CAJ | 학술논문

目的观察低剂量环磷酰胺(CYC)抑制荷瘤小鼠体内微环境对改善细胞毒性T淋巴细胞(CTL)瘤体内存留、分布和功能的作用.方法体外制备黑色素瘤抗原特异性CTL并进行荧光染料2-羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)标记和培养.建立C57BL/6小鼠荷瘤模型并分为生理盐水腹腔注射(NSI)+ CTL组和环磷酰胺腹腔注射(CYCI)+ CTL组,各40只,每组小鼠分别腹腔注入等量生理盐水(NS)或环磷酰胺(CYC).检测注射前、后小鼠脾脏调节性T细胞(Tregs)和血清白细胞介素(IL)-10、转化生长因子(TGF)-β水平.注射后第4天回输CTL,流式细胞检测对比两组瘤体内CTL荧光强度和细胞周期.实时定量聚合酶链反应(Real-time PCR)检测对比两组瘤体内干扰素(IFN)-γ、IL-2、颗粒酶、穿孔素mRNA表达.激光共聚焦观测两组CFSE阳性CTL分布.对比肿瘤体积.结果两组小鼠于CYC或NS注射后第4天Tregs/CD4+(0.05 ±0.00比0.27±0.07,P＜0.05)和IL-10[(26.89 ±0.12) ng/L比(47.64±1.40) ng/L,P＜0.05]显著降低;TGF-β第7天降至最低[(4.00 ±0.12) ng/L比(9.80 ±0.36) ng/L,P＜0.05].CTL回输后第3天始CYCI+CTL组瘤体内荧光强度比NSI+ CTL组明显增高且持久(91.90±1.37比39.70±2.10,P＜0.05);两组各期细胞比例差异无统计学意义(P＞0.05).两组瘤体内颗粒酶、穿孔素、IL-2和IFN-γ表达差异均有统计学意义(P＜0.05);两组间肿瘤生长抑制自CTL回输后第6～15天尤为显著(P＜0.05).结论采用低剂量CYC对体内微环境进行提前干预,可同时降低Treg、IL-10和TGF-β水平,使回输CTL在肿瘤内的归巢存留明显增强,杀伤效率提高,肿瘤生长抑制.
목적 관찰저제량배린선알(CYC)억제하류소서체내미배경대개선세포독성T림파세포(CTL)류체내존류、분포화공능적작용.방법 체외제비흑색소류항원특이성CTL병진행형광염료2-최기형광소이작산염호박선아알지(CFSE)표기화배양.건립C57BL/6소서하류모형병분위생리염수복강주사(NSI)+ CTL조화배린선알복강주사(CYCI)+ CTL조,각40지,매조소서분별복강주입등량생리염수(NS)혹배린선알(CYC).검측주사전、후소서비장조절성T세포(Tregs)화혈청백세포개소(IL)-10、전화생장인자(TGF)-β수평.주사후제4천회수CTL,류식세포검측대비량조류체내CTL형광강도화세포주기.실시정량취합매련반응(Real-time PCR)검측대비량조류체내간우소(IFN)-γ、IL-2、과립매、천공소mRNA표체.격광공취초관측량조CFSE양성CTL분포.대비종류체적.결과 량조소서우CYC혹NS주사후제4천Tregs/CD4+(0.05 ±0.00비0.27±0.07,P＜0.05)화IL-10[(26.89 ±0.12) ng/L비(47.64±1.40) ng/L,P＜0.05]현저강저;TGF-β제7천강지최저[(4.00 ±0.12) ng/L비(9.80 ±0.36) ng/L,P＜0.05].CTL회수후제3천시CYCI+CTL조류체내형광강도비NSI+ CTL조명현증고차지구(91.90±1.37비39.70±2.10,P＜0.05);량조각기세포비례차이무통계학의의(P＞0.05).량조류체내과립매、천공소、IL-2화IFN-γ표체차이균유통계학의의(P＜0.05);량조간종류생장억제자CTL회수후제6～15천우위현저(P＜0.05).결론 채용저제량CYC대체내미배경진행제전간예,가동시강저Treg、IL-10화TGF-β수평,사회수CTL재종류내적귀소존류명현증강,살상효솔제고,종류생장억제.
Objective To observe the role of improving the persistence,distribution and function of cytotoxic T lymphocytes (CTL) in tumor by inhibiting the tumor microenvironment with low-dose cyclophosphamide (CYC) administration.Methods Melanoma antigen-specific CTL were prepared in vitro,labeled with carboxyfluorescin diacetate succinimidyl ester (CFSE) and cultivated.Melanoma-bearing mice models were established and randomly divided into two groups:NSI + CTL group and CYCI + CTL group.The same amount of CYC or normal saline (NS) was intraperitoneally injected in mice,respectively.The levels of regulatory T cells (Tregs),transforming growth factor (TGF)-β and interleukin (IL)-10 were detected at different time points after injection.The cultured CTL were transfused at the 4th day after injection.The fluorescence intensity and cell cycle of CTL were analyzed by flow cytometry (FCM) and the morphological distribution of CTL were observed using laser scanning confocal microscopy.Perforin,granzyme B,IL-2,and interferon (IFN)-γwere assessed by real-time quantitative polymerase chain reaction (Real-time PCR).Tumor volumes in each group were compared.Results In CYCI + CTL group,Tregs (0.05 ±0.00 vs.0.27±0.07,P＜0.05) and IL-10 [(26.89±0.12) ng/L vs.(47.64±1.40) ng/L,P＜ 0.05] were clearly declined on the 4th day after CYC injection,but a reduction in TGF-β was delayed until the 7th day [(4.00 ± 0.12) ng/L vs.(9.80 ± 0.36) ng/L,P ＜ 0.05].The fluorescence intensity of CTL in tumors of CYCI + CTL group was significantly higher and lasted longer than that in NSI + CTL group from the 3rd day (91.90 ± 1.37 vs.39.70 ± 2.10,P ＜0.05).However,no significant difference existed in the cell cycle of lymphocytes (P ＞ 0.05).The amounts of granzyme B (P ＜ 0.05),perforin (P ＜ 0.05),IL-2 (P ＜ 0.05),and IFN-γ (P ＜ 0.05) in tumors of CYCI + CTL group were greater than in NSI + CTL group.The difference in tumor growth between the groups was most evident from the 6th to the 15th day.Conclusion Tumor immunosuppressive microenvironment was successfully inhibited by low-dose CYC administration,with decreases in the levels of Tregs,IL-10,and TGF-β simultaneously.As a result,more recruitment into tumor tissues and more sustainable effective time of the transferred CTL in vivo lead to the strenthened killing efficacy.