脂多糖致小鼠急性肺损伤组织病理变化与角质细胞生长因子受体表达的关系

目的 :观察急性肺损伤 (ALI)时小鼠肺组织损伤程度与角质细胞生长因子受体 (KGFR)表达水平的关系 ,为基因治疗急性肺损伤提供依据。方法 :建立脂多糖 (LPS)造成的小鼠肺损伤模型和Ⅱ型肺泡上皮细胞 (A5 49细胞 )损伤模型 ,通过病理分析、反转录聚合酶链反应 (RT PCR)和荧光定量聚合酶链反应 (FQ PCR)观察肺组织损伤程度与KGFR表达水平的关系。结果 :LPS造成轻度、中度和重度肺损伤时肺组织KGFR表达水平逐步下降 ,各种损伤程度之间KGFR表达存在显著差异 (P <0 .0 5 )。A5 49细胞的KGFR表达变化不明显 (P >0 .0 5 )。结论 :随着肺损伤程度的加重 ,肺组织KGFR表达水平逐次减低 ,ALI对肺泡上皮细胞的损害严重影响KGFR的表达     

角质细胞生长因子促进大鼠胰腺导管上皮细胞体外增殖

目的 探讨不同浓度的角质细胞生长因子(KGF)对大鼠胰腺导管上皮细胞增殖的影响,寻求最佳浓度.方法 免疫细胞化学染色及RT-PCR方法鉴定SD大鼠胰腺导管上皮细胞;不同浓度的KGF刺激胰腺导管上皮细胞增殖,寻求最佳浓度.结果 大鼠胰腺导管上皮细胞表达Nestin和CK19,不表达Insulin及Glucagon;不同浓度KGF刺激胰腺导管上皮细胞2d后,细胞均有不同程度增殖,20 μg/L KGF作用2d时,细胞数为0.35±0.03,显著高于对照组的0.27±0.02(P ＜0.01).结论 KGF在本实验的剂量范围内可促进胰腺导管上皮细胞增殖,20 μg/L为促进增殖的最佳浓度.     

多囊卵巢综合征卵泡颗粒细胞提前对黄体生成素反应

目的： 探讨多囊卵巢综合征（PCOS）卵泡液中激素和颗粒细胞黄体生成素（LH）受体mRNA表达的关系。方法: 对PCOS组12例和对照组15例患者于月经周期的第7-10 d手术获取卵泡和卵泡液，采用微粒子化学发光法检测卵泡液中卵泡刺激素（FSH）、LH、孕酮（P）、雌二醇（E2）和胰岛素（insulin）的水平，采用ELISA检测卵泡液中雄烯二酮（A）的水平，对颗粒细胞和卵泡膜细胞LH受体mRNA进行RT-PCR半定量检测。结果: PCOS组卵泡液中LH［（3.8±2.1 vs 1.7±0.8)U/L, P<0.01］、A［(600.0±373.4 vs 212.4±205.4)μg/L, P<0.05］和颗粒细胞LH受体mRNA的表达(0.29±0.16 vs 0.12±0.13, P<0.01)显著高于对照组， PCOS组卵泡的颗粒细胞提前表达LH受体mRNA; 对照组卵泡直径小于 7 mm 时，RT-PCR检测不到颗粒细胞LH受体mRNA表达，而PCOS组卵泡直径达到 4 mm 时，发现颗粒细胞LH受体mRNA已提前表达。颗粒细胞LH受体mRNA的表达与卵泡液中LH(r=0.67,P<0.01)、胰岛素(r=0.51,P<0.05)及卵泡膜细胞LH受体mRNA表达的水平(r=0.6,P<0.01)呈正相关。结论: PCOS的卵泡液中存在高水平的LH，PCOS卵泡颗粒细胞提前对LH反应，颗粒细胞和卵泡膜细胞合成A和P增强，这可能是PCOS卵泡发育停滞的原因之一。     

角质细胞生长因子

角质细胞生长因子(KGF)是由成纤维细胞及其他间质细胞分泌的生长因子。按照氨基酸序列的同源性,KGF属于成纤维细胞生长因子(FGF)家族,又称FGF-7。与其他FGF不同,KGF特异地调节上皮源性细胞的生长和发育,这是由于KGF受体主要存在于上皮细胞的缘故。KGF的这一独特性质,使得它被认为在间质-上皮相互作用过程中起一种副泌介质的角色。     

维甲酸对高氧暴露新生大鼠肺组织角化细胞生长因子及其受体表达的影响

目的: 探讨高氧暴露新生大鼠肺组织结构的变化和角化细胞生长因子(KGF)及其受体(KGFR)的表达情况及维甲酸(RA)对其的影响。方法: 将出生24 h内SD大鼠90只随机分为3组,Ⅰ组:空气+生理盐水(NS);Ⅱ组:高氧+NS;Ⅲ组:高氧+RA。Ⅱ、Ⅲ组持续暴露于85% O₂中,Ⅰ组置于空气中;Ⅲ组每天腹腔注射RA,Ⅰ、Ⅱ组每天腹腔注射NS。分别于生后3、7、14 d取肺标本,用HE染色法观察肺组织结构变化及辐射状肺泡计数(RAC);RT-PCR检测KGF和KGFR mRNA表达强度和免疫组织化学法检测KGF蛋白表达水平。结果: (1)生后第14 d,Ⅱ、Ⅲ组较Ⅰ组RAC值显著减少(P<0.05),但Ⅲ组较Ⅱ组明显增高(P<0.05)。(2)与Ⅰ组相比,Ⅱ组3 d时,KGF mRNA表达明显增强(P<0.05);其后开始下降,7 d仍高于Ⅰ组(P<0.05);14 d时较Ⅰ组有所降低(P<0.05);Ⅲ组各时点表达量均高于同期Ⅱ组(P<0.05)。3 d时,各组KGFR mRNA表达量无明显差异;7 d、14 d时,Ⅱ、Ⅲ组表达量明显低于Ⅰ组(均P<0.05),且Ⅱ组和Ⅲ组之间无显著差异(P>0.05)。(3)KGF阳性细胞主要分布在部分肺泡壁及肺泡周围血管内皮细胞和间质细胞。KGF蛋白表达强度与其mRNA表达变化相似。结论: RA可促进肺组织KGF表达,改善高氧所致肺发育受阻。对未成熟肺高氧损伤有一定的保护作用。     

人非小细胞肺癌中KGF mRNA的表达及意义

目的研究人非小细胞肺癌(non-small cell lung cancer,NSCLC)角化细胞生长因子(keratinocyte growth factor,KGF)mR-NA的表达,及其在NSCLC发生过程中肿瘤细胞与间质细胞间的相互作用。方法采用原位杂交和免疫组化法检测KGF mR-NA与Ki-67在50例NSCLC的表达,并与正常组织对照。结果KGF mRNA的表达除在NSCLC某些实质细胞内观察到外,主要见于NSCLC的纤维母细胞和血管平滑肌细胞胞质。肿瘤组织KGF mRNA表达的阳性率86%明显高于正常肺组织的24%(P<0·05)。有淋巴结转移者比无淋巴结转移者的表达更强,且与肺癌的分化相关,分化程度越低,KGF mRNA表达越强。在50例肺癌中Ki-67表达的分布与KGF mRNA相似。结论NSCLC存在KGF mRNA高表达。KGF可通过旁分泌、自分泌两种方式发挥作用。KGF可能与NSCLC的发生有一定的相关性。     

猪卵泡闭锁过程中颗粒细胞凋亡现象的检测

目的　验证猪卵泡闭锁过程中是否存在细胞凋亡及其发生的范围。　方法　用透射电镜观察猪各类卵泡的超微结构 ,对颗粒细胞DNA进行琼脂糖凝胶电泳图谱分析 ,对颗粒细胞涂片进行了DNA末端原位标记(TUNEL)及HE染色检测。　结果　猪的闭锁卵泡中存在大量的凋亡颗粒细胞 ,而健康卵泡中凋亡颗粒细胞则很少 ;对颗粒细胞涂片进行DNA末端标记和常规HE染色检测所得到的颗粒细胞凋亡比例分别为 :健康卵泡TUNEL法为 9% ,HE法为 3 6 % ;闭锁卵泡TUNEL法为 39 2 % ,HE法为 34 % ,尽管两种方法检测的结果之间有差异 ,但两者之间的相关系数达到 0 94。　结论　猪卵泡闭锁过程中确实存在细胞凋亡现象 ,并且凋亡细胞主要集中于颗粒层 ;同时DNA末端标记和常规HE染色检测所得到的颗粒细胞凋亡比例结果说明在某些情况下可以采用简单易行的HE法替代TUNEL法进行细胞凋亡趋势判定     

M1型巨噬细胞源外泌体增强卵泡膜细胞的活力

目的:探讨M1型巨噬细胞源外泌体对卵泡膜细胞活力的影响及其作用机制。方法:采用脂多糖(LPS)处理Raw264.7小鼠巨噬细胞,构建M1型巨噬细胞模型及巨噬细胞-卵泡膜细胞共培养体系。超速离心法分离巨噬细胞分泌的外泌体;通过电镜、Western blot和纳米流式检测仪鉴定外泌体。体外分离培养小鼠卵泡膜细胞,与PKH67荧光标记的外泌体共孵育,观察卵泡膜细胞摄取外泌体的情况。CCK-8法检测细胞活力,流式细胞术分析细胞周期分布,q PCR及Western blot检测细胞周期蛋白依赖性激酶抑制因子1B(CDKN1B)的表达。结果:在巨噬细胞-卵泡膜细胞共培养体系中,LPS诱导的M1型巨噬细胞通过外泌体增强卵泡膜细胞活力。电镜、Western blot及纳米流式检测结果显示,巨噬细胞源外泌体被成功分离。PKH67标记的外泌体与卵泡膜细胞共孵育实验证实卵泡膜细胞能摄取大量巨噬细胞源外泌体。这些外泌体可增强卵泡膜细胞的活力,使卵泡膜细胞G0/G1期比例下降,S期比例升高,且卵泡膜细胞CDKN1B的m RNA及蛋白相对表达量均明显低于对照组。结论:卵泡膜细胞能摄取巨噬细胞分泌的外泌体。M1型巨噬细胞源外泌体通过抑制CDKN1B的表达而增强卵泡膜细胞的活力。     

大鼠卵泡细胞的程序发育和类胰岛素生长因子Ⅱ的表达

用孕马血清，人绒毛膜促性腺激素刺激２０ｄ雌性大鼠，然后在ｈＣＧ刺激的不同时程处死动物，用细胞生物学方法和分子生物学原位杂交方法，观察卵泡细胞形态学的改变，以及卵泡内的类胰岛素生物因子－Ⅱ在该动物卵泡细胞的动态表达。在ＰＭＳＧ刺激４８ｈ后，卵泡开始发育成对促性腺激素有应答反应或对其依赖的卵泡、内泡膜细胞和颗粒细胞增殖、甘油三脂滴大量堆积在内泡膜细胞浆中，外泡膜细胞不含ＴＧＤ，同时ＩＧＦ－Ⅱ基因开始在     

LC3蛋白在小鼠卵泡颗粒细胞中的表达及定位研究

目的:探讨自噬相关蛋白微管相关蛋白1轻链3(microtubule associated protein 1 light chain 3,LC3)在小鼠卵泡颗粒细胞中的表达及定位。方法:昆明小鼠腹腔注射孕马血清促性腺激素(pregnant mare serum gonadotropin,PMSG)前及注射后的第1、2、3、4、5天,采用免疫组织化学染色方法检测自噬相关蛋白LC3和凋亡相关蛋白cleaved caspase-3在卵泡中表达及共定位情况;促卵泡激素(follicle stimulating hormone,FSH)处理颗粒细胞后,Western blot检测LC3和cleaved caspase-3在颗粒细胞中的水平,免疫荧光方法对LC3在颗粒细胞中的亚细胞定位进行研究。结果:在小鼠卵泡发育的各个阶段,LC3蛋白主要在颗粒细胞中表达;免疫荧光显示在闭锁卵泡的颗粒细胞中cleaved caspase-3和LC3的蛋白水平较高;在PMSG腹腔注射后第1、2天,颗粒细胞cleaved caspase-3和LC3-II的蛋白水平减少(P0.05);在PMSG腹腔注射后第3、4、5天,颗粒细胞的cleaved caspase-3和LC3-II蛋白水平依次增加;LC3-II和cleaved caspase-3的蛋白水平具有相关性(r~2=0.8299,P0.05)。FSH处理后,LC3-II定位于颗粒细胞胞浆并呈点状分布。结论:LC3主要在卵泡颗粒细胞中表达,其表达具有细胞特异性和区域特异性,提示自噬在卵泡发育过程中主要在颗粒细胞中发生。卵巢颗粒细胞自噬和凋亡具有相关性,均呈促性腺激素依赖性。     

滤泡树突状细胞与生发中心反应研究的新进展

生发中心是在T细胞依赖性抗体应答过程中于外周淋巴组织内形成的一个特殊的结构。在GC内,受抗原刺激而活化的B细胞进行克隆扩增、IgV区基因的体细胞高度突变、亲和力成熟以及同类型转换,最终形成记忆性B细胞或是产生Ig的浆细胞。在GC内B细胞增殖的同时,也启动了凋亡机制,以确保最终形成的记忆B细胞或浆细胞对抗原的高度特异性。FDCs是参与再次免疫应答的重要细胞,它主要是通过表面的FcR和CR将免疫复合物结合在细胞膜上,并选择性的将抗原递呈给表达高亲和力BCR的B细胞,使之激活并产生抗体或形成记忆B细胞。因此,FDCs在生发中心反应、免疫记忆的维持、B细胞的分化、成熟以及记忆B细胞的形成具有极其重要的作用。但最近的研究对FDCs及其结合的免疫复合物的重要性提出了质疑,认为FDCs在生发中心反应、B细胞的分化、成熟以及记忆B细胞的形成中的作用很可能是非特异性的,并对驻留在FDCs表面的免疫复合物的其它潜在功能进行了讨论。     

滤泡树突状细胞在HIV感染中的作用研究

目的体外实验探讨滤泡树突状细胞（folliculardendriticcells，FDC）在HIV感染中的作用。方法提取人扁桃体FDC，用FDC培养上清液或将FDC放入隔膜装置中，与感染了HIV的外周血T淋巴细胞或扁桃体T淋巴细胞共培养，测定HIV-1P24抗原及tatmRNA水平。结果加入FDC培养上清组[（33．20±3．Z4）w4m1]HIV病毒水平高于加入外周血T淋巴细胞培养上清对照组[（15．89±0．04）w4m1]，P〈0．05；FDC与扁桃体T淋巴细胞共培养上清液组[（82．02±1．64）ngml]及FDC单独培养上清液组[（6．54±0．34）w4ml]HTV病毒水平显著高于扁桃体T淋巴细胞上清液对照组，P〈0．01；且FDC与扁桃体T淋巴细胞共培养上清液组亦高于FDC单独培养上清液组，P〈0．01；FDC组tat mRNA表达水平高于对照组。结论FDC不仅可通过直接接触增强HIV在外周血T淋巴细胞或扁桃体T淋巴细胞中的感染，亦可通过间接接触增强HIV的感染，但间接接触的作用弱于直接接触。     

Disentangling Tfr cells from Treg cells and Tfh cells: How to untie the Gordian knot

T follicular regulatory (Tfr) cells are a subpopulation of Treg cells that have adopted the T follicular helper cell program to localize to the B‐cell follicle. Because of the difficulties in generating mouse models in which Tfr cells are selectively affected, determining where and how Tfr cells regulate the germinal center response remains to be resolved. In this issue of the European Journal of Immunology, Dent and colleagues [Eur. J. Immunol. 2016. 46: 1152–1161] describe a simple, elegant mouse model to conditionally delete Tfr cells without impacting on the Treg‐ and Tfh‐cell populations. Their initial studies suggest that Tfr cells have a more complex role than previously thought, particularly with respect to the regulation of immunoglobulin isotype switching to IgA.     

Detection of calbindin-D immunoreactivity in human follicular dendritic cells.

The calcium-binding protein calbindin-D (28 kD) has been analysed immunohistochemically in different human lymphoid tissues. Combined immunohistochemical staining showed that calbindin-D (28 kD) is expressed by only a proportion of dendritic cells within the light zone of germinal centres, where antigens in the form of immune complexes are trapped and presented to B lymphocytes. All other cell types including macrophages, interdigitating cells, and various lymphocyte populations were negative. The expression of calbindin-D in this functionally relevant subset of follicular dendritic cells could have a role in the regulation of proliferation and selection of memory B-cells by modulating the concentration of calcium ions. Calbindin-D may be a useful marker for analysing in situ the phases of follicular development in different physiological and pathological conditions.     

A novel molecular interaction for the adhesion of follicular CD4 T cells to follicular DC

Nectins and Nectin‐like molecules (Necl) play a critical role in cell polarity within epithelia and in the nervous and reproductive systems. Recently, immune receptors specific for Nectins/Necl have been described. Since the expression and distribution of Nectins/Necl is often subverted during tumorigenesis, it has been suggested that the immune system may use these receptors to recognize and eliminate tumors. Here we describe a novel immunoreceptor, Washington University Cell Adhesion Molecule, which is expressed on human follicular B helper T cells (TFH) and binds a Nectin/Necl family member, the poliovirus receptor (PVR), under both static and flow conditions. Furthermore, we demonstrate that PVR is abundantly expressed by follicular DC (FDC) within the germinal center. These results reveal a novel molecular interaction that mediates adhesion of TFH to FDC and provide the first evidence that immune receptors for Nectins/Necl may be involved the generation of T cell‐dependent antibody responses.     

类风湿关节炎患者外周血Tfh及Th9的变化及临床意义

目的 观察类风湿关节炎(RA)患者外周血中滤泡辅助性T细胞(Tfh)及T辅助细胞9(Th9)的变化,并与病情活动性及脏器受累等临床资料进行相关性分析,探讨Tfh及Th9在RA发病过程中可能的免疫学发病机制.方法 选择36例RA患者和22例健康对照.根据病情活动度不同将病例组分为病情高度活动组(22例)、病情中度活动组(14例),流式细胞仪检测RA和正常对照组外周血单个核细胞( PBMCs)中CD4-FITC、CXCR5-PE、ICOS-APC标记的CD4+ CXCR5+ ICOS+(Tfh)及CD8-FITC、CD3-APC、IL9-PE标记的CD3+CD8-IL-9+( Th9)比例.分析Tfh及Th9与RA患者的血沉(ESR)、C反应蛋白(CRP)、类风湿因子(RF)、关节压痛数、肿胀数及骨质破坏等指标的相关性;分析Tfh与Th9的关系.结果 RA患者的Tfh表达率明显高于对照组(Z=-6.082,P=0.000),RA患者Th9的表达率亦高于对照组(0.989±0.498 vs 0.213 ±0.084,t=13.063,P=0.000);RA重度活动组Tfh表达率亦高于中度活动患者的表达率(3.880±1.255 vs 2.678±1.022,t=2.990,P=0.005),且两组Tfh的表达率均高于对照组(P均＜0.01);RA重度活动患者Th9表达率高于中度患者(1.181±0.523 vs 0.686±0.254,t=4.043,P=0.000),且两组Th9的表达率亦均高于对照组(P均＜0.01); Tfh 细胞数与RA患者DAS28(r=0.571,P=0.000)、ESR(r=0.375,P=0.029)、CRP(r=0.357,P=0.032)、关节压痛数(r=0.598,P=0.000)、RF(r=0.421,P=0.023)及抗CCP滴度(r=0.421,P=0.023)正相关;与病程、晨僵、关节肿胀数、骨质破坏、心电图异常无相关性.Th9表达的百分率与RA患者的DAS28( r=0.461,P=0.005)、ESR(r=0.347,P=0.042)、CRP(r=0.384,P=0210)、关节压痛数(r=0.341,P=0.042)、关节肿胀数(r=0.347,P=0.038)及RF(r=0.379,P=0.025)正相关,与病程、晨僵时间、抗CCP滴度、心电图异常及骨质破坏无相关性;Tfh与Th9在外周血中的表达率呈正相关(r=0.727,P=0.000).结论 RA患者外周血Tfh及Th9的比例显著升高,且与疾病活动度及相关炎症指标明显相关,提示Tfn及Th9可能参与RA的发病及病情发展.     

Follicular dendritic cells and B cell chemotaxis

B cells isolated from germinal centers (GC) of immune mice 2–5 days after antigen (Ag) challenge migrate in response to chemotactic signals, whereas GC B cells isolated at other times and resting B cells do not. Since B cells are in direct contact with follicular dendritic cells (FDC) in GC we reasoned that FDC might play a role in enabling B cells to become chemotactically active. Resting B cells were co-cultured with FDC either with or without anti-μ-dextran (anti-μ-dex) as an Ag surrogate and/or recombinant interleukin (rIL)-4 as a T cell surrogate. After 3 days, the B cells were isolated and their migration to chemotactic factors contained in zymosan-activated serum assessed in microchemotaxis chambers. B cells incubated alone or with anti-μ-dex or rIL-4 showed minimal migration, which could be increased if both anti-μ-dex and rIL-4 were present. However, maximal migration was obtained when B cells were cultured with FDC, and this was not increased by addition of anti-μ-dex and/or rIL-4, indicating that the FDC signal was a primary signal and did not require pre-activation of the B cells. Checkerboard analysis using variation in concentration and location of the chemoattractant in chemotaxis chambers indicated that both chemotaxis and chemokinesis occurred. B cell migration began within 6 h of culture, peaked by 48 h and decreased thereafter. Removal of FDC or interference with FDC-B cell contact ablated or significantly decreased induction of B cell migration. Furthermore, induction did not require functional T cells. These data indicate that FDC can induce resting B cells to become responsive to chemotactic signals.     

Follicular dendritic cells and the maintenance of IgE responses

Antigen (Ag) is retained for long periods of time in secondary lymphoid tissues in the form of immune complexes on follicular dendritic cells (FDC). Ag retained on FDC is thought to play a role in maintaining antibody (Ab) responses in vivo. A model for study of Ab production induced by retained Ag in vitro is the spontaneous Ab response. In this response, specific Ab production is induced spontaneously (no exogenous Ag needed) in cultures derived from secondary lymphoid tissues containing persisting Ag. Specific IgG is spontaneously induced and we reasoned that FDC may also play a role in the maintenance of specific IgE responses. To test this hypothesis, we monitored spontaneous antiovalbmin (OVA) IgE production in cultures of lymph node (LN) fragments from OVA-immunized mice. In addition, highly enriched preparations of OVA bearing FDC were added to OVA-specific memory cells in an attempt to stimulate OVA-specific IgE production. Months after secondary immunization, anti-OVA IgE responses were spontaneously induced when fragments from draining LN were placed into culture. Furthermore, FDC bearing OVA from draining LN induced anti-OVA IgE production when incubated with spleen cells from OVA-immune mice whereas identical cultures with FDC bearing environmental Ag from non-draining LN of the OVA immune animals did not. The anti-OVA IgE responses were elicited only in cultures containing OVA-immune memory cells indicating that specific memory cells were critical for these anti-OVA IgE responses. Removal of FDC from cultures with an FDC-specific mAb dramatically decreased anti-OVA IgE production. These studies demonstrate that FDC can induce specific memory T and B cells to produce IgE and help support the concept that FDC-associated antigen may be involved in the long-term maintenance of specific IgE responses.