趋化因子CXCL9和CXCL10介导的胃癌免疫细胞浸润的探讨

目的:探讨不同刺激因子对胃癌中CXCL9和CXCL10表达的影响，及不同血样中免疫细胞经不同方式活化后，其膜表面CXCR3的表达情况，建立并评价CXCR3和CXCL9、CXCL10介导的靶向抗胃癌免疫治疗新技术。方法:采用IFN-γ、TNF-α、Poly（I∶C）、LPS和PHA刺激胃癌细胞（BGC-823、SGC-7901及MKN-28），ELISA法测定肿瘤细胞分泌CXCL9和CXCL10的情况。将免疫细胞分别活化为CIK和CAPRI，流式细胞术检测活化前后免疫细胞表面CXCR3的表达情况。四甲基偶氮唑蓝（MTT）法评价刺激因子［IFN-γ、TNF-α、Poly（I∶C）、LPS和PHA］本身对肿瘤细胞增殖的影响。Transwell趋化实验比较趋化因子CXCL9和CXCL10及其受体CXCR3的表达差异对免疫细胞靶向归巢至肿瘤的数量的影响。结果:胃癌细胞株基础状态无明显CXCL9和CXCL10的表达，IFN-γ可以显著升高CXCL9和CXCL10的表达水平，TNF-α、Poly（I∶C）、LPS和PHA均可升高CXCL9和/或CXCL10的表达水平，与IFN-γ有一定的协同作用，其中Poly（I∶C）效果最强。IFN-γ、TNF-α、Poly（I∶C）、LPS和PHA等刺激因子本身在单独或联合应用时对3种肿瘤细胞均无明显增殖抑制作用。相同刺激条件下，胃癌高中分化细胞株的CXCL9和CXCL10表达显著高于低分化细胞株。CIK、CAPRI两种方式活化后细胞膜表面CXCR3显著升高。CIK、CAPRI两种活化方式刺激CXCR3的升高效能相当，无统计学差异。3种血样［健康足月新生儿脐带血、健康成人外周血单个核细胞（PBMC）、肿瘤患者PBMC］在相同活化方式刺激条件下CXCR3的表达无统计学差异。免疫活性细胞经由CXCL9、CXCL10与CXCR3介导，可以更多数量归巢至肿瘤。结论:趋化因子CXCL9和CXCL10及其受体CXCR3的高表达可以有效促进免疫细胞靶向归巢至肿瘤。

Chemokine -driven lymphocyte infiltration in human gastric cancer

Objective:The chemokine receptor CXCR3 and its corresponding ligands,including monokine induced by IFN-γ(CXCL9) and interferon-γ inducible protein 10kDa (CXCL10),are known to impact immunocytes infiltration.However,the optimal mode that regulates this infiltration requires further understanding.Here we investigated that how different regulators effect the expression level of CXCR3 or CXCL9/10.Methods:For regulation studies,BGC-823,SGC-7901 and MKN-28 gastric cancer cells were stimulated with IFN-γ,TNF-α,Poly （I∶C）,LPS and PHA.CXCL9/CXCL10 released from cells was measured by ELISA.Flow cytometry was used to assess the expression levels of CXCR3 on immunocytes before and after culture.The affinity of immunocytes to gastric cancer cells was determined by transwell migration assay.Results:CXCL9 and CXCL10 were prominently produced from 3 gastric cancer cell lines with impact of stimulants used respectively,except PHA,while they can not be detected in the culture medium of unstimulated cells.This effect was further enhanced by combination of IFN-γ and the rest reagents.Moderately or well differentiated gastric cancer cells produced more chemokines than poorly differentiated cells with the same stimulants.There were higher expression levels of CXCR3 on immune cells after activation.And various sources of blood samples had difference of CXCR3 expression.Moreover,there was no significant difference between the two patterns(CIK and CAPRI group) in shaping immunocytes surface receptor portrait.Chemotaxis assay showed enhanced transmigration of immunocytes to gastric cancer cells.Conclusion:Increase of the intratumoral CXCL9/10 concentration and the surface express level of CXCR3 on immunocytes leads to higher affinity of immune cell to gastric cancer cells.CXCR3 and CXCL9/CXCL10 might be the potential therapeutical candidates to strengthen the efficiency of gastric cancer immunotherapy.