人胰腺癌基因治疗的实验性研究
摘要
胰腺癌是人类恶性肿瘤中诊断和治疗均很困难的一种肿瘤。由于确诊时大多数病例已是晚期，因而手术切除机率极低，其它非手术治疗效果亦差，故胰腺癌预后差，死亡率高。本研究的目的是从基因水平研究胰腺癌的实验性治疗，为临床上人胰腺癌的基因治疗提供理论和实验依据。
我们的研究分两部分：1．构建能表达反义癌基因(c-myc和ki-ras)的重组逆转录病毒载体，观察这些重组载体对人胰癌细胞生长的抑制作用和恶性表型的逆转作用；2．构建能表达“自杀”基因HSV-TK的重组逆转录病毒载体介导的药物致敏对人胰腺癌细胞的杀伤作用。
1．重组逆转录病毒载体
(1)表达反义c-myc的重组逆转录病毒载体：将c-myc基因的第3外显子及其旁侧序列约长1.35Kb的片段正向和反向插入逆转录病毒载体pXT1，连接后构成2个重组载体pXT1Sm(正义)和pXT1Am(反义)。经PA317细胞包装和转染PC-2细胞，G418筛选后获转化细胞系PC-2／S-c-myc(正义)和PC-2／AS-c-myc(反义)。
(2)表达反义Ki-ras的重组逆转录病毒载体：将Ki-ras第4B外显子及其旁侧序列2.4kb长的片段插入逆转录病毒载体pBabe-puro，经连接后构成两个重组载体pBabe／s-Ki-ras(正义)和pBabe／As-Ki-ras(反义)。经PA317细胞包装和转染PC-2细胞，puromycin筛选后获转化细胞系PC-2／S-Ki-ras(正义)和PC-2／AS-Ki-ras(反义)。
(3)表达HSV-TK基因的重组逆转录病毒载体：将HSV-TK基因插入逆转录病毒载体后构成重组载体NTK，经PA317细胞包装后转染PC-2细胞，形成转化细胞系PC-2／NTK。
Human pancreatic carcinoma (HPC) is one of the malignant tumor which are very difficult in both diagnosis and treatment. Because of the lack of early detecting method, most of the HPC cases diagnosed are in advanced stage of the disease. Tumor resectability is therefore very low, and other non-operative approaches are also not encouraging. The prognosis of HPC is very poor and the mortality is very high. The aim of our researches was to investigate the gene therapy of HPC experimentally, and try to pave the theoretical and practical ground for the clinical gene therapy of HPC.
Our studies included two parts: A). Constructing the recombinant retroviral vectors expressing antisense c-myc or antisense Ki-ras, and observing the effects of recombinant retroviral vectors on retardation of HPC cell growth in vitro and reversion of malignant phenotype of HPC. B). Constructing a recombinant retroviral vector expressing the "suicide" gene (HSV-TK) , and examining the killing effect of the HSV- TK gene on HPC cells ( tumor cells "suicide").
1. Construction of the recombinant retroviral vectors
(1).A 1.35Kb c-myc fragment containing the third exon and adjoining flanking intron sequences was inserted into the retroviral vector pXT1 in both sense and antisense orientation, the recombinant vectors were defined as pXT1Sm (sense) and pXTlAm (antisense).The recombinant vector DNA was introduced into PA317 amphotropic packaging cells. The HPC cell line (PC-2 )
我们的研究分两部分：1．构建能表达反义癌基因(c-myc和ki-ras)的重组逆转录病毒载体，观察这些重组载体对人胰癌细胞生长的抑制作用和恶性表型的逆转作用；2．构建能表达“自杀”基因HSV-TK的重组逆转录病毒载体介导的药物致敏对人胰腺癌细胞的杀伤作用。
1．重组逆转录病毒载体
(1)表达反义c-myc的重组逆转录病毒载体：将c-myc基因的第3外显子及其旁侧序列约长1.35Kb的片段正向和反向插入逆转录病毒载体pXT1，连接后构成2个重组载体pXT1Sm(正义)和pXT1Am(反义)。经PA317细胞包装和转染PC-2细胞，G418筛选后获转化细胞系PC-2／S-c-myc(正义)和PC-2／AS-c-myc(反义)。
(2)表达反义Ki-ras的重组逆转录病毒载体：将Ki-ras第4B外显子及其旁侧序列2.4kb长的片段插入逆转录病毒载体pBabe-puro，经连接后构成两个重组载体pBabe／s-Ki-ras(正义)和pBabe／As-Ki-ras(反义)。经PA317细胞包装和转染PC-2细胞，puromycin筛选后获转化细胞系PC-2／S-Ki-ras(正义)和PC-2／AS-Ki-ras(反义)。
(3)表达HSV-TK基因的重组逆转录病毒载体：将HSV-TK基因插入逆转录病毒载体后构成重组载体NTK，经PA317细胞包装后转染PC-2细胞，形成转化细胞系PC-2／NTK。
Human pancreatic carcinoma (HPC) is one of the malignant tumor which are very difficult in both diagnosis and treatment. Because of the lack of early detecting method, most of the HPC cases diagnosed are in advanced stage of the disease. Tumor resectability is therefore very low, and other non-operative approaches are also not encouraging. The prognosis of HPC is very poor and the mortality is very high. The aim of our researches was to investigate the gene therapy of HPC experimentally, and try to pave the theoretical and practical ground for the clinical gene therapy of HPC.
Our studies included two parts: A). Constructing the recombinant retroviral vectors expressing antisense c-myc or antisense Ki-ras, and observing the effects of recombinant retroviral vectors on retardation of HPC cell growth in vitro and reversion of malignant phenotype of HPC. B). Constructing a recombinant retroviral vector expressing the "suicide" gene (HSV-TK) , and examining the killing effect of the HSV- TK gene on HPC cells ( tumor cells "suicide").
1. Construction of the recombinant retroviral vectors
(1).A 1.35Kb c-myc fragment containing the third exon and adjoining flanking intron sequences was inserted into the retroviral vector pXT1 in both sense and antisense orientation, the recombinant vectors were defined as pXT1Sm (sense) and pXTlAm (antisense).The recombinant vector DNA was introduced into PA317 amphotropic packaging cells. The HPC cell line (PC-2 )
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