成年肌萎缩脊髓侧索硬化症转基因鼠脊髓细胞增殖的研究

目的观察成年肌萎缩脊髓侧索硬化症(ALS)转基因模型鼠脊髓内源性细胞的增殖和存活情况。方法对ALS转基因鼠进行BrdU注射,分别于不同时间点取材,冷冻切片,应用免疫荧光染色技术检测脊髓内细胞的增殖、分布和存活情况。结果成年ALS鼠脊髓的中央管、灰质、白质均可检测到BrdU阳性细胞,经常可检测到形状相似、成对出现的细胞。在灰质内,BrdU阳性细胞主要分布于脊髓前角。在白质内,BrdU阳性细胞散在分布。前角ALS鼠脊髓内BrdU阳性细胞数量较野生型鼠多。BrdU末次注射后1d、14d均可检测到BrdU阳性细胞,但细胞数量逐渐减少,28d ALS鼠脊髓内仍可检测到少量BrdU阳性细胞,主要分布于中央管部位。增殖细胞核抗原(PCNA)阳性细胞的分布和变化规律与BrdU阳性细胞一致。在95d龄ALS鼠(BrdU末次注射1d后),脊髓中Nestin阳性细胞较野生型鼠多,多数Nestin阳性细胞分布于脊髓前角,某些细胞呈BrdU/Nestin双标阳性。在BrdU注射后14d、28d脊髓中,未检测到BrdU/Nestin双标阳性细胞。结论神经退行性病变可激活ALS转基因鼠脊髓的细胞增殖,且细胞存活可达28d。     

大鼠视网膜干细胞的增殖和多向分化**

背景：国内对视网膜干细胞的体外分离培养及鉴定仍处于初步探索阶段。 目的：体外分离、培养及鉴定新生大鼠视网膜干细胞,探讨其多向分化潜能。 方法：用神经干细胞无血清培养方法分离和培养新生24 h的SD大鼠睫状体细胞,第6代视网膜干细胞经胎牛血清诱导分化,应用免疫细胞化学方法检测视网膜干细胞的分化特性。 结果与结论：体外培养的细胞球具有连续克隆能力,Nestin抗原阳性,BrdU 标记结果显示悬浮细胞团主要由分裂增殖的细胞组成,并表达胚胎发育早期视网膜内原始细胞的特异性抗原Chx-10;诱导分化后的细胞表达星形胶质细胞特异性标志物GFAP、神经元特异性标志物NSE、感光细胞标志物Opsin、双极细胞特异性抗原PKC和节细胞特异性抗原β-tubulin,实验初步证实培养的视网膜干细胞具有神经干细胞特性,能自我更新和增殖分化成为感光细胞类型的细胞。 关键词：视网膜干细胞;细胞培养;无血清;细胞分化;大鼠 doi:10.3969/j.issn.1673-8225.2012.06.015     

胎儿海马神经干细胞的体外培养及神经元前体细胞的纯化

目的　从人胎儿海马分离培养具有自我更新和多向分化能力的神经干细胞 ,并从中纯化神经元前体细胞。　方法　利用无血清培养从胚胎 4个月的胎儿海马区分离细胞 ,并在体外连续传代培养 ;采用免疫荧光细胞化学技术检测神经上皮干细胞蛋白 (nestin)的表达、经BrdU孵育后的BrdU表达 ;比较两种诱导分化方法所获得的神经元和胶质细胞的比例差异 ;利用单细胞克隆技术纯化神经元前体细胞。　结果　分离的细胞具有连续克隆能力 ,在体外培养了 16个月 ,传了 4 3代 ;细胞冻存、复苏后仍保持干细胞特性 ;培养的细胞呈Nestin阳性 ,在BrdU孵育后呈BrdU阳性 ,诱导分化后的细胞能够表达Tubulin、NeuN或GFAP ;利用无血清诱导所得到的分化细胞中神经元的比例约占 80 % ,而血清诱导的分化细胞中胶质细胞的比例则大于 90 %。利用单细胞克隆技术可从神经球中纯化的细胞表达Nestin ,并且全部分化成神经元。　结论　从胎儿海马区分离的细胞具有自我更新能力和多向分化潜能 ,属于神经干细胞 ,从中可纯化出神经元前体细胞     

成年肌萎缩脊髓侧索硬化症转基因鼠脊髓增殖细胞的分化

目的 探讨成年肌萎缩脊髓侧索硬化症(ALS)转基因模型鼠脊髓内增殖细胞的类型及分化情况. 方法 对ALS转基因鼠发病期进行BrdU标记,分别于不同时间点取材,冷冻切片,应用免疫荧光双标及三标染色技术检测ALS转基因鼠病变进展过程中脊髓内增殖细胞的分化情况. 结果 成年ALS转基因鼠发病期脊髓的中央管、灰质、白质均未检测到BrdU/DCX双标记阳性细胞和BrdU/NeuN双标记阳性细胞.灰质、白质和中央管周围检测到大量NG2阳性细胞,阳性细胞数量随病变进展逐渐减少,NG2阳性细胞多呈BrdU阳性表达;可检测到少量BrdU/A2B5双标记阳性细胞;ALS转基因鼠发病期脊髓BrdU/GFAP双标记阳性细胞较多,部分双阳性细胞呈Nestin阳性,而野生型鼠脊髓内未检测到BrdU/GFAP双标记阳性细胞.结论 神经退行性病变激活ALS转基因鼠脊髓内源性增殖细胞向神经胶质细胞方向分化,未检测到向神经元方向分化,内源性增殖细胞尚不能有效地促进退行性病变的修复.     

新生鼠基底前脑神经干细胞的分离和培养

目的　探讨基底前脑神经干细胞的增殖及多向分化潜能。方法　利用无血清培养技术 ,加表皮生长因子 (EGF)和碱性成纤维生长因子 (FGF 2 )刺激生长 ,在体外进行神经干细胞的克隆培养和血清诱导分化 ;采用免疫组化研究神经干细胞的增殖特性及多向分化潜能 ,并用 5 溴 2 脱氧尿苷 (BrdU)标记证实其增殖能力。结果　从新生鼠基底前脑成功分离出神经干细胞 ,该细胞具有连续增殖能力 ,可以传代培养 ,表达神经上皮干细胞蛋白抗原 ,可以分化成神经元和胶质细胞 ;BrdU标记结果阳性。结论　新生鼠基底前脑存在具有自我更新能力和多分化潜能的神经干细胞     

新生小鼠海马神经干细胞体外培养条件下的增殖与分化

背景：神经干细胞移植可用于中枢神经损伤的临床治疗。 目的：观察新生小鼠海马神经干细胞在体外培养条件下的增殖和分化。 方法：从新生昆明小鼠海马取材,采用机械分离法对原代细胞进行无血清培养,采用机械法复合酶消化法对原代细胞进行传代,以体积分数为10%胎牛血清诱导分化。免疫荧光行巢蛋白、β-微管蛋白Ⅲ和胶质纤维酸性蛋白染色,对培养的细胞进行鉴定。CCK-8法检测神经干细胞增殖能力。 结果与结论：从新生小鼠海马分离得到的细胞具有连续传代形成克隆球的能力,克隆球呈巢蛋白免疫反应阳性;加入胎牛血清可诱导分化为β-微管蛋白Ⅲ和胶质纤维酸性蛋白阳性细胞。提示实验成功建立了体外培养新生小鼠海马组织分离和培养神经干细胞的方法,培养的神经干细胞在体外培养条件下保持着自我增殖和分化的潜能。     

体外培养获取高纯度神经前体细胞

目的　获取较高纯度的神经前体细胞 (NPC)。方法　加入 30ng/mlbFGF培养E12天大鼠皮质、海马和隔区的NPC ,用免疫组化与免疫荧光组化方法将其显示。结果　加bFGF组的NPC不断分裂 ,形成大量的分裂球并维持到第 4代 ;绝大部分细胞 (约 98%以上 )表达Nestin ,约有 6 0 %为BrdU和Nestin免疫荧光双标阳性细胞。对照组的分裂球维持时间较短 ,随培养时间延长细胞向外迁移生长 ;第 2代约有 6 0 %细胞表达Nestin ,40 %为BrdU和Nestin免疫荧光双标阳性细胞。结论　bFGF对E12天大鼠脑组织的NPC有明显的促分裂作用 ,选用胎龄较小的胚脑组织加入适量的bFGF培养可获取较高纯度的NPC。     

食蟹猴胚胎干细胞的培养及向神经细胞诱导分化

目的 建立食蟹猴胚胎干细胞系体外培养体系,并诱导其向神经细胞分化,为在体移植实验奠定基础。方法 用小鼠胚胎成纤维细胞作为滋养层,长期培养食蟹猴胚胎干细胞。在无血清培养基中添加小鼠重组Noggin的方式诱导其向神经细胞分化,并对分化各阶段细胞进行免疫组化染色检测分化效果。结果 食蟹猴胚胎干细胞在滋养层上成克隆样生长,可以长期扩增超过20代并保持胚胎干细胞的特性。诱导向神经细胞分化约14d,即可形成呈玫瑰花环样的神经前体细胞结构,可见大量Nestin阳性细胞,及部分Tuj-1阳性细胞;分化约21d时,可见大量Nestin阳性细胞以及大量Tuj-1阳性细胞;分化超过35d,可见GFAP阳性细胞,而Tuj-1阳性细胞减少。结论 成功建立食蟹猴胚胎干细胞的培养体系,在此基础上诱导其分化可获得大量神经前体细胞,尤其是早期神经细胞。     

新生小鼠小肠Cajal间质细胞增殖的实验研究

目的 探讨在新生小鼠小肠Cajal间质细胞(interstitial cells of Cajal,ICC)的发育过程中是否出现增殖.方法 采用新生2 d(P2d)、14 d(P14d)和24 d(P24d)的小鼠小肠,应用BrdU腹腔注射,24 h后取材,Kit和BrdU免疫荧光染色.结果 P2d小鼠小肠,可见大量Kit/BrdU双重标记阳性细胞,这些细胞的形态与成熟ICC基本相似,这些细胞在P14d时减少,而在P24d消失.统计学分析发现,不同时间点之间Kit/BrdU双标阳性细胞数量存在显著差别(P＜0.05).结论 在小肠生后发育过程中,出现了ICC增殖,而这种增殖在发育过程中逐渐减弱,最后在成年前消失.     

成年小鼠颈动脉体细胞的培养方法及低氧对其特性的影响

目的:建立体外分离培养小鼠颈动脉体细胞的方法。方法:摘取小鼠双侧颈总动脉分叉,采用机械剪切和混合酶消化颈动脉体(CB),培养24 h后固定细胞,通过免疫荧光染色鉴定细胞类型,并在低氧培养3 d后,观察TH阳性细胞数量的变化。结果:原代培养的CB细胞绝大部分为单细胞,有少数呈簇状生长的细胞群。所分离的原代CB细胞中,主要有TH+、GFAP+和Nestin+三种类型的细胞组成;低氧培养3 d后TH+细胞数量较常氧组明显增加。结论:从分离步骤和消化方式等方面优化小鼠CB细胞的原代培养方法,而低氧可以增加CB细胞中TH+细胞的数量,可能与促进CB细胞中神经干细胞样细胞的增殖有关。     

rhG-CSF促进体外培养神经干细胞的增殖

目的 探讨重组人粒细胞集落刺激因子(rhG-CSF)对小鼠胚胎神经干细胞(NSCs)增殖的作用.方法 分离培养小鼠NSCs,通过向培养基中添加G-CSF(10、30、60、100和200μg/L),四甲基偶氮唑蓝(MTT)比色法以及5-溴-2′-脱氧尿苷(BrdU) 免疫荧光染色标记检测NSCs的增殖.免疫印迹法检测NSCs增殖时信号转导与转录激活因子3(STAT3)和磷酸化信号转导和转录激活因子3(p-STAT3)的表达.结果 神经干细胞表达G-CSF受体,细胞活性随G-CSF的浓度不同表现出一定的剂量依赖性,当G-CSF的浓度为100μg/L时细胞的活性最高.G-CSF处理组的细胞增殖活性明显高于对照组.进一步证实,在G-CSF处理后,p-STAT3的表达则在5 min开始升高,30 min 到达峰值,之后开始下降,至75 min 接近正常水平.加入G-CSF受体的抗体后,p-STAT3的活化现象消失,并且细胞的增殖也明显低于G-CSF作用组,和对照组接近.结论 rhG-CSF可促进小鼠NSCs的增殖,其作用机制可能是通过细胞表面的G-CSF-R促进STAT3的活化.G-CSF可能作用于内源性NSCs的活化和增殖.     

Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain

The neurosphere assay can detect and expand neural stem cells (NSCs) and progenitor cells, but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall, we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 mum coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay, the neural colony forming cell assay (N-CFCA), and label-retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis, with the number of neurosphere-forming cells detected in individual 400 mum sections varying from a minimum of eight to a maximum of 891 depending upon the rostral-caudal coordinate assayed. Moreover, the greatest variability occurred in the rostral portion of the lateral ventricles, thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 +/- 276) or colonies (4275 +/- 124) we detected along the neuraxis did not differ significantly, LRC numbers were significantly reduced (1186 +/- 188, 7 month chase) in comparison to both total colonies and neurospheres. Moreover, approximately two orders of magnitude fewer NSC-derived colonies (50 +/- 10) were detected using the N-CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU+/Ki-67+) or competent to divide (BrdU+/Mcm-2+), and proliferate upon transfer to culture, it is unclear whether this technique selectively detects endogenous NSCs. Overall, caution should be taken with the interpretation and employment of all these techniques.     

Induction of B lymphocyte colony growth in vitro by thymus-derived stimulating factor.

Murine lymphoid cells which were stimulated in liquid culture containing thymus culture fluid (Thy-CF) and seeded in a soft agar culture system, proliferated and developed into B cell colonies. Two types of colonies were formed: large colonies within the upper layer and small flat colonies on the surface of the upper layer. Thy-CF prepared from cells of normal hydrocortisone-treated mice had a higher cloning potential than Thy-CF prepared from untreated mice. At concentrations of Thy-CF in culture medium greater than 35%, Thy-CF prepared from normal mice had an inhibitory effect on colony formation. Cells of nude mice were also able to form B cell colonies if thymocytes of normal mice were mixed with lymphoid cells in the culture medium. Thymocytes elaborate a B lymphocyte colony-stimulating factor which, with the help of T cells, triggers a B cell population into colony formation and immunoglobulin production.     

Novel approach for the generation of recombinant African swine fever virus from a field isolate using GFP expression and 5-bromo-2'-deoxyuridine selection

Generation of African swine fever virus (ASFV) recombinants has so far relied mainly on the manipulation of virus strains which had been adapted to growth in cell culture, since field isolates do not usually replicate efficiently in established cell lines. Using wild boar lung cells (WSL) which allow for propagation of ASFV field isolates, a novel approach for the generation of recombinant ASFV directly from field isolates was developed which includes the integration into the viral thymidine kinase (TK) locus of an ASFV p72-promoter driven expression cassette for enhanced green fluorescent protein (EGFP) embedded in a 16 kbp mini F-plasmid into the genome of the ASFV field strain NHV. This procedure enabled the monitoring of recombinant virus replication by EGFP autofluorescence. Selection for the TK-negative (TK(-)) phenotype of the recombinants on TK(-) Vero (VeroTK(-)) cells in the presence of 5-bromo-2'-deoxyuridine (BrdU) led to efficient isolation of recombinant virus due to the elimination of TK(+) wild type virus by BrdU-phosporylation in infected VeroTK(-) cells. The recombinant NHV-dTK-GFP produced titres of both cell-associated and secreted viral progeny in WSL cells similar to parental NHV indicating that insertion of large heterologous sequences into the viral TK locus and EGFP expression do not impair viral replication in these cells. In summary, a novel method has been developed for generation of ASFV recombinants directly from field isolates, providing an efficacious method for further manipulations of wild-type virus genomes.     

A colony assay system that detects B cell progenitors in fresh and cultured bone marrow

This report describes a colony assay system, based on methods used to grow myeloid precursors in semisolid medium, in which B cell progenitors can be grown. The formation of these B cell progenitor colonies is dependent upon soluble mediators from a stromal cell line known to support B lymphopoiesis. In initial experiments a double layer culture system was employed in which target cells in methylcellulose medium were separated from an adherent layer of S17 stromal cells by an agar interface. Target cells were harvested from Dexter type long-term bone marrow cultures at a time after transfer to the lymphoid Whitlock-Witte conditions, when myeloid progenitors were depleted and mature B cells had not yet appeared. On day 15 of culture a colony could be identified that contained several hundred tightly clustered lymphoid cells. There was a linear relationship between the number of cells plated and the number of colonies that developed. Identically appearing colonies were also observed in agar using fresh bone marrow cells as targets with either an underlayer of S17 cells or S17 conditioned medium to potentiate colony growth. Lymphoid colonies derived from fresh bone marrow appeared on days 6 and 14 of growth. A proportion of the cells from the fresh or cultured marrow derived colonies expressed the B220 antigen and cytoplasmic mu heavy chains, but surface IgM was never observed. Cell depletion experiments on antibody coated plates demonstrated the colony forming unit to be B220 antigen positive, surface IgM negative, and replating experiments indicated the colonies were lymphoid restricted in their differentiative potential.     

Proliferative heterogeneity in the human prostate: evidence for epithelial stem cells

Clonal analysis of human prostate epithelial cells was undertaken in order to identify stem cells. Two types of colony were distinguished, termed type I and type II. Type I colonies were relatively small and irregular and contained a loose mixture of differentiated and undifferentiated cells. In contrast, type II colonies were large, round, and homogeneous, consisting almost exclusively of small undifferentiated and dividing cells. The colony-forming efficiency was 5.8% +/- 1.8 for freshly isolated epithelial cells. There were approximately 10 times as many type I as type II colonies and about 1 in 200 of the plated cells was capable of forming a type II colony. In three-dimensional culture on Matrigel, the type II colonies produced structures reminiscent of prostate epithelium, with luminal cells expressing markers of prostate epithelial differentiation, including the androgen receptor. On the basis of their proliferative characteristics and pluripotency, the type II colonies may be the progeny of stem cells and the type I colonies of a more differentiated transit-amplifying population.     

Development of natural killer cells from lymphohematopoietic progenitors of murine fetal liver

We established a clonal culture system which supports the growth of murine immature natural killer (NK) cells. When we plated day 14 fetal thymocytes in methylcellulose media containing interleukin 2 (IL-2), IL-7 and steel factor (SF), we observed diffuse colonies which could not be classified into known colony types. Cells in the colonies were blast-like and expressed Thy-1 and CD25 but not lineage-specific markers. Cells in the colonies developed into NK1.1+ cells in fetal thymus organ culture indicating that the colonies consist of immature NK cells. We then examined the colony-forming ability of fetal liver cells. The combination of IL-2, IL-7 and SF with or without IL-11 supported formation of few immature NK cell colonies from purified progenitors. Interestingly, addition of IL-11 to the culture stimulated formation of mixed colonies consisting of immature NK cells, B cells, macrophages and/or mast cells. The clonal origin of the mixed NK cell colonies was confirmed by micromanipulation of the colony-forming cells. This culture assay should facilitate the analysis of the pathway and cytokine regulation of NK cell development.     

Bone marrow T cell colony-forming cells: studies of their origin and use in monitoring T cell-depleted bone marrow grafts.

Peripheral blood T cell colony forming cells (T-CFC) are mature T cells. When blood was fractionated into sheep red blood cell receptor positive (E+) and negative (E-) fractions, T cell colony growth was largely restricted to the E+ population. When bone marrow was similarly fractionated, many colonies grew from the E- cells but myeloid colonies also grew making interpretation of colony numbers difficult. We have, therefore, also assessed T cell proliferation during culture as an expansion index (EI) by determining the absolute number of T cells pre and post culture. This data shows that T cell expansion is on average nine times greater in the E- marrow fraction than in the E+ fraction. Studies are presented suggesting that this is because marrow E- cells contain appropriate accessory cells and that high numbers of T cells inhibit T cell growth. The cells giving rise to bone marrow T cell colonies thus appear to be contaminating mature T cells rather than pre-thymic progenitor cells. We have measured T cell expansion in culture as a sensitive assay of T cell contamination following procedures to remove T cells from bone marrow grafts for the prevention of graft versus host disease (GVHD).     

Colony growth in vitro of mitogen-stimulated mouse B lymphocytes.

The ability of mouse B-mitogen-induced lymphocytes to grow and develop into colonies in a soft agar system was studied. Prerequisite conditions for the colony formation of mouse lymphocytes from inguinal lymph nodes of strains ICR C3H/eB and C3H were their suspension in a liquid medium and stimulation with polyclonal B-cell activators such as bacterial lipopolysaccharide (LPS), purified protein derivative (PPD) or dextran sulphate (DxS) prior to being seeded on a soft agar culture medium. After 3-5 days of culture, colonies of 50-350 cells or more per clone developed. A linear relationship was found between the number of cells seeded and the number of colonies growing. Of the cells seeded, only a limited population of the mitogen-stimulated lymphocytes has the potential to divide and to develop into colonies. The largest number of colonies was obtained by culturing lymph node cells of ICR mice and using LPS as mitogens. Two sublines of C3H were found to respond differently to LPS: C3H/HeJ mice were low responders while C3H/eB mice were high responders. Experiments with inbred, congenitally athymic nude (nu/nu) mice known to be deficient in T cells showed that LPS-stimulated lymphocytes were capable of forming colonies. The morphology of the colony cells, as well as the fact that they stain positively for cell-membrane immunoglobulins, suggest that the colonies developed from B lymphocytes.     

Culture and expansion of the human embryonic stem cell line HS181, evaluated in a double-color system

An approach of using RFP-transfected human foreskin fibroblasts (hFS-RFP) to support the growth of GFP expressing human embryonic stem cells (hES; HS181-GFP) is reported. The two-color system was applied to detect interactions between hFS and human embryonic stem cells (hES). After overnight culture, the hES cell colonies showed a behavior of "pushing away" the underlying feeder cells. This phenomenon occurred with both a low and high density of feeders. The density of the feeder cell layer, however, influenced the growth pattern of hES cell colonies. At a high feeder cell density, the hES colonies were more pointed and aligned with the direction of the fibroblasts, whereas less dense feeder layers allowed a more rounded and flat hES colony formation. Not surprisingly, a small fraction of mitotically inactivated feeder cells reattached after passage and remained viable in the cultures for up to four subsequent passages. The prospect of using the two-color system for detection of possible fusion events between hES cells and feeder cells was assessed by screening a large number of cell cultures for double RFP/EGFP expressing cells. The results indicate that fusion events are extremely rare (<10(-6)), or alternatively that after fusion the dual expression of both EGFP and RFP is not easily detected for other reasons. In summary, a two-color system allows analysis of colony formation and also helps to identify and follow the differentiation of cells.