV were located uniformly, the diameters were about 20-25 nm, and the figure were polyhedron. The successful packaging of rAAV establishes the foundation for later experiment using this vector in vivo and in vitro.Part 2Study on rAAV-wt-p53 drug gene therapy of bladder cancer in vitro Objective: bladder cancer cell transfected with wild-type p53 mediated by rAAV in vitro to observe the effect on apoptosis and growth of cell, and analyze the synergistic effect combination with cisplatin on bladder cancer cell.Methods: 1. Bladder cancer cell 5637 is the cell line contains mutant p53 gene. In order to confirm this gene mutation, The part of p53 cDNA contains mutant site was amplified by RT-PCR and then sequenced the PCR product to confirm p53 mutation.2. 5637 cells were transected with rAAV-EGFP under different dose and observed cell under fluorescence microscope 96 hours later.3. 5637 cells were transfected with rAAV-wt-p53 or rAAV-EGFP, used un-transfected 5637 cells as control group, 96 hours later 5637 cells protein were extracted and western blot was performed to detect the proteins from different cell groups.
4. 5637 cells were transfected with rAAV-wt-p53 or rAAV-EGFP, 72 hours later the apoptosis rate was determined by flow cytometry with CellQuest software.5. 5637 cells were seeded in 96-well tissue culture plates. The cells were infected with either rAAV-wt-p53 or rAAV-TGFP or the same volume PBS and incubated. After infecting trypisnized counted every three well cells of different groups on the 1st, 2nd, 3rd, 4th, 5th days respectively. Drew the growth curve of the cells.6. 5637 cells were seeded in 96-well tissue culture plates. Added different dose viral particle of rAAV-wt-p53 and different dose of cisplatin alone or combined with another. After 72 hours the cells were assessed by MTT and calculated the IC50 of cisplatin and rAAV-wt-p53 alone and combined with another, then calculated the synergic index with the Isobole equation to analyze the synergistic effect of these two drugs.Results: 1. RNA was successful extracted from 5637 cells and cDNA was obtained by RNA reversed transcription. The product of PCR with primers of p53 was sequenced and found the codon 280 has changed from AGA to ACA. This confirms the mutation of p53 in 5637 cells.2.The 5637 cells were found to express green fluorescence protein under fluorescence microscope after infection with rAAV-EGFP. The percentage of emission green fluorescent cell were increased alone with increase dose of rAAV-EGFP.3. p53 protein of 5637 cells were detected by western blot. Untreated cells showed expression of p53 protein. Following transduction with rAAV-wt-p53 we observed an increased concentration of p53 protein in 5637 cells, whereas the concentration of p53 protein in 5637 cell after transduction with rAAV-EGFP was the same with the untreated 5637 cell.
4. The result of apoptosis rate of 5637 cells treated with rAAV-wt-p53 or rAAV-EGFP indicated that: The rate of apoptotic cells of cells treated with rAAV-wt-p53 was increased compared with cells treated with rAAV-EGFP or untreated cells.5. The influence of rAAV-wt-p53 on 5637 cell growth was assessed. rAAV-wt-p53 could inhibit the growth of 5637 cell and presented dose-effect relationship. But the rAAV-EGFP has no obviously inhibitory effect on cell growth. The drug synergism between cisplatin and rAAV-wt-p53 was analyzed, the synergic index D is less than 1. These two drugs have coordinate repression on cell growth.Part 3The study of rAAV-wt-p53 gene therapy of bladder cancer of nude mice in vivoObjective: To construct bladder cancer animal model using nude mice and study the therapeutical effect of rAAV-wt-p53 and cisplatin on bladder cancer model in vivo to observe the effect of anti-tumor and assess the interaction between two therapies.Methods: l.Cell suspension of 5637 cells were injected into the subcutaneously of right scapular region of nude mice, The number of injected cells were 5><106. The nude mice were raised under SPF condition and observed the xenograft tumor growth.2. Bearing tumor nude mice were randomly divided into 4 groups: normal group, cisplatin treatment group, rAAV-wt-p53 treatment group and cisplatin combined with rAAV-wt-p53 treatment group. 4 weeks later of treatment, the nude mice were killed by dislocate vertebrae cevicales. The xenografts tumors were weighted and fixed for HE stain. The liver tissue and nephridial tissue were
also fixed for HE stain. The drug interaction between cisplatin and rAAV-wt-p53 was analyzed by Jing-Zheng formula: Q=Eab/(Ea+Eb-Ea>
引文
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