三色荧光标记鉴定早孕蜕膜及外周免疫细胞组成

目的建立早孕妇女蜕膜免疫细胞高纯度的分离方法,流式细胞术多色荧光直接标记分析早孕蜕膜及外周血主要免疫细胞的构成比。方法经胶原酶消化、Percoll密度梯度离心、短期培养结合的方法分离纯化蜕膜免疫细胞,采用三色、双色及单色荧光直接标记流式细胞术分别检测早孕妇女和对照组中蜕膜、外周血CD3-CD56+CD16-和CD3-CD56+CD16+NK细胞、NKT和γδT细胞、T细胞和单核细胞的阳性率。结果与外周血相比,蜕膜免疫细胞以CD3-CD56+CD16-数量最多,约占免疫细胞总数的(67.02±18.33)%,T细胞占(11.05±7.22)%,单核细胞占(5.28±0.29)%,NKT细胞占(2.35±1.62)%;早孕妇女外周血T淋巴细胞较正常生育期妇女明显下降(P<0.05),而早孕妇女外周血中NK细胞、NKT细胞、单核细胞数量与对照组相比无显著差别。结论早孕妇女蜕膜中具有与外周血不同的免疫细胞组成,应用多色荧光标记的流式细胞术能够简单、直接地鉴定各免疫细胞亚群。     

小剂量高渗氯化钠羟乙基淀粉40对自然杀伤细胞和血小板CD41的影响

目的 观察术中高渗氯化钠羟乙摹淀粉40注射液(HSS40)对恶性肿瘤患者体内自然杀伤细胞(NK细胞)和血小板活化分子CD41影响.方法 将76例手术患者随机分两组:输血组(A组)38例、HSS40组(B组)38例.于麻醉前1 h、术后1、3、7 d抽取外周血,细胞检测仪检测CD56和CD41含量;以乳酸脱氢酶释放法检测NK细胞活性.结果 组间比较:CD56术后第3、7天B组高于A组,差异显著(25.560±11.026比15.648±6.729;29.040±10.221比15.035±6.758,P<0.01),NK细胞活性术后第7天两组比较差异有统计学意义(19.939±6.994比15.307±5.107,P<0.05);CD4,术后l d B组明显低于A组(7.740 4-4.101比10.752 4-5.493,P<0.01).组内比较:A组术后第3天NK细胞活性下降(P<0.05),术后第7天下降明显,与术前比较差异有统计学意义(P<0.01),B组术后第7天NK细胞活性与术前比较差异有统计学意义(P<0.05),CD56术后第3天有所上升(P<0.05),术后第7天上升明显,与术前比较差异有统计学意义(P<0.01).两组CD41术后1～7 d均明显高于术前水平(P<0.01).结论 手术和输血可导致术后NK细胞活性降低,血小板CD41含量明显升高,术中输注HSS40,术后NK细胞活性及数目不同程度升高,且降低血小板CD41含量.     

小剂量高渗氯化钠羟乙基淀粉40对自然杀伤细胞和血小板CD41的影响

目的 观察术中高渗氯化钠羟乙摹淀粉40注射液(HSS40)对恶性肿瘤患者体内自然杀伤细胞(NK细胞)和血小板活化分子CD41影响.方法 将76例手术患者随机分两组:输血组(A组)38例、HSS40组(B组)38例.于麻醉前1 h、术后1、3、7 d抽取外周血,细胞检测仪检测CD56和CD41含量;以乳酸脱氢酶释放法检测NK细胞活性.结果 组间比较:CD56术后第3、7天B组高于A组,差异显著(25.560±11.026比15.648±6.729;29.040±10.221比15.035±6.758,P<0.01),NK细胞活性术后第7天两组比较差异有统计学意义(19.939±6.994比15.307±5.107,P<0.05);CD4,术后l d B组明显低于A组(7.740 4-4.101比10.752 4-5.493,P<0.01).组内比较:A组术后第3天NK细胞活性下降(P<0.05),术后第7天下降明显,与术前比较差异有统计学意义(P<0.01),B组术后第7天NK细胞活性与术前比较差异有统计学意义(P<0.05),CD56术后第3天有所上升(P<0.05),术后第7天上升明显,与术前比较差异有统计学意义(P<0.01).两组CD41术后1～7 d均明显高于术前水平(P<0.01).结论 手术和输血可导致术后NK细胞活性降低,血小板CD41含量明显升高,术中输注HSS40,术后NK细胞活性及数目不同程度升高,且降低血小板CD41含量.     

不良IVF结局患者NK细胞信号通路调节FoxP3细胞表达的研究

目的探讨IVF不良结局患者叉头样转录因子P3(FoxP3)细胞表达情况及NK细胞信号通路调节机制。方法选取我院自2018年1月至2019年1月间收治的75例IVF不良结局患者作为研究对象,依据失败类型分成3组:实施IVF≥3次均未获得妊娠者(反复失败组)25例、实施IVF后仅生化妊娠患者(生化妊娠组)25例、实施IVF后妊娠12周内胚胎停止发育/自然流产者(流产胚停组)25例;选取同期生育1次健康未孕女性25例作为对照组。所有受检者均于卵泡末期采集静脉血液,EDTA抗凝后密度梯度离心法分离血液中的单个核细胞(PBMC),再用免疫磁珠法分离血液中的NK细胞、CD4+CD25-Treg细胞,流式细胞分析各组NK细胞亚群活性;实时定量PCR检测4组受检者CD4+CD25-Treg细胞中FoxP3 mRNA表达情况,并分析肿瘤坏死因子-α(TNF-α)诱导的NK细胞对于CD4+CD25-Treg细胞表达FoxP3的影响。结果与健康对照组比较,IVF不良结局各组CD56+、CD56+CD16+NK细胞水平均显著升高(P<0.05);IVF不良结局组中流产胚停组CD56+NK细胞水平显著高于其他两组(P<0.05)。IVF不良结局各组CD4+CD25-Treg细胞FoxP3 mRNA表达水平均显著低于健康对照组(P<0.05),且不良结局组中反复失败组、生化妊娠组CD4+CD25-Treg细胞FoxP3 mRNA表达水平显著低于流产胚停组(P<0.05)。体外刺激实验显示TNF-α诱导NK细胞下调CD4+CD25-Treg细胞FoxP3的表达(P<0.05)。结论IVF不良结局患者外周血中CD56+、CD56+CD16+NK细胞水平显著升高,而Treg细胞中Fox3 mRNA水平显著降低,且NK细胞能够下调Treg细胞中FoxP3的表达。     

早孕期蜕膜免疫细胞谱及其与妊娠结局的相关性

目的探讨早孕期蜕膜免疫细胞谱变化及稽留流产对妊娠免疫细胞谱的影响。方法选取2017年9月至2018年9月深圳中山泌尿外科医院收治的行人工流产的正常早孕患者(人工流产组,n=34),行清宫术的稽留流产患者(稽留流产组,n=17)及同期于本院IVF助孕成功的正常妇女(正常内膜组,n=27)作为研究对象。运用免疫组化及数字化分析系统定量检测正常妇女子宫内膜及流产组蜕膜组织中各免疫细胞的检测阳性率。结果早孕期妊娠前后的免疫细胞谱有如下改变:妊娠早期人工流产组蜕膜CD56⁺自然杀伤(NK)细胞、CD68⁺巨噬细胞、CD83⁺成熟树突状细胞数均显著高于正常内膜组(P<0.001),而CD8⁺T细胞、Foxp3⁺调节性T(Treg)细胞及CD1a⁺未成熟树突状细胞在两组之间则无显著差异(P>0.05)。在不良妊娠结局稽留流产组患者蜕膜组织中CD68⁺巨噬细胞、CD8⁺T细胞及Foxp3⁺调节性T(Treg)细胞数均显著高于人工流产组(P<0.05),而CD56⁺自然杀伤(NK)细胞、CD83⁺成熟树突状细胞及CD1a⁺未成熟树突状细胞与人工流产组相比无显著差异(P>0.05)。结论早孕期蜕膜中NK细胞、巨噬细胞及成熟树突状细胞的适度增加有利于母胎免疫耐受的形成,但异常高表达的蜕膜巨噬细胞、CD8⁺T细胞及Foxp3⁺Treg细胞可能与不良妊娠结局有关。     

复发性流产患者主动免疫治疗后淋巴细胞免疫表型的变化

目的探讨原因不明复发性流产(URSA)患者行淋巴细胞主动免疫治疗(LIT)后淋巴细胞免疫表型的变化对治疗效果的评估价值。方法采用流式细胞术分析URSA患者LIT前后外周血T淋巴细胞、B淋巴细胞、NK细胞和调节性T细胞免疫表型的变化(P0.05)。结果 25例URSA患者经LIT后成功妊娠16例,治疗后所有URSA患者(n=25)和妊娠成功组(n=16)外周血CD3+T细胞、CD4+HLA-DR+T细胞比例较治疗前均明显增加,CD4+T和CD3-CD56+NK细胞比例明显降低(P0.05);而治疗前后B细胞和Treg细胞、CD56bright CD16-NK、CD56dimCD16+NK、CD3+CD56+NKT及CD69+NK细胞比例则无明显变化(P0.05)。结论 LIT后外周血T细胞、NK细胞的比例发生了明显变化,CD4+T细胞、CD3-CD56+NK细胞比例降低和CD3+T细胞、活化CD4+T细胞增加也许有利于维持妊娠,T细胞和NK细胞的免疫表型有望作为LIT疗效评估的一个重要指标。     

T淋巴细胞亚群检测在肾移植排斥反应与巨细胞病毒感染鉴别诊断中的应用

目的:探讨外周血淋巴细胞亚群的检测在人类同种异体肾移植术后急性排斥反应与巨细胞病毒(CMV)感染的诊断与鉴别诊断中的价值。方法:采用三色流式细胞技术对47例肾移植术后肾功能正常、16例急性排斥反应、11例CMV感染三组患者外周血淋巴细胞中CD3+、CD3+CD4+、CD3+CD8+细胞的百分比进行检测,并计算出CD3+CD4+细胞与CD3+CD8+的比值,分析不同T淋巴细胞亚群在三组患者中的差别,并行t检验。结果:肾功能正常组、急性排斥反应组与CMV感染组外周血淋巴细胞中CD3+细胞的百分比分别为(70.33±10.96)%、(74.46±8.78)%和(74.06±12.94)%,差异无统计学意义;CD3+CD4+细胞的百分比分别为(40.85±9.58)%、(50.85±8.43)%和(28.62±9.40)%,急性排斥反应组及CMV感染组与肾功能正常组相比,差异均有统计学意义(t=3.871;P<0.01。CD3+CD8+淋巴细胞的百分比分别为(27.08±9.40)%、(20.15±5.47)%和(46.32±15.51)%,急性排斥反应组及CMV感染组与肾功能正常组相比,差异均有统计学意义(t=2.787,P<0.01;t=5.346,P<0.01);CD3+CD4+/CD3+CD8+分别为1.78±0.98、2.88±0.76和0.69±0.31,急性排斥反应组及CMV感染组与肾功能正常组相比,差异均有统计学意义(均P<0.01)。结论:检测外周血淋巴细胞亚群的变化,特别是CD3+CD4+/CD3+CD8+比值,对肾移植术后急性排斥反应及CMV感染具有鉴别诊断价值。     

基于肾虚邪伏理论的滋肾育胎方治疗不明原因复发性流产患者的效果及其对自然杀伤细胞表达水平的影响

目的探讨肾虚邪伏理论指导下的自拟滋肾育胎方治疗不明原因复发性流产(URSA)患者的效果及其对激素水平和自然杀伤(NK)细胞表达水平的影响。方法选取2020年2月至2022年3月我院收治的URSA患者94例, 数字表法随机分为对照组及观察组。对照组47例给予地屈孕酮治疗, 观察组47例给予联合基于肾虚邪伏理论的自拟滋肾育胎方治疗, 比较两组患者的中医证候评分、激素水平以及NK细胞表达水平。结果治疗后, 对照组总有效率为68.09%, 观察组总有效率为87.23%, 妊娠保胎总成功率对照组(61.70%)低于观察组(85.11%), 组间对比差异有统计学意义(P<0.05)。两组患者激素雌二醇( E2 )水平、血绒毛膜促性腺激素(HCG)以及孕酮(P)水平较治疗前均上升, 且观察组均比对照组高(P<0.05)。两组NK细胞CD16和CD56表达水平均较治疗干预前下降, 其中CD16水平观察组较对照组低(P<0.05), CD56表达水平组间对比差异无统计学意义(P>0.05)。结论基于肾虚邪伏理论的滋肾育胎方联合地屈孕酮片治疗URSA有较好的疗效, 能改善性激素水平...     

胃癌患者及其手术前后红细胞免疫功能与淋巴细胞功能之间的相关性研究

目的探讨胃癌患者及其手术前后红细胞免疫功能的变化及与淋巴细胞免疫功能之间的相关性.方法72例胃癌患者,20例正常人为对照,检测血红细胞C3b受体花环(RBC-C3bRR)和免疫复合物花环(RBC-ICR),T淋巴细胞亚群、B细胞含量;NK细胞活性、血清循环免疫复合物(CIC)、血清免疫球蛋白和补体.结果(1)胃癌患者RBC-C3bRR显著降低,RBC-ICR显著升高(P＜0.01).(2)术后组RBC-C3bRR显著升高,RBC-ICR显著降低(P＜0.01).(3)胃癌患者CD3+、CD4+、CD4+/CD8+,NK细胞活性,B细胞,IgG、IgA均显著降低,CIC显著升高(P＜0.05或P＜0.01);手术后CD3+、CD4+、CD4+/CD8+、NK细胞活性,免疫球蛋白IgA均有显著提高(P＜0.01);胃癌患者术前组及手术前后RBC-C3bRR变化组与CD3+、CD4+、NK细胞组均呈显著正相关(P＜0.01).结论(1)胃癌患者红细胞免疫粘附功能、淋巴细胞免疫功能低下.(2)手术可使胃癌患者红细胞免疫、淋巴细胞免疫功能明显改善.(3)红细胞免疫粘附功能与T淋巴细胞、NK细胞活性之间关系密切.     

氟比洛芬酯联合吗啡镇痛对胃癌患者T淋巴细胞亚群及自然杀伤细胞的影响

目的 观察氟比洛芬酯联合吗啡镇痛对胃癌根治术患者罔术期外周血T淋巴细胞亚群及自然杀伤(NK)细胞的影响.方法 40例择期全麻下行胃癌根治术患者随机分为氟比洛芬酯组(A组)和吗啡组(B组),每组20例,分别于术前0.5 h静注氟比洛芬酯或安慰药英脱利匹特,术后距第一次给药6 h再次静注氟比洛芬酯或英脱利匹特.两组患者术后均行患者自控静脉镇痛(PCIA).于麻醉前、手术开始后2 h、术后24、48、120 h五个时点用流式细胞仪检测T淋巴细胞亚群(CD3+、CD4+、CD8+)及NK细胞(CD3+CD6+CD56+).结果 与麻醉前比较,两组CD3+、CD4+、CD4+/CD8+和NK细胞在手术2 h、术后24、48 h均明显降低(P<0.05);术后120 h CD3+CD16+CD56+仍未恢复至麻醉前水平(P<0.05).与B组比较,A组CD3+、CD4+、CD4+/CD8+在术后24 h下降幅度较小(P<0.05),而NK细胞则在手术2 h和术后24 h下降幅度较小(P<0.05).结论 胃癌根治术患者围术期用氟比洛芬酯联合吗啡镇痛较单用吗啡镇痛对T淋巴细胞亚群和NK细胞有保护作用.     

The Role of Lymphocyte Subset in Predicting Allograft Rejections in Kidney Transplant Recipients

BackgroundAllograft rejection remains a significant challenge in managing post-transplant recipients despite the improvement in immunologic risk assessment and immunosuppressive therapy. Published literature including animal studies has demonstrated that the cells responsible for rejection are beyond the innate T and B cells, and other studies revealed evidence supporting natural killer (NK) cells’ role in kidney allograft injury. This study aims to find the association between the peripheral blood lymphocyte subset counts, primarily NK cells, and the kidney allograft biopsy findings.MethodsThis is a prospective cross-sectional study among a total of 100 kidney allograft biopsies in 61 kidney transplant recipients. The peripheral blood for the lymphocyte subset was sent just before the allograft biopsy. The patients' immunosuppression and other laboratory investigations were managed as per clinical practices by the attending nephrologist.ResultsOverall, the mean age of our patients was 43.72 ± 10.68 years old, and 55.7% of recipients were male. Higher counts of T cells (CD4+; 658.8 ± 441.4 cells/µL; P = .043) and NK cells (CD3-CD16+CD56+; 188 [interquartile range = 133.0-363.0 cells/µL]; P = .002) were associated with higher risk of allograft rejection in the initial analysis. Patients with an allograft age <12 months had significantly higher total T cells, CD4+ T cells, and NK cells in the rejection groups. However, after assessing factors associated with rejection in the multivariate analysis, we only found that being ABO-incompatible and having >497 CD4+ cells/µL had a higher odds of allograft rejection.ConclusionsHigher CD4± counts were associated with a higher risk of allograft rejection. However, there was no significant increase in CD8±, CD19±, and NK cells count in our cohort with allograft rejection.     

不明原因复发性流产患者子宫内膜/早孕蜕膜组织HOXA-10基因的表达

目的探讨不明原因复发性流产(URSA)与子宫内膜/早孕蜕膜组织HOXA-10基因表达的关系。方法采用原位杂交技术,以HOXA-10反义核苷酸为探针检测51例URSA患者及38例正常生育(NF)组的分泌期子宫内膜/早孕蜕膜组织HOXA-10 mRNA的表达水平,并以灰度阳性单位(PU值)表示。结果NF组子宫内膜分泌早期HOXA-10mRNA表达的PU值腺体为(6.66±0.11),间质为(6.76±0.15);分泌中期分别为(10.95±0.90)及(11.46±1.08);分泌晚期分别为(11.05±1.12)及(11.54±1.10);蜕膜组织分别为(11.55±1.14)及(11.93±1.92);分泌中晚期及蜕膜组织的PU值显著高于分泌早期。URSA组不同时期子宫内膜及早孕蜕膜组织中HOXA-10 mRNA的表达水平基本一致,无明显的分泌中晚期及早孕期峰,并且分泌中晚期及早孕蜕膜组织的PU值明显低于NF同期组。两组各期腺体和间质的PU值无显著差异。结论子宫内膜HOXA-10基因在分泌中晚期及早孕蜕膜组织中的高表达可能和孕卵着床及妊娠维持密切相关,其表达缺陷可能是导致URSA发生的重要原因之一。     

Relationship between perceived social support and immune function

Although a previous meta analysis showed some substantial relationships between social support and immune function, there is still no knowledge about the effects of social support on natural killer (NK) cell number. In this study we examined the direct relationships between peripheral lymphocyte subpopulations and several aspects of social support. We administered the Japanese version of the Stress and Coping Inventory (SCI) by Rahe (1994) to 98 male workers with a written informed consent. Blood samples were collected at 10.00 hours. Lymphocytes subsets were measured by flowcytometry using CD3, CD16, and CD56 antibodies. Partial correlation coefficient controlled by age and smoking between social support and immune cells revealed that there were weak but significant correlations between perceived social support and the numbers of CD3‐/CD16+and CD3‐/CD56+ NK cells (r = 0.25, 0.26). There was no correlation between social support and percentage of NK cells. Positive correlation of perceived social support with NK cell numbers suggested that perceived social support has a direct effect on NK cells and that increased social support might be accompanied by high natural immunity. Further investigation should be undertaken to elucidate why only perceived social support was correlated to NK cells. Copyright © 2003 John Wiley & Sons, Ltd.     

肾移植术后外周血自然杀伤细胞的CD158b表达及意义

目的探讨肾移植术后外周血自然杀伤细胞(NK细胞)的CD158b表达及意义。方法测定62例患者肾移植前、术后第1d、术后第7d、肾功能正常时以及疑有排斥反应时外周血NK细胞的CD158b表达水平。结果62例中,术后38例肾功能恢复正常,观察期内无排斥反应发生,移植前后外周血CD3-CD16/CD56 细胞(NK细胞)及CD3-CD16/CD56 CD158b 细胞稳定,NK细胞中CD158b 细胞的比例也稳定;24例术后7～14d发生急性排斥反应,其外周血CD3-CD16/CD56 细胞呈上升趋势,CD3-CD16/CD56 CD158b 细胞呈下降趋势,NK细胞中CD158b 细胞的比例也呈下降趋势,经单因素方差分析,各指标在不同测定时点的差异均有统计学意义(P<0.05,P<0.01)。结论干扰NK细胞表达CD158b的因素较少,在临床上做出排斥反应诊断前,患者外周血中NK细胞的CD158b表达即呈下降趋势,因此术后监测NK细胞的CD158b表达可为评价患者的免疫状况提供依据。     

Th1/Th2 balance and CD45-positive T cell subsets in primary nephrotic syndrome

T cells are involved in the pathogenesis of nephrotic syndrome (NS). The aim of the study was to determine whether the activity of T-helper-1 (Th1) and T-helper-2 (Th2) cells and the distribution of the lymphocyte subsets, namely CD45RA+CD4+ (”naive” helper T cells, suppressor-inducer), CD45RA+CD8+ (”naive” suppressor T cells, suppressor-effector), CD45RO+CD4+ (”memory” helper T cells), are predictive for steroid sensitivity in children with primary NS. These parameters were assessed at the onset of disease, before initiation of steroid therapy. Two groups of NS children were retrospectively formed according to steroid sensitivity (SS) or resistance (SR). The activity of Th1 and Th2 cells was defined by the production of interleukin-2 (IL-2), interferon-γ, IL-4, and IL-10 in the supernatants of CD4+ T cell cultures activated with autologous monocytes presenting tetanus toxoid (TT). Peripheral lymphocyte subsets were determined using double- or triple-color flow cytometry. In SS children with NS we found a decreased proliferative response of CD4+ T cells to TT stimulation, cytokine synthesis indicating the predominance of Th2 activity, and an increased percentage of activated suppressor-inducer (CD45RA+ CD4+CD25+, 5.18±0.8, P<0.001) and suppressor-effector (CD45RA+CD8+CD25+, 2.05±0.6, P<0.01) cells, with the concomitant reduction of activated memory cells (CD45RO+CD4+CD25+, 0.2±0.1, P<0.001). In children with SRNS we found an increased proliferative response of CD4+ T cells to TT, a rise in activated memory (CD45RO+CD4+CD25+, 3.82±0.7, P<0.01) and suppressor-inducer peripheral T cells (CD45RA+ CD4+CD25+, 3.85±0.6, P<0.01), but a low percentage of activated suppressor-effector (CD45RA+CD8+ CD25+, 0.5±0.2, P<0.05) T cells. We conclude that prior to treatment the distribution of lymphocyte subpopulations in peripheral blood together with Th1 and Th2 cell activity provides a useful tool for evaluating the likelihood of steroid sensitivity in patients with primary NS. Received: 3 November 1998 / Revised: 1 September 1999 / Accepted: 8 September 1999     

Expression of CD158b on peripheral blood lymphocytic cell after kidney transplantation

OBJECTIVE: To investigate the expression of CD158b on peripheral blood lymphocytes after kidney transplantation. METHODS: Sixty two kidney transplant patients were divided into two groups (normal group and rejection group) according to pathologic results and clinical situation. Blood samples were assessed for percentage of CD3+; CD19+; CD3-CD16/56+; CD3+CD158b+; CD19+CD158b+, and CD3-CD16/56+CD158b+ subsets. RESULTS: The percentages of CD3+ cells preop as well as at 1 and 7 postoperative and the day acute rejection happened were 60.06 +/- 4.67, 40.43 +/- 4.11, 31.67 +/- 4.04, and 39.21 +/- 5.20, respectively. The percentages of CD3-CD16/56+ were 21.65 +/- 1.79, 33.84 +/- 5.45, 38.10 +/- 4.86, and 39.53 +/- 4.80, respectively. The percentages of CD3+CD158b+ were 1.46 +/- 0.31, 1.88 +/- 0.70, 2.03 +/- 1.04, and 0.65 +/- 0.12, respectively. The percentages of CD3-CD16/56+CD158b+ were 5.87 +/- 1.24, 3.57 +/- 0.57, 2.82 +/- 0.45, and 1.60 +/- 0.33, respectively. CONCLUSIONS: The percentage of CD3+ cells in the normal and the rejection groups decreased significantly. The percentages of CD158b+T cells decreased significantly after acute rejection. The percentage of CD158b+NK cells decreased significantly after kidney transplantation, decreasing gradually after acute rejection. The percentage of CD158b+ total T cells decreased significantly following acute rejection. The percentage of CD3-CD16/56+CD158b+ of total NK cells decreased significantly after kidney transplantation and after acute rejection. Because few factors interfere with the expression of CD158b on NK cells, monitoring of this marker may be accurate and sensitive.