Role of ion channels in lymphocytes

Summary Ion channels, and ion fluxes in general, appear to regulate a wide variety of processes important to lymphocyte function in normal and disease states. These include resting ionic homeostasis and the more complex signaling events involved in activation, proliferation, cytotoxic function, and volume regulation. The wider application of patch-clamp and microfluorimetry techniques to lymphocytes has helped to clarify some issues and raised many more. It seems likely that rapid progress will be made in our understanding of these areas through a combination of immunological, biochemical, and electrophysiological approaches.     

Hepatocyte progenitors in man and in rodents--multiple pathways, multiple candidates

In severe injury, liver-cell progenitors may play a role in recovery, proliferating, and subsequently differentiating into mature liver cells. Identifying these progenitors has major therapeutic potential for ex vivo pharmaceutical testing, bioartificial liver support, tissue engineering and gene therapy protocols. Potential liver-cell progenitors have been identified from bone marrow, peripheral blood, cord blood, foetal liver, adult liver and embryonic stem cells. Differences and similarities are found among cells isolated from rodents and humans. This review will discuss identifying markers and differentiation potential in in vitro and in vivo models of these putative progenitors in both humans and rodents.     

两种不同纯度的嗅鞘细胞在体外存活与生长的比较

比较80%和50%两种纯度的嗅鞘细胞在体外培养条件下的存活与生长,为嗅鞘细胞体内移植选用最佳纯度提供依据。分离、培养并纯化成年SD大鼠嗅鞘细胞,将纯化的嗅鞘细胞和收集到的贴壁成纤维细胞进行约80%和50%的比例混合后接种,继续培养1d或3d。所有细胞于不同时间点行P75免疫细胞化学荧光染色,以鉴定嗅鞘细胞的初始及其后培养过程中数量和纯度的变化。观察细胞状态,计数细胞总数和P75免疫阳性细胞数目,求得各组嗅鞘细胞数量和纯度并行统计学分析。结果显示:两种不同纯度嗅鞘细胞在培养1d时形态正常,3d时纯度为50%的嗅鞘细胞胞体依然饱满,突起更加纤长,数量和纯度亦无明显下降。而纯度为80%的嗅鞘细胞在培养3d时已出现胞体萎缩和突起变短,数量从1d时每一观察区域内的35±13显著下降为23±5(P=0.02),但纯度未发生明显变化。结果表明50%纯度嗅鞘细胞的存活及生长状态优于80%者,提示细胞体内移植时应考虑选取50%纯度的嗅鞘细胞。     

人骨髓间充质干细胞在体内外分化为心血管组织

目的验证人骨髓间充质干细胞(MSC)分化为心血管组织的潜能。方法用5-氮胞杂苷诱导MSC向心肌细胞分化,用VEGF-B诱导向血管内皮细胞分化。异丙肾上腺素法制成NOD/SCID小鼠心肌损伤模型,经尾静脉注入标记的MSC。免疫荧光法检测MSC的体内、外分化。结果在体外,经诱导的MSC表达肌凝蛋白重链和肌钙蛋白I,表达Ⅷ因子相关抗原和CD31。在体内,标记的MSC表达阳性的肌凝蛋白重链和Ⅷ因子相关抗原。结论MSC可分化为心肌和血管内皮细胞,是再生医学的理想种子细胞。     

Role of L-selectin in the development of autoimmune diabetes in non-obese diabetic mice

Autoimmune diabetes is characterized by an early mononuclear infiltration of pancreatic islets and later selective autoimmune destruction of insulin-producing beta cells. Lymphocyte homing receptors have been considered candidate targets to prevent autoimmune diabetes. L-selectin (CD62L) is an adhesion molecule highly expressed in naive T and B cells. It has been reported that blocking L-selectin in vivo with a specific antibody (Mel-14) partially impairs insulitis and diabetes in autoimmune diabetes-prone non-obese diabetic (NOD) mice. In the present study we aimed to elucidate whether genetic blockade of leukocyte homing into peripheral lymph nodes would prevent the development of diabetes. We backcrossed L-selectin-deficient mice onto the NOD genetic background. Surprisingly NOD/L-selectin-deficient mice exhibited unaltered islet mononuclear infiltration, timing of diabetes onset and cumulative incidence of spontaneous diabetes when compared to L-selectin-sufficient animals. CD4, CD8 T cells and B cells were present in islet infiltrates from 9-week-old L-selectin-sufficient and -deficient littermates. Moreover, total splenocytes from wild-type, heterozygous or NOD/L-selectin-deficient donor mice showed similar capability to adoptively transfer diabetes into NOD/SCID recipients. On the other hand, homing of activated, cloned insulin-specific autoaggressive CD8 T cells (TGNFC8 clone) is not affected in NOD/L-selectin-deficient recipients. We conclude that L-selectin plays a small role in the homing of autoreactive lymphocytes to regional (pancreatic) lymph nodes in NOD mice.     

体外诱导成熟树突状细胞的研究

目的：探讨在体外从干细胞中诱导出成熟DC的适宜环境。方法：分别将人胎肝、骨髓和脾细胞以及小鼠骨髓和脾细胞在体外用GM－CSF和TNF－α诱导,观察了第3、5、7天DC的收获情况。进一步用S－P免疫细胞化学染色检测了mBmDC和fLDC有关分子表达。结果：在体外通过GM－CSF和TNF－α的作用,小鼠骨髓、人胎肝、骨髓细胞呈典型的毛刺状胞浆突起,第5天的收获率分别为：39.5％、67.2％、12.9％,而基本不能从脾细胞中诱导出成熟DC,获得的DC能与MAbCD80、MAbCD40呈强阳性反应。结论：在GM－CSF和TNF－α的共同作用下,能在体外从人胎肝、 骨髓和小鼠骨髓干细胞中获得成熟DC,其DC能表达高高水平的B7－1和CD40分子,这为大量获得DC提供了一种简便可行的手段,为研究DC的生物学特征及其抗原提呈功能提供了丰富的材料,为开展以DC为基础的各种免疫治疗研究奠了良好的基础。     

干细胞标志物在免疫细胞化学中应用的研究现状

干细胞以其自我更新和分化为成熟组织细胞的能力成为当前科研与临床应用的热点,但干细胞的鉴定目前尚无公认一致的方法,随着干细胞相关的分子标记物的发现以及功能的明晰,免疫细胞化学染色法在各种干细胞的鉴定中以其简便、快速、准确的优势发挥越来越重要的作用。本文就应用免疫细胞化学鉴定干细胞的主要分子标记进行综述。     

大鼠肝卵圆细胞增殖模型建立及其表面标记检测

本文拟建立大鼠卵圆细胞增殖模型,分离纯化卵圆细胞并对其特性进行研究。给SD大鼠喂饲2-乙酰氨基芴(2-AAF),剂量分别为5、10、15、20mg／kg,连续给药6d,第7天行三分之二肝切除术,术后继续给药1周,并于术后每隔3d取肝组织行常规组织学观察、细胞增殖试验及免疫组化染色。经免疫磁珠标记C-kit阳性细胞以纯化卵圆细胞,再经免疫细胞化学及RT-PCR对其进行鉴定。结果显示,10、15、20mg／kg三种剂量2-AAF均可成功建立卵圆细胞增殖模型,卵圆细胞增殖于第8天较明显,至第11天达高峰,第14天有所减少。卵圆细胞胞核Brdu染色阳性,卵圆细胞表达特异性抗原OV6、肝细胞标记白蛋白、胆管细胞标记CK19、甲胎蛋白以及连接蛋白43,同时还表达造血干细胞标记C-kit。实验表明,2-AAF剂量为10～20mg／kg时可获得较理想的大鼠卵圆细胞增殖模型,增生的卵圆细胞为具有双向分化潜能的肝干细胞,前体细胞。     

人外周血树突状细胞-乳腺癌细胞融合细胞

目的：探讨树突状细胞—肿瘤细胞融合细胞的形态特性，为研制融合细胞疫苗提供形态学依据。方法：将免疫磁珠法分离的人外周血树突状细胞与人乳腺癌细胞株MCF7融合，瑞氏—姬姆萨染色观察；扫描电镜观察树突状细胞、融合细胞的表面超微结构。结果：树突状细胞与MCF细胞按10：1比例融合后，一个乳腺癌细胞可以与一个或多个树突状细胞相融合；扫描电镜下可见分离的树突状细胞表面有突起，树突状细胞/MCF7融合细胞具两种亲代细胞的表面超微结构特点。结论：树突状细胞与人乳腺癌细胞融合后无明显的形态改变。     

Recruitment of dendritic cells to pathological niches in inflamed liver

The liver is a specialized organ for host defense and immunity. Recruitment of dendritic cells (DCs) is crucial to host defense in a granulomatous liver disease in mice. In response to danger signals, DC precursors are mobilized de novo into the circulation. Myeloid DC (mDC) precursors are recruited to perisinusoidal spaces and activated to form granulomas. Recruited mDCs subsequently extravasate into Disse’s space and migrate to the portal area to induce portal tract-associated lymphoid tissue (PALT). Some mDCs are remobilized into draining hepatic lymph nodes (LNs) to prime antigen-specific CD4⁺ helper T cells. Kupffer cell-derived CCL3/MIP-1α attracts mDC precursors to the sinusoidal granulomas, whereas PALT composed cell-derived CCL21/SLC attracts activated mDCs to the T-cell zone of PALT. Inflammatory cytokines modulate this sinusoid-portal migration through IL-1R/TLR signaling. Recruited mDCs themselves also produce several chemokines and cytokines that modulate T-cell responses. A unique trafficking of circulating mDC precursors within the inflammation-associated, newly formed compartments (“pathological niches”) is strictly regulated by both homeostatic and inducible chemokines and determines the final efficiency of the immunity in this organ.     

多能干细胞制备方法的研究进展及问题

多能干细胞为研究疾病的分子机制、药物筛选及再生医学应用提供理想的细胞来源,然而,当前多能干细胞的来源或制备方法面临众多瓶颈和挑战,本文回顾了多能干细胞制备方法的最新研究进展,为多能干细胞的后续研究提供思路。     

早期胚胎中干细胞自组织的研究进展

哺乳动物早期胚胎发育中,特定结构的形成是精细而复杂的自组织过程。由于胚胎体积小,且不易获得, 加之自组织存在时序性,因此体内自组织研究尤为困难。但胚胎相关干细胞研究的进展,为体外研究早期胚胎发 育自组织现象提供了极大的便利。现就自组织的基本原理、胚胎早期的自组织现象和胚胎相关干细胞自组织研究 的进展作一综述,以期能清晰呈现早期胚胎中干细胞自组织过程及其调控机制。     

对胎儿胃粘膜三种内分泌细胞的观察

目的探讨胎儿胃粘膜三种内分泌细胞的发生过程。为胚胎学发展提供资料。方法收集2-10个月胎儿53例,新生儿3例,利用免疫组织化学方法,检测胃窦粘膜中胃泌素细胞（G）,生长抑素细胞（SS）和5-羟色胺细胞（5-HT）,计数进行组间比较。结果胎龄5-9个月胎儿胃窦粘膜中G、SS、5-HT细胞迅速增多,以G细胞数目最多,足月胎儿及新生儿G细胞减少。结论胃粘膜三种内分泌细胞随胎龄增多,在胎儿生长发育旺盛期6-8月数目最多。     

Types of parvalbumin-containing retinotectal ganglion cells in mouse

The calcium-binding protein parvalbumin (PV) occurs in the retinal ganglion cells (RGCs) of various vertebrate species. In the present study, we aimed to identify the types of PV-containing RGCs that project to the superior colliculus (SC) in the mouse. We injected retrograde tracer dextran into the mouse SC to label RGCs. PV-containing RGCs were first identified by immunocytochemistry and then neurons double-labeled with dextran and PV were iontophoretically injected with a lipophilic dye, DiI. Subsequently, confocal microscopy was used to characterize the morphologic classification of the PV-immunoreactive (IR) retinotectal ganglion cells on the basis of dendritic field size, branching pattern, and stratification within the inner plexiform layer. Among the 8 different types of PV-containing RGCs in the mouse retina, we found all 8 types of RGCs projecting to the SC. The RGCs were heterogeneous in morphology. The combined approach of using tracer injection and a single cell injection after immunocytochemistry on a particular protein will provide valuable data to further understand the functional features of the RGCs which constitute the retinotectal pathway.     

胚胎干细胞移植治疗缺血性心脏疾病的研究进展

缺血性心脏疾病是威胁人类健康的头号杀手之一。冠状动脉阻塞引起心肌梗死,伴随心肌细胞大量死亡,损失的心肌细胞将被没有收缩功能的疤痕组织所替代,最终导致心力衰竭。近年来,移植外源性干细胞替代受损心肌的治疗策略得到人们越来越多的关注,并取得诸多进展。其中,胚胎干细胞具有无限增殖和多向分化的特点,在向心肌细胞分化、与宿主心肌细胞整合和心肌电信号传导方面具有优势,因此,在移植治疗心肌梗死方面具有广阔的应用前景。然而,胚胎干细胞最终应用于临床治疗晚期心脏疾病仍面临许多问题。本文就胚胎干细胞及其来源的细胞移植应用于缺血性心脏疾病治疗的研究进展作一综述。     

皮肤树突状细胞亚群研究进展

皮肤树突状细胞(DC)作为重要的抗原提呈细胞,在机体免疫应答或自身耐受的发生中扮演着非常重要的角色.皮肤免疫系统中定居着多种DC亚群,主要包括表皮层中的郎格汉斯细胞(LC)与真皮层中的各种真皮DC亚群.健康皮肤中的DC亚群主要有表皮LC、真皮DC(dDC)和浆细胞DC(pDC),dDC又分为Langerin+ dDC及Langerin-dDC等.但在炎症性皮肤,如过敏性皮炎、银屑病等病变皮肤中则存在着炎症性DC亚群.DC由于其复杂的异质性群体,导致了其各亚群的特殊化功能.皮肤DC亚群的特殊化功能,为皮肤性疾病的临床治疗及新型疫苗的研发设计等都提供了良好的新策略.     

人外周血初始B细胞和记忆性B细胞亚群的特征和功能

B淋巴细胞是免疫系统的重要免疫成份，主要功能是介导体液免疫应答。在人外周血中，按照B淋巴细胞的发育阶段及功能的不同，可将B淋巴细胞分为初始成熟B细胞、记忆B细胞和浆细胞。记忆B细胞又可分为IgM记忆B细胞和类型转换的记忆B细胞。近年来的研究表明，B淋巴细胞的亚群远比人想象中的复杂，因此对人B细胞各亚群的起源、发育和功能进行更深入的研究，将有助于治疗自身免疫性疾病，在慢性感染性疾病的治疗过程中找到新的策略，并指导研发安全有效的疫苗。     

Cell polarity and spindle orientation in the distal epithelium of embryonic lung

A proper balance between self‐renewal and differentiation of lung‐specific progenitors at the distal epithelial tips is absolutely required for normal lung morphogenesis. Cell polarity and mitotic spindle orientation play a critical role in the self‐renewal/differentiation of epithelial cells and can impact normal physiological processes, including epithelial tissue branching and differentiation. Therefore, understanding the behavior of lung distal epithelial progenitors could identify innovative solutions to restoring normal lung morphogenesis. Yet little is known about cell polarity, spindle orientation, and segregation of cell fate determinant in the embryonic lung epithelium, which contains progenitor cells. Herein, we provide the first evidence that embryonic lung distal epithelium is polarized and highly mitotic with characteristic perpendicular cell divisions. Consistent with these findings, mInsc, LGN, and NuMA polarity proteins, which control spindle orientation, are asymmetrically localized in mitotic distal epithelial progenitors of embryonic lungs. Furthermore, the cell fate determinant Numb is asymmetrically distributed at the apical side of distal epithelial progenitors and segregated to one daughter cell in most mitotic cells. These findings provide evidence for polarity in distal epithelial progenitors of embryonic lungs and provide a framework for future translationally oriented studies in this area. Developmental Dynamics 240:441–445, 2011. © 2011 Wiley‐Liss, Inc.     

Hepatic progenitor cells, stem cells, and AFP expression in models of liver injury

Adult hepatocytes and liver-cell progenitors play a role in restoring liver tissue after injury. For the study of progenitor cells in liver repair, experimental models included (a) surgical removal of liver tissue by partial hepatectomy; (b) acute injury by carbontetrachloride; (c) acute injury by d-galactosamine (GalN) and N-nitrosomorpholine (NNM); and (d) chemical hepatocarcinogenesis by feeding NNM in low and high doses. Serological and immunohistological detection of alpha-fetoprotein gene expression served to follow pathways of cellular differentiation. Stem cells were not required in models of surgical removal of parenchyma and in carbon tetrachloride intoxication of adult hepatocytes. In contrast, regeneration of liver occurred through biliary epithelial cells in injuries induced by GalN and NNM. These biliary epithelial cells, collectively called oval cells, are most probably derived from the canals of Hering. Proliferating bile duct cells reached a level of differentiation with reactivation of foetal genes and significant alpha-1-fetoprotein (AFP) synthesis signalling a certain degree of retrodifferentiation with potential stemness. Due to the same embryonic origin of bile ducts and hepatocytes, biliary epithelium and its proliferating progeny (oval cells) have a defined role in liver regeneration as a transit and amplification compartment. In their early proliferation stage, oval cells were heavily engaged in DNA synthesis ([3H]thymidine labelling). Pulse-chase experiments during experimental hepatocarcinogenesis exhibited their development into hepatocytes with high risk for transformation and leading to foci of altered hepatocytes. Hepatocellular carcinomas may arise either from proliferating/differentiating oval cells or from adult hepatocytes; both cell types have stem-like properties. AFP-positive and AFP-negative carcinomas occurred in the same liver. They may represent random clonal origin. The heterogeneity of phenotypic marker (AFP) correlated with a process of retrodifferentiation.