经尿道灌注HSV-tk基因治疗大鼠膀胱癌的实验研究
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摘要
膀胱肿瘤是泌尿外科常见疾病，大部分移行细胞癌可以通过经尿道膀胱肿瘤切除和膀胱灌注BCG治疗。不幸的是，多数病人肿瘤会复发,因此人们从未停止对膀胱癌治疗的探索。在现有治疗研究中，基因治疗受到广泛关注。目前，药物敏感基因(或称自杀基因)是肿瘤基因治疗中研究较多目的基因之一。其通过目的基因酶蛋白的表达，使无毒或低毒的药物前体转化为强毒性物质，达到杀死肿瘤细胞的目的，同时，也引起邻近未转染细胞的死亡，即旁观者效应。其中，HSV-tk/GCV系统是较常用的一种，其编码的胸苷激酶能将核苷类似物(常用GCV或ACV)磷酸化，在细胞内代谢为毒性产物，后者能抑制细胞DNA聚合酶，将肿瘤细胞杀死。现在该基因被广泛应用于各种肿瘤基因治疗研究，其抗癌作用的有效性和安全性已得到初步证实。
在本实验室，刘为池，张荣贵对HSV-tk基因治疗膀胱肿瘤进行了广泛研究，发现其在膀胱肿瘤体外和皮下肿瘤模型实验中发挥了治疗作用。国外学者也观察到HSV-tk对裸鼠膀胱肿瘤的治疗作用。
膀胱解剖位置特殊，容易由体外通过尿道接近，由尿道膀胱灌注可以用于膀胱肿瘤基因治疗。为研究经尿道灌注HSV-tk基因治疗膀胱肿瘤，建立适当的动物原位膀胱肿瘤模型是必要的。由N-甲基－亚硝基脲(MNU)膀胱灌注诱导的膀胱肿瘤与人类膀胱肿瘤发生特点及病理学特征非常相似，可以认为是膀胱肿瘤研究的较理想模型之一。
本实验中，用MNU诱导的大鼠原位膀胱肿瘤模型，以逆转录病毒载体，脂质体/DNA复合物，裸质粒DNA形式，通过膀胱灌注转导HSV-tk基因。
实验方法和结果总结如下：
1. 大鼠膀胱肿瘤的诱导:雌性Wistar大鼠 ,通过MNU 2.5mg膀胱灌注诱导肿瘤，每两周一次，共4次。膀胱肿瘤在8-10周时产生，成瘤率100%，82%为膀胱移行细胞癌。HSV-tk基因治疗开始于第一次MNU灌注后第13周。
2.大鼠随机分为四组，每组20只。裸质粒组：质粒DNA 50ug 膀胱灌注，逆转录病毒组：病毒上清1×106cfu (0.5ml)膀胱灌注；脂质体组：质粒DNA 50ug+ 脂质体150ug 膀胱灌注；对照组：0.5ml生理盐水膀胱灌注。第二天所有动物给予GCV40mg/kg/d腹腔内注射，连续6天。
3. 从膀胱肿瘤组织和肾脏组织中提取RNA，用HSV-tk特异引物，通过RT-PCR,
发现从逆转录病毒组和脂质体/DNA复合物组膀胱肿瘤组织RNA样品中可检测到HSV-tk基因扩增产物。证实：经尿道膀胱灌注可以向肿瘤细胞转染HSV-tk基因。
4.肿瘤生长情况观察：治疗前后，膀胱肿瘤超声检查大小，及以称量膀胱总重量作为肿瘤发生率、肿瘤大小的指标。观察发现治疗组：逆转录病毒组和脂质体组与对照组有显著差别，裸质粒组与对照组无显著差别。
5. 通过DNA 电泳，TUNEL 法凋亡检测，发现诱导凋亡可能是自杀基因治疗的重要机制之一。
本研究表明：在原位膀胱肿瘤模型中，逆转录病毒载体和脂质体通过尿道膀胱灌注可以成功转导HSV-tk基因；HSV-tk/GCV系统对膀胱肿瘤生长有抑制作用。
Bladder cancer is one of the conditions most frequently treated by urologists. Most bladder tumors are superficial transitional cell carcinomas , which can be treated with transurethral resection of the bladder tumor (TUR- BT) and with intravesical instillation of Bacilli Calmette -Guerin (BCG).Unfortunately , tumors recur in the majority of patients .
Although a number of new treatment strategies have been tested recently , the combination of the herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir (GCV) is most studied because HSV-tk, as suicide genes, can induce cell death only in dividing cells such as those found in tumors .HSV-tk converts the protoxic nucleoside analog GCV into a highly toxic phosphorylated GCV that acts as a chain terminator of DNA synthesis and an inhibitor of DNA polymerase , selectively killing dividing cells . Many studies have examined gene therapy for cancer using the HSV-tk/GCV system in vitro and in vivo.
The bladder would seem to be an especially appropriate organ for this technique because it is easily approached from outside the body . Accordingly, the transurethral intravesical instillation is less invasive than either direct puncture of the tumor or organ or systemic administration .
Most animal studies are currently performed by using transplanted tumors produced by implantation of cell lines . Subcutaneous heterotopic graft models are often used because they are relatively easy to generate. Unfortunately , this approach completely ignores the histologic and anatomic specificity of the organs under investigation .In contrast , N-methyl -N-nitrosourea (MNU )-induced tumors are very similar to human bladder transitional cell carcinomas and are considered to be excellent models of clinical bladder cancer.
In this study , we describe our use of the MNU-induced rat bladder tumor to evaluate in vivo retrovirus -mediated and Liposome-mediated gene transfer .An retrovirus vector containing HSV-tk gene or Liposome/DNA complexes was transurethrally instilled into the bladder , after which the distribution of the gene was examined by reverse transcriptase -poly-merase chain reaction ( RT-PCR).
The methods and results are listed as follows:
1. The orthotopic rat bladder cancer were induced in femal Wistar rats by intravascular administration of the carcinogen ,MNU. The administration way of a total dose of 10 mg on 4 fraction of 2.5 mg at 2 -weekly intervals produced a 100% incidence of bladder tumors after 8 weeks . Histopathological characteristics of bladder tumor induced by MNU were basically similar to those of human bladder tumor .
2. The orthotopic rat bladder tumor models were instillated retrovirus vector containing HSV-tk gene or Liposome/DNA complexes or naked DNA plasmid , saline. GCV was given the next day after instillation for 6 days.
3. We harvested RNA from kidney tissue and bladder tumor after 48 hours since the rats' instillation and subjected the RNA to RT-PCR ,using HSV-tk -specific primers . A 681 -base pair HSV-tk -specific band was obtained exclusively, which conformed that the distribution of gene after instillation into the bladder was transferred into the tumor cells by retrovirus vector or liposome and expressed successfully.
4. The tumor volume were examinated by ultrasound before and after HSV-tk/GCV system administration. Tumor incidence and tumor weight , defined as the weight of the tumored bladder , were determined on day 6 and 10 after GCV intraperitoneal injection for 6 days . Significant difference was observed between retrovirus vector or liposome -treated rats and control rats .
5. DNA extraction, electrophoresis and immunohistochemistry technique were used to observe apoptosis of the bladder tumor cells treated by HSV-tk /GCV system . Inducing apoptosis of tumor cells might be the important mechanism of HSV-tk/ GCV system.
Our study show that retrovirus and liposome could transfer the HSV-tk gene effectively in the orthotopic rat bladder tumor models and HSV-tk/GCV system can acts as a candidate tr
在本实验室，刘为池，张荣贵对HSV-tk基因治疗膀胱肿瘤进行了广泛研究，发现其在膀胱肿瘤体外和皮下肿瘤模型实验中发挥了治疗作用。国外学者也观察到HSV-tk对裸鼠膀胱肿瘤的治疗作用。
膀胱解剖位置特殊，容易由体外通过尿道接近，由尿道膀胱灌注可以用于膀胱肿瘤基因治疗。为研究经尿道灌注HSV-tk基因治疗膀胱肿瘤，建立适当的动物原位膀胱肿瘤模型是必要的。由N-甲基－亚硝基脲(MNU)膀胱灌注诱导的膀胱肿瘤与人类膀胱肿瘤发生特点及病理学特征非常相似，可以认为是膀胱肿瘤研究的较理想模型之一。
本实验中，用MNU诱导的大鼠原位膀胱肿瘤模型，以逆转录病毒载体，脂质体/DNA复合物，裸质粒DNA形式，通过膀胱灌注转导HSV-tk基因。
实验方法和结果总结如下：
1. 大鼠膀胱肿瘤的诱导:雌性Wistar大鼠 ,通过MNU 2.5mg膀胱灌注诱导肿瘤，每两周一次，共4次。膀胱肿瘤在8-10周时产生，成瘤率100%，82%为膀胱移行细胞癌。HSV-tk基因治疗开始于第一次MNU灌注后第13周。
2.大鼠随机分为四组，每组20只。裸质粒组：质粒DNA 50ug 膀胱灌注，逆转录病毒组：病毒上清1×106cfu (0.5ml)膀胱灌注；脂质体组：质粒DNA 50ug+ 脂质体150ug 膀胱灌注；对照组：0.5ml生理盐水膀胱灌注。第二天所有动物给予GCV40mg/kg/d腹腔内注射，连续6天。
3. 从膀胱肿瘤组织和肾脏组织中提取RNA，用HSV-tk特异引物，通过RT-PCR,
发现从逆转录病毒组和脂质体/DNA复合物组膀胱肿瘤组织RNA样品中可检测到HSV-tk基因扩增产物。证实：经尿道膀胱灌注可以向肿瘤细胞转染HSV-tk基因。
4.肿瘤生长情况观察：治疗前后，膀胱肿瘤超声检查大小，及以称量膀胱总重量作为肿瘤发生率、肿瘤大小的指标。观察发现治疗组：逆转录病毒组和脂质体组与对照组有显著差别，裸质粒组与对照组无显著差别。
5. 通过DNA 电泳，TUNEL 法凋亡检测，发现诱导凋亡可能是自杀基因治疗的重要机制之一。
本研究表明：在原位膀胱肿瘤模型中，逆转录病毒载体和脂质体通过尿道膀胱灌注可以成功转导HSV-tk基因；HSV-tk/GCV系统对膀胱肿瘤生长有抑制作用。
Bladder cancer is one of the conditions most frequently treated by urologists. Most bladder tumors are superficial transitional cell carcinomas , which can be treated with transurethral resection of the bladder tumor (TUR- BT) and with intravesical instillation of Bacilli Calmette -Guerin (BCG).Unfortunately , tumors recur in the majority of patients .
Although a number of new treatment strategies have been tested recently , the combination of the herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir (GCV) is most studied because HSV-tk, as suicide genes, can induce cell death only in dividing cells such as those found in tumors .HSV-tk converts the protoxic nucleoside analog GCV into a highly toxic phosphorylated GCV that acts as a chain terminator of DNA synthesis and an inhibitor of DNA polymerase , selectively killing dividing cells . Many studies have examined gene therapy for cancer using the HSV-tk/GCV system in vitro and in vivo.
The bladder would seem to be an especially appropriate organ for this technique because it is easily approached from outside the body . Accordingly, the transurethral intravesical instillation is less invasive than either direct puncture of the tumor or organ or systemic administration .
Most animal studies are currently performed by using transplanted tumors produced by implantation of cell lines . Subcutaneous heterotopic graft models are often used because they are relatively easy to generate. Unfortunately , this approach completely ignores the histologic and anatomic specificity of the organs under investigation .In contrast , N-methyl -N-nitrosourea (MNU )-induced tumors are very similar to human bladder transitional cell carcinomas and are considered to be excellent models of clinical bladder cancer.
In this study , we describe our use of the MNU-induced rat bladder tumor to evaluate in vivo retrovirus -mediated and Liposome-mediated gene transfer .An retrovirus vector containing HSV-tk gene or Liposome/DNA complexes was transurethrally instilled into the bladder , after which the distribution of the gene was examined by reverse transcriptase -poly-merase chain reaction ( RT-PCR).
The methods and results are listed as follows:
1. The orthotopic rat bladder cancer were induced in femal Wistar rats by intravascular administration of the carcinogen ,MNU. The administration way of a total dose of 10 mg on 4 fraction of 2.5 mg at 2 -weekly intervals produced a 100% incidence of bladder tumors after 8 weeks . Histopathological characteristics of bladder tumor induced by MNU were basically similar to those of human bladder tumor .
2. The orthotopic rat bladder tumor models were instillated retrovirus vector containing HSV-tk gene or Liposome/DNA complexes or naked DNA plasmid , saline. GCV was given the next day after instillation for 6 days.
3. We harvested RNA from kidney tissue and bladder tumor after 48 hours since the rats' instillation and subjected the RNA to RT-PCR ,using HSV-tk -specific primers . A 681 -base pair HSV-tk -specific band was obtained exclusively, which conformed that the distribution of gene after instillation into the bladder was transferred into the tumor cells by retrovirus vector or liposome and expressed successfully.
4. The tumor volume were examinated by ultrasound before and after HSV-tk/GCV system administration. Tumor incidence and tumor weight , defined as the weight of the tumored bladder , were determined on day 6 and 10 after GCV intraperitoneal injection for 6 days . Significant difference was observed between retrovirus vector or liposome -treated rats and control rats .
5. DNA extraction, electrophoresis and immunohistochemistry technique were used to observe apoptosis of the bladder tumor cells treated by HSV-tk /GCV system . Inducing apoptosis of tumor cells might be the important mechanism of HSV-tk/ GCV system.
Our study show that retrovirus and liposome could transfer the HSV-tk gene effectively in the orthotopic rat bladder tumor models and HSV-tk/GCV system can acts as a candidate tr
引文
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