高生物安全性人内皮抑素载体抑瘤作用的研究

The antitumor effect of the high bio-safety vector containing human endostatin

目的 :探索利用内皮抑素 (Endostatin)进行肿瘤抗血管基因治疗的效果与安全性。方法 :以pVAX1为载体 ,构建含IgGγ链信号肽序列和Endostatin基因的重组载体pVAX sEN。重组子经KpnI、EcoRI双酶切及测序鉴定。建立小鼠肝癌细胞H2 2动物模型 ,分别瘤内注射重组载体pVAX sEN、空载体、生理盐水(NS) ,每周 2次 ,测量肿瘤体积 ,ELISA检测肿瘤局部Endosatin的表达量 ,流式细胞术检测肿瘤细胞的凋亡。同时 ,10 0 μg高剂量pVAX sEN裸DNA注射实验动物 ,观察动物的一般状况 ,2 0d后处死动物取其内脏病理切片H E染色。结果 :测序结果表明正确构建了含有信号肽及Endostatin的真核表达载体。瘤内注射pVAX sEN后 ,ELISA检测到实验组肿瘤局部Endostatin表达量为 (2 0 1± 3 1)ng/ml,而空载体及NS对照组瘤内未见Endostatin特异性表达 ;瘤体测量证明pVAX sEN可以抑制肿瘤的生长 ,重组载体组肿瘤的平均体积为 (0 4± 0 3)cm3 ,显著低于对照组 [空载体和NS对照组分别为 (1 6± 0 4 )cm3 、(1 9± 0 6 )cm3 ,P <0 0 5 ]。pVAX sEN可以增加肿瘤细胞的凋亡 ,实验组、空载体和NS对照组肿瘤细胞的凋亡率分别为 (5 1 9± 3 16 ) %、(2 4 6± 4 4 3) %、(18 8± 1 2 ) % ,实验组与两组差异均有统计学意义 (P <0 .0 1)。

Objective:To explore a safe and efficient way in gene therapy on tumor using endostatin.Methods:Endostatin gene with signal sequence of human IgG γ chain was amplified by PCR and cloned into pVAX1 plasmid to construct a recombinant plasmid named as pVAX-sEN.The recombinant plasmid was detected with EcoRⅠ/KpnⅠ and DNA sequencing.BalB/c mice bearing with hepatoma cells H22 were treated with naked pVAX-sEN ,pVAX1 and NS respectively.The tumor size was measured to observe the antitumor effect.Expression of endostatin in the treated site was assayed with ELISA.The apoptosis rate of the tumor cells was detected by flow cytometry.The 100μg naked pVAX-sEN was injected into the experimental mice to observe general effect.Results:DNA sequencing showed that endostatin gene together with the signal sequence were correctly cloned.The dose of endostatin expressed in the experiment tumor site was(201±3.1)ng/ml.There was no specific expression in tumor site of the control group.The tumor size demonstrated the recombinant plasmid could inhibit the growth of H22.The average tumor volume was(0.4±0.3)cm 3 in the gene therapy group but (1.6±0.4)cm 3 and (1.9±0.6)cm 3 in the pVAX1 and NS control group(P<0.05).The apoptotic rate in tumor cells in the experiment,pVAX1 and NS groups were(51.9±3.16)%?(24.6±4.43)%?(18.8±1.2)% respectively which was statistically significant (P<0.01).The general condition of the BalB/c mice was good and there was no inflammatory and other damnification in pathological slice after the high dose of pVAX-sEN was injected into the mice.Conclusion:pVAX-sEN could distinctly inhibit the growth of H22 in vivo and promote tumor cell apoptosis.There was no side effect after high dose of pVAX-sEN was injected into the experimental mice which showed pVAX-sEN was a safe and efficient vector in gene therapy.