腺病毒介导hIL-24抑制裸鼠肝癌荷瘤生长及其机制研究

为了研究腺病毒hIL-24抑制SMMC-7721肝癌细胞裸鼠荷瘤的生长抑制及其作用机制,构建了重组腺病毒载体pAdeasy-1-pTrack-CMV-hIL-24(Ad-hIL-24),经PacI线性化后转染QBI-293A细胞,多轮感染后收获高效价腺病毒重组病毒子。用SMMC-7721肝癌细胞使16只裸鼠致瘤,然后随机NS分成干预组、5-Fu组、Ad组和Ad-hIL-24组,进行抗肿瘤试验。四组均使用瘤体内注射干预用药,100μL/只NS组、Ad组(107pfu)和Ad-hIL-24组(107pfu)隔日一次,共注射5次;5-Fu组20μg/kg,连续注射5d。用药前、用药后1周和2周分别测皮下瘤体的长径(L)、短径(W),计算出瘤体体积,治疗开始后第15天,将裸鼠脱颈处死,摘取瘤体称重,计算出抑瘤率。显微镜观察细胞生长形态、免疫组化法测caspase3蛋白表达、P53及P27核内抑癌基因表达、CD34染色标记测定的MVD及VEGF蛋白表达。与NS组比较,Ad-hIL-24组与5-Fu组的抑瘤率分别为68.52%(P<0.01)和65.64%(P<0.01);镜下Ad-hIL-24组肿瘤组织的caspase3蛋白表达细胞数较其它3组呈显著性升高(P<0.01),P27核内抑癌基因表达细胞数也较NS组明显增高(P<0.01),CD34染色标记测定的CD34及VEGF较NS组和Ad组明显减少(P<0.001),P53未见明显变化。结论Ad-hIL-24可以显著抑制裸鼠SMMC-7721荷瘤的生长,其机制可能通过激活caspase途径、上调P27抑癌基因表达、抑制血管形成等多途径发挥抑瘤作用。

The Study of the Growth-Suppression and Mechanisms of Hepatocelluar Carcinoma Tumor in Nude Mice

Study effect and mechanisms of growth-suppression of hepatocelluar carcinoma (HCC) in nude mice. The construction of the pAdeasy-1-pTrack-CMV-hIL-24 recombined adenovirus vector (Ad-hIL-24) was completed and lineared with PacI. Ad-hIL-24 were transfected into QBI-293 cells and obtained. 16 nude mice of the subcutaneous tumor models were established with SMMC-7721 HCC and were randomly divided into NS, 5-Fu, Ad and Ad-hIL-24 groups. Then 100 microL NS, Ad (10(7) pfu) and Ad-hIL-24 (10(7) pfu) for each one were given respectively QOD, and 5-Fu (20 microg/kg) were injected Q.D., for 5 times, with intratumor injections. After 15 d, 16 mice were sacrificed and subcutaneous tumors were taken out. The volumes (before administration, 1 week and 2weeks after administration) were measured and the weights of tumor were weighed and ratios of tumor-suppression were calculated. The morphological changes of apoptotic tumor cells were observed under microscope. Caspase3, P53 and P27, CD34 and VEGF were tested in immunohistochemistry. In tumor subcutaneous model, compared with NS group, the ratios of tumor-suppression of Ad-hIL-24 group and 5-Fu group were 68.52% (P < 0.01) and 65.64 (P < 0.01), respectively. Caspase3 protein in Ad-hIL-24 group was higher than other 3 groups significantly (P < 0.01). The expression of P27 also differed from NS group (P < 0.01). CD34 and VEGF protein in Ad-hIL-24 group can inhibit neovascularization obviously (P < 0.001), compared with NS and Ad groups. Ad-hIL-24 inhibits the growth of SMMC-7721 HCC on nude mice's. The mechanisms of tumor-suppression may be multi-pathways such as the induction of caspase3 pathway, P27 activities and the antiangiogenic mechanism.