双自杀基因联合放疗对大肠癌治疗的实验研究
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摘要
自杀基因是目前基因治疗研究的一个热点。目前国外大多数研究还集中在单自杀基因的治疗。单自杀基因的治疗有容易控制、肿瘤的靶向作用和诱导免疫等优点,但也有很多不足之处。大量的实验研究表明,不同的恶性肿瘤细胞对两种自杀基因/前药的治疗反应不同,其介导的旁观者效应也明显不同。然而,双自杀基因(CD-TK基因)的产物具有两种酶的活性,利用其互补作用,可以大大提高其细胞毒作用。本实验探索利用携带CD、TK双自杀基因融合基因的复制缺陷型腺病毒载体:Ad.CDglyTK联合放射对人结直肠癌细胞和荷瘤鼠进行治疗。腺病毒载体是目前最为有效的基因转导载体,可以接受大的转移基因插入,腺病毒可以生长到高效价(1012-1013 表达颗粒/mL)。再利用CD/5-FC和TK/GCV可能存在的的放射增敏作用进一步协同杀伤肿瘤细胞,将二者有机的结合在一起,发挥二者的协同作用,也将放疗、化疗、基因治疗有机的结合起来。我们试图寻找出一种新颖的治疗恶性肿瘤方案,为临床治疗癌症提供理论以及实践的依据。
方法:
1.携带CDglyTK基因的腺病毒粘粒载体(pAxcAwt/CDglyTK)的构建。
从pWZLneoCDglyTK质粒中回收含有目的基因的DNA片段,继而再将其插入带有腺病毒基因组的粘粒载体pAxcAwt,扩增后挑选插入正确的克隆。
2.含CDglyTK基因的腺病毒载体(Ad.CDglyTK)的构建。
本应用磷酸钙共沉淀法及DNA-TPC,将插入CDglyTK基因的腺病毒粘粒载体导入293细胞,制备重组腺病毒。并将酶切鉴定为正确的病毒克隆进一步扩增、纯化和测定滴度。同时对其是否含有野生型腺病毒进行检测。
3.研究重组腺病毒载体介导双自杀基因治疗放射增敏作用。
将LoVo、HCT-8、SGC-7901三种细胞转染重组腺病毒,然后进行不同剂量的离体Co60放疗实验,与放疗联合5-FC(5-氟胞嘧啶)、GCV(丙氧鸟苷)前药治疗相比较。测量三种细胞转染后细胞倍增时间;检测三种细胞转染后融合基因蛋白的表达;MTT法计算细胞生长存活率;观察体外旁观者效应的产生;用细胞集落形成实验生成细胞存活曲线。计算细胞集落形成数量,测量细胞存活曲线的D0、Dq值,比较放射增强比(SER),分析HSV-tk/GCV、CD/5-FC双自杀基因系统治疗后对肿瘤细胞的放射增敏作用。
4.研究双自杀基因联合放疗对荷大肠癌裸鼠治疗效果及与细胞凋亡的关系
首先建立荷瘤小鼠模型,然后进行荷瘤裸鼠前药治疗实验,期间治疗组给予CO60放射治疗。检测肿瘤组织标本,用流式细胞仪检测凋亡细胞,透射电镜下观察转染细胞的超微结构, DNA凝胶电泳法检测细胞凋亡,探讨CDglyTK基因联合放疗对荷大肠癌裸鼠治疗效果的机理。
主要结果:
1.从pWZLneoCDglyTK质粒中回收的DNA片段为目的基因,插入粘粒载体pAxCAwt后获得了插入正确的克隆,并予以扩增备用。
2.探索出了稳定、有效的重组腺病毒构建方法,以此方法构建了含CDglyTK基因的腺病毒载体(Ad.CDglyTK)。扩增后重组腺病毒滴度高达1012pfu/ml,体外使用安全有效。
3.通过Western Blot方法检测肿瘤细胞转染重组腺病毒后融合基因蛋白的表达;通过对细胞存活曲线的D0、Dq值、SER的分析证实HSV-tk/GCV、CD/5-FC双自杀基因系统治疗后对肿瘤细胞有放射增敏作用。
4.研究结果表明双自杀基因联合放疗对荷大肠癌裸鼠治疗比单纯放疗、双前药治疗明显有效,荷瘤鼠生存时间明显延长。通过流式细胞仪、电镜分析双自杀基因联合放疗疗效与细胞凋亡有关。
主要结论:
1.DNA末端蛋白复合物可以使重组腺病毒的制备效率大大提高,降低野生型腺病毒的出现概率。实验获得的重组腺病毒滴度高、纯度好、毒性低。
2.实验所构建的携带CDglyTK基因的重组腺病毒载体,能高效转染大肠癌细胞。转染的肿瘤细胞能表达CDglyTK基因的融合蛋白,通过双前药治疗能获得明显的细胞杀伤作用和旁观者效应。
3.双自杀基因联合放疗能获得最大的肿瘤杀伤,二者有协同作用,并且有放射增敏作用。二者作用的机制可能是通过促进肿瘤细胞的凋亡获得的。
Suicide gene therapy has been a new hot spot of cancer gene therapy research. Previous studies focused mainly on single suicide gene therapy, which have virtues of easy control and induce immunity as well as tumor target function. However, every knife has two edges, suicide gene therapy has some deficiencies too. Many experiments have demonstrated that different malignant tumors have various responses to different system of suicide gene/prodrug and the bystander effect has distinctness too. On the other side, the product of double suicide gene has the activities of two enzymes. Cytotoxicity would strongly increased by the complementary of double suicide gene expressing products. In this experiment, we probe to effects of fusion gene combined with radiotherapy to human colorectal cells and nude mice bearing human colorectal carcinoma. For this purpose, we construct a replication-defective adenovirus vector (Ad.CDglyTK). Adenovirus vector is the most effective vector at present, which can receive an insertion of large gene. We can get a high titer also (1012-1013pfu/ml) through this method. Combination the irradiation enhancement effect of CD/5-FC and TK/GCV we can kill the tumor cells more strongly. At the same time, it unites the radiotherapy/ chemotherapy with gene therapy into one. We try to find a novel way and to provide a foundation of clinical malignant tumor’s therapy.
Methods:
1 The construction of cosmid vector inserting CDglyTK gene
(pAxcAwt/CDglyTK)
DNA fragment reclaimed from the plasmid of pWZLneoCDglyTK. Then the DNA was inserted into cosmid vector pAxcAw. The recombinant cosmid was amplified and was confirmed to be the correct gene . At last the correct clones containing target gene in right direction were selected.
2 The construction of replication-defective recombinant adenovirus coding for CDglyTK gene (Ad.CDglyTK)
Human embryonic kidney 293 cells cultured in a 6cm dish were transfected with DNA-TPC and pAxcAwt/CDglyTK by the calcium phosphate method. After 8-18 days, virus clones were isolated and propagated to for restriction analysis. The desired Ad vectors were purified by density gradient ultracentrifuge and titrated in 293 cells. Whether there has a little wild type adenovirus was also examined at the same time.
3 Transfection of Ad.CDglyTK to carcinoma cells and its effects of enhancement of radiosensitization in vitro
Three kinds of gastrointestinal cells(LoVo, HCT-8, SGC-7901) were
isolated and cultured. Transfection efficiency of Ad.CDglyTK on these cells was examined at various titrations by immunofluorescence staining. And then Co60 radiotherapy of different dose in vitro were completed compared with prodrug treatment of 5-FC or GCV. Cell doubling time was tested after three kinds of cells were transfected by recombinant adenovirus. Fusion protein expression was evaluated by Western blot respectively. The cell survival curve was calculated by cell cloning forming test. Cell survival rate was examined by MTT assay. Bystander effect in vitro was observed. D0,Dq of the cell survival curve were measured and the SER(Sensitizing Enhancement Ratio) were compared in different groups.
4 To research into the therapeutic effect of double suicide gene combining radiotherapy to nude mice bearing human colorectal carcinoma
First, the model of nude mice bearing carcinoma was established. Then the prodrug experiment was completed. In the period of prodrug treatment, Co60 therapy to nude mice in some groups was completed on the time. In the end, tumor of nude mice was examined. Cell apoptosis was checked through FCM method, DNA gel electrophoresis and transmission electron microscope .
Results:
1 The segment of DNA obtained from pWZLneoCDglyTK plasmid was identical to that expected. After the target DNA was inserted into cosmid vector pAxcAw, the recombinant cosmid was amplified and confirmed to be the correct gene.
2 An efficient and reliable method of constructing recombinant Ad vectors was established. Replication-defective adenovirus vectors coding for
方法:
1.携带CDglyTK基因的腺病毒粘粒载体(pAxcAwt/CDglyTK)的构建。
从pWZLneoCDglyTK质粒中回收含有目的基因的DNA片段,继而再将其插入带有腺病毒基因组的粘粒载体pAxcAwt,扩增后挑选插入正确的克隆。
2.含CDglyTK基因的腺病毒载体(Ad.CDglyTK)的构建。
本应用磷酸钙共沉淀法及DNA-TPC,将插入CDglyTK基因的腺病毒粘粒载体导入293细胞,制备重组腺病毒。并将酶切鉴定为正确的病毒克隆进一步扩增、纯化和测定滴度。同时对其是否含有野生型腺病毒进行检测。
3.研究重组腺病毒载体介导双自杀基因治疗放射增敏作用。
将LoVo、HCT-8、SGC-7901三种细胞转染重组腺病毒,然后进行不同剂量的离体Co60放疗实验,与放疗联合5-FC(5-氟胞嘧啶)、GCV(丙氧鸟苷)前药治疗相比较。测量三种细胞转染后细胞倍增时间;检测三种细胞转染后融合基因蛋白的表达;MTT法计算细胞生长存活率;观察体外旁观者效应的产生;用细胞集落形成实验生成细胞存活曲线。计算细胞集落形成数量,测量细胞存活曲线的D0、Dq值,比较放射增强比(SER),分析HSV-tk/GCV、CD/5-FC双自杀基因系统治疗后对肿瘤细胞的放射增敏作用。
4.研究双自杀基因联合放疗对荷大肠癌裸鼠治疗效果及与细胞凋亡的关系
首先建立荷瘤小鼠模型,然后进行荷瘤裸鼠前药治疗实验,期间治疗组给予CO60放射治疗。检测肿瘤组织标本,用流式细胞仪检测凋亡细胞,透射电镜下观察转染细胞的超微结构, DNA凝胶电泳法检测细胞凋亡,探讨CDglyTK基因联合放疗对荷大肠癌裸鼠治疗效果的机理。
主要结果:
1.从pWZLneoCDglyTK质粒中回收的DNA片段为目的基因,插入粘粒载体pAxCAwt后获得了插入正确的克隆,并予以扩增备用。
2.探索出了稳定、有效的重组腺病毒构建方法,以此方法构建了含CDglyTK基因的腺病毒载体(Ad.CDglyTK)。扩增后重组腺病毒滴度高达1012pfu/ml,体外使用安全有效。
3.通过Western Blot方法检测肿瘤细胞转染重组腺病毒后融合基因蛋白的表达;通过对细胞存活曲线的D0、Dq值、SER的分析证实HSV-tk/GCV、CD/5-FC双自杀基因系统治疗后对肿瘤细胞有放射增敏作用。
4.研究结果表明双自杀基因联合放疗对荷大肠癌裸鼠治疗比单纯放疗、双前药治疗明显有效,荷瘤鼠生存时间明显延长。通过流式细胞仪、电镜分析双自杀基因联合放疗疗效与细胞凋亡有关。
主要结论:
1.DNA末端蛋白复合物可以使重组腺病毒的制备效率大大提高,降低野生型腺病毒的出现概率。实验获得的重组腺病毒滴度高、纯度好、毒性低。
2.实验所构建的携带CDglyTK基因的重组腺病毒载体,能高效转染大肠癌细胞。转染的肿瘤细胞能表达CDglyTK基因的融合蛋白,通过双前药治疗能获得明显的细胞杀伤作用和旁观者效应。
3.双自杀基因联合放疗能获得最大的肿瘤杀伤,二者有协同作用,并且有放射增敏作用。二者作用的机制可能是通过促进肿瘤细胞的凋亡获得的。
Suicide gene therapy has been a new hot spot of cancer gene therapy research. Previous studies focused mainly on single suicide gene therapy, which have virtues of easy control and induce immunity as well as tumor target function. However, every knife has two edges, suicide gene therapy has some deficiencies too. Many experiments have demonstrated that different malignant tumors have various responses to different system of suicide gene/prodrug and the bystander effect has distinctness too. On the other side, the product of double suicide gene has the activities of two enzymes. Cytotoxicity would strongly increased by the complementary of double suicide gene expressing products. In this experiment, we probe to effects of fusion gene combined with radiotherapy to human colorectal cells and nude mice bearing human colorectal carcinoma. For this purpose, we construct a replication-defective adenovirus vector (Ad.CDglyTK). Adenovirus vector is the most effective vector at present, which can receive an insertion of large gene. We can get a high titer also (1012-1013pfu/ml) through this method. Combination the irradiation enhancement effect of CD/5-FC and TK/GCV we can kill the tumor cells more strongly. At the same time, it unites the radiotherapy/ chemotherapy with gene therapy into one. We try to find a novel way and to provide a foundation of clinical malignant tumor’s therapy.
Methods:
1 The construction of cosmid vector inserting CDglyTK gene
(pAxcAwt/CDglyTK)
DNA fragment reclaimed from the plasmid of pWZLneoCDglyTK. Then the DNA was inserted into cosmid vector pAxcAw. The recombinant cosmid was amplified and was confirmed to be the correct gene . At last the correct clones containing target gene in right direction were selected.
2 The construction of replication-defective recombinant adenovirus coding for CDglyTK gene (Ad.CDglyTK)
Human embryonic kidney 293 cells cultured in a 6cm dish were transfected with DNA-TPC and pAxcAwt/CDglyTK by the calcium phosphate method. After 8-18 days, virus clones were isolated and propagated to for restriction analysis. The desired Ad vectors were purified by density gradient ultracentrifuge and titrated in 293 cells. Whether there has a little wild type adenovirus was also examined at the same time.
3 Transfection of Ad.CDglyTK to carcinoma cells and its effects of enhancement of radiosensitization in vitro
Three kinds of gastrointestinal cells(LoVo, HCT-8, SGC-7901) were
isolated and cultured. Transfection efficiency of Ad.CDglyTK on these cells was examined at various titrations by immunofluorescence staining. And then Co60 radiotherapy of different dose in vitro were completed compared with prodrug treatment of 5-FC or GCV. Cell doubling time was tested after three kinds of cells were transfected by recombinant adenovirus. Fusion protein expression was evaluated by Western blot respectively. The cell survival curve was calculated by cell cloning forming test. Cell survival rate was examined by MTT assay. Bystander effect in vitro was observed. D0,Dq of the cell survival curve were measured and the SER(Sensitizing Enhancement Ratio) were compared in different groups.
4 To research into the therapeutic effect of double suicide gene combining radiotherapy to nude mice bearing human colorectal carcinoma
First, the model of nude mice bearing carcinoma was established. Then the prodrug experiment was completed. In the period of prodrug treatment, Co60 therapy to nude mice in some groups was completed on the time. In the end, tumor of nude mice was examined. Cell apoptosis was checked through FCM method, DNA gel electrophoresis and transmission electron microscope .
Results:
1 The segment of DNA obtained from pWZLneoCDglyTK plasmid was identical to that expected. After the target DNA was inserted into cosmid vector pAxcAw, the recombinant cosmid was amplified and confirmed to be the correct gene.
2 An efficient and reliable method of constructing recombinant Ad vectors was established. Replication-defective adenovirus vectors coding for
引文
Bett AJ, Haddara W, Prevec L, et al. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3[J]. Proc Natl Acad Sci USA,1994,91(35):8802-8806.
2 Mizuguchi H, Kay MA. Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method. Hum Gene Ther, 1998, 9: 2577-2583.
Tashiro F, Niwa H, Miyazaki J. Constructing adenoviral vectors by using the circular form of the adenoviral genome cloned in a cosmid and the Cre-loxP recombination system. Hum Gene Ther. 1999,10(11): 1845 -1852.
He TC, Zhou S, da Costa LT, et al. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA. 1998,95(5):2509- 2514.
Collins J, Hohn B. Cosmids: a type of plasmid gene-cloning vector that is packageable in vitro in bacteriophage lambda heads. Proc Natl Acad Sci USA. 1978,75(9):4242-4246.
Miyake S, Makimura M, Kanegae Y, et al. Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl Acad Sci U S A. 1996 ,93(3):1320-1324.
魏道严,糜军,陈诗书. 细菌内同源重组法高效制备含CD、TK融合自
杀基因重组体腺病毒. 癌症,2000,19(9):399-403.
2 Mizuguchi H, Kay MA. Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method. Hum Gene Ther, 1998, 9: 2577-2583.
Tashiro F, Niwa H, Miyazaki J. Constructing adenoviral vectors by using the circular form of the adenoviral genome cloned in a cosmid and the Cre-loxP recombination system. Hum Gene Ther. 1999,10(11): 1845 -1852.
He TC, Zhou S, da Costa LT, et al. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA. 1998,95(5):2509- 2514.
Collins J, Hohn B. Cosmids: a type of plasmid gene-cloning vector that is packageable in vitro in bacteriophage lambda heads. Proc Natl Acad Sci USA. 1978,75(9):4242-4246.
Miyake S, Makimura M, Kanegae Y, et al. Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl Acad Sci U S A. 1996 ,93(3):1320-1324.
魏道严,糜军,陈诗书. 细菌内同源重组法高效制备含CD、TK融合自
杀基因重组体腺病毒. 癌症,2000,19(9):399-403.