观察融合穿膜肽的HSP70基因联合CIK细胞对裸鼠胃癌移植瘤的治疗效应。方法：采用编码11个精氨酸（11R）的穿膜肽序列融合HSP70基因序列，构建重组腺病毒载体AdCMV-HSP70s、AdCMV-HSP70（无穿膜肽对照）和AdCMV-EGFV（空载体对照）。Western blotting、ELISA法检测重组腺病毒介导的HSP70基因在胃癌SCG-7901细胞中的表达，MTT检测重组腺病毒感染后HSP70分泌性表达对胃癌细胞的抑制作用。裸鼠移植瘤模型的抗瘤实验检测HSP70基因联合CIK细胞对胃癌移植瘤的抗瘤效应。结果：与AdCMV-HSP70感染细胞比，AdCMV-HSP70s感染细胞的HSP70表达量明显增加[胃癌SGC-7901细胞：（360.72±20.89） vs（121.01±15.94）ng/ml，P<0.05 ；胃黏膜上皮GES-1细胞：（188.62±10.82） vs（135.00±13.96） ng/ml，P<0.05]。融合11R的HSP70蛋白能够穿膜并分泌到细胞外后对胃癌细胞产生一定的增殖抑制作用[MOI=100 pfu/cell时，细胞存活率（66.33±4.33）% vs（101.33±7.64）%，P<0.01]。AdCMV-HSP70s+CIK联合治疗对胃癌移植瘤的抑制率显著高于AdCMV-HSP70+CIK组[（66.5±7.3)% vs (43.6±5.6)%, P<0.05]，该组移植瘤组织间质中CD3+T细胞浸润数量也明显多于后者。结论：融合穿膜肽可明显促进HSP70蛋白的表达和分泌，和CIK细胞联合治疗能增强抗瘤免疫效应，从而显著抑制裸鼠胃癌移植瘤。

To evaluate the efficacy of cell-penetrating peptide-fused HSP70 gene therapy in combination with the use of cytokine-induced killer (CIK) cells for gastric cancer in a xenograft nude mouse model. Methods: CMV promoter-driven adenoviral vectors expressing wild-type HSP70 (AdCMV-HSP70) and HSP70 fused with a cell-penetrating peptide of 11 arginines (AdCMV-HSP70s) were constructed. In in vitro experiments, human gastric cancer SGC-7901 and GES-1 cells were infected with these two viral vectors respectively. At 48 h after infection, cell viability was assessed by MTT assays and HSP70 protein content by Western blotting. In in vivo experiments, Sgc-7901 cells were injected subcutaneously into BALB/c nude mice. Cancer cell-challenged mice were treated every 48 h for a total of five times with AdCMV-HSP70 or AdCMV-HSP70s, either each alone or in combination with a single dose of 1×107 CIK cells (tail vein injection) prepared from normal BALB/c mice. Thirty-five days after treatment, animals were sacrificed. The number and size of tumors formed were assessed. Results: HSP70 protein content after AdCMV-HSP70s infection was significantly higher than that after AdCMV-HSP70 infection in both SGC cells (360.72±20.89 ng/ml vs 121.01±15.94 ng/ml, P<0.05) and GES-1 cells (188.62±10.82 ng/ml vs 135.00±13.96 ng/ml, P<0.05). Fusion of 11 arginine peptide into HSP70 led to significant inhibition of gastric cancer cell proliferation; at low multiplicity of infection (MOI) of 100 pfu/ml, the cell viability was (66.33±4.33)% in AdCMV-HSP70s-infected SGC-7901 cells, significantly lower than that (101.33±7.64%) in AdCMV-HSP70-infected SGC-7901 cells (P<0.05). CIK cell administration resulted in immune reconstruction in nude mice carrying xenograft gastric cancer cells. Adenoviral delivery of HSP70 enhanced infiltration of CD3+ T cells into tumor tissues and induce antitumor immune response. CIK cells in combination with AdCMV-HSP70s showed a significantly higher tumor inhibition rate (66.5%) than CIK cells in combination with AdCMV-HSP70 (43.6%) in nude mince challenged with gastric cancer cells (P<0.05). Conclusion: In nude mice carrying human gastric cancer cells, HSP70 may not only inhibit gastric cancer cell proliferation but also induce antitumor immune response. These effects can be enhanced by fusion of a cell-penetrating peptide with the HSP70 molecule and implantation of cytokine-induced killer cells.

国家自然科学基金资助项目(No.81370552，No.81172303)；南京军区医学科技创新项目(No.10MA127)