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目的: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。
OBJECTIVE: To observe the therapeutic effect of HSP70 gene combined with CIK cells on gastric cancer xenografts in nude mice. Methods: The recombinant adenoviral vector AdCMV-HSP70s, AdCMV-HSP70 (without transmembrane peptide control) and AdCMV-EGFV (empty vector control) were constructed by fusion of the transmembrane peptide sequence encoding 11 arginine (11R) . Western blotting was used to detect the expression of recombinant adenovirus-mediated HSP70 gene in gastric cancer SCG-7901 cells by ELISA. The inhibitory effect of HSP70 expression on gastric cancer cells was detected by MTT assay. Antitumor Effects of Transplanted HSP70 Gene Combined with CIK Cells on Transplanted Tumor Tumor in Nude Mice. Results: Compared with AdCMV-HSP70 infected cells, the expression of HSP70 was significantly increased in AdCMV-HSP70s-infected cells [SGC-7901 cells: (360.72 ± 20.89) vs (121.01 ± 15.94) ng / ml, GES-1 cells: (188.62 ± 10.82) vs (135.00 ± 13.96) ng / ml, P <0.05]. The fusion protein 11R HSP70 could penetrate the membrane and secrete it to the outside of the cells, which could inhibit the proliferation of gastric cancer cells [(66.33 ± 4.33)% vs (101.33 ± 7.64)% at MOI = 100 pfu / <0.01]. The inhibitory rate of AdCMV-HSP70s combined with CIK on gastric cancer was significantly higher than that in AdCMV-HSP70 + CIK group (66.5 ± 7.3% vs 43.6 ± 5.6%, P <0.05) CD3 + T cell infiltration in the number is also significantly more than the latter. Conclusion: The fusion membrane penetrating peptide can significantly promote the expression and secretion of HSP70 protein, and CIK cell combination therapy can enhance the anti-tumor immune effect, which can significantly inhibit the nude mice gastric cancer xenografts.