靶向重组腺病毒介导Hsp70基因表达对大鼠胰腺癌移植瘤的抑制作用

Recombinant adenovirusmediated Hsp70 gene expression inhibits tumor growth in a rat xenograft model of pancreatic cancer

目的:观察重组腺病毒Ad5-pCEA-Hsp70介导的Hsp70基因表达对大鼠胰腺癌移植瘤生长的影响,并分析其相关机制.方法:建立大鼠胰腺癌皮下移植瘤模型,随机分为3组,分别给予Ad5-pCEA-Hsp70、Ad5-control以及PBS进行治疗.对比不同时间点3组肿瘤大小,评价抑瘤效果:通过ELISA的方法检测外周血Hsp70蛋白含量及细胞因子INF-γ、TNF-α、IL-6的含量;取动物的移植瘤常规HE染色,观察淋巴细胞浸润情况;分离动物脾脏单核细胞,通过流式细胞仪检测CD83+细胞比例,并观察脾淋巴细胞对体外培养的大鼠胰腺癌细胞的杀伤能力.结果:治疗后第4、6、8周,Ad5-pCEA-Hsp70组肿瘤体积分别与Ad5-control组及PBS组相比(724.4mm3±81.6mm3vs901.3mm3±103.9mm3、987.5mm3±126.0mm3;681.3mm3±64.9mm3vs1270.6mm3±131.6mm3、1398.5mm3±193.0mm3;648.0mm3±65.9mm3vs1487.0mm3±243.0mm3、1660.0mm3±167.0mm3),差异均具有统计学意义(均P<0.01).Ad5-pCEA-Hsp70组动物外周血Hsp70蛋白及细胞因子INF-γ、TNF-α、IL-6的含量均显著高于Ad5-control组和PBS组(均P<0.01).HE染色发现,与Ad5-control组和PBS组相比,Ad5-pCEA-Hsp70组有较多的淋巴细胞浸润.经流式细胞仪检测,Ad5-pCEA-Hsp70组脾单核细胞中CD83+细胞比例显著高于Ad5-control组及PBS组(10.8±1.3%vs5.1±0.6%、4.8±0.6%;均P<0.01).脾淋巴细胞介导的CTL实验中,当效靶细胞比为1:1时,3组动物之间并无统计学差异(P>0.05),但随着效靶细胞比的增加,Ad5-pCEA-Hsp70组对胰腺癌细胞的杀伤能力较Ad5-control组和PBS组明显增加(P<0.05,P<0.01).结论:重组腺病毒Ad5-pCEA-Hsp70介导的Hsp70基因表达对大鼠胰腺癌动物模型的肿瘤生长具有抑制作用,其机制与促进树突状细胞(dendritic cells,DC)的成熟、诱导细胞因子INF-γ、TNF-α、IL-6的分泌、促进淋巴细胞浸润有关.

AIM： To observe the effect of recombinant ad- enovirus Ad5-pCEA-Hsp70-mediated Hsp70 gene expression on tumor growth in a rat xeno- graft model of pancreatic cancer, and to analyze the underlying mechanism. METHODS： A rat xenograft model of pancreatic cancer was established, and model animals were randomly divided into three groups, which weregiven Ad5-pCEA-Hsp70, Ad5-control and PBS treatment, respectively. Antitumor effect was evaluated by comparing tumor size at differ- ent time points among the three groups. ELISA was used to detect the peripheral blood levels of Hsp70 protein, INF-γ, TNF-α and IL-6. HE stain- ing was used to detect lymphocyte infiltration. Animal spleen mononuclear cells were isolated to determine the proportion of CD83＋ cells by flow cytometry. Cell-killing ability of spleen lymphocytes was observed in vitro. RESULTS： At 4, 6, and 8 weeks after treatment, tumor volume in the Ad5-CEA-Hsp70 group was significantly lower than that in the Ad5-con- trol group and PBS group （724.4mm3 ±81.6 mm3 vs 901.3 mm3 ± 103.9 mm3, 987.5 mm3 ± 126.0 mm3; 681.3 mm3 ±64.9 mm3 vs 1 270.6 mm3 ± 131.6 mm3, 1 398.5 mm3 ＋ 193.0 mm3; 648.0 mm3 ± 65.9 mm3 vs 1 487.0 mm3 ± 243.0 mm3, 1 660.0 mm3 ± 167.0 mm3; all P 〈 0.01）. The levels of Hsp70 protein and cytokines INF-γ, TNF-α and IL-6 in peripheral blood in the Ad5-pCEA-Hsp70 group were significantly higher than those in the Ad5-control group and PBS group （all P 〈 0.01）. Compared to the Ad5-control group and PBS group, Ad5-pCEA-Hsp70 group had more lym- phocytic infiltration. The proportion of CD83＋ cells in the Ad5-pCEA-Hsp70 group was signifi- cantly higher than that in the Ad5-control group and PBS group （10.8% ± 1.3% vs 5.1%± 0.6%, 4.8% ± 0.6%; both P 〈 0.01）. In the lymphocyte- mediated CTL experiment, when the cell ratio of effect： target was 1：1, there was no significant difference in the cell killing ability among the three groups （P 〉 0.05）, but with the increase in the effect： target cell ratio, the cell killing ability in the Ad5-pCEA-Hsp70 group was significantly increased （P 〈 0.05, P 〈 0.01）. CONCLUSION： Hsp70 gene expression medi- ated by recombinant adenovirus Ad5-pCEA- Hsp70 could inhibit tumor growth in a rat xeno- graft model of pancreatic cancer via mechanisms that are related to the promotion of dentritic cell maturation, induction of cytokine secretion, andpromotion of lymphocyte infiltration.