重组新城疫病毒rClone30-CD/5-FC系统的抗肿瘤活性

Antitumor activity of the recombinant rClone30-CD/5-FC system

本研究提取大肠杆菌JM109菌株的全基因组,克隆获得CD基因,将CD基因和IRES-EGFP基因构建到pEE12.4表达载体上,形成pEE12.4IE-CD真核表达载体。用该表达载体转染人肝癌细胞后,以不同浓度的5-氟胞嘧啶(5-flucytosine,5-FC)处理细胞,采用噻唑蓝(MTT)法测定CD/5-FC系统对人肝癌细胞HepG2的杀伤效果。当前导药物5-FC的浓度分别为10、20和30 mmol.L 1时,CD/5-FC系统对HepG2细胞的杀伤效率分别为18.07%、42.98%和62.20%,与对照组相比较差异具有统计学意义。急性毒性实验结果表明,小鼠尾静脉注射不同剂量5-FC,组间剂量比为1∶0.5,采用改良寇氏法测定5-FC对小鼠的LD50为507 mg.kg 1,95%可信限为374～695 mg.kg 1。再以LD50实验中最大LD0剂量为基准,组间剂量差值为10 mg.kg 1,测得最大安全剂量为200 mg.kg 1,并对体重、体征指标进行跟踪观察。这些检测结果可为动物实验奠定基础。将CD基因连于新城疫病毒(NDV)rClone30载体中并建立肿瘤动物模型,抑制肿瘤实验结果表明,重组新城疫病毒rClone30-CD/5-FC系统具有较高的体内抗肿瘤活性。综上所述,克隆获得的CD基因具有生物学活性,重组rClone30-CD/5-FC系统是一种潜在的治疗肿瘤的方法。

5-Flucytosine （5-FC） could be changed to 5-fluorouracil （5-FU） by cytosine deaminase （CD）, the latter is able to kill cancer cells. However, there is no efficient method to deliver the CD gene into the tumor cells, which hampers the application of the suicide gene system. In this experiment, for the first time, the NDV has been utilized as a vector to deliver the CD gene into the cancer cells, the virus can infect the cancer cells specifically, replicate and assemble, while the cytosine deaminase is expressed. Then the CD converts the prodrug 5-FC into 5-FU to achieve the purpose of inhibiting tumor. Firstly, the whole genome of E. coli JM109 was extracted, and the CD gene was obtained by cloning method. Then the CD and IRES-EGFP were ligated into the pEE12.4 expression vector to become a recombinant pEE12.4IE-CD eukaryotic expression plasmid. The human liver cancer cells were transfected with the plasmid. The cells were treated with different concentrations of 5-FC, MTT method was used to determine the killing effect of CD/5-FC system on the human liver cancer cells. The cell deaths were 18.07%, 42.98% and 62.20% respectively when the concentrations of prodrug were at 10, 20 and 30 mmol·L？1. In 5-FC acute toxicity experiment, Kunming mice were injected with different concentrations of 5-FC at intervals of 1∶0.5. The LD50 of 5-FC through iv injection was determined by improved Karber’s method, the LD50 was 507 mg·kg？1 and the 95% confidence limit was 374？695 mg·kg？1. According to the maximum LD0 dose of the LD50, the maximum safe dose was 200 mg·kg？1. Body weight and clinic symptoms of the experimental animals were observed. These results laid the foundation to verify the antitumor effect and safety of CD/5-FC system in animal models. The CD gene was ligated into the NDV （rClone30） carrier, then the tumor-bearing animal was established to perform the tumor inhibiting experiment. The result showed that the recombinant rClone30-CD/5-FC system has a high antitumor activity in vivo. To summarize, CD gene has been cloned and its bioactivity has been confirmed in the mammalian cells. It is the first time in this study to utilize the recombinant NDV to deliver the CD gene into the tumor cells; our result proves the rClone30-CD/5-FC system is a potential method for cancer therapy.