小鼠胚胎干细胞来源的神经干细胞的稳定传代体系探索

目的探讨体外诱导、获取均一的神经干细胞群(NSCs)的有效方法,并建立神经干细胞体外稳定传代扩增体系。方法首先采用无血清的诱导培养基贴壁诱导mESCs形成神经上皮祖细胞(NPCs)。然后将经添加表皮生长因子(EGF)和成纤维细胞生长因子-2(FGF2)的无血清培养基短暂悬浮培养后的NPCs再贴壁培养,诱导形成NSCs。通过细胞系46C监测NPCs的形成,同时对分化细胞进行定量PCR和免疫荧光染色,在不同水平检测细胞分化效果。结果 mESCs神经诱导5 d出现大量Sox1+的NPCs;NPCs悬浮培养后,进一步诱导可得到形态均一的NSCs。第2代和第6代NSCs的神经干细胞标志定量PCR检测结果为:Pax6、Nestin、Mash1、BLBP高表达。第8代NSCs免疫荧光染色显示90%以上的细胞均为Nestin、RC2和Pax6阳性。结论成功诱导mESC生成神经干细胞群,并且可以在体外连续稳定的传代。     

增强型绿色荧光蛋白基因转染小鼠胚胎干细胞及其无血清神经细胞诱导

目的构建表达绿色荧光蛋白的小鼠胚胎干细胞(MES)克隆，观察基因转染对MES细胞增殖和无血清诱导为nestin阳性神经前体细胞(NPCs)的影响。方法原代分离小鼠胚胎成纤维细胞并经丝裂霉素-C处理作为饲养层细胞。质粒的转化、抽提、纯化。电穿孔法基因转染，MES细胞克隆的NBT／BCIP染色，NPCs的nestin免疫组织化学染色。结果EGFP基因转染后可得到表达绿色荧光蛋白的MES细胞克隆，采用N2或ITSF无血清条件培养基可将其定向诱导为nestin阳性的并能发绿色荧光的NPCs。基因转染后的MES细胞增殖、神经前体细胞诱导率和未转染组相比均无明显下降，但NPCs绿色荧光的表达较诱导前明显下降。结论基因转染对ES细胞的增殖和神经分化无明显影响。     

定向诱导人尿液细胞源性多能干细胞向多巴胺能神经元的分化

目的探讨定向诱导人尿液来源诱导性多能干细胞(U-iPS)向多巴胺(DA)能神经元分化。方法单层贴壁法体外培养U-iPS,通过时向性添加TGF-β及BMP信号通路抑制剂,SHH细胞因子,GSK3β抑制剂Chir99021将U-iPS诱导为DA能神经前体细胞(NPCs),通过添加BDNF/GDNF等细胞因子将其进一步分化为DA能神经元。在诱导分化的4个时间点(第0天、7天、14天及25天)采用RT-qPCR和免疫荧光检测多能干细胞标志物Oct4与Nanog的表达;NPCs标志物Nestin、Pax6和Foxa2的表达;DA神经前体细胞标志物Lmx1a和En1的表达;神经元标志物Tuj1的表达以及DA能神经元标志物TH的表达情况。以人皮肤成纤维细胞来源的iPS(F-iPS)向DA能神经元的诱导分化作为对照进行分化效率的比较。结果 U-iPS在第0天表达多能干标志物Oct4和Nanog;诱导第7天能转化为Nestin、Pax6及Foxa2阳性的NPCs;第14天转化为Lmx1a和En1阳性的DA能神经前体细胞;第25天分化得到的神经元样细胞表达神经元标志物Tuj1,其中部分细胞表达DA能神经元标志物TH。各时间点神经类细胞标志物的表达与F-iPS诱导分化得到的神经类细胞标志物的表达差异无统计学意义(P0.05)。结论 U-iPS细胞具有向DA能神经元分化的潜力,能在体外经过诱导分化得到TH阳性的DA能神经元,且与F-iPS向DA能神经元分化的效率无明显差异。     

缺氧对新生大鼠海马神经干细胞增殖和分化的影响

目的探讨单纯性缺氧对新生大鼠海马神经干细胞(NSCs)增殖和分化的影响。方法将36只新生1d大鼠随机均分为N组(正常对照组)、Q2组(缺氧2h组)、Q5组(缺氧5h组)。将海马组织在无血清培养液(均含有碱性成纤维生长因子bFGF、表皮生长因子EGF和白细胞抗原B27)中,分别进行原代培养、单克隆培养和传代培养,单克隆培养细胞进行巢蛋白(Nestin)和Hoechst33342免疫荧光双标染色;传代培养的细胞传三代诱导分化,诱导分化3d的细胞分别行神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)和Hoechst3342免疫荧光双标染色,计数NSE阳性细胞比例,进行统计学分析。结果单克隆培养的细胞均呈Nestin阳性表达,诱导分化的细胞部分呈NSE或GFAP阳性表达。Q2组、N组、Q5组细胞原代培养克隆球直径达2μm的时间分别为7d、10d、13d。传3代后,对各组细胞进行诱导分化,Q2组细胞诱导分化为神经元的比例明显高于N组,Q5组的细胞诱导分化为神经元的比例明显低于N组。结论短时间缺氧可促进在体海马神经干细胞增殖和诱导其向神经元分化,长时间缺氧则抑制在体神经干细胞增殖和向神经元分化。     

Desert Hedgehog诱导大鼠胚胎中脑神经前体细胞定向分化为多巴胺能神经元

目的 研究Desert Hedgehog(DHH)对胚胎中脑神经前体细胞(NPCs)向多巴胺能神经元分化的诱导作用。方法 大鼠DHH病毒上清感染COS7、NIH/3T3和9L细胞, 用免疫荧光、实时定量PCR和Western blot方法检测DHH表达。分离培养大鼠胚胎中脑神经前体细胞，免疫荧光鉴定神经前体细胞的增殖分化特性。条件细胞表达DHH成功后分别与上述NPCs共培养10d，免疫荧光检测酪氨酸羟化酶(TH)的表达。结果 DHH在COS7、NIH/3T3和9L细胞中成功表达；同时，分离培养的大鼠胚胎中脑神经前体细胞具有良好的增殖分化特性，但两者共培养10d后偶见TH阳性的细胞。结论 DHH编码的蛋白不能或不能单独诱导与其共培养的NPCs定向分化为多巴胺能神经元。     

巢蛋白的表达模式及其功能

巢蛋白 (Nestin)属于第Ⅵ类中间丝蛋白 ,主要由胚胎期和成年的神经前体细胞一过性表达 ,在中枢神经系统各种损伤条件下 ,脑中反应性星形胶质细胞和神经元又再现其表达。然而 ,本实验室首次观察到 ,在正常成年大鼠和成人基底前脑存在一个有别于胆碱能神经元的Nestin免疫反应阳性神经元簇 ,并证明了这些Nestin -IR神经元的Nestin表达受卵巢雌激素的影响。Nestin的功能与维持神经前体细胞的正常形态结构有关 ,也可能与其它的中间丝蛋白如神经丝共聚形成多聚体 ,此外 ,尚可能在影响其它蛋白的功能方面发挥作用。     

MiR-125a-5p靶向APC激活经典WNT信号通路促进hiPSCs分化为多巴胺能神经前体细胞

目的通过调节miR-125a-5p的表达实现人诱导性多能干细胞(hiPSCs)向多巴胺(DA)能神经前体细胞及DA能神经元的高效转化。方法应用MicroRNAs高通量测序与分析,miR-125a-5p的筛选、靶基因(APC)的预测与验证;miR-125a-5p前体miR-125a慢病毒载体的构建与包装,miR-125a-5p过表达hiPSCs细胞株的构建、筛选及其向多DA能神经前体细胞与DA能神经元的诱导分化;免疫细胞化学、实时定量PCR(qRT-PCR)及蛋白印迹(WB)法检测miR-125a-5p过表达hiPSCs与对照hiPSCs向DA能神经前体细胞及DA能神经元诱导分化过程中神经前体细胞标志物Nestin、底板细胞标志物Foxa2、DA能神经前体细胞标志物En1、DA能神经前体细胞分选标志物Corin、神经元标志物Tuj1及DA能神经元标志物TH的表达。结果 MiR-125a-5p在hiPSCs向DA能神经前体细胞及神经元诱导分化过程中的表达呈递增趋势,miR-125a-5p靶向调节经典WNT信号通路关键因子APC,稳定过表达miR-125a-5p的hiPSCs细胞株能向DA能神经前体细胞及DA能神经元分化,其诱导分化效率与对照hiPSCs相比在神经前体细胞(NPCs)阶段FOXA2、EN1、CORIN的表达明显增高,神经元阶段TH、DAT和Nurr1的表达明显增高,提示过表达miR-125a-5p的hiPSCs具有更好的向DA能神经前体细胞及DA能神经元的诱导分化潜能。结论 MiR-125a-5p能通过靶向下调APC的表达激活经典WNT信号通路活性,促进hiPSCs向DA能神经前体细胞及DA能神经元的诱导分化。     

大鼠视神经损伤后大胶质细胞去分化及预变性腓总神经的诱导

目的研究视神经损伤后神经胶质细胞的去分化及其诱导。方法成年雄性SD大鼠,分为正常对照组、损伤组、移植组和压榨移植组,在视神经伤后3d、7d、14d和28d 4个不同时相点取视神经,用HE方法计数细胞数量,用免疫组织化学、免疫印迹、原位杂交组织化学结合计算机图像分析检测巢蛋白(Nestin)、胶质纤维酸性蛋白(GFAP)、髓鞘碱性蛋白(MBP)、神经丝(NF)、脑源性神经营养因子(BDNF)、Nogo-A及Nogo-A mRNA的表达,用免疫荧光双标组织化学法检测Nestin-GFAP和Nestin-MBP的共表达。结果损伤组细胞数量仅在伤后7d明显增多,Nestin、MBP、Nogo-A及其mRNA表达上调,GFAP、NF、BDNF下调,出现一些Nestin-GFAP双标细胞和少量Nestin-MBP双标细胞;与损伤组相比,移植组和压榨移植组一些时相点的细胞数量不同程度地增多,Nestin、GFAP、BDNF和NF表达上调,MBP、Nogo-A及其mRNA表达下调,Nestin-GFAP双标细胞有所增加,且28d组高于14d组。结论视神经损伤后,主要是星形胶质细胞发生去分化,大胶质细胞呈现一些不利于神经再生的基因表达变化;移植预变性周围神经能进一步诱导星形胶质细胞去分化,大胶质细胞的一些基因表达变化有利于神经再生。     

Sertoli细胞对大鼠大脑皮质神经前体细胞的诱导分化作用

目的:研究Sertoli细胞对体外培养大鼠皮质神经前体细胞生长发育的促进作用,及向多巴胺能神经元分化的诱导作用.方法:将大鼠Sertoli细胞与14 d大鼠皮质神经前体细胞体外共培养,免疫细胞化学检测神经干细胞标记物巢蛋白,神经元、星形胶质细胞和小胶质细胞标记物βⅢ-微管蛋白(βⅢ-tubulin)、胶质纤维酸性蛋白(GFAP)和半乳糖脑苷脂(GalC),及多巴胺能神经元标记物酪氨酸羟化酶(TH)的表达情况.结果:随着共培养时间的延长,神经前体细胞中GFAP阳性细胞数量逐渐减少,而β-Ⅲ tubulin 阳性细胞数增多,并出现TH阳性神经元.结论:Sertoli细胞抑制体外培养的大鼠皮质神经前体细胞的生长,但具有显著促神经元分化作用,并能够诱导与其共培养的大鼠皮质神经前体细胞分化为多巴胺能神经元.     

毛囊神经干细胞促进受损大鼠视神经大胶质细胞去分化

目的 培养毛囊神经干细胞,研究其对受损视神经大胶质细胞去分化的影响。 方法 取约90g雄性SD大鼠触须垫毛囊隆突区贴块,无血清培养或经15%胎牛血清诱导培养毛囊隆突区细胞,用免疫荧光细胞化学和PCR鉴定;以含增强型绿色荧光蛋白的腺相关病毒（rAAV2EGFP）稳定转染该细胞。成年雄性SD大鼠54只,分为正常对照组、损伤组、移植组,移植组为视神经损伤后移植上述毛囊神经干细胞。转染细胞移植组术后7d、14d、30d,取视神经荧光显微镜下观察;损伤组和未转染细胞移植组术后7d取术侧视神经行Affymetrix基因芯片及实时荧光定量PCR检测,术后7d、14d取术侧视神经行HE、免疫组织化学检测。 结果 成功培养出神经前体细胞标记分子阳性的毛囊神经干细胞,诱导后该细胞可表达成熟神经细胞标记分子。毛囊神经干细胞移植到受损视神经30 d后,仍能存活和迁移。与损伤组相比,移植组差异表达基因有240条,其中一些与去分化相关,如干细胞、凋亡、增殖、信号转导、转录、分化发育、细胞黏附等相关基因,实时荧光定量PCR与基因芯片结果基本相符。移植组视神经远侧段细胞数较损伤组明显增多,巢蛋白、髓鞘碱性蛋白(MBP)、细胞外信号调节激酶1/2(ERK1/2)表达增加,胶质纤维酸性蛋白(GFAP)表达减少,且视神经近侧段神经丝(NF)表达增加。 结论 毛囊神经干细胞通过调节受损视神经中的一些基因表达,促进了其大胶质细胞去分化并向有利于神经再生方向变化。     

Langerhans cells - dendritic cells of the epidermis

Langerhans cells (LC) are dendritic cells of the epidermis. They are highly specialized leukocytes that serve immunogenic and tolerogenic purposes. Here, we review some aspects of LC biology, emphasizing those areas where LC are or may turn out to be special.     

Langerhans cells: antigen presenting cells of the epidermis

While epidermis in the skin provides an excellent barrier to the environment, it is an incomplete one. Some antigenic material can penetrate through the stratum corneum (or be introduced pathologically) where strategically placed epidermal Langerhans cells reside. In this review, we have assembled relevant data concerning the antigen presenting potential of epidermal Langerhans cells. Strong circumstantial evidence derived from in vitro studies of epidermal cell suspensions enriched for Langerhans cells indicates that Langerhans cells possess this capability. In vivo studies with intact skin indicate that critical numbers of functioning Langerhans cells are essential for successful induction of contact hypersensitivity by epicutaneously applied haptens. And within the past several months, experiments with purified preparations of epidermal Langerhans cells have proven that these cells, and perhaps they alone among epidermal cells, possess the capacity of processing and presenting haptenic determinants to the immune system. The challenge for the future is to determine the extent to which this unique property of Langerhans cells affords physiologic protection to the skin and under what pathologic circumstances altered Langerhans cell function leads to disease.     

Mast cells: the forgotten cells of renal fibrosis

BACKGROUND/AIMS: Mast cells, when activated, secrete a large number of fibrogenic factors and have been implicated in the development of fibrotic conditions of the liver, lung, and skin. There is evidence that renal fibrosis is closely linked with a chronic inflammatory cell infiltrate within the interstitium, but a potential role for mast cells in this process has yet to be defined. Therefore, the numbers of mast cells in normal and fibrotic kidneys with various pathologies were investigated. METHODS: Mast cells were quantified in renal transplants showing acute and chronic rejection and cyclosporin toxicity, kidneys removed for chronic pyelonephritis, and renal biopsies from patients with IgA nephropathy, membranous nephropathy, and diabetic nephropathy. Mast cells were stained using two methods: acid toluidine blue detected less than 30% of the mast cells revealed by immunohistochemistry for mast cell tryptase. RESULTS: Mast cells were scarce or absent in normal kidney (median, 1.6 mast cells/mm2) but numerous throughout the cortex and medulla in all specimens that showed fibrosis. They were almost entirely confined to the renal interstitium. Mast cells were present in large numbers in biopsies from patients with membranous nephropathy (median, 21.7 mast cells/mm2) and diabetic nephropathy (median, 29.2 mast cells/mm2), which were selected on the basis of showing chronic injury. In 24 unselected IgA nephropathy biopsies there was a close correlation between numbers of mast cells and the extent of interstitial fibrosis (r = 0.771; p < 0.0001). In renal transplant biopsies, mast cells were associated with allograft fibrosis in chronic rejection (median, 27.1 mast cells/mm2) and chronic cyclosporin toxicity (median, 10.6 mast cells/mm2) but not acute rejection (median, 2.7 mast cells/mm2) or acute cyclosporin toxicity (median, 2.0 mast cells/mm2). There was no detectable increase in mast cell numbers during acute rejection in those transplants that subsequently progressed to chronic rejection. In some biopsies the mast cells were largely intact, but in most cases some or all were degranulated. CONCLUSIONS: An increased number of mast cells is a consistent feature of renal fibrosis, whatever the underlying pathology, and the number of mast cells correlates with the extent of interstitial fibrosis. This suggests that mast cells might play a pathogenetic role in the fibrotic process.     

Hepatoma cells inhibit the differentiation and maturation of dendritic cells and increase the production of regulatory T cells

Tumor cells may escape from the immune responses because of defective differentiation of dendritic cells (DC). Recent studies have found an increased number of regulatory T cells (Treg) in both peripheral blood and tissues from patients with hepatocellular carcinoma. In the present study, we used tumor culture supernatants (TSN) from hepatoma-derived cell lines to investigate whether TSN interfere with the differentiation of human monocyte-derived DC and/or their ability to increase Treg. The results showed that exposure to TSN significantly inhibited the differentiation of monocytes into DC with retained CD14 molecule and reduced expression of CD1a. These TSN-exposed immature DC also produced significant amount of immunosuppressive cytokine IL-10 and displayed an increased expression of co-stimulatory molecules. Upon stimulation with LPS, however, the TSN-exposed DC failed to undergo full maturation, with a blockage of the upregulation of co-stimulatory molecules on their surface and a switch to an IL-10(high)IL-12(low)TNF-alpha(low) phenotype. Moreover, exposure of DC to TSN selectively inhibited their capacity to stimulate the proliferation of allogeneic CD8(+) T cells, but promoted the generation of CD4(+)CD25(hi)Foxp3(+) Treg cells. These findings, together with previous clinical studies showing that CD4(+)CD25(hi) Treg cells are concentrated within hepatocellular carcinoma tissue, suggest that the local tumor microenvironment may favor the induction of Treg cells through inhibiting the differentiation and maturation of DC.     

Endocrine cells and parietal cells in the stomach of the developing rat

Gastrin-immunoreactive cells were fairly numerous in the pancreas and upper duodenum of the rat at about the time of birth. A minor population of these cells stained with antibodies directed against the N-terminal region of gastrin-34 as well as with antibodies directed against the C-terminal region. The remainder of the cells stained with the C-terminally directed antibodies only. Within a fortnight after birth all gastrin-immunoreactive cells disappeared from the pancreas and were greatly reduced in number in the duodenum; those that remained were probably CCK cells. Gastrin cells were rare in the antrum at birth and remained rare during the first days after birth. They increased in number, slowly until after weaning (15-20 days of age) and then more rapidly, until 25-30 days of age when the gastrin cell density reached that in adult rats. At the time of birth the gastrin concentration in serum was low; the subsequent increase during the first 2 weeks paralleled the development of the antral gastrin cell system. Adult postprandial serum gastrin concentrations were reached 12 days after birth. Somatostatin cells were rare in both the antral and oxyntic mucosa at birth. They increased gradually in number until about a month after birth when the cell density reached that seen in adult rats. In the oxyntic mucosa the ECL and A-like cells are the predominant endocrine (argyrophil) cell types. They were not detected until about 4 days after birth. Their number increased slowly until about 30 days of age. They did not stain argyrophil until about 2-4 weeks after birth. Parietal cells were few at birth; ultrastructurally they appeared to be in an active state and histochemically they were shown to contain carbonic anhydrase. The pH of the gastric content of newborn rats was close to 5; 15-17 days after birth the pH was about 4 in freely fed rats. In fasted rats shortly after birth the pH was about 4. Two weeks later it was around 2, which is the pH measured in older rats. Hence, the full capacity for acid secretion is probably not established until weaning. Fasting greatly lowers the serum gastrin concentration and the histidine decarboxylase activity of the ECL cells in adult rats. Before weaning, fasting produced these effects only to a minor degree.(ABSTRACT TRUNCATED AT 400 WORDS)     

从G-CSF动员的外周血细胞悬液培养的成纤维细胞样细胞的特性分析

目的： 研究从G-CSF动员的外周血细胞（PBC）悬液培养的成纤维细胞样（F-L）细胞的特性。 方法： 取PBC中贴壁细胞分4组培养：①RPMI-1640组；②L-DMEM组；③粒细胞集落刺激因子（G-CSF）组；④白细胞介素-3（IL-3）组。流式细胞仪分析各组培养的F-L细胞特性。 结果： 培养2-3周后，4组均能收获到F-L细胞，细胞贴壁生长，但不融合，不能连续传代培养，③④组细胞数量明显多于①②组，4组F-L细胞表型相似：CD33+、CD11c+、CD64+、CD14+、CD45+、HLA-DR+、CD86+、CD34-、CD38-、CD3-、CD19-、CD56-、CD29-、CD44-、CD105-；与单核细胞（PB-M）表型差异仅CD38表达不同而与间质干细胞（MSC）或树突状细胞（DC）表型明显不同。 结论： 从PBC培养的F-L细胞为巨噬细胞，不是MSC或DC；G-CSF、IL-3能提高F-L细胞培养数量，不改变PB-M向巨噬细胞分化的分化方向。     

Human natural killer cells suppress the proliferation of B cells

We have investigated the suppressive effect of human natural killer (NK) cells on autologous B-cell proliferation. Removal of NK cells by anti-NK-cell monoclonal antibodies (CD16, Leu 11b; Leu 7) increased by 2-3-fold the proliferative response of purified B cells activated by anti-mu and B-cell growth factor (BCGF). The inhibitory effect of NK cells was observed using recombinant IL-2 or semi-purified BCGF-I as sources of BCGF. Moreover NK cells, highly purified by centrifugation on a Percoll discontinuous density gradient, suppressed the proliferative response of purified autologous B cells activated by anti-mu and BCGF. These results show a suppressive effect of human NK cells on B-cell proliferation in vitro.     

Human plasmacytoid-derived dendritic cells and the induction of T-regulatory cells

Suppression by T-regulatory (Tr) cells is essential for the induction of T-cell tolerance and the prevention of autoimmune diseases, organ rejection, and graft-versus-host disease. Increasing attention has been devoted to understand the role of dendritic cells (DC) in the control of Tr-cell differentiation. Here we review the recent evidence that cluster designation (CD)40-ligand activated plasmacytoid-derived DCs (DC2) have the ability to induce primary Tr-cell differentiation. We propose that in addition to the regulatory functions of immature myeloid DC, Tr-cell induction by DC2 represents a nonredundant mechanism for the safeguard of peripheral T-cell tolerance. DC2 can be used as tool to drive potent antigen specific Tr-cell differentiation and expansion in vitro and in vivo.     

TCRγδ cells: Mysterious cells of the immune system

γδ cells participate in pathogenic infections and autoimmune conditions, yet, almost a decade after their discovery, little is known regarding their TCR repertoire or effector functions. Unlike MHC-restricted antigen recognition employed by TCRαβ cells, TCRγδ cells can recognize whole unprocessed antigens in an MHC-independent manner. The nature of positive and negative selection used to shape the repertoire of TCRγδ cells is unclear, especially in the nonlymphoid tissues where these cells predominate. While TCRγδ cells express an activated phenotype and are present in pathological conditions, their roles in immunological protection is unknown. This review will focus on our efforts to study these issues of TCRγδ biology.