新生大鼠肺成纤维细胞的原代培养改良法及细胞鉴定

探讨新生大鼠肺成纤维细胞原代培养的改良方法及细胞鉴定。用胰酶消化组织块结合的方法提取新生大鼠肺成纤维细胞,并纯化细胞,对肺成纤维细胞进行形态学观察,用HE染色及免疫组化染色法对细胞进行鉴定,并用MTT法测定细胞生长曲线。倒置相差显微镜下观察选用改良法获得的细胞,3 d后可见组织块周边有少许细胞,5 d后组织块周围有大量细胞爬出,生长迅速,10 d接近融合。经改良后的方法纯化细胞,细胞活性状态较好的为3~5代,5代以后的细胞增殖能力下降。对第3代肺成纤维细胞进行HE染色,镜下可见形态典型的成纤维细胞,免疫组化结果显示波形蛋白(Vi-mentin)阳性表达,细胞角蛋白(cytokeratin)阴性表达。MTT法检测第3代细胞于3~5 d处于对数生长期。胰酶消化组织块结合法是一种可靠快速的肺成纤维细胞分离纯化的培养方法,使用这种方法可得到具有典型形态特征且活性较好的肺成纤维细胞,初学者容易掌握。     

成年SD大鼠心肌原代成纤维细胞分离及培养的优化方案

目的:摸索及优选成年SD大鼠心肌原代成纤维细胞的体外分离、培养及鉴定的实验方法。方法:将成年SD大鼠心脏剪成小组织块,采用以下四种方案(A:0.08%胰酶+0.1%胶原酶II消化15 min,B:0.2%胶原酶II消化15 min,C:0.2%胶原酶II消化60min,D:0.2%胶原酶II消化90 min)提取成年大鼠心脏原代成纤维细胞,再通过差速贴壁分离方法培养原代成纤维细胞。采用倒置显微镜观察成纤维细胞的基本形态特征,并进行Vimentiin免疫荧光染色对培养的原代细胞进行荧光鉴定;采用台盼兰染色对培养的原代成纤维细胞存活率进行鉴定;采用细胞计数对培养的成纤维细胞生长趋势进行鉴定。结果:四种方法均能培养成纤维细胞,但单酶消化60 min可一次性提取较多细胞,并且细胞状态佳,3 d即可传代。72 h成纤维细胞Vimentin免疫荧光染色阳性率高达97%。台盼兰染色可见其细胞死亡率明显降低,并且细胞计数可见细胞生长状态极佳。结论:单酶消化60 min是提取成年SD大鼠心肌原代成纤维细胞的高效、快速、稳定的实验方法,为心脏疾病的基础及临床研究提供了较为理想的细胞学实验模型。     

原代培养的二倍体上皮细胞和成纤维细胞的纯化

人体包皮原代培养物所长出的两种细胞—上皮细胞和成纤维细胞,通过本文介绍的纯化方法可以分别收集提供不同实验的需要。如正常上皮细胞在肿瘤实验及烧伤病人植皮等方面都有一定的实用价值。而成纤维细胞在转化试验及干扰素等的制备物中都是有用的实验材     

树鼩原代皮肤成纤维细胞的分离培养技术

建立稳定的树鼩(Tupaiabelangeri)皮肤成纤维细胞的体外培养体系,可为有关此类细胞的实验和疾病树鼩细胞模型提供技术支持。取树鼩大腿内侧皮肤用组织块贴壁法和胶原酶Ⅰ消化法分离皮肤细胞,胰蛋白酶差别消化法纯化细胞;用MEM(10%FBS)完全培养基和含低血清生长添加物(LSGS)的培养基培养细胞;免疫荧光和蛋白印迹法鉴定细胞,并测定细胞的生长、冻存和复苏特性。经树鼩皮肤细胞分离效果比较,胶原酶消化法比组织块贴壁法更适合用于树鼩原代皮肤细胞分离;对分离及冻存复苏后细胞生长状况观察比较发现,添加了LSGS的MEM培养基更利于细胞存活、生长;细胞形态观察、免疫荧光和蛋白印迹检测鉴定所分离的细胞为树鼩皮肤成纤维细胞。成功建立了树鼩原代皮肤细胞的分离、纯化方法,并优化了该细胞的培养条件。     

成年大鼠嗅鞘细胞与嗅觉神经成纤维细胞的原代培养及鉴定

目的 建立一种原代提取嗅鞘细胞与嗅觉神经成纤维细胞混合培养的方法.方法 自2.5月龄SD大鼠嗅球最外两层分离嗅鞘细胞和嗅觉神经成纤维细胞进行混合培养,并不进行纯化,分别于7 d、10 d、14 d行免疫细胞化学鉴定,并计算各个时间点嗅鞘细胞的纯度.结果 体外培养的嗅鞘细胞主要呈两极或多极状,而嗅觉神经成纤维细胞则成扁平的像成纤维细胞的形态,免疫细胞化学结果显示嗅鞘细胞呈p75 NGFR阳性,嗅觉神经成纤维细胞呈fibronectin阳性,两种细胞都呈vimentin阳性,在7 d、10 d、14 d各个时间点嗅鞘细胞分别占混合培养的34.1%、25.6%、8.6%.结论 从成年大鼠嗅球最外两层分离的培养中主要包含嗅鞘细胞和嗅觉神经成纤维细胞,嗅鞘细胞在混合培养中所占的比例随培养时间的延长而逐渐降低.     

成纤维细胞生长因子9抑制PDGF-BB诱导的肺血管平滑肌细胞表型转化

该研究探讨了成纤维细胞生长因子9(fibroblast growth factor 9,FGF9)在肺动脉高压(pulmonary arterial hypertension,PAH)及肺血管平滑肌细胞表型转化中的作用.建立野百合碱(monocrotaline,MCT)诱导的大鼠肺动脉高压模型,Western blot...     

ICR胎鼠成纤维细胞培养方法的优化

目的确定胎鼠成纤维细胞（mouse embryonic fibroblasts,MEF）最优制备方法,为MEF的制备节省时间与原材料。方法取胎鼠组织,采用组织块培养法、胰酶消化培养法、胰酶消后组织块培养法进行分离培养MEF,比较观察MEF纯度、生长速度、细胞形态等指标,筛选最节省时间与原料的一种培养方法。结果组织块培养法所得MEF生长速度慢,细胞体积小,杂细胞少,纯化所需传代次数少。胰酶消化培养法的MEF生长速度快,体积较大,杂细胞数量多,纯化所需传代次数较多。胰酶消化后组织块培养法的消化后组织块的成纤维细胞生长速度介于前两种方法之间,细胞体积和形态与组织块法培养结果相似,杂细胞数量较少,纯化所需传代次数少。结论胰酶消化结合组织块培养法能最大限度的利用材料,在短时间内可制备大量MEF,满足核移植试验与作为饲养层细胞的需求。因此,胰酶消化结合组织块培养法是MEF最优培养方法。     

RA滑膜成纤维细胞的原代培养及鉴定

目的:改良体外分离、培养类风湿性关节炎滑膜成纤维细胞的方法,并进行鉴定。方法:取关节镜手术中获得RA患者滑膜组织进行机械分离、胶原酶消化后直接将所有消化产物置于细胞培养皿两次贴壁培养,差速消化法纯化成纤维细胞,倒置显微镜观察细胞形态、流式细胞术及免疫细胞化学的方法鉴定细胞纯度。结果:胶原酶消化后直接贴壁结合差速消化纯化法分离获得的原代滑膜细胞中呈梭形的成纤维样细胞占98%以上,细胞核呈椭圆形位于细胞中央,偶见少量圆形的滑膜巨噬细胞。流式细胞术显示98%以上的滑膜细胞具有vimeintin+CD68的成纤维细胞特征。免疫细胞化学提示滑膜细胞vimentin表达阳性、CD68不表达。结论:成功分离获得了纯度和活性很高的人滑膜成纤维样细胞,方法更简便,效率更高,为后续类风湿性关节炎滑膜侵袭机制的研究奠定了基础。     

内皮细胞培养中清除成纤维细胞的新方法

在原代细胞培养中很容易混杂成纤维细胞,并且随着传代次数的增加,由于成纤维细胞生长迅速,在培养细胞中所占的比例也日渐增加。本文介绍一种清除成纤维细胞的方法,对各种非成纤维细胞的原代培养有一定参考价值。以往是用0.1％的胶原酶消化法来清除成纤维细胞。但由于胶原酶价格昂贵,故并不     

Regulation of alpha-smooth muscle actin gene expression in myofibroblast differentiation from rat lung fibroblasts

Myofibroblasts express alpha-smooth muscle actin and have a phenotype intermediate between fibroblasts and smooth muscle cells. Their emergence can be induced by cytokines such as transforming growth factor beta; but the regulatory mechanism for induction of alpha-smooth muscle actin gene expression in myofibroblast differentiation has not been determined. To examine this mechanism at the level of the alpha-smooth muscle actin promoter, rat lung fibroblasts were transfected with varying lengths of the alpha-smooth muscle actin promoter linked to the chloramphenicol acetyl transferase reporter gene and treated with transforming growth factor beta1. The results show that the shortest inducible promoter was 150 base pairs long, suggesting the presence in this region of cis-elements of potential importance in transforming growth factor beta1 induced myofibroblast differentiation. Transfection of "decoy" oligonucleotides corresponding to sequences for four suspected regulatory factors demonstrated that only the transforming growth factor beta control element is involved in the regulation of transforming growth factor beta1-induced alpha-smooth muscle actin expression in myofibroblast differentiation. Consistent with this conclusion is the finding that a mutation in the transforming growth factor beta control element caused a significant reduction in promoter activity. These observations taken together show that alpha-smooth muscle actin promoter regulation during myofibroblast differentiation is uniquely different from that in smooth muscle cells and other cell lines. Since myofibroblasts play a key role in wound contraction and synthesis of extracellular matrix, clarification of this differentiation mechanism should provide new insight into fibrogenesis and suggest future novel strategies for modulation of wound healing and controlling fibrosis.     

Phenotype-dependent expression of alpha-smooth muscle actin in visceral smooth muscle cells

Alpha-Smooth muscle actin is one of the molecular markers for a phenotype of vascular smooth muscle cells, because the actin is a major isoform expressed in vascular smooth muscle cells and its expression is upregulated during differentiation. Here, we first demonstrate that the phenotype-dependent expression of this actin in visceral smooth muscles is quite opposite to that in vascular smooth muscles. This actin isoform is not expressed in adult chicken visceral smooth muscles including gizzard, trachea, and intestine except for the inner layer of intestinal muscle layers, whereas its expression is clearly detected in these visceral smooth muscles at early stages of the embryo (10-day-old embryo) and is developmentally downregulated. In cultured gizzard smooth muscle cells maintaining a differentiated phenotype, alpha-smooth muscle actin is not detected while its expression dramatically increases during serum-induced dedifferentiation. Promoter analysis reveals that a sequence (-238 to -219) in the promoter region of this actin gene acts as a novel negative cis-element. In conclusion, the phenotype-dependent expression of alpha-smooth muscle actin would be regulated by the sum of the cooperative contributions of the negative element and well-characterized positive elements, purine-rich motif, and CArG boxes and their respective transacting factors.     

Carnitine transport in cultured muscle cells and skin fibroblasts from patients with primary systemic carnitine deficiency

Summary l-Carnitine transport was studied in cultured muscle cells and skin fibroblasts of patients with primary systemic carnitine deficiency and control subjects. In both cell culture types, two systems for carnitine transport were identified. The kinetic parameters for carnitine transport were remarkably similar in cultured muscle cells and skin fibroblasts. Normal rates and kinetic properties of carnitine transport were observed for both cell lines from patients with systemic carnitine deficiency. These studies do not rule out a defect in carnitine transport in vivo. This study was supported by research grants AM27451 and NS06277 from the National Institutes of Health and by a Research Center Grant from the Muscular Dystrophy Association.     

A function for filamentous alpha-smooth muscle actin: retardation of motility in fibroblasts

Actins are known to comprise six mammalian isoforms of which beta- and gamma-nonmuscle actins are present in all cells, whereas alpha-smooth muscle (alpha-sm) actin is normally restricted to cells of the smooth muscle lineages. alpha-Sm actin has been found also to be expressed transiently in certain nonmuscle cells, in particular fibroblasts, which are referred to as myofibroblasts. The functional significance of alpha-sm actin in fibroblasts is unknown. However, myofibroblasts appear to play a prominent role in stromal reaction in breast cancer, at the site of wound repair, and in fibrotic reactions. Here, we show that the presence of alpha-sm actin is a signal for retardation of migratory behavior in fibroblasts. Comparison in a migration assay of fibroblast cell strains with and without alpha-sm actin revealed migratory restraint in alpha-sm actin-positive fibroblasts. Electroporation of monoclonal antibody (mAb) 1A4, which recognizes specifically the NH2-terminal Ac-EEED sequence of alpha-sm actin, significantly increased the frequency of migrating cells over that obtained with an unrelated antibody or a mAb against beta-actin. Time- lapse video microscopy revealed migratory rates of 4.8 and 3.0 microns/h, respectively. To knock out the alpha-sm actin protein, several antisense phosphorothioate oligodeoxynucleotide (ODNs) were tested. One of these, 3'UTI, which is complementary to a highly evolutionary conserved 3' untranslated (3'UT) sequence of alpha-sm actin mRNA, was found to block alpha-sm actin synthesis completely without affecting the synthesis of any other proteins as analyzed by two-dimensional gel electrophoresis. Targeting by antisense 3'UTI significantly increased motility compared with the corresponding sense ODN. alpha-Sm actin inhibition also led to the formation of less prominent focal adhesions as revealed by immunofluorescence staining against vinculin, talin, and beta1-integrin. We propose that an important function of filamentous alpha-sm actin is to immobilize the cells.     

Passages in culture and stimulation conditions influence protein expression of primary fibroblasts

Fibroblasts (Fb) are key effector cells in systemic sclerosis (SSc). Fb stimulation with transforming growth factor beta 1 (TGF-β1) is considered as a positive control in studies assessing fibrogenesis. The lack of standardization of TGF-β1 stimulation might be responsible for discrepancies in experiments performed in different conditions. Using quantitative proteomics analysis, we evaluated the impact of changes in experimental conditions on proteomic profiles of primary Fb. Principal component analysis (PCA) identified several groups of differentially expressed proteins influenced by cell passage, culture medium, and both concentration and duration of exposure to TGF-β1 stimulation. Bioinformatics analysis revealed that late passages expressed proteins involved in senescence. TGF-β1 concentration and time of stimulation were correlated with the expression of proteins involved in the fibrogenesis and inflammatory processes. These data underline the need for standardization of culture conditions to allow inter-data comparisons in future in vitro studies, especially when using “omics” approaches.     

Multiple roles of alpha-smooth muscle actin in mechanotransduction

Alpha-smooth muscle actin (SMA), an actin isoform that contributes to cell-generated mechanical tension, is normally restricted to cells of vascular smooth muscle, but SMA can also be expressed in certain non-muscle cells, most notably myofibroblasts. These cells are present in healing wounds, scars, and fibrocontractive lesions where they contribute to fibrosis. In myofibroblasts, cell-generated traction forces associated with SMA contribute to matrix remodeling, but exogenous mechanical forces can also increase SMA expression. Force-induced SMA utilizes a feed-forward amplification loop involving a priori SMA in focal adhesions, the binding of the p38 MAP kinase to SMA filaments, activation of Rho and binding of serum response factor to the CArG-B box of the SMA promoter. Thus, in addition to its importance as a structural protein in tissue remodeling and contraction, SMA may serve as a mechanotransducer, based on its ability to physically link mechanosensory elements and to enhance its own, force-induced expression.     

Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts

Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilament bundles and the expression of alpha-SM actin, the actin isoform typical of vascular SM cells. Myofibroblasts have been proposed to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. We show here that the subcutaneous administration of transforming growth factor- beta 1 (TGF beta 1) to rats results in the formation of a granulation tissue in which alpha-SM actin expressing myofibroblasts are particularly abundant. Other cytokines and growth factors, such as platelet-derived growth factor and tumor necrosis factor-alpha, despite their profibrotic activity, do not induce alpha-SM actin in myofibroblasts. In situ hybridization with an alpha-SM actin probe shows a high level of alpha-SM actin mRNA expression in myofibroblasts of TGF beta 1-induced granulation tissue. Moreover, TGF beta 1 induces alpha-SM actin protein and mRNA expression in growing and quiescent cultured fibroblasts and preincubation of culture medium containing whole blood serum with neutralizing antibodies to TGF beta 1 results in a decrease of alpha-SM actin expression by fibroblasts in replicative and non-replicative conditions. These results suggest that TGF beta 1 plays an important role in myofibroblast differentiation during wound healing and fibrocontractive diseases by regulating the expression of alpha-SM actin in these cells.