CAJ | 학술논문

目的观察CD160胞外段(即可溶性CD160分子)对荷瘤小鼠CD4+T淋巴细胞的活性和免疫功能的影响.方法构建小鼠CD160分子胞外段(sCD160)的重组表达载体psCD160,转染中国仓鼠卵巢(CHO)细胞,Western blot检测培养上清中的sCD160水平.建立TC-1宫颈癌细胞荷瘤小鼠模型,流式细胞术(FCM)检测CD160在脾CD4+T细胞上的表达;免疫磁珠分选荷瘤3周小鼠脾CD4+T细胞,与负载肿瘤细胞抗原肽复合物的树突状细胞(DC)共培养6d,FCM检测CD4+T细胞上CD160的表达.负载抗原肽复合物的DC分别在转染pcDNA3.1或psCD160的CHO培养上清中孵育18h后,与分选的CD4+T细胞共培养6d,羧基荧光素二醋酸盐琥珀酰亚胺脂(CFSE)标记法检测CD4+T细胞增殖,酶联免疫吸附实验(ELISA)检测培养上清中白细胞介素-2(IL-2)和γ-干扰素(IFN-γ)的浓度.结果转染psCD160的CHO细胞表达并分泌可溶性CD160分子.荷瘤3周小鼠CD4+T细胞上CD160分子的阳性率与对照组比较差异无统计学意义[(5.288±2.723)％比(3.213±1.461)％,P＞0.05],在体外被负载特异性抗原肽复合物的DC再活化后,其CD160阳性率显著增高达(17.590±7.287)％(P＜0.05).与未经sCD160作用的DC组相比,经sCD160封闭的负载特异性抗原肽复合物的DC再活化的CD4+T细胞中增殖细胞百分数[(17.980 ±5.337)％比(12.610 ±4.431)％,P＜0.05]、上清中的IL-2和IFN-γ浓度[(383.9±83.67) ng/L比(237.5±54.64) ng/L,(730.8±135.8) ng/L比(451.9±149.5) ng/L,P＜0.05]均显著升高.结论肿瘤特异性CD4+T细胞在体外再活化后CD160的表达升高,可溶性CD160阻断其作用可有效增强CD4+T细胞增殖和细胞因子分泌的活性.
목적 관찰CD160포외단(즉가용성CD160분자)대하류소서CD4+T림파세포적활성화면역공능적영향.방법 구건소서CD160분자포외단(sCD160)적중조표체재체psCD160,전염중국창서란소(CHO)세포,Western blot검측배양상청중적sCD160수평.건립TC-1궁경암세포하류소서모형,류식세포술(FCM)검측CD160재비CD4+T세포상적표체;면역자주분선하류3주소서비CD4+T세포,여부재종류세포항원태복합물적수돌상세포(DC)공배양6d,FCM검측CD4+T세포상CD160적표체.부재항원태복합물적DC분별재전염pcDNA3.1혹psCD160적CHO배양상청중부육18h후,여분선적CD4+T세포공배양6d,최기형광소이작산염호박선아알지(CFSE)표기법검측CD4+T세포증식,매련면역흡부실험(ELISA)검측배양상청중백세포개소-2(IL-2)화γ-간우소(IFN-γ)적농도.결과 전염psCD160적CHO세포표체병분비가용성CD160분자.하류3주소서CD4+T세포상CD160분자적양성솔여대조조비교차이무통계학의의[(5.288±2.723)％비(3.213±1.461)％,P＞0.05],재체외피부재특이성항원태복합물적DC재활화후,기CD160양성솔현저증고체(17.590±7.287)％(P＜0.05).여미경sCD160작용적DC조상비,경sCD160봉폐적부재특이성항원태복합물적DC재활화적CD4+T세포중증식세포백분수[(17.980 ±5.337)％비(12.610 ±4.431)％,P＜0.05]、상청중적IL-2화IFN-γ농도[(383.9±83.67) ng/L비(237.5±54.64) ng/L,(730.8±135.8) ng/L비(451.9±149.5) ng/L,P＜0.05]균현저승고.결론 종류특이성CD4+T세포재체외재활화후CD160적표체승고,가용성CD160조단기작용가유효증강CD4+T세포증식화세포인자분비적활성.
Objective To investigate the effects of CD160 extracellular domain (namely soluble CD160) on the immunoactivity of CD4 + T cells from the tumor-bearing mice.Methods Recombinant expression vector psCD160,expressing CD160 extracellular domain (sCD160),was constructed and transfected into CHO cells,and the sCD160 expression of CHO cells transfected with psCD160 was verified by Western blotting.The expression of CD160 on splenetic CD4+T cells was detected by flow cytometry (FCM) after C57BL/6 mice were inoculated with TC-1 cervical cancer cells.CD4 +T cells were separated from splenocytes with immunomagnetic beads 3 weeks after the inoculation of TC-1 cells and were co-cultured with dendritic cells (DCs) for 6 days,which were loaded with tumor cells peptide complex,then FCM was performed to detect CD160 on CD4 + T cells.The sorted CD4 + T cells were co-cultured with DCs from four different treatment groups for 6 days,which were loaded separately with different tumor cells peptide complex in the presence of culture supernatants of CHO cells transfected with pcDNA3.1 or psCD160.The proliferation of CD4 + T cells was assayed by carboxyfluorescein succinimidyl amino ester (CFSE) labelling in combination with flow cytometry,and the concentrations of interleukin-2 (IL-2) and γ-interferon (IFN-γ) were measured by enzyme linked immunosorbent assay (ELISA).Results Soluble CD160 was expressed and secreted into culture supernatants of CHO cells transfected with psCD160.On the week 3 after the inoculation with TC-1 cells,the positive percentage of CD160 on CD4 + T cells of tumor-bearing mice had no statistically significant difference as compared with that of control mice [(5.288 ± 2.723) ％ vs.(3.213 ± 1.461) ％,P ＞ 0.05].After reactivation by DCs loaded with specific antigen complex,the positive percentage of CD160 on CD4 +T cells was significantly rose to (17.590 ± 7.287) ％ (P ＜ 0.05).Compared with those of CD4 + T cells activated by DCs in the absence of sCD160,the proliferation percentage [(17.980 ± 5.337) ％ vs.(12.610 ± 4.431) ％,P ＜ 0.05] and the concentrations of IL-2 and IFN-γ [(383.9 ±83.67) ng/L vs.(237.5 ±54.64) ng/L,(730.8 ± 135.8) ng/L vs.(451.9 ± 149.5) ng/L,P ＜ 0.05] in supernatants of CD4 +T cells activated by DCs loaded with specific antigen complex in the presence of sCD160 significantly increased.Conclusion After reactivation in vitro,tumor specific CD4 +T cells up-regulate the expression of CD160.Soluble CD160,by blocking the pathway of CD160,enhances the activities on proliferation and secretion of CD4 + T cells.