经动脉导入脂质体联合转铁蛋白介导的p53基因治疗肝癌的实验研究

Experimental research on treating hepatic carcinoma by trans-arterial delivery of p53 gene mediated by lipsome combined transferring

目的探讨经导管动脉内联合应用转铁蛋白对脂质体介导的p53基因治疗肝癌的转染效率及治疗效果的影响。方法25只荷瘤兔（VX2肝癌模型兔）采用随机数字表法分为5组，每组5只。取0、100、200、300、400μg的转铁蛋白（Tf）分别与阳离子脂质体（LipofectAMINE）和真核细胞表达质粒（pCMV-myc—053）的复合体混合后，分别注入5组兔VX2肝癌模型的肿瘤供血动脉内，48h后提取肿瘤组织蛋白，采用免疫斑点法（Western blot）和免疫组化法检测基因的转染及其表达水平。对5个不同Tf剂量组053表达水平的比较，采用x^2检验。另取10只荷瘤兔分为2组，1组经肿瘤供血动脉注入Tf200μg及LipofectAMINE45μl和pCMV—myc—p53质粒15μg形成的复合体，另1组仅注入后两者的复合体，观察2组兔的肝功能、肿瘤体积和生存期。对2组实验兔同一时间点的肝功能检测结果采用成组t检验；肿瘤体积检查结果采用配对t检验；生存期采用单一样本均数t检验。结果Western blot半定量分析显示，含Tf组的实验兔053基因的转染及表达水平均较不含Tf组高。免疫组化染色显示，含0、100、200、300、400μg Tf的5组实验兔，053基因高度表达的阳性率分别为58．33％、69．44％、80．00％、83．33％、81．67％，含Tf组与不含Tf组之间差异有统计学意义（总体x^2=42．37，P〈O．01）；随着Tf的量从0增加到200μg，p53基因高度表达的比例逐渐增加，但Tf自200μg增加到400μg时，053基因高度表达比例差异无统计学意义（x^2分割：前3组x^2=4．8161，P〈0．05，后3组x^2=0．67，P〉0．05）。含Tf及不含Tf组实验兔术前及术后各时间点肝功能结果比较的差异无统计学意义（P值均〉0．05），术后第3、7、10、14、20、25、30天2组实验兔平均肿瘤体积分别为（4．33±0．72）、（5．65±0．85）、（7．12±0．99）、（8．44±1．23）、（9．21±1．51）、（10．32±1．78）、（11．55±1．90）cm^3和（4．36±0．66）、（4．86±0．68）、（5．12±0．73）、（5．72±0．93）、（6．13±1．26）、（6．68±1．38）、（7．02±1．55）cm^3，在同一时间点差异有统计学意义（t=4．59，P〈0．05），2组实验兔的平均生存时间分别为（28．6±2．2）及（35．8±3．1）d，差异有统计学意义（t=1．52，P〈0．05）。结论经动脉途径联合应用Tf是安全的，可明显提高脂质体介导的p53基因的转染效率和治疗效果。

Objective To investigate the trans-arterial delivery of p53 gene transfection efficiency and therapy effect on hepatic carcinoma in combination with transferrin mediated by liposome. Methods Twenty-five VX2 experimental rabbits were randomly divided into five groups, and the different doses of transferrin combined with p53-LipofectAMINE complex were delivered into the hepatic arteries of the VX2 hepatic carcinoma models. The tissue protein of the carcinoma was extracted after 48 h and the transfection efficiency and expression rate of p53 gene were analyzed by western blot and immune histochemical techniques, to inspect the expression proportion of p53 with different doses transferring. Another ten VX2 were divided into two groups, recombinant plasmid p53-LipofectAMINE complex and transferrin-p53-LipofectAMINE complex were delivered into the hepatic arteries in two groups respectively. The liver function, size of the tumor and survival time of the animals was compared between the two groups, and results were analyzed statistically. Results Semiquantitative analysis by Western Blot showed that the transfection and expression efficiency of p53 gene combined with transferrin were higher than those without it. By immune histochemical techniques, the p53 gene＇s positive rate of highly expression with various doses of transferrin were found to be different, and there was remarkable difference between the groups with and without transferring. They were 58. 33% , 69. 44% , 80. 00% , 83.33% , 81.67% respectively, there was remarkable difference between the groups with and without transferring （Totality： x^2 = 42. 37 ,P 〈 0. 01 ）. The p53 gene＇s positive rate of expression increased gradually as the doses of transferrin increasing from 0 up to 200 μg, but the differences of positive rate had no statistical significance as the doses of transferrin increasing from 200 up to 400 μg （ x^2 section ： 3 groups as former x^2 = 4. 82, P 〈 0. 05,3 groups as latter x^2 = 0.67, P 〈 0.05 ）. There was no statistical difference in the liver function at points of time between VX2 rabbits with and without transferring. But the tumors＇ sizes had significant difference at various points of time. Conclusion Liposome-mediated p53 gene on treating hepatic carcinoma by trans-arterial gene delivery combined with transferrin is safe, and it can markedly enhance transfection efficiency and improve the therapy effect of p53 gene.