重组溶瘤腺病毒rAd-E1A-CDglyTK的构建和体内、外抑杀瘤作用的实验研究

目的构建重组溶瘤腺病毒r Ad-E 1A-CDgly TK,并研究其在体内、体外杀瘤作用的应用。方法选取2011年2月~2013年12月北华大学附属医院收治的卵巢癌患者100例,随机均分为治疗组与对照组（n=50）,治疗组采取患者的癌症细胞SKOV 3细胞,将病毒质粒经介质传染给癌症细胞并进行PCR体外扩增,经紫外线分光光度法检测病毒颗粒浓度,以TCID 50法检测感染性滴度,并计算病毒比活性,保证植入的病毒含有E 1A基因片段;对照组患者进行体内植入重组溶瘤腺病毒r Ad-E 1A-CDgly TK,观察抑制杀瘤疗效。结果经PCR体外扩增,得到DNA片段,进行凝胶电泳,可知植入的重组溶瘤腺病毒含有E 1A基因片段。通过紫外线分光光度法测得治疗组病毒颗粒浓度比上测得的感染性滴度得到病毒比活性为3.56%,显著高于对照组2.01%（P〈0.05）。进行体外抑瘤实验中,治疗组注入病毒成分为r Ad-E 1A-CDgly TK,其对SKOV 3细胞的生长抑制率为（16.59±1.57）,显著高于含有r AdCDgly TK的对照组生长抑制率（10.80±1.47）（P〈0.05）。体内抑瘤实验中,治疗组生长抑制率为（26.12±8.11）,显著高于对照组（1.69±4.12）,差异有统计学意义（P〈0.05）。结论重组溶瘤腺病毒r Ad-E 1A-CDgly TK具有较好的抑制杀瘤作用,其中体内杀瘤强于体外杀瘤,疗效显著,值得推广使用。

Objective To build recombinant adenovirus soluble tumor rAd-E1A-CDglyTK, and to study the application of the in vivo and in vitro killing tumor effect.Methods 100 cases of patients with ovarian cancer randomly divided into the treatment group and the control group, 50 cases in each group, the treatment group was taken the patient＇s cancer cell SKOV3 cell, the virus plasmid by the medium transmission to cancer cells in vitro and PCR amplification, the ultraviolet spectrophotometry to detect virus particles concentration, degree of TCID50 method in detecting infectious droplets, and calculate the virus than activity, to ensure the implant virus contains E1A gene fragment, the control group patients were implanted with recombinant adenovirus rAd - soluble tumor E1A - CDglyTK, to observe inhibiting tumor response.Results To get DNA fragments by PCR amplification in vitro, it was implanted recombinant adenovirus containing soluble tumor E1A gene fragment for gel electrophoresis. The degree of infectious droplets get virus activity of the treatment group virus particles concentration ratio by ultraviolet spectrophotometry measured（3.56%） was higher than the control group（2.01%）. The treatment group was injected the virus components in rAd - E1A - CDglyTK in vitro tumor suppression experiments, the SKOV3 cells growth inhibition rate （16.59±1.57） was significantly higher than the control group containing rAd-CDglyTK growth inhibition rate （10.80±1.47）. The treatment group growth inhibition rate （26.12±8.11） was high than the control group （1.69±4.12） in vivo tumorsuppression experiments, the difference had statistically significant （P〈0.05）.Conclusion Recombinant adenovirus rAd - soluble tumor E1A-CDglyTK has good inhibitory effect of killing tumor, killing tumor in the body is stronger than killing tumor in vitro, the curative effect is distinct, it should use to be promoted.