大鼠CD4~＋CD25-T细胞的Foxp3基因转染及其表达

背景:调节性T细胞在维持机体免疫应答稳态和免疫耐受方面具有非常重要的作用,但外周血中CD4+CD25+调节性T细胞含量极低,且增殖能力较差。目的:以携带Foxp3基因的慢病毒EGFP+载体转染大鼠CD4+CD25-T细胞,观察其在大鼠CD4+CD25-T细胞中的表达。方法:以免疫磁珠两步法分选大鼠CD4+CD25-T细胞,用携带大鼠Foxp3基因的慢病毒载体体外转染分选的细胞,以转染Foxp3基因的CD4+CD25-T细胞为实验组,EGFP空白质粒组及CD4+CD25-T细胞为阴性对照组,CD4+CD25+T细胞为阳性对照组。荧光显微镜和RT-PCR分别从蛋白和mRNA水平检测Foxp3基因的表达。结果与结论:成功完成了免疫磁珠的分选,获得了纯度较高的CD4+CD25-T细胞和CD4+CD25+T细胞,细胞存活率为(94±2)%,慢病毒转染的CD4+CD25-T细胞高表达Foxp3基因。表明以携带Foxp3基因的慢病毒载体系统可有效介导Foxp3基因在大鼠CD4+CD25-T细胞中高表达。

Impact of duration of adjuvant chemotherapy in radically resected patients with T4bN1-3M0/TxN3bM0 gastric cancer

AIM To provide evidence regarding the postoperative treatment of patients with T4 b N1-3 M0/Tx N3 b M0 gastric cancer, for which guidelines have not been established. METHODS Patients who had undergone curative resection between 1996 and 2014 with a pathological stage of T4 b N1-3 M0/Tx N3 b M0 for gastric cancer were retrospectively analyzed; staging was based on the 7 th edition of the American Joint Committee on Cancer staging system. The clinicopathological characteristics, administration of adjuvant chemotherapy, and patterns of recurrence were studied. Univariate and multivariate analyses of prognostic factors were conducted. The chemotherapeutic agents mainly included fluorouropyrimidine, platinum and taxanes, used as monotherapy, doublet, or triplet regimens. Patterns of first recurrence were categorized as locoregional recurrence, peritoneal dissemination, or distant metastasis.RESULTS The 5-year overall survival(OS) of the whole group(n = 176) was 16.8%, and the median OS was 25.7 mo(95%CI: 20.9-30.5). Lymphovascular invasion and a node positive rate(NPR) ≥ 0.8 were associated with a poor prognosis(P = 0.01 and P = 0.048, respectively). One hundred forty-seven(83.5%) of the 176 patients eventually experienced recurrence; the most common pattern of the first recurrence was distant metastasis. The prognosis was best for patients with locoregional recurrence and worst for those with peritoneal dissemination. Twelve(6.8%) of the 176 patients did not receive adjuvant chemotherapy, while 164(93.2%) patients received adjuvant chemotherapy. Combined chemotherapy, including doublet and triplet regimens, was associated with a better prognosis than monotherapy, with no significant difference in 5-year OS(17.5% vs 0%, P = 0.613). The triplet regimen showed no significant survival benefit compared with the doublet regimen for 5-year OS(18.5% vs 17.4%, P = 0.661). Thirty-nine(22.1%) patients received adjuvant chemotherapy for longer than six months; the median OS in patients who received adjuvant chemotherapy for longer than six months was 40.2 mo(95%CI: 30.6-48.2), significantly longer than the 21.6 mo(95%CI: 19.1-24.0) in patients who received adjuvant chemotherapy for less than six months(P = 0.001).CONCLUSION Patients with T4 b N1-3 M0/Tx N3 b M0 gastric cancer showed a poor prognosis and a high risk of distant metastasis. Adjuvant chemotherapy for longer than six months improved outcomes for them.

Analysis of tumor-infiltrating gamma delta T cells in rectal cancer

AIM: To investigate the regulatory effect of Vδ1 T cells and the antitumor activity of Vδ2 T cells in rectal cancer.METHODS: Peripheral blood, tumor tissues and para-carcinoma tissues from 20 rectal cancer patients were collected. Naïve CD4 T cells from the peripheral blood of rectal cancer patients were purified by negative selection using a Naive CD4+ T Cell Isolation Kit II (Miltenyi Biotec). Tumor tissues and para-carcinoma tissues were minced into small pieces and digested in a triple enzyme mixture containing collagenase type IV, hyaluronidase, and deoxyribonuclease for 2 h at room temperature. After digestion, the cells were washed twice in RPMI1640 and cultured in RPMI1640 containing 10% human serum supplemented with L-glutamine and 2-mercaptoethanol and 1000 U/mL of IL-2 for the generation of T cells. Vδ1 T cells and Vδ2 T cells from tumor tissues and para-carcinoma tissues were expanded by anti-TCR γδ antibodies. The inhibitory effects of Vδ1 T cells on naïve CD4 T cells were analyzed using the CFSE method. The cytotoxicity of Vδ2 T cells on rectal cancer lines was determined by the LDH method.RESULTS: The percentage of Vδ1 T cells in rectal tumor tissues from rectal cancer patients was significantly increased, and positively correlated with the T stage. The percentage of Vδ2 T cells in rectal tumor tissues from rectal cancer patients was significantly decreased, and negatively correlated with the T stage. After culture for 14 d with 1 μg/mL anti-TCR γδ antibodies, the percentage of Vδ1 T cells from para-carcinoma tissues was 21.45% ± 4.64%, and the percentage of Vδ2 T cells was 38.64% ± 8.05%. After culture for 14 d, the percentage of Vδ1 T cells from rectal cancer tissues was 67.45% ± 11.75% and the percentage of Vδ2 T cells was 8.94% ± 2.85%. Tumor-infiltrating Vδ1 T cells had strong inhibitory effects, and tumor-infiltrating Vδ2 T cells showed strong cytolytic activity. The inhibitory effects of Vδ1 T cells from para-carcinoma tissues and from rectal cancer tissue were not significantly different. In addition, the cytolytic activities of Vδ2 T cells from para-carcinoma tissues and from rectal cancer tissues were not significantly different.CONCLUSION: A percentage imbalance in Vδ1 and Vδ2 T cells in rectal cancer patients may contribute to the development of rectal cancer.

T-regulatory lymphocytes in peripheral blood of gastric and colorectal cancer patients

AIM: To assess the absolute number of T-regulatory cells (Tregs; CD4+CD25+Foxp3+) in the peripheral blood of gastric and colorectal cancer patients. METHODS: We enrolled 70 cancer patients (33 gastric cancer, 37 colorectal cancer) and 17 healthy volunteers. The CD3+CD4+ lymphocytes and CD4+CD25+Foxp3+ Tregs in the peripheral blood were analyzed with flow cytometry. The absolute numbers of Tregs were calculated based on the CD4+CD25+Foxp3+ cells percent-age of CD3+CD4+ cells and the absolute numbers of CD3+CD4+ cells per microliter. RESULTS: The mean number of CD4+CD25+Foxp3+ cells per microliter in colorectal cancer patients was 15.7 (SD: 21.8), for gastric cancer patients 12.2 (SD: 14.3), and for controls 17.5 (SD: 11.4). The absolute number of Tregs was significantly lower in gastric cancer patients than in controls (P = 0.026). There was no statistically significant difference for gastric vs colorectal cancer or colorectal cancer vs controls. The absolute number of Tregs was also significantly depressed in N+ vs Ncancer patients [22.0 (27.7) vs 10.1 (9.0), P = 0.013], and in the subgroup of gastric cancer patients [30.3 (27.6) vs 9.6 (8.0), P = 0.003]. No statistical difference was observed in the proportion of Tregs in the CD4+ population between the groups. CONCLUSION: The absolute number of Tregs in peripheral blood of gastric cancer but not colorectal cancer patients was significantly decreased in comparison with that in healthy controls.

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。

外周血DNT细胞和T细胞亚群检测在乙型肝炎病毒感染者诊断中的意义

目的探讨乙型肝炎病毒感染者外周血CD3+CD4-CD8-T细胞(DNT)和T细胞亚群的变化及意义。方法使用流式细胞仪检测136例乙型肝炎病毒感染者,包括33例无症状携带者、28例急性乙型肝炎患者、28例轻度慢性乙型肝炎患者、25例中度慢性乙型肝炎患者、22例重度慢性乙型肝炎患者和39例健康人外周血DNT细胞及T细胞亚群。结果健康人群和急性乙型肝炎患者外周血DNT细胞比例分别为(4.82±3.43)%和(4.75±2.71)%,显著低于无症状携带者[(5.43±3.31)%,P<0.05]和慢性乙型肝炎患者(P<0.05);轻度慢性乙型肝炎患者DNT细胞比例为(7.97±4.12)%,显著低于重度慢性乙型肝炎患者[(11.36±5.01)%,P<0.05];中度慢性乙型肝炎患者DNT细胞比例为(8.41±4.93)%,也显著低于重度慢性乙型肝炎患者(P<0.05);健康人、急性乙型肝炎患者和无症状携带者之间T淋巴细胞亚群分布无明显差异,但随着慢性乙型肝炎患者病情加重,外周血CD3+、CD3+CD4+CD8-细胞比例降低(P<0.05),CD3+CD4-CD8+细胞比例升高(P<0.05)。结论外周血DNT细胞比例的升高与乙型肝炎病毒感染者慢性化及慢性乙型肝炎患者的疾病进程有关。

不裂开下唇及下颌骨的舌癌联合根治术在T3-T4期舌癌中的应用

目的:本研究探讨不裂开下唇及下颌骨的舌癌联合根治术及游离皮瓣舌缺损修复术用于T3-T4期舌癌患者时对术后外观、咬合的影响及临床应用。方法:回顾分析我院2013年1月至2016年12月间收治的T3-T4期舌癌患者210例(原发灶>4cm,侵犯或不侵犯口底,未侵犯下颌骨及骨膜,无手术禁忌症,T3期153例,T4期57例),其中不裂开下唇及下颌骨行舌癌根治术143例(不裂开组),裂开下唇及下颌骨行舌癌根治术67例(裂开组)。将两组资料的手术时间、出血量、术后皮瓣危象发生率、口底裂开发生率、口底瘘发生率、恢复经口进食时间、胃管拔除时间、住院天数、外形满意度、咬合、张口度、术后放疗致骨髓炎发生率、术后24个月局部复发率等指标进行对比,分析两种手术方式效果。结果:与裂开组相比,不裂开组平均手术时间无延长,平均失血无增加,平均经口进食、胃管拔除与住院时间更短,所有患者均未使用钛板、钛钉等固定用高值耗材,患者面部均无瘢痕,张口度影响更小,咬合功能保留更好,术后皮瓣危象、皮瓣坏死,皮瓣与残舌、前口底、舌根裂开,口底瘘的发生率无增加,差异均无统计学意义(P>0. 05)。需要术后行放疗的患者中,不裂开组患者放疗致下颌骨骨髓炎与伤口感染发生率更低,差异有统计学意义(P=0. 014)。不裂开组术后大于24个月口腔局部复发率与总体肿瘤复发率无增加,差异无统计学意义(P>0. 05)。结论:在严格的术前评估下,不裂开下唇及下颌骨行舌癌联合根治及游离皮瓣舌缺损修复术的手术方式在符合条件的T3-T4期舌癌患者中的应用,可以达到与裂开下唇及下颌骨行舌癌根治术同样的根治性效果,不仅面部外形保留满意,张口度、咬合功能恢复更好,创伤更小,术后并发症发生率、复发率无增加,手术时间无延长,术中失血无增加,平均经口进食、胃管拔除和住院时间缩短,并且不裂开组术后放疗后发生放射性骨髓炎、伤口感染可能性更低,故该方法值得在临床在推广。

HIV感染合并肺部感染患者 CD3+CD4+-T细胞表达的研究

目的探讨α干扰素(Interferon-α,IFN-α)、干扰素诱导基因56(Interferon-stimulated gene 56,ISG56)、粘病毒抵抗蛋白A(Myxovirus resistance protein A,MxA)、免疫调控受体程序性死亡分子1(Programmed death-1,PD-1)和T细胞免疫球蛋白及免疫酪氨酸样抑制基序(T cell immunoglobulin and ITIM domain,TIGIT)对人类免疫缺陷病毒(Human immunodeficiency virus,HIV)合并肺部感染患者CD3^+CD4^+-T细胞表达的影响。方法选取2016年12月-2017年12月于余姚市市人民医院就诊的52例HIV合并肺部感染患者作为研究组进行研究,另选择同期48例HIV未合并肺部感染患者作为对照组,检测患者外周血CD3^+CD4^+-T细胞表面的IFN-α、ISG56、MxA、PD-1和TIGIT指标水平情况。结果研究组CD3^+CD4^+-T细胞比例高于对照组(P<0.05);研究组IFN-α、ISG56、MxA分别为(15.82±5.31)、(0.74±0.22)、(0.83±0.14)pg/ml低于对照组(P<0.001);研究组TIGIT和PD-1的CD3^+CD4^+-T细胞分别为(23.61±12.83)%、(51.32±13.74)%高于对照组(P<0.05);研究组TIGIT CD3^+CD4^+-T细胞表面百分数与CD4+-T细胞绝对数呈负相关(P=0.027);与病毒载量呈正相关(P=0.001);研究组PD-1CD3^+CD4^+-T细胞表面百分数与CD4+-T细胞绝对数呈负相关(P=0.026);与病毒载量无相关性(P=0.711)。结论HIV合并肺部感染患者,CD3^+CD4^+-T细胞占比上升,IFN-α、ISG56、MxA降低,TIGIT和PD-1升高,相关指标变化证明当受到感染时机体免疫功能会发生紊乱,且与HIV疾病进展具有一定程度的相关性,具有一定的临床价值。

CD4^＋T细胞及其相关前炎症细胞因子基因多态性与胃癌发生的关系

目的探讨CD4^＋T细胞及其前炎症细胞因子基因多态性与胃癌发生的关系。方法①采用PCR—RFLP法检测170例胃癌患者及110例健康对照者肿瘤坏死因子（TNF）．B＋252、白细胞介素（IL）-10启动子-1082和IL-4内含子3基因多态性。②流式细胞术检测63例胃癌患者和31例健康对照者T细胞亚群和共刺激分子CD28。结果①非贵门胃癌组TNF—β＋252^＊A等位基因频率和IL-10启动子-1082A等位基因频率高于健康对照组（P〈0．05或0．01）。IL-4内含子3基因多态性与非贲门癌患者无明显相关（P〉0.05）。贲门癌组IL-4内含子3RP2基因频率明显高于健康对照组,RP1基因频率低于健康对照组（均P〈0．01）。②胃癌组外周血总T细胞、CD4^＋T细胞和CD8^＋T细胞均较健康对照组显著减少（P〈0．001或0．05）；CD28表达率胃癌组较健康对照组显著增高（P〈0.001）。术后1周总T细胞和CD4^＋T细胞明显上升（P〈0．05）。CD28表达率明显下降（P〈0．05）。结论CD4^＋T细胞亚群异常与胃癌发生关系密切。TNF—β＋252^＊A等位基因和IL-10启动子-1082A等位基因可能是胃癌发生的易感基因，IL-4内含子3-RP2等位基因与贲门癌的发生有一定相关。

大麻素受体2与子宫颈癌组织内浸润T细胞亚群的关系

目的研究大麻素受体2(cannabinoid receptor 2,CB 2)在人子宫颈癌组织中的表达及其与肿瘤内浸润T细胞亚群,包括FOXP 3+T、CD 4+T、CD 8+T细胞的关系。方法选取2016年德州市中医院病理科保存的人体手术切除标本90例,纳入30例正常人宫颈组织为对照A组、30例人高级别宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)Ⅱ~Ⅲ级组织为对照B组、30例人子宫颈癌组织为观察组。采用免疫组织化学法及实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,RT-PCR)检测CB 2在各组中表达情况,采用间接免疫荧光双标法检测观察组组织内浸润FOXP 3+、CD 4+T、CD 8+T细胞数目。结果 CB 2蛋白及mRNA在3组中均有表达,且在观察组中表达最高、对照B组中次之、对照A组中表达量最低,各组间差异均有统计学意义(P<0.05);观察组CB 2蛋白及mRNA的表达均与组织内浸润的CD8+T呈负相关(P<0.05)。结论 CB 2在人子宫颈癌组织内存在高表达现象,且与组织内浸润CD 8+T细胞数量减少有一定相关性。