重组质粒pIRES-GM-CSF-IL-21的构建及其治疗小鼠肝癌移植瘤的疗效

目的 构建重组质粒plRES-GM-CSF-IL-21，观察其在小鼠体内的抑瘤作用。方法 将体外培养的肝癌H22细胞接种于小鼠肝左叶，成瘤后，将小鼠分为5组，每组10只，分别尾静脉注射重组质粒plRES-GM-CSF-IL-21、plRES-GM-CSF、plRES-IL-21、plRES和PBS，观察各组荷瘤小鼠的肿瘤生长情况以及GM-CSF和IL-21的抗肿瘤效应。采用酶联免疫吸附法检测小鼠血清γ干扰素(lFN-γ)和白细胞介素2(IL-2)的含量。采用四甲基偶氮唑蓝法检测小鼠脾脏自然杀伤细胞(NK)和细胞毒T淋巴细胞(CTL)的活性。结果 H22组、H-22/Neo组、H22/GM-CSF组、H22/IL-21组和H22/GM-CSFIL-21组小鼠的肿瘤重量分别为(1.591±0.280)g、(1.489±0.155)g、(0.603±0.223)g、(0.583±0.290)g和(0.303±0.323)g，H22/GM-CSF-IL-21组、H22/IL-21组和H22/GM-CSF组小鼠的肿瘤重量明显低于H22组和H22/Neo组(P＜0.01)，H22/GM-CSF-IL-21组小鼠的肿瘤重量明显低于H22/GM-CSF组和H22/IL-21组(P＜0.05)。与H22/GM-CSF组和H22/IL-21组比较，H22/GM-CSF-IL-21组小鼠血清中IFN-γ和IL-2的浓度显著升高(P＜0.01)，而H22组和H22/Neo组小鼠血清中IFN-Y和IL-2的浓度显著降低(P＜0.01)。与H22/GM-CSF组和H22/IL-21组比较，H22/GM-CSF-IL-21组小鼠脾脏CTL和NK细胞的杀伤活性显著增强(P＜0.01)，而H22组和H22/Neo组小鼠脾脏CTL和NK细胞的杀伤活性显著降低(P＜0.01)。结论 重组质粒plRES-GM-CSF-IL-21对小鼠肝癌移植瘤具有明显的抗肿瘤效应，并能促进小鼠体内IFN-γ和IL-2的分泌，增强脾脏中NK和CTL细胞的活性，其效果优于单—GM-CSF或II-21基因治疗。

Construction of recombinant plasmid plRES-GM-CSF-IL-21 and its therapeutic effect on orthotopically transplanted liver cancer in mice

Objective To construct a recombinant plasmid plRES-GM-CSF-IL-21, and to investigate its antitumor effect on tumors in the mice. Methods Fifty Bal b/c mice were included in this study. Cultured hepatoma H22 cells were inoculated in the left lobe of the liver to develop orthotopically transplanted liver tumor models. The mice with orthotopically transplanted liver tumor were randomly divided.into 5 groups (n ＝ 10) : ( 1 ) Each mouse received injection of recombinant plasmid plRES-GM-CSF-IL-21 ;(2) Received injection of plasmid plRES-GM-CSF; (3) plRES-IL-21 ; (4) Received injection of ampty plasmid plRES (H22/neo group); (5) Received injection of PBS (H22 group ) via the tail vein,respectively. Therefore, the anti-rumor effect was induced by both GM-CSF and IL-21, or by either of them alone. The serum levels of IFN-γand IL-2 were detected by ELISA, and the cytotoxicity of spleen NK and CTL cells were tested by MTT colorimetry. Results Comparing with the H22 and H22/Neo groups, the tumor weight in the mice of H22/GM-CSF group was (0.603 ±0.223)g, H22/IL21-treated group (0.583 ±0.290)g and H22/GM-CSF-IL21-treated group (0.303 ±0. 323)g, significantly lower than that in the H422 group(1.591 ±0.280)g]and H22/Neo group(1.489 ±0. 155) g]. Among them the tumor growth was most significantly inhibited in the H22/GM-CSF-IL-21 group (0. 303 ± 0. 323 ) g, compared with that of H22 and H22/neo groups (P ＜0.01 ). But there was no significant difference between the tumor weights of the H22/GM-CSF group and H22/IL-21 group, and between the tumor weights of the H22 and H22/Neo groups (P ＞0.05). The levels of IFN-γand IL-2 in peripheral blood of mouse models treated with H22/GM-CSFIL-21 were significantly increased than that in the H22/GM-CSF group and H22/IL-21 group (all P ＜0.01), but significantly decreased in the H22group and H22/Neo group (P ＜ 0. 01 ). The anti-tumor activity of splenic NK cells and CTLs in the H22/GM-CSF-IL21 group was significantly enhanced ( P ＜0.01 ), compared with the significantly decreased in the H22 and H22/Neo groups. ConclusionsOur results demonstrate apparent antitumor effect of the plasmid pIRES-GM-CSF-IL-21 on the orthotopically transplanted liver tumor in mice. The combination of both pIRES-GM-CSF and IL-21 is more effective than that of plRES/IL21 or plRES/GM-CSF treatment alone. In adition, the plasmid pIRES-GM-CSF-IL-21 can also promote the secretion of IFN-γand IL-2 in vivo, and enhance the cytotoxic activity of splenic NK and CTLs against the transplanted liver tumor.