丹参酮Ⅱ-A调控Th17/Treg免疫平衡影响骨关节炎软骨退变大鼠中TLR4相关通路表达的机制研究

目的探讨丹参酮Ⅱ-A(tanshinoneⅡ-A,TSⅡ-A)对骨关节炎(osteoarthritis,OA)软骨退变大鼠中调节性T细胞(regulatory T,Treg)/辅助性T细胞17(Th17)的影响,以及其对Toll样受体4(Tolllike receptor 4,TLR4)信号的调控。方法以关节腔注射木瓜蛋白酶构建大鼠OA模型。造模成功后,分为TSⅡ-A低剂量组(TSⅡ-A-L)、TSⅡ-A高剂量组(TSⅡ-A-H)、阳性对照药物硫酸氨基葡萄糖(glucosamine sulfate,DGS)组进行药物干预;酶联免疫吸附试验检测大鼠血清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-6(interleukin-6,IL-6)水平;脱氧核糖核苷酸末端转移酶介导的缺口末端标记试剂盒(TUNEL)检测大鼠软骨组织中的细胞凋亡;流式细胞仪检测脾组织中Treg、Th17的比重,实时荧光定量聚合酶链式反应(qRT-PCR)检测大鼠膝关节软骨组织中TLR4、髓样分化因子88(myeloid differentiation factor 88,MyD88)、以及核转录因子(nuclear factorκB,NF-κB)、蛋白聚糖(aggrecan)和Ⅱ型胶原(CollagenⅡ)、叉状头/翅膀状螺旋转录因子3(forkhead/winged helix transcription factor 3,Foxp3)、IL-17、B淋巴细胞瘤-2(B cell lymphoma-2,Bcl-2)和Bcl-2相关X蛋白(Bcl2-associated X protein,Bax)的mRNA的表达;蛋白质印迹法检测软骨组织中TLR4、MyD88、p-NF-κB、Bcl-2和Bax的表达。结果与Sham组相比,其余各组大鼠脾组织中Treg细胞的比重、膝关节软骨组织中aggrecan和CollagenⅡ以及Foxp3、Bcl-2的表达明显下降,血清中TNF-α和IL-6的含量、软骨组织中的细胞凋亡率、脾组织中Th17细胞的比重、膝关节软骨组织中IL-17、TLR4、MyD88、p-NF-κB和Bax的表达明显升高;与OA组相比,TSⅡ-A-L组、TSⅡ-A-H组、DGS组大鼠脾组织中Treg细胞的比重、膝关节软骨组织中aggrecan和CollagenⅡ以及Foxp3、Bcl-2的表达明显升高,血清中TNF-α和IL-6的含量、软骨组织中的细胞凋亡率、脾组织中Th17细胞的比重、膝关节软骨组织中IL-17、TLR4、MyD88、p-NF-κB和Bax的表达明显下降;与TSⅡ-A-L组相比,TSⅡ-A-H组大鼠脾组织中Treg细胞的比重、膝关节软骨组织中aggrecan和CollagenⅡ以及Foxp3、Bcl-2的表达明显升高,血清中TNF-α和IL-6的含量、软骨组织中的细胞凋亡率、脾组织中Th17细胞的比重、膝关节软骨组织中IL-17、TLR4、MyD88、p-NF-κB和Bax的表达明显下降,差异均具有统计学意义(P均<0.05),TSⅡ-A-H组与DGS组间各指标的差异不具有统计意义(P>0.05)。结论丹参酮Ⅱ-A(TSⅡ-A)能明显抑制OA大鼠关节软骨组织中TLR4、MyD88、p-NF-κB的表达,降低软骨组织中的细胞凋亡率,这可能与改善Treg/Th17免疫失衡有关。

Effects of estrogen on CD4^+ CD25^+ regulatory T cell in peripheral blood during pregnancy

Objective To investigate the effects of estrogen(E_2)level on regulatory T cells(Treg)in peripheral blood during pregnancy.Methods:A total of 30 healthy non-pregnant women were selected as control group,90 pregnant women of early,middle and late pregnancy and 30 postpartum women at 1 month after parturition were selected as experimental groups including early pregnancy group,middle pregnancy group and late pregnancy group;the proportions of CD4^+CD25^+Treg and CD4^+CD25^+CD127^-Treg among CD4 T cells were detected by flow cytometry;the serum estrogen content in peripheral blood was detected by electrochemical immune luminescence method.Results:E_2 level was coincident with the change of Tregs number during pregnancy.The estrogen content in peripheral blood increased gradually from early pregnancy to late pregnancy,then decreased significantly after parturition,and the level at 1 month after parturition down to the level in non-pregnancy group(P>0.05);the level of E_2 in pregnancy groups were significantly higher than those in non-pregnancy group(P<0.01);and there were significant differences among early pregnancy group,middle pregnancy group and late pregnancy group(P<0.05).The proportions of CD4^+CD25^+Treg and CD4^+CD25^+CD127^-Treg in pregnancy groups were significantly higher than those in non-pregnancy group(P<0.05),but decreased significantly after parturition,and there was no significant difference between non-pregnancy group and postpartum women group(P>0.05):the proportions in middle and late pregnancy groups were significantly higher than those in early pregnancy group(P<0.05).but decreased slightly in late pregnancy group,there was no significant difference between late pregnancy group and middle pregnancy group(P>0.05).There was correlation between Tregs number with estrogen level during pregnancy.The proportion of CD4^+CD25^+Treg and CD4^+CD25^+CD 127^-Treg were positively correlated with estrogen level.Conclusions:High proportion of CD4^+CD25^+Trcg and CD4^+CD25^+CD127^-Treg is closely related to the high level of E,during pregnancy.It suggested that high level of estrogen may induce an increase of CD4^+CD25^+Treg in peripheral blood.and then influence the immune function of pregnant women.The results of this experiment might play an important role of estrogen in immune-modulation during pregnancy.

Analysis of CD4^+CD25^+ Regulatory T Cells and Foxp3 mRNA in the Peripheral Blood of Patients with Asthma

The changes of CD4^＋CD25^＋ regulatory T cells （CD4^＋CD25^＋ Treg） and Foxp3 mRNA in peripheral blood mononuclear cells （PBMCs） from patients with asthma were investigated in order to elucidate the possible roles of CD4^＋CD25^＋ Treg in the development of asthma. The peripheral blood samples were collected from 29 healthy controls （normal control group） and 78 patients with asthma which included 30 patients in exacerbation group, 25 patients in persistent group, and 23 patients in remission group. By using flow cytometry and RT-PCR, the CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA in PBMCs were detected. The CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA in PBMCs of exacerbation and persistent groups were lower than that of remission and normal control groups （P〈0.05）. Although the CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA of remission group were also lower than that of normal control group, there was no significant difference between them （P〉0.05）. As compared with persistent group, exacerbation group had lower CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA （P〈0.05）. It was indicated that the decrease of CD4^＋CD25^＋ Treg ratio and its function in PBMCs may be responsible for pathogenesis of asthma.

Depletion of CD4^+CD25^+ regulatory T cells can promote local immunity to suppress tumor growth in benzo[a]pyrene-induced forestomach carcinoma

AIM： To elucidate the distribution of CD4^＋CD25^＋ regulatory T cells （Tregs） in different lymphoid tissues and its local enhancement on tumor growth before and after depletion of CD4^＋CD25^＋ Tregs. METHODS： Female ICR mice were garaged with benzo[a]pyrene （BaP） to induce forestomach carcinoma. CD4^＋CD25^＋ Tregs were intraperitoneally depleted with monoclonal antibody PC61. These mice were divided into BaP-only, BaP ＋ IgG, BaP ＋ PC61, and control groups. The forestomach of mice was dissected for histological analysis, and tunnel test was performed for apoptosis of tumor cells. CD4^＋CD25^＋ Tregs were sorted from different lymphoid tissues and expression of Foxp3, IL-10, and chemokine receptors was analyzed by flow cytometry, semi-quantitative and veal-time polymerase chain reaction. RESULTS： The mice gavaged with only BaP showed increased forestomach papilloma and carcinoma at wk 16 and 32. The proportion of CD4^＋CD25^＋ Tregs was significantly higher in peri-stomach regional lymph nodes than in other lymphoid tissues. These CD4^＋CD25^＋ Tregs in regional lymph nodes expressed higher levels of Foxp3 and IL-10, enriched in the CD62L-subset, and CCR1 and CCR5 chemokine receptors. In mice gavaged with BaP ＋ PC61, the number of tumor nodules and tumor volume decreased significantly with massive infiltrating cells and apoptosis of tumor cells. In the draining regional lymph nodes, the number of CD4^＋CD25^＋ Tregs also decreased significantly. CONCLUSION： Inducible and activated CD4^＋CD25^＋ Tregs in the draining regional lymph nodes suppress host local immunity during tumor growth. Depletion of CD4^＋CD25^＋ Tregs can promote host local immunity to suppress tumor growth.

Influence of Danshen Injection on airway inflammation and CD4^+ CD25^+ regulatory T cells of asthmatic rats

Objective： To investigate the influence of Danshen Injection on airway inflammation and CD4^＋CD25^＋ regulatory T cells（CD4^＋CD25^＋ Tr） of asthmatic rats, and elucidate the possible mechanism of Danshen Injection in treatment of asthma. Methods： 30 Wister rats were randomly divided into control group, asthma group and Danshen Injection treated group. Bronchoalveolar lavage fluids （BALF） were collected, and cytology studies were conducted. Lung tissues were obtained and pathologic analyses were done with hematoxylin and eosin stain （HE）. Flow cytometry was used to detect the CD4^＋CD25^＋ Tr ratio in peripheral blood mononuclear cells （PBMCs）. Results： Total cell, the percentage of lymphocytes, neutrophils and eosinophils （Eos） in BALF of Danshen Injection-treated group were lower than that in asthma group （P〈0.05, P〈0.01）. Compared with asthma group, less infiltration of inflammatory cells in lung tissues was observed in Danshen Injection-treated group. CD4^＋CD25^＋ Tr of asthma group was lower than that of control and Danshen Injection treated group （P〈0.05）. Conclusion： Danshen Injection can suppress airway inflammation of asthmatic rats, probably by increasing the number of CD4^＋CD25^＋ Tr.

CD28/CTLA-4/B7 and CD40/CD40L costimulation and activation of regulatory T cells

Costimulatory signals are crucial for T cell activation. Attempts to block costimulatory pathways have been effective in preventing unwanted immune reactions. In particular, blocking the CD28/cytotoxic T lymphocyte antigen(CTLA)-4/B7 interaction(using CTLA-4Ig) and the CD40/CD40 L interaction(using anti-CD40 L antibodies) prevents T cell mediated autoimmune diseases, transplant rejection and graft vs host disease in experimental models. Moreover, CTLA-4Ig is in clinical use to treat rheumatoid arthritis(abatacept) and to prevent rejection of renal transplants(belatacept). Under certain experimental conditions, this treatment can even result in tolerance. Surprisingly, the underlying mechanisms of immune modulation are still not completely understood. We here discuss the evidence that costimulation blockade differentially affects effector T cells(Teff) and regulatory T cells(Treg). The latter are required to control inappropriate and unwanted immune responses, and their activity often contributes to tolerance induction and maintenance. Unfortunately, our knowledge on the costimulatory requirements of Treg cells is very limited. We therefore summarize the current understanding ofthe costimulatory requirements of Treg cells, and elaborate on the effect of anti-CD40 L antibody and CTLA-4Ig treatment on Treg cell activity. In this context, we point out that the outcome of a treatment aiming at blocking the CD28/CTLA-4/B7 costimulatory interaction can vary with dosing, timing and underlying immunopathology.

Isolation and identification of CD4^+CD25^+ regulatory T cells in rat

Objective： To establish a stable and high efficient method for collection of CD4^＋CD25^＋ regulatory T cells from rats in vitro. Methods： CD4^＋CD25^＋ regulatory T cells were isolated from the rat splenic cells through two steps by magic cell sorting （MACS） system. The first step was negative selection of CD4^＋T cells by cocktail antibodies and anti-IgG magic microbeads, and the second step was positive selection of CD25^＋T cells by anti-CD25 PE and anti-PE magic microbeads. The purity and viability of separated cells were measured by flow cytometry （FACS） and Trypan blue staining. The suppressive ability of seperated cells on the proliferation of CD4^＋CD25^- T cells was assessed by cell proliferation assay. Results： The purity of negatively enriched CD4^＋ T cells was 79%-87% （83.6%±2.5% ） , and the purity of positively enriched CD4^＋CD25^＋ T cells was 86%- 93% （ 90.2±1.8% ） with the viability of 92%～95% （92.8% ± 3.4% ）. The enriched cells significantly suppressed the proliferation of CD4^＋CD25^- T cells in mixed lymphocyte culture （P 〈 0.05）. Conclusion： An effective method can be established for enrichment of CD4^＋CD25^＋ regulatory T cells in two steps by MACS, with satisfied cell purity, viability and function.

Effect of CD4^+CD25^+ regulatory T cells in the development of anterior chamber-associated immune deviation

AIM: To investigate whether CD4(+)CD25(+) regulatory T (Treg) cells play a role in the development of anterior chamber-associated immune deviation (ACAID). METHODS: The dynamic changes in the frequency of CD4(+)D25(+) T cells, CD4(+)D25(+) FoxP3(+) T cells and CD4(+)CD25(+) PD-1(+) T cells from spleens of mice with ACAID were analyzed by flow cytometry. Foxp3 mRNA expression in purified CD4(+)CD25(+) T cells was analyzed using real-time PCR. The suppressive effect of purified CD4(+)CD25(+) T cells on the proliferation of CD4(+)CD25(-) T cells was evaluated by [H-3] thymidine incorporation. A blocking experiment was performed to further address the role of CD4(+)CD25(+) T cells in ACAID. The expression of IL-10 in purified CD4(+)CD25(+) T cells was evaluated by ELISA. RESULTS: Increased frequencies of CD4(+)CD25(+) T cells, CD4(+)CD25(+) Foxp3(+) T cells and CD4(+)CD25(+) PD-1(+) T cells were observed in ACAID. The CD4(+)CD25(+) T cells from mice with ACAID showed enhanced suppressive effect on the proliferation of CD4(+)CD25(-) T cells. Treatment of BALB/c mice with anti-CD25 antibody after injection of OVA into the anterior chamber significantly inhibited the induction of ACAID. Furthermore, purified CD4(+)CD25(+) T cells from ACAID mice secreted IL-10. CONCLUSION: Our results demonstrate that Treg cells are induced in the mice undergoing ACAID. These Treg cells may play a role in the development of ACAID.

CD4^+CD25^(high) Regulatory Cells in Peripheral Blood of NSCLC Patients

The proportion and changes of CD4^＋CD25^high regulatory T cells （Trs） in peripheral blood of non-small cell lung cancer （NSCLC） patients were analyzed and their clinical significance explored. The peripheral blood was collected from 61 patients with NSCLC and 15 healthy controls. By using monoclonal antibodies, the blood samples were evaluated with the flow cytometry for lymphocyte subsets （CD3^＋, CD4^＋ and CD8^＋） and CD4^＋CD25^high Tr cells. The results showed that the proportion of CD4^＋CD25^high Tr cells in NSCLC group was significantly higher than in control group [（4.36 ±2.07） % vs （2.04±1.03） %, P〈0.01]. The proportion of CD4^＋CD25^ high Tr cells in late stage was higher than that in early stage [stages Ⅰ ＋Ⅱ （2.264±0.6） %; stage Ⅲ（3.284± 1.38） %; stage IV （6.06 4±4.08） %] （P〈0.05）. Kaplan-Meier survival analysis revealed that the prognosis of the patients who had higher proportion of CD4^＋CD25^high Tr cells in peripheral blood was worse （P=0.0026）. In conclusion, the relative increase in CD4^＋CD25^high Tr cells in peripheral blood may be related to im- munosuppression and tumor progression in patients with NSCLC. This finding suggests that CD4^＋CD25^high Tr cells in peripheral blood of NSCLC may be positive for prognosis analysis. The use of depletion of the CD4^＋CD25^high Tr cell therapy to treat NSCLC patients may be an effective strategy.

Regulatory T cells suppress autoreactive CD4^+ T cell response to bladder epithelial antigen

AIM： To investigate the role of regulatory T （Treg） cells in CD4^＋ T cell-mediated bladder autoimmune infammation. METHODS： Urothelium-ovalbumin （URO-OVA）/OT-II mice, a double transgenic line that expresses the membrane form of the model antigen （Ag） OVA as a self-Ag on the urothelium and the OVA-specific CD4^＋ T cell receptor specifc for the I-Ab/OVA323-339 epitope in the periphery, were developed to provide an autoimmune environment for investigation of the role of Treg cells in bladder autoimmune infammation. To facilitate Treg cell analysis, we further developed URO-OVA^GFP-Foxp3/OT-II mice, a derived line of URO-OVA/OT-II mice that express the green fuorescent protein （GFP）-forkhead box protein P3 （Foxp3） fusion protein. RESULTS： URO-OVA/OT-II mice failed to develop bladder infammation despite the presence of autoreactive CD4^＋ T cells. By monitoring GFP-positive cells, bladder infltration of CD4^＋ Treg cells was observed in URO-OVA^GFP-Foxp3/OT-II mice. The infiltrating Treg cells were functionally active and expressed Treg cell effector molecule as well as marker mRNAs including transforming growth factor-β, interleukin （IL）-10, fibrinogen-like protein 2, and glucocorticoid-induced tumor necrosis factor receptor （GITR）. Studies further revealed that Treg cells from URO-OVA^GFP-Foxp3/OT-II mice were suppressive and inhibited autoreactive CD4^＋ T cell proliferation and interferon （IFN）-g production in response to OVA Ag stimulation. Depletion of GITR-positive cells led to spontaneous development of bladder infammation and expression of inflammatory factor mRNAs for IFN-γ, IL-6, tumor necrosis factor-α and nerve growth factor in URO-OVA^GFP-Foxp3/OT-II mice. CONCLUSION： Treg cells specifc for bladder epithelial Ag play an important role in immunological homeostasis and the control of CD4^＋ T cell-mediated bladder autoimmune infammation.

地塞米松对哮喘小鼠CD4^+ CD25^+调节性T细胞及IL-4 TGF-β水平的影响

目的探讨地塞米松对哮喘小鼠CD4^+、CD25^+调节性T细胞(regulatory T cells,Treg)及IL-4、TGF-β水平的影响。方法 30只雄性BALB/c小鼠随机分为正常对照组、哮喘组及地塞米松组3组各10只;利用鸡卵白蛋白腹腔注射、雾化吸入制备哮喘模型;使用流式细胞仪分别检测各组小鼠脾脏单个核细胞CD4^+、CD25^+Treg细胞占CD4^+T细胞的百分比;使用Western Blot方法分析IL-4在小鼠肺组织中的表达情况;使用酶联免疫吸附试验方法检测各组小鼠血清中TGF-β的水平。结果哮喘组小鼠脾单个核细胞CD4^+、CD25^+Treg细胞百分比及TGF-β水平较正常对照组降低(均P<0.05),哮喘组IL-4水平较正常对照组增高(P<0.05),地塞米松组与正常对照组比较,差异无统计学意义(P>0.05)。结论哮喘小鼠CD4^+、CD25^+Treg数量的减少,功能的下降,可能参与了哮喘的发病过程,地塞米松可能通过上调CD4^+、CD25^+Treg比例,调节T细胞亚群失衡来发挥抗炎作用。

哮喘患者外周血单个核细胞CD4^+CD25^+调节性T细胞及IL-4分泌水平的检测

目的:探讨CD4+CD25+调节性T细胞在哮喘发病机制中的作用,为哮喘的临床治疗提供新的思路和方法。方法:分离哮喘患者和正常人外周血单个核细胞(PBMC),用流式细胞仪分析CD4+CD25+调节性T细胞在外周血单个核细胞中的比例;酶联免疫吸附法(ELASA)检测外周血PBMC培养上清IL-4的分泌状况。结果:哮喘组外周血CD4+CD25+调节性T细胞的百分率为(3.2±1.5)%,健康对照组为(6.5±2.5)%,哮喘组明显低于对照组(P<0.05);哮喘组外周血PBMC培养上清IL-4分泌水平为(57.3±13.5)ng/L,健康对照组为(28.6±7.2)ng/L,哮喘组明显高于对照组(P<0.05)。结论:CD4+CD25+调节性T细胞可能参与了哮喘的发生与发展。

初发系统性红斑狼疮患者调节性T细胞与IL-4、IFN-γ的关联性

目的初步探讨调节性 T 细胞与 Th1/Th2型细胞的相关性及其在系统性红斑狼疮(SLE)发病机制中的作用。方法采用流式细胞术检测54例 SLE 患者(包括31例初发病例和23例激素治疗病例)及20名正常对照外周血 CD4^+CD25^+T 细胞和 CD4^+CD25^(high)T 细胞比率,ELISA 法检测血清白细胞介素-4(IL-4)和干扰素-γ(IFN-γ)水平。结果 SLE 患者外周血 CD4^+CD25^(high)T 细胞比率显著低于正常对照组(P<0.05)。初发组 CD4^+CD25^+T 细胞及CD4^+CD25^(high)T 细胞的比率均显著低于正常对照(P<0.05,P<0.01)。初发组(P<0.01,P<0.01,P<0.05)与治疗组(P<0.01,P<0.05,P<0.01)IL-4与 IFN-γ水平及 IL-4/IFN-γ比值较正常对照组显著增高。初发 SLE 组 IFN-γ水平显著高于治疗组(P<0.05)。初发 SLE 患者 CD4^+CD25^+T 细胞比率与 IL-4/IFN-γ比值呈正相关(r=0.241,P=0.040)。结论调节性 T 细胞可能参与了 SLE 的发病,并对 Th1/Th2的平衡有一定的影响。

川崎病患者血中CD4^+CD25^+调节性T细胞与血清IL-4、IFN-γ水平关系

目的探讨CD4+CD25+调节性T细胞在川崎病(KD)发病机制中的作用。方法 KD组患儿32例;对照组30例中分为两组:非KD发热组15例为急性呼吸道感染的发热患儿,健康组15例为健康体检患儿。KD患儿分别于静注丙种球蛋白(IVIG)前和IVIG治疗后热退2~3天取外周血,用流式细胞仪测定CD4+CD25+调节性T细胞比例,用酶联免疫吸附法(ELISA)检测血清干扰素-γ(IFN-γ)、白细胞介素-4(IL-4)水平并分析其关系。结果急性期KD患儿外周血CD4+CD25+调节性T细胞比例[(4.40±1.31)%]与非KD发热组[(6.37±1.99)%]和健康对照组[(7.45±3.18)%]相比均明显降低(P<0.01),而IVIG治疗后热退2~3天,CD4+CD25+调节性T细胞比例[(6.88±2.50)%]明显升高,接近于正常水平。同时在KD急性期,血清IFN-γ、IL-4水平均明显升高,在IVIG治疗后热退2~3天无明显下降。且在KD急性期血清IFN-γ、IL-4水平与CD4+CD25+调节性T细胞比例成负相关性(r=-0.838,P<0.01)。在静注IVIG后热退2~3天,血清IFN-γ、IL-4水平与CD4+CD25+调节性T细胞比例无明显相关性(r分别为-0.04和-0.072,P>0.05)。结论 CD4+CD25+调节性T细胞可能通过对体内Th1和Th2细胞的调控参与了KD的发病机制。

胃癌患者血清CA72-4、CEA、CA19-9水平与CD4^(+)CD25^(+)Treg细胞比例的相关性及临床意义

目的探讨胃癌患者血清糖类抗原72-4(CA72-4)、癌胚抗原(CEA)、糖类抗原19-9(CA19-9)水平与外周血CD4^(+)CD25^(+)调节性T(Treg)细胞比例的相关性和临床意义。方法回顾性分析2020年1月至2022年12月在西安长安医院就诊的胃癌患者68例作为胃癌组,另选取同期体检的健康志愿者50例作为对照组。检测并比较两组患者血清CA72-4、CEA、CA19-9水平及外周血CD4^(+)CD25^(+)Treg细胞比例。比较胃癌组不同临床资料患者的CA72-4、CEA、CA19-9水平及CD4^(+)CD25^(+)Treg细胞比例,采用Pearson相关分析胃癌组CA72-4、CEA、CA19-9水平与CD4^(+)CD25^(+)Treg细胞比例的相关性。结果胃癌组血清CA72-4、CEA、CA19-9水平及外周血CD4^(+)CD25^(+)Treg细胞比例比对照组高,差异均有统计学意义(P<0.05)。胃癌组TNM分期为Ⅲ~Ⅳ期患者的CA72-4、CEA、CA19-9水平及CD4^(+)CD25^(+)Treg细胞比例比Ⅰ~Ⅱ期患者高,高~中分化程度患者CA72-4、CEA、CA19-9水平比低分化程度患者低,淋巴结转移患者的CA72-4、CEA、CA19-9水平及CD4^(+)CD25^(+)Treg细胞比例比淋巴结未转移患者高,差异均有统计学意义(P<0.05)。胃癌组血清CA72-4、CEA、CA19-9水平与CD4^(+)CD25^(+)Treg细胞比例均呈正相关(r=0.587,0.678,0.696,P<0.05)。结论胃癌患者血清CA72-4、CEA、CA19-9水平及外周血CD4^(+)CD25^(+)Treg细胞比例明显升高且存在正相关性,与胃癌的发生、发展及肿瘤浸润转移密切相关;CD4^(+)CD25^(+)Treg细胞比例随肿瘤负荷的变化而变化,对胃癌患者病情评估、监测机体免疫状态及判断预后具有一定临床价值。

Anti-inflammatory effect of miR-125a-5p on experimental optic neuritis by promoting the differentiation of Treg cells

Methylprednisolone pulse treatment is currently used fo r optic neuritis.It can speed visual recovery,but does not improve the ultimate visual outcomes.Recent studies have repo rted that miR-125 a-5 p has immunomodulatory effects on autoimmune diseases.However,it remains unclear whether miR-125 a-5 p has effects on optic neuritis.In this study,we used adeno-associated virus to overexpress or silence miR-125 a-5 p in mice.We found that silencing miR-125 a-5 p increased the latency of visual evoked potential and aggravated inflammation of the optic nerve.Ove rexpression of miR-125 a-5 p suppressed inflammation of the optic nerve,protected retinal ganglion cells,and increased the percentage of Treg cells.Our findings show that miR-125 a-5 p exhibits anti-inflammatory effects through promoting the diffe rentiation of Treg cells.

哮喘合并COPD患者血清TLR4、HMGB1、Treg表达与肺功能和病情严重程度的关系

目的探究哮喘合并慢性阻塞性肺疾病(ACO)患者血清Toll样受体4(TLR4)、高迁移率族蛋白B1(HMGB1)、调节性T细胞(Treg)表达情况与肺功能、病情严重程度的关系。方法选取2019年10月—2022年10月南京医科大学第四附属医院收治的ACO患者117例为ACO组,根据慢性阻塞性肺疾病全球倡议(GOLD)分级分为两个亚组:轻中度组(1~2级,n=68)和重度组(3~4级,n=49);另选取同期收治的哮喘患者50例为哮喘组,慢性阻塞性肺疾病(COPD)患者52例为COPD组。比较各组血清TLR4、HMGB1、Treg细胞亚群(Treg、Th17、Treg/Th17)表达情况及肺功能指标[第1秒用力呼气容积占预计值百分比(FEV_(1)%pred)、FEV_(1)/用力肺活量(FEV_(1)/FVC)、最大呼气中段流量占预计值百分比(MMEF%pred)],采用Pearson或Spearman相关分析ACO患者TLR4、HMGB1、Treg表达水平与其肺功能、病情严重程度的相关性。结果ACO组TLR4[(4.13±1.15)ng/mL vs(3.56±0.84)ng/mL vs(2.89±0.95)ng/mL]、HMGB1[(6.63±2.54)μg/L vs(4.78±1.06)μg/L vs(3.64±0.97)μg/L]、Th17[(5.07±1.63)%vs(4.14±1.35)%vs(1.96±0.64)%]水平均高于COPD组和哮喘组,COPD组均高于哮喘组(均P<0.05);ACO组Treg[(0.85±0.23)%vs(1.34±0.41)%vs(3.17±0.96)%]、Treg/Th17[(0.19±0.06)vs(0.35±0.12)vs(1.83±0.37)]、FEV_(1)%pred[(33.67±9.78)vs(51.49±13.17)vs(75.82±16.34)]、FEV_(1)/FVC[(60.73±7.59)vs(63.26±8.45)vs(79.14±13.82)]和MMEF%pred[(44.27±8.65)vs(59.23±11.34)vs(71.65±15.87)]水平均低于COPD组和哮喘组,COPD组均低于哮喘组(均P<0.05)。ACO组中,轻中度组血清TLR4[(3.81±1.03)ng/mL vs(4.58±1.16)ng/mL]、HMGB1[(5.87±1.87)μg/L vs(7.69±2.75)μg/L]、Th17[(4.78±1.47)%vs(5.48±1.72)%]水平均低于重度组,Treg[(1.03±0.21)%vs(0.61±0.19)%]、Treg/Th17[(0.24±0.05)vs(0.11±0.03)]水平均高于重度组(均P<0.05)。相关性分析示,ACO患者TLR4、HMGB1、Th17与FEV_(1)%pred、FEV_(1)/FVC、MMEF%pred呈负相关,与GOLD分级均呈正相关(均P<0.05);Treg、Treg/Th17与FEV_(1)%pred、FEV_(1)/FVC、MMEF%pred均呈正相关,与GOLD分级均呈负相关(均P<0.05)。结论ACO患者血清TLR4、HMGB1表达水平上调,并存在明显的Treg/Th17失衡;TLR4、HMGB1、Treg表达水平与ACO患者肺功能、病情严重程度存在相关性,临床或可通过检测上述指标水平评估患者病情及肺功能。

Mechanism of T cell hyporesponsiveness to HBcAg is associated with regulatory T cells in chronic hepatitis B

AIM： To study the mechanisms of hyporesponsiveness of HBV-specific CD4^＋ T cells by testing TH1 and TH2 commitment and regulatory T cells. METHODS： Nine patients with chronic hepatitis B were enrolled. Peripheral blood mononuclear cells were stimulated with HBcAg or HBsAg to evaluate their potential to commit to TH1 and TH2 differentiation. HBcAg-specific activity of regulatory T cells was evaluated by staining with antibodies to CD4, CD25, CTLA-4 and interleukin-10. The role of regulatory T cells was further assessed by treatment with anti-interleukin-10 antibody and depletion of CD4^＋CD25^＋ cells. RESULTS： Level of mRNAs for T-bet, IL-12R β2 and IL-4 was significantly lower in the patients than in healthy subjects with HBcAg stimulation. Although populations of CD4^＋CD25^highCTLA-4^＋ T cells were not different between the patients and healthy subjects, IL-10 secreting cells were found in CD4^＋ cells and CD4^＋CD25^＋ cells in the patients in response to HBcAg, and they were not found in cells which were stimulated with HBsAg. Addition of anti-IL-10 antibody recovered the amount of HBcAgspecific TH1 antibody compared with control antibody （P 〈 0.01, 0.34% ± 0.12% vs 0.15% ± 0.04%）. Deletion of CD4^＋CD25^＋ T cells increased the amount of HBcAgspecific TH1 antibody when compared with lymphoo/tes reconstituted using regulatory T cells （P 〈 0.01, 0.03% ± 0.02% vs 0.18% ± 0.05%）.CONCLUSION： The results indicate that the mechanism of T cell hyporesponsiveness to HBcAg includes activation of HBcAg-induced regulatory T cells in contrast to an increase in TH2-committed cells in response to HBsAg.

Hepatocellular carcinoma and macrophage interaction induced tumor immunosuppressionvia Treg requires TLR4 signaling

AIM: To investigated the interaction between toll-like receptor 4 (TLR4)-activated hepatoma cells and macrophages in the induction of tumor-immune suppression mediated by CD4+CD25high family of transcription factor P3 (FOXP3) regulatory T cells (Tregs). METHODS: The proportion of FOXP3+ Tregs was identified in peripheral blood and tumor tissues of 60 hepatocellular carcinoma (HCC) patients. TLR4 expression was examined in tumor tissues and cell lines. The correlation was examined between FOXP3+ Tregs in peripheral blood and TLR4 expression of HCC tissues. Following activation of TLR4 in H22 murine hepatoma cells pre-incubated with lipopolysaccharide (LPS) and co-cultured with macrophage cell line RAW246.7, the synthesis of cytokines tumor necrosis factor-α, CCL22, and interleukin (IL)-10 by the two cell lines was detected and analyzed. RESULTS: FOXP3+ Tregs were enriched in tumor sites, and circulating FOXP3+ Tregs were increased in HCC patients in correlation with multiple tumor foci and up-regulated TLR4 expression in HCC tissues. Semi-quantitative analysis indicated that TLR4 was over-expressed in HCC compared with the matched normal tissues. Cell cultivation experiments indicated that the mRNAs of IL-10 and CCL22 were significantly up-regulated in the RAW246.7 cell line when co-cultured with LPS preincubated H22 cells. CONCLUSION: In hepatoma cell lines, TLR4 may indirectly facilitate the recruitment of Tregs to the tumor site and promote intrahepatic metastasis through its interaction with macrophages.

肾癌患者中外周血淋巴细胞亚群、T-reg、MDSC的表达及临床意义

目的 采用流式细胞术分析肾癌患者MDSC、T-reg、CD_(3)^(+)CD4^(+)T细胞、CD_(3)^(+)CD_(8)^(+)T细胞和CD3-CD_(1)^(+)6CD_(5)^(+)6细胞的表达情况,以及各项指标与肾癌进展的相关性,探讨各项指标在肾癌治疗预后中的相关价值。方法 选取2021年9月至2022年11月住院的肾癌患者44例为肾癌组,选取29例健康人作为对照组。清晨,空腹采集3 mL静脉血。所有肾癌患者均已通过组织病理学诊断。采用全血9色荧光11参数流式细胞术检测肾癌患者和健康体检者MDSC、Treg、CD_(3)^(+)CD_(4)^(+)T细胞、CD_(3)^(+)CD_(8)^(+)T细胞、CD_(3)^(+)CD_(4)^(+)T细胞/CD_(3)^(+)CD_(8)^(+)T细胞比值和CD3-CD_(1)^(+)6CD_(5)^(+)6细胞表达水平。收集患者的临床数据:包括年龄、性别、BMI、临床分期、肿瘤大小、病理类型。结果 肾癌组与对照组之间性别、年龄、BMI差异均无统计学意义(P>0.05)。肾癌组与对照组之间外周血CD_(3)^(+)CD_(4)^(+)、CD_(3)^(+)CD_(8)^(+)、CD_(3)^(+)CD_(4)^(+)/CD_(3)^(+)CD_(8)^(+)、NK、T-reg、PMN-MDSC、M-MDSC水平差异有统计学意义(P<0.05)。肾癌患者CD_(3)^(+)CD_(4)^(+)、CD_(3)^(+)CD_(8)^(+)的表达水平低于对照组(P<0.05),T-reg、PMN-MDSC、M-MDSC的表达水平高于对照组(P<0.05)。肾癌周血中患者外的CD_(3)^(+)CD_(4)^(+)、CD_(3)^(+)CD_(8)^(+)、CD_(3)^(+)CD_(4)^(+)/CD_(3)^(+)CD_(8)^(+)、NK、T-reg、PMN-MDSC、M-MDSC的表达水平与性别、年龄、BMI、病理学类型水平无显著相关(P>0.05)。CD_(3)^(+)CD_(8)^(+)T、CD_(3)^(+)CD_(4)^(+)/CD_(3)^(+)CD_(8)^(+)、NK与肿瘤大小、临床分期之间无明显相关性(P>0.05),CD_(3)^(+)CD_(4)^(+)、M-MDSC、PMN-MDSC、Treg与肿瘤大小、临床分期有相关性(P<0.05)。经Logistic回归分析结果显示:外周血CD_(3)^(+)CD_(4)^(+)淋巴细胞低表达,T-reg、PMN-MDSC、M-MDSC的高表达可能与肾癌患者分期相关(P<0.05)。通过构建ROC曲线结果显示:按照临床分期进行分组,“0”为Ⅰ期、Ⅱ期组,“1”为,Ⅲ期、Ⅳ期组,T-reg、PMN-MDSC、M-MDSC血清指标水平检测肾细胞Ⅲ期、Ⅳ期患者的AUC均>0.70,均有一定的评估价值。T-reg、PMN-MDSC、M-MDSC、M-MDSC+Treg、PMN-MDSC+Treg与参考线比较差异有统计学意义(P<0.05),其中PMN-MDSC+Treg联合检测的差异性最为显著(P<0.05)。结论 肾癌患者与对照组相比CD_(3)^(+)CD_(4)^(+)T细胞、CD_(3)^(+)CD_(8)^(+)T、CD3-CD_(1)^(+)6CD_(5)^(+)6细胞有明显降低,CD4/CD8比值升高,PMN-MDSC、M-MDSC和Treg细胞明显升高,提示机体的免疫功能受损。CD_(3)^(+)CD_(4)^(+)、PMN-MDSC、M-MDSC、T-reg在肿瘤大小、临床分期中表达水平不同,肿瘤直径越大,临床分期越晚,PMN-MDSC、M-MDSC、T-reg表达水平越高。外周血MDSC、T-reg与肾癌临床分期相关,外周血MDSC、T-reg对于肾癌的发生、发展和预后可能有一定的提示意义。