胸苷激酶系统血管靶向抗肝癌效应的机制

Therapeutic efficacy and mechanism of HSV-tk for targeting angiogenesis against hepatocellular carcinoma

目的探讨KDR为启动子的HSV-tk重组腺病毒对肿瘤血管内皮细胞的特异性杀伤效能及机制.方法采用pAdeasy系统,构建受KDR启动子或巨细胞病毒(CMV)启动子调控并可表达HSV-tk基因的重组腺病毒,体外分别感染人脐静脉血管内皮细胞系(HUVEC)和肝癌细胞系HepG2,并以MTT法检测其细胞增殖情况.人肝癌皮下移植瘤裸鼠随机分成更昔洛韦组(I),空载体病毒组(II),重组腺病毒CMV-tk组(III)及重组腺病毒AdKDR-tk组(IV).各治疗组瘤内分别注入重组腺病毒液及空载体病毒液.重组腺病毒治疗组在病毒给予24h后分别ipGCV,连续10d.对照组,ipGCV.观察瘤体大小及免疫组化法定量测定肿瘤微血管密度.结果病毒滴度均为1×1013pfu/L.感染复数(MOI)为100的条件下,GCV浓度由0增至50mg/L时感染含AdKDR-tk的HUVEC细胞和HepG2细胞存活率由(90.7±4.5)%和(91.8±4.3)%分别下降至(28.9±5.7)%和(75.4±2.9)%(P<0.01),而感染含AdCMV-tk的HUVEC细胞和HepG2细胞存活率分别下降至(17.6±2.5)%和(23.2±5.7)%(P>0.05).体内试验中,与对照组比较,Ⅲ,IV组经治疗后肿瘤的生长均受到了明显的抑制,其抑瘤率分别为(14.7±3.2)%和(23.6±5.6)%(P<0.05).组织内的平均血管密度(MVD),Ⅰ组为37.4±8.6,Ⅱ组为30.6±7.8,Ⅲ组为27.6±7.1,IV组为10.7±4.1.其中,Ⅲ组与Ⅱ组(P<0.05),IV组与Ⅱ组(P<0.01),IV组与Ⅲ组(P<0.01)之间比较均有统计学差异.结论KDR启动子介导的HSV-tk具有特异性杀伤肿瘤血管内皮细胞的作用.

AIM： To explore the therapeutic efficacy and mechanism of HSV-tk for targeting angiogenesis against hepatocellular carcinoma. METHODS： By using pAdeasy system, recombinant adenovirus containing kinase domain receptor （KDR） or cytomegalovirus （CMV） promoter-controlled HSV-tk gene （AdKDR- tk and AdCMV-tk） was constructed. The virus was used to infect KDR-expressed human umbilical venous endothelial cells （HUVEC） and KDR-unexpressed HepG2. Following administration of ganciclovir （GCV） , the survival rate of gene-transfected HUVEC and HepG2 was evaluated by using MTT method. Hepatocarclnomas were transplanted in 32 Balb/c mice with HepG2 cells, which were subsequently divided into 4 groups： GCV group （ Ⅰ ）, Ad group （Ⅱ）, AdCMV-tk group （Ⅲ） and AdKDR-tk group （Ⅳ）. Then selective administration of recombinant adenovirus or Ad intratumorally was performed in all rats. GCV was given at a dose of 100 mg/（kg · d） ip in the following days and for 10 d. Microvessel density （MVD） of tumor in all the treated animals was checked with the immunohistochemical methods and tumor burden was assessed 10 d before and after the last GCV administration. RESULTS： The pAdeasv system produced a high titer of the recombinant adenovirus [ （ 1 × 10^13 ） pfu/L]. When the multiplicity of infection （MOI） was 100, with increasing GCV concentration from 0 up to 50 mg/L, the survival rate of AdKDR- tk-transfected HUVEC and HepG2 decreased from （90. 7 ± 4.5）% and （91.8 ±4.3）% to （28.9±5.7）% and （75.4± 2.9 ） %, respectively （ P 〈 0.01 ）, while the survival rate of Ad- CMV-tk-transfected HUVEC and HepG2 declined from 100% to （17.6 ±2.5）% and （23.2 ±5.7）%, respectively （P〉0.05）. Compared with group Ⅰ , there was a decrease of tumor weight by （14.7 ±3.2）% in group Ⅲ and （23.6 ±5.6）% in group Ⅳ, respectively ; and there was a distinct difference between group Ⅲ and Ⅳ （P〈0.05）, The median MVD was 37.4 ±8.6,30.6± 7.8,27.6 ± 7.1,10.7 ± 4.1 （microvessels/mm^2） in group Ⅰ, Ⅱ, Ⅲ and Ⅳ, respectively; and there were significent differences between group Ⅲ and Ⅱ （P 〈 0. 05）, Ⅳ and Ⅱ （ P 〈 0.01 ）, Ⅳ and Ⅲ （P 〈 0.01 ）. CONCLUSION： KDR promoter-HSV-tk gene may effectually restrain the growth of tumor via targeting angiogenesis of hepatocellular carcinoma with treatment of GCV.