定期无偿献血者NK细胞免疫功能的研究

目的 分析定期无偿全血捐献者、血小板捐献者的NK细胞数量以及细胞毒活性,评价定期捐血对机体NK细胞免疫功能的影响.方法 采用流式细胞术对三组人群(定期无偿全血、血小板和首次捐血者,后者为对照组)外周血中NK细胞群的数量进行分析.采用效/靶细胞混合培养的方法,计算出NK细胞对靶细胞的杀伤率.结果 全血捐献者NK细胞数量及其对靶细胞的杀伤率与对照组的差异无统计学意义(P>0.05);血小板捐献者NK细胞数量较对照组明显增高(P<0.05),而NK细胞杀伤活性明显降低(P<0.05).结论 本研究结果说明,全血捐献者献血间隔期较长(>3个月),机体有足够的时间来恢复NK细胞数量及其细胞毒性,而血小板捐献者献血间隔期短,机体通过NK细胞数量的增加来调节由于其功能降低对机体造成的影响.     

恶性肿瘤患者外周血调节性T细胞及Th1、Th2细胞的检测和临床意义

目的:通过检测恶性肿瘤患者外周血中的淋巴细胞亚群、CD4+CD25+调节性T细胞(Treg细胞)以及Th1、Th2细胞的数量,了解恶性肿瘤患者的免疫状态。方法:采用流式细胞仪检测90例恶性肿瘤患者(肿瘤组)及60例健康体检者(对照组)外周血中的淋巴细胞亚群、Treg细胞和以及Th1和Th2细胞数量。结果:肿瘤组外周血CD3+T细胞、CD4+T细胞和NK细胞的数量均显著低于对照组(P<0.05),而Treg细胞显著高于对照组(P<0.05)。肿瘤组分泌IFN-γ的Th1细胞显著低于对照组(P<0.05),而分泌IL-4的Th2细胞显著高于对照组(P<0.05)。结论:恶性肿瘤患者Treg细胞数量增多,Th1细胞数量减少,Th2细胞数量增加,这可能是其免疫功能处于抑制状态的重要原因。     

肺炎支原体肺炎患儿外周血和肺泡灌洗液中T细胞及细胞因子变化研究

目的探讨肺炎支原体肺炎(MPP)患儿外周血和肺泡灌洗液(BALF)中T细胞亚群及细胞因子变化情况,以探讨该病可能的免疫损伤机制。方法选择43例MPP患儿(MPP组)为研究对象,并选取同期40例普通肺炎患儿(普通肺炎组),以及因异物入院行软式支气管镜术的25例患儿(对照组)作为对照。流式细胞仪检测外周血和BALF中T细胞亚群构成比,ELISA法检测白细胞介素(IL)-6、IL-10水平,并进行统计学分析。结果 MPP组患儿外周血及BALF中CD3~+CD8~+T细胞水平显著高于普通肺炎组及对照组(P0.05);MPP组外周血及BALF中CD3~+CD4~+T细胞、CD3~+CD4~+CD25~+T细胞水平显著低于普通肺炎组及对照组(P0.05);MPP组患儿血清及BALF中IL-6水平显著高于普通肺炎组及对照组(P0.05),IL-10水平显著高于对照组(P0.05);MPP患儿血清与BALF中IL-6(r=0.635,P=0.000)、IL-10(r=0.396,P=0.009)水平呈显著性正相关。结论肺炎支原体肺炎患儿外周血及BALF中T细胞失衡表达,细胞因子IL-6、IL-10显著升高。     

定期单采血小板捐献者部分免疫指标变化的研究

目的了解定期单采血小板捐献者外周血T淋巴细胞亚群及免疫球蛋白IgG、IgM、IgA水平的变化。方法按捐献间隔期将87名男性单采血小板捐献者分为对照组:首次单采血小板捐献者;实验1组:2次单采血小板间隔45～73 d的捐献者;实验2组:2次单采血小板间隔29～44 d的捐献者。分别用流式细胞术和免疫比浊法检测其外周血T淋巴细胞亚群和免疫球蛋白IgG、IgM、IgA的水平,比较3组捐献者上述指标的差异。结果外周血中CD3+CD4+T淋巴细胞、CD3+CD4+T淋巴细胞/CD3+CD8+T,在实验1组中为(51.33±10.47)%和1.36±0.43,在实验2组中为(50.48±9.50)%和1.34±0.51,在对照组中为(55.18±9.84)%和1.61±0.64。外周血中CD3+CD8+T淋巴细胞、IgG、IgM、IgA,在实验1组中为(39.42±7.14)%、(13.13±2.56)g/L、(1.37±0.60)g/L、(2.55±0.70)g/L,在实验2组中为(40.72±9.24)%、(13.13±2.58)g/L、(1.31±0.70)g/L、(2.44±0.70)g/L,在对照组中为(37.32±8.30)%、(11.55±1.95)g/L、(1.08±0.40)g/L和(2.20±0.91)g/L。3组间上述指标的差异均无统计学意义(P>0.05)。结论定期捐献单采血小板不会影响外周血T淋巴细胞亚群和IgG、IgM、IgA的水平。     

定期双份单采血小板捐献者部分免疫指标变化的研究

目的了解定期捐献双份机采血小板捐献者免疫功能的变化。方法随机抽取本站男性单采血小板捐献者42例,其中首次捐献单采血小板捐献者为对照组,共22例;双份血小板捐献者为实验组,共20例。分别用流式细胞术和免疫比浊法检测其外周血T淋巴细胞亚群和免疫球蛋白IgG、IgM、IgA的水平。结果实验组外周血中CD3+CD4+、CD3+CD8+、CD4+/CD8+分别为(57.23±10.7)%、(40.65±6.89)%、(1.77±0.41)%,对照组上述指标分别为(55.18±9.84)%、(37.32±8.30)%、(1.61±0.64)%,实验组与对照组比较差异无统计学意义(P>0.05);实验组外周血中IgG、IgM、IgA分别为(12.25±2.13)g/L、(1.39±0.48)g/L、(2.68±0.62)g/L,对照组上述指标分别为(11.55±1.95)g/L、(1.08±0.40)g/L、(2.20±0.91)g/L,实验组与对照组比较差异无统计学意义(P>0.05)。结论定期捐献双份单采血小板不会影响捐献者外血T淋巴亚群和免疫球蛋白IgG、IgM、IgA的水平。     

外周血Th17及CD4~+CD25~+FoxP3~+调节性T细胞失衡与不明原因反复自然流产的相关性研究

目的观察不明原因反复自然流产(URSA)孕妇外周血Th17与CD4+CD25+FoxP3+调节性T细胞比例的变化,探讨Th17/T细胞比例失衡在URSA孕妇发病机制中的作用。方法选择36例URSA孕妇为实验组,健康早孕妇女40例为对照组,应用流式细胞仪检测Th17与CD4+CD25+FoxP3+T细胞的比例,应用ELISA法检测IL-6、TGFβ-和IL-17的水平。结果 URSA孕妇外周血中,CD3+CD8-IL-17+T细胞占CD3+T淋巴细胞的百分比高于对照组,P<0.05;CD4+CD25+FoxP3+T细胞占CD4+T淋巴细胞的百分比明显低于对照组,P<0.05。相关细胞因子测定结果显示,IL-6、IL-17水平在URSA孕妇血清中均明显升高,TGF-β水平在2组间比较,差异无统计学意义。结论外周血Th17与CD4+CD25+FoxP3+调节性T细胞的比例及相关细胞因子数量的异常可能下调母胎免疫耐受功能,参与URSA的发生。     

长期单采血小板献血者甲状旁腺激素水平改变及其对献血者免疫功能的影响

目的 探讨长期单采血小板献血者甲状旁腺激素(PTH)水平的改变及其对献血者免疫功能的影响.方法 采用简单随机抽样法按性别构成比为1∶1,选择2015年10月7日至2015年12月5日于烟台市中心血站行单采血小板捐献的30例献血者作为研究对象,纳入研究组(n=30).研究组纳入标准:①献血品种为单采血小板;②捐献单采血小板5年以上,且每年捐献单采血小板次数≥10次;③献血前相关体检和血液学检查结果均符合《献血者健康检查要求》(GB18467-2011).排除标准:①献血者献血前相关体检和血液学检查结果中,任何一项不符合《献血者健康检查要求》(GB 18467-2011);②不愿意参加试验者.按性别、年龄匹配后,随机选择同期于本站首次捐献全血的献血者30例纳入对照组(n=30).对照组纳入标准:①首次献血;②献血品种为全血;③献血前相关体检和血液学检查结果均符合《献血者健康检查要求》(GB18467-2011).排除标准:①献血者献血前相关体检和血液学检查结果中,任何一项不符合《献血者健康检查要求》(GB 18467-2011);②不愿意参加试验者.采用酶联免疫吸附试验(ELISA)检测研究组献血者献血前,献血后1、24 h及7d,以及对照组献血者献血前的血浆PTH水平和免疫球蛋白(Ig)M、IgG、IgA水平;采用流式细胞术(FCM)检测研究组献血者献血前,献血后1、24 h及7d,以及对照组献血者献血前的CD3+T细胞、CD4+T细胞、CD8+T细胞、CD4+ CD8+T细胞等T细胞亚群表达水平;并采用统计学方法比较上述各指标水平变化情况.两组献血者年龄、性别构成比等一般临床资料比较,差异均无统计学意义(P＞0.05).献血者本人认可对献血与健康相关性进行研究的必要性,自愿配合本项研究,并与之签署知情同意书.结果 ①研究组献血者献血前PTH水平与对照组相比,水平增高,但差异无统计学意义(P＞0.05);血浆IgM、IgG、IgA水平,以及CD3+T细胞绝对数、CD4+T细胞绝对数、CD8+T细胞绝对数、CD4+ CD8+T细胞绝对数、CD4+T细胞百分比、CD8+T细胞百分比、CD4+ CD8+T细胞百分比、CD3+CD4+T细胞/CD3+ CD8+T细胞比值,与对照组比较,差异均无统计学意义(P＞0.05).②研究组献血者献血后1 h PTH水平为(72.47±7.25)ng/L,较采集前的(54.70±6.59)ng/L和对照组的(51.90±7.62)ng/L均显著增高,且差异有统计学意义(t=9.937、10.712,P＜0.01);研究组献血者献血后24 h及7 d PTH水平与采集前及对照组相比,差异无统计学意义(P＞0.05);研究组献血者献血后1、24 h和7d的IgM、IgG、IgA水平与采集前及对照组比较,差异均无统计学意义(P＞0.05).③研究组献血者献血后1、24 h和7d的CD3+T细胞绝对数、CD4+T细胞绝对数、CD8+T细胞绝对数、CD4+ CD8+T细胞绝对数、CD4+T细胞百分比、CD8+T细胞百分比、CD4+ CD8+T细胞百分比、CD3+CD4+T细胞/CD3+ CD8+T细胞比值,与采集前和对照组相比,差异无统计学意义(P＞0.05).结论 长期单采血小板献血者PTH水平有短暂升高,但未发现其对献血者机体免疫功能产生影响,我国目前的单采血小板捐献模式不会影响无偿献血者的健康.     

慢性特发性血小板减少性紫癜患者活化T细胞CD30表达与凋亡关系

本研究检测慢性特发性血小板减少性紫癜(ITP)患者外周血单个核细胞中CD3+数、CD3+/CD30+T细胞比值和活化后T细胞凋亡率及外周血血小板计数,探讨2组间的差异和患者活化T淋巴细胞CD30表达率与凋亡率和血小板计数的相关性。以40例慢性ITP患者为研究对象,24例健康志愿者为正常对照,体外培养刺激T细胞活化后运用流式细胞仪检测外周血单个核细胞悬液中CD3+数、CD3+/CD30+T细胞比值及CD3+T细胞凋亡率,同步检测血小板计数,分析ITP组与对照组各指标间的差异和相关性。结果表明:ITP组外周血CD3+细胞数与对照组无明显差异(p0.05)。ITP组活化T细胞表面CD30表达率明显高于对照组(p0.01)。ITP组活化T细胞凋亡率明显低于对照组(p0.01)。活化后T细胞表面CD30表达与其凋亡率呈负相关(r=-0.786,p0.01)。活化T细胞凋亡率与血小板计数呈正相关(r=0.680,p0.01)。结论:慢性ITP患者活化T细胞CD30的高表达导致了T细胞活化后凋亡受阻,在体内蓄积引起免疫功能紊乱,使血小板减少。     

原因不明复发性流产患者外周血NK细胞亚群及细胞因子水平的研究

目的分析原因不明复发性流产患者调节性T(Treg)细胞分泌的细胞因子、外周血自然杀伤(NK)细胞亚群及辅助性T(Th)相关细胞因子水平。方法选取该院2018年2月至2019年2月收治的41例原因不明复发性流产患者作为实验组;选取同期于该院进行检查的41例健康早孕者作为对照组。检测两组人群Treg细胞分泌的细胞因子(TGF-β、IL-10)、NK细胞亚群(CD56~+CD16~-、CD56~+CD16~+、CD56~-CD16~+)及Th1型细胞因子IL-2、IFN-γ,Th2型细胞因子IL-4、IL-10水平。结果实验组Treg细胞分泌的TGF-β、IL-10水平显著低于对照组(P0.05)。实验组CD56~+CD16~-、CD56~-CD16~+NK细胞亚群百分比显著低于对照组(P0.05),CD56~+CD16~+NK细胞亚群百分比显著高于对照组(P0.05)。实验组外周血IL-2、IFN-γ细胞因子水平显著高于对照组(P0.05);实验组IL-4、IL-10等细胞因子水平显著低于对照组(P0.05)。结论外周血Treg细胞分泌的细胞因子、NK细胞亚群及Th相关细胞因子水平可作为诊断原因不明复发性流产患者的重要参考指标。     

紫癜性肾炎患儿外周血CD4+CD25+调节性T细胞的临床意义

目的 研究紫癜性肾炎(HSPN)患儿外周血CD4+CD25+调节性T细胞数量、相关细胞因子的水平及Foxp3的表达,探讨它们在HSPN发病机制中的作用.方法 分别采集40例HSPN患儿及20例健康儿童外周静脉血,通过流式细胞仪分析CD4+CD25+调节性T细胞百分率,ELISA测定血浆IL-10和TGF-β1水平,荧光定量PCR(Real-time PCR)检测T细胞Foxp3mRNA的表达.结果 HSPN患儿外周血CD4+T,CD25+T,CD4+CD25+T/CD4+T均低于对照组(P<0.05);T细胞Foxp3mRNA的表达明显降低,血浆IL-10水平也明显低于对照组(P<0.05),血浆TGF-β1水平较对照组明显升高(P<0.01);CD4+CD25+调节性T细胞百分率随HSPN病理分级加重有降低趋势,二者呈负相关(r＝-0.966,P<0.01).结论 HSPN患儿外周血存在细胞免疫功能失调,CD4+CD25+调节性T细胞数量减少或功能异常,导致免疫抑制效应不足,参与并促进了儿童HSPN的发生发展,其外周血水平与HSPN病理分级有相关性.     

Conditional vascular cell adhesion molecule 1 deletion in mice: impaired lymphocyte migration to bone marrow

We generated vascular cell adhesion molecule (VCAM)-1 "knock-in" mice and Cre recombinase transgenic mice to delete the VCAM-1 gene (vcam-1) in whole mice, thereby overcoming the embryonic lethality seen with conventional vcam-1-deficient mice. vcam-1 knock-in mice expressed normal levels of VCAM-1 but showed loss of VCAM-1 on endothelial and hematopoietic cells when interbred with a "TIE2Cre" transgene. Analysis of peripheral blood from conditional vcam-1-deficient mice revealed mild leukocytosis, including elevated immature B cell numbers. Conversely, the bone marrow (BM) had reduced immature B cell numbers, but normal numbers of pro-B cells. vcam-1-deficient mice also had reduced mature IgD+ B and T cells in BM and a greatly reduced capacity to support short-term migration of transferred B cells, CD4+ T cells, CD8+ T cells, and preactivated CD4+ T cells to the BM. Thus, we report an until now unappreciated dominant role for VCAM-1 in lymphocyte homing to BM.     

Interferon-gamma preferentially reduces memory/effector CD8 T lymphocytes in healthy subjects.

To evaluate the influence of interferon-gamma (IFN-gamma) on leukocyte dynamics, with a focus on naive and memory T cells, we studied 6 healthy subjects twice in a placebo-controlled trial: once after the administration of recombinant human IFN-gamma (rhIFN-gamma; 100 microg/m2 subcutaneously) and at least 4 weeks later, after the administration of saline solution. Additionally, we studied the expression of adhesion molecules on T lymphocytes after in vitro incubation of whole blood with rhIFN-gamma. IFN-gamma induced a significant depletion in the number of T lymphocytes (P < .05 vs control), which was more severe in the CD8+ cell subset than in the CD4+ T cell subset. The numbers of naive CD4+ T cells and memory CD4+ T cells were equally affected by IFN-gamma, whereas within the CD8+ T cell subset, memory/effector cells disappeared preferentially as compared with naive cells (P < .05 vs control). In addition, IFN-gamma induced a decrease in B cells, NK cells, and monocytes. After an initial increase, granulocyte counts decreased significantly as compared with controls. These effects appeared not to be caused by the minimal rise in plasma cortisol levels (P < .05 vs control). In vitro, IFN-gamma did not up-regulate the expression of CD11a, NKI L16, CD11b, LFA-3, or VLA-4. We conclude that the administration of a single dose of IFN-gamma to healthy subjects profoundly affects the numbers of several leukocyte subsets in the peripheral blood compartment.     

哮喘患者外周血CD4^＋CD25^＋调节性T细胞的测定及临床意义

目的探讨CD4^＋CD25^＋调节性T细胞在哮喘患者外周血的变化及意义。方法采用流式细胞术检测60例哮喘患者和40例健康对照者外周血中CD4^＋CD25^＋调节性T细胞的表达量,同时测定20例急性发作期哮喘患者的肺功能。结果哮喘患者外周血CD4^＋CD25^＋调节性T细胞占CD4^＋T细胞的百分比明显低于健康对照组,差异有统计学意义（P〈0．01）;急性发作期哮喘患者外周血CD4^＋CD25^＋调节性T细胞明显低于非急性发作期患者,差异也有统计学意义（P〈0．01）。结论CD4^＋CD25^＋调节性T细胞参与了哮喘的发生。     

Removal by white cell-reduction filters of activated platelets expressing CD62

White cell (WBC)-reduction filters that remove more than 99 percent of the WBCs from platelet concentrates are rapidly being introduced into routine use. Using activation-dependent monoclonal antibodies and flow cytometry, platelet activation was evaluated before and after WBC reduction in 10 platelet concentrates prepared manually from whole blood obtained from five male and five female regular volunteer blood donors. In general no significant increases were found in platelet activation markers after WBC reduction using filters. However, if platelets were activated during preparation, increased numbers of platelets were found expressing the activation marker CD62, and this correlated with the decrease in the platelet count after WBC reduction. These observations may explain increased platelet loss following WBC reduction in some platelet components.     

传染性非典型肺炎患者外周血CD4+T细胞 CD38的表达及意义

目的 探讨传染性非典型肺炎（急性重症呼吸综合征，SARS）患者外周血CD4＋T细胞CD38的表达的变化及其与病程和病情严重程度的关系。方法 用双色流式细胞术测定157例SARS临床诊断病例、37例同期发生的肺炎支原体感染患者及38名健康成人外周血淋巴细胞中CD4＋T细胞的百分率（CD4％）、CD4＋T细胞CD38阳性率（CD38／CD4％）及CD38平均荧光强度（CD38MF）进行检测。结果 SARS病例CD4％与健康对照组相比均无统计学意义，但其CD38／CD4％及CD4＋T细胞CD38 MF显著低于健康对照组（P〈0．05，P〈0．05）；SARS重症患者CD38／CD4％及CD38MF显著低于轻症患，有统计学意义（P〈0．01，P〈0．001）；SARS轻症患者CD4％、CD38／CD4％及CD38MF与健康对照组相比无统计学意义；SARS临床诊断病例CD4％显著高于肺炎支原体感染者，有统计学意义（P〈0．01），而CD38／CD4％及CD38MF显著低于肺炎支原体感染，有统计学意义（P〈0．01，P〈0．01）。结论 SARS患者CD4＋T细胞CD38的表达降低，降低的水平与SARS病情严重程度相关。CD4＋T细胞CD38的表达在SARS诊断病例及肺炎支原体感染中的变化存在统计学意义，CD4＋T细胞CD38的表达的检测有助于SAILS与肺炎支原体感染的鉴别。     

Monitoring of peripheral blood CD34+ cell counts on the first day of apheresis is highly predictive for efficient CD34+ cell yield.

The purpose of this study was to evaluate the correlation of preleukapheresis circulating CD 34+ cells/micro L, white blood cells (WBC), and platelet counts on the first day of apheresis with the yield of collected CD 34+ cell counts in 40 patients with hematological malignancies (n = 29) and solid tumors (n = 11). The median numbers of apheresis cycles, numbers of CD 34+ cells, peripheral blood (PB) mononuclear cells, and total nucleated cells collected were 2 (range, 1-4), 5.5 x 106/kg (range, 0.05-33.78), 2.59 x 108/kg (range, 0.04-20.68), and 7.36 x 108/kg (range, 0.15-28.08), respectively. There was a strong correlation between the number of preleukapheresis circulating CD 34+ cells/micro L and the yield of collected CD 34+ cells per kilogram (r = 0.962, p < 0.001). The threshold levels of PB C 34+ cell/micro L to obtain > or =1 x 106/kg and > or =2.5 x 106/kg CD 34+ cell in one collection were 12/micro L and 34/ micro L, respectively. Fifteen of 17 (88%) patients who had > or =34 CD 34+ cells/ micro L in the PB before collection reached the level of > or =2.5 x 106/kg in a single apheresis. Despite a low r value, WBC and platelet counts on the first day of apheresis also correlated with the yield of collected daily CD 34+ cells per kilogram (r = 0.482, p < 0.01 and r = 0.496 p < 0.01, respectively). These data suggest that preleukapheresis circulating CD 34+ cells/ micro L correlated significantly better with the yield of collected CD 34+ cells than WBC and platelet counts on the first day of apheresis. Using a value of 34/micro L preleukapheresis circulating CD 34+ cells as a guide for the timing of peripheral blood stem cells collections can be time saving and cost-effective.     

Increased numbers of T lymphocytes with gamma delta-positive antigen receptors in a subgroup of individuals with pulmonary sarcoidosis.

Individuals with sarcoidosis were evaluated for preferential usage of T cells with the gamma delta-positive (+) type of T cell antigen receptor. Compared with normal subjects (n = 19), the group with sarcoidosis had increased numbers of CD3+ alpha beta-negative (-) T cells in the blood (normal, 58 +/- 12 cells/microliters; sarcoid, 192 +/- 45 cells/microliters, P less than 0.05) and in the epithelial lining fluid of the lung (normal, 78 14 cells/microliters; sarcoid, 240 +/- 60 cells/microliters, P less than 0.04) and a concomitant elevated number of blood and lung CD3+ gamma delta+ T cells, owing to a striking increase in the number of CD3+ gamma delta+ T cells in a subgroup (7 of 20) of sarcoid individuals. The elevated numbers of sarcoid blood gamma delta+ T lymphocytes were mostly Ti gamma A+ and delta TCS1-, a pattern also seen in normal individuals, consistent with the majority of gamma delta+ T cells expressing one gamma-chain variable region, V gamma 9. The observation of an increase in the total gamma delta+ T cell numbers in a sarcoid subgroup suggests that various specific stimuli may trigger the expansion of different T cell subpopulations within different groups of individuals with sarcoidosis.     

Recovery of mononuclear cell subsets after bone marrow transplantation: overabundance of CD4+CD8+ dual-positive T cells reminiscent of ontogeny.

Patients after bone marrow transplantation are immunodeficient for months to years. To understand better the pathogenesis of this immunodeficiency, we studied quantitative reconstitution of blood monocytes, natural killer (NK) cells, T cells, and B cells at 2-22 months post-transplant. The results indicate monocyte and NK counts generally recover within 2 months, followed by CD4-CD8+ T cell, B cell, and finally (after > 1 year) CD4+CD8- T cell numbers. Dual-positive CD4+CD8+ T cells (which were barely detectable in normal adults), CD4-CD8+ T cells and B cells transiently reached supranormal levels during recovery. Both CD4+CD8- and CD4-CD8+ T cells were larger than controls throughout the 2-year follow up. Comparison with neonatal and infant mononuclear cell subsets suggested the reconstitution of CD4+CD8+ T cells and B cells is similar to ontogeny. In contrast, the reconstitution of CD4+CD8- T cells did not resemble ontogeny.