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目的通过观察胃癌源性的外泌体对HLA-DR~(~(neg))leukocytes中Dicer1、PTEN基因表达的影响,探讨肿瘤源性外泌体调控机体免疫抑制功能的机制。方法提取胃癌患者血清外泌体并鉴定,免疫磁珠法分选HLA-DR~(neg)leukocytes,吖啶橙染色后的外泌体与CFSE染色后的HLA-DR~(neg)leukocytes在37℃,5%CO_2条件下共培养12 h;采用QRT-PCR和Western blot方法分别检测共培养后胃癌患者与健康对照组中Dicer1、PTEN基因和蛋白的表达。结果成功分离胃癌患者外周血外泌体;同时观察到外泌体被HLA-DR~(neg)leukocytes有效摄取。QRT-PCR检测结果显示Dicer1 m RNA、PTEN m RNA的相对表达量分别是健康对照组的0.46±0.19、0.48±0.27倍(P<0.05);Western blot结果显示Dicer1、PTEN蛋白表达量是健康对照组的0.15±0.11、0.33±0.19倍(P<0.05)。结论胃癌来源的外泌体可以抑制HLA-DR~(neg)leukocytes中Dicer1、PTEN基因的表达,为今后进一步研究肿瘤来源的外泌体的免疫调控作用提供线索。
OBJECTIVE: To investigate the effect of gastric cancer-derived exosomes on the expression of Dicer1 and PTEN in HLA-DR-negative leukocytes, and to explore the mechanism of tumor-derived exosomes regulating the immunosuppression in vivo. Methods Serum exosomes of patients with gastric cancer were extracted and identified. HLA-DR-leukemic cells were sorted by immunomagnetic beads method. Acridine orange-stained exosomes and CFSE-stained HLA-DR- C and 5% CO 2 for 12 h. The expression of Dicer1 and PTEN gene and protein in gastric cancer patients and healthy controls were detected by QRT-PCR and Western blot respectively. Results The peripheral blood exosomes were successfully separated from gastric cancer patients. Meanwhile, exosomes were effectively upregulated by HLA-DR-neg leukocytes. The results of QRT-PCR showed that the relative expression levels of Dicer1 m RNA and PTEN m RNA were 0.46 ± 0.19 and 0.48 ± 0.27 times respectively (P <0.05) in the healthy control group. The Western blot results showed that the expression of Dicer1 and PTEN protein were healthy controls 0.15 ± 0.11, 0.33 ± 0.19 fold (P <0.05) of the group. Conclusion Exosomes derived from gastric cancer can inhibit the expression of Dicer1 and PTEN in HLA-DRneg leukocytes and provide clues for the further study on the immune regulation of tumor-derived exosomes.