A comparative study of myostatin,follistatin and decorin expression in muscle of different origin

Muscle regeneration supports muscle function in aging, and plays a role in the functional impairment caused by progressive neuromuscular diseases. Major substances controlling this process are growth factors and the extracellular matrix (ECM). Thus, follistatin is known to antagonize the function of several members of the TGF-β family of secreted signaling factors, including myostatin—the most powerful inhibitor of muscle growth characterized to date. Decorin—a small leucine-rich proteoglycan—traps myostatin and modulates its activity towards myogenic cells in the ECM. In addition, there are few reports concerning the regenerative muscle process of masseter muscles, which are of branchial arch origin, in mdx mice. Thus, in order to clarify the muscle regenerative process of masseter muscle, gene and protein expression of myostatin, follistatin and decorin were examined using the tibialis anterior (TA)muscle as a positive control. In both muscles, a gradual increase in mRNA myostatin, follistatin and decorin expression was detected, with the increase being greater in TA muscle than in masseter muscle. At 2 weeks, both muscles exhibited normal skeletal muscle cells. At 3 weeks, masseter muscle demonstrated scant areas of necrosis, whereas large necrotic zones were seen in TA muscle. At 4 weeks, the formation of necrotic tissue and presence of follistatin protein was observed clearly in masseter muscle. This result indicates that follistatin production is stimulated in the presence of necrosis. Interestingly, both muscles showed the same process of muscular formation, but with different time frames, which could be related to muscle origin.     

In vitro magnesium- and calcium-induced sialidase activity in caprine intestinal mucosa cells

Sialidase activity identical to 0.0196 mg per ml of liberated sialic acids (2.11 Units) was detected in small intestinal contents of goats. Mg++ and Ca++ induced in vitro detection in mucosa cells of both small and large intestines. Magnesium alone induced sialidase activities identical to 0.0128 mg per ml of liberated sialic acids (1.38 Units) and 0.0166 mg per ml (1.79 Units) in small and large intestinal mucosal cells, respectively. Mg++ and Ca++ together induced higher sialidase activities identical to 0.0191 mg per ml (2.06 Units) and 0.0194 mg per ml (2.09 Units) in small and large intestinal mucosal cells, respectively. The enzyme activity was slightly higher in large than in small intestinal mucosal cells. The results are discussed in relation to control of African animal trypanosomiasis.     

Gene expression profiling in an in vitro model of angiogenesis

In the present study we have used a novel, comprehensive mRNA profiling technique (GeneCalling) for determining differential gene expression profiles of human endothelial cells undergoing differentiation into tubelike structures. One hundred fifteen cDNA fragments were identified and shown to represent 90 distinct genes. Although some of the genes identified have previously been implicated in angiogenesis, potential roles for many new genes, including OX-40, white protein homolog, KIAA0188, a homolog of angiopoietin-2, ADAMTS-4 (aggrecanase-1), and stanniocalcin were revealed. Support for the biological significance was confirmed by the abrogation of the changes in the expression of angiogenesis inhibitors and in situ hybridization studies. This study has significantly extends the molecular fingerprint of the changes in gene expression that occur during endothelial differentiation and provides new insights into the potential role of a number of new molecules in angiogenesis.     

Heterologous expression of wild-type and mutant β-cardiac myosin changes the contractile kinetics of cultured mouse myotubes

The properties of myosin expressed in muscle are a major determinant of muscle performance. In this study we used a novel approach to examine the functional impact of changes in myosin heavy chain (MHC) isoform expression, as well as the consequences of expressing the mutant MHC implicated in familial hypertrophic cardiomyopathy (FHC). Cultured mouse myoblasts that normally express fast embryonic myosin were untransfected, or stably transfected with a plasmid expressing either wild-type (cWT) or mutant (D778G or G741R) β-cardiac myosin. After differentiation for 5–7 days, cWT or mutant β-cardiac myosin was expressed at 25 % of total myosin in the myotube. We measured time-to-peak shortening (ttp), time for half-relaxation ( t _0.5), the maximum velocity of shortening ( V _max) at 1 Hz stimulation, and the tetanic fusion frequency. Expression of cWT β-cardiac myosin significantly increased ttp and t _0.5 and decreased the fusion frequency compared with untransfected myotubes. However, when we compared myotubes expressing mutant β-cardiac myosin with those expressing cWT β-cardiac myosin, we found that ttp and t _0.5 were significantly decreased, and V _max was increased for the D778G mutant, whereas ttp, t _0.5 and V _max were unchanged for the G741R mutant. The fusion frequency was increased for both mutant myosins. Our data support the conclusion that the impact of the slower myosin isoform dominates when both slow and fast isoforms are present. This work suggests that FHC associated with either D778G or G741R mutation in MHC is an 'energy cost' disease, but that the phenotype of D778G is more severe than that of G741R.     

核心蛋白聚糖对鼠肌腱细胞I型胶原及其mRNA的影响

目的:探讨核心蛋白聚糖(Decorin,DCN)对大鼠肌腱细胞I型胶原的影响,为进一步临床应用提供理论依据。方法:50μg/μL的DCN作用大鼠肌腱细胞12h后,采用免疫细胞化学,免疫荧光方法观察DCN对大鼠肌腱细胞I型胶原蛋白的影响,采用半定量反转录聚合酶链反应(RT-PCR)方法检测DCN对大鼠肌腱细胞I型胶原mRNA表达的影响。结果:DCN在早期能够明显减少肌腱细胞的I型胶原蛋白的合成,免疫荧光定量分析实验组和对照组相差显著;DCN早期还能够显著下调肌腱细胞I型胶原蛋白mRNA水平的表达。结论:DCN在早期对大鼠肌腱细胞I型胶原的形成起着抑制作用。     

核心蛋白聚糖对鼠肌腱细胞Ⅰ型胶原及其mRNA的影响

目的：探讨核心蛋白聚糖（Decorin，DCN）对大鼠肌腱细胞Ⅰ型胶原的影响，为进一步临床应用提供理论依据。方法：50μg/μL的DCN作用大鼠肌腱细胞12h后，采用免疫细胞化学，免疫荧光方法观察DCN对大鼠肌腱细胞Ⅰ型胶原蛋白的影响，采用半定量反转录聚合酶链反应（RT-PCR）方法检测DCN对大鼠肌腱细胞Ⅰ型胶原mRNA表达的影响。结果：DCN在早期能够明显减少肌腱细胞的Ⅰ型胶原蛋白的合成，免疫荧光定量分析实验组和对照组相差显著；DCN早期还能够显著下调肌腱细胞Ⅰ型胶原蛋白mRNA水平的表达。结论：DCN在早期对大鼠肌腱细胞Ⅰ型胶原的形成起着抑制作用。     

In vitro metabolism of moxidectin in Haemonchus contortus adult stages

We studied the implication of cytochrome P450 enzymes in the in vitro metabolism of moxidectin (MXD) in homogenates of Haemonchus contortus adult stages (susceptible isolate, Weybridge, UK). After homogenisation in a phosphate buffer, 2 ml of homogenates (equivalent to 1 g of nematodes) were incubated with 5 microg [14C] MXD at 37 degrees C for 24 h. MXD and its metabolites were separated by HPLC with radiodetection on-line. Only one metabolite was detected and its production was inhibited by carbon monoxide. This result demonstrates that the cytochrome P450 system is implicated in the metabolisation of MXD in H. contortus susceptible to milbemycin. Furthermore, this metabolite did not match those previously described in vertebrates.     

成人脂肪基质细胞体外扩增生长和超微结构

背景：脂肪基质细胞在体外能诱导分化为多种细胞,但脂肪基质细胞开始诱导反应的最佳时间尚未明确。 目的：通过成人脂肪基质细胞的体外扩增生长曲线和超微结构特征推测诱导分化反应的最佳时期。 方法：将取自吸脂术的脂肪组织消化、离心、提取细胞,并体外培养、传代。倒置相差显微镜下观察细胞形态学变化;细胞计数法测定脂肪基质细胞生长曲线;免疫组化和免疫荧光法检测CD44、CD29、CD34的表达;透射电镜观察脂肪基质细胞的超微结构。 结果与结论：脂肪基质细胞生长迅速,传代到第3代5-7 d呈对数生长,第8天细胞呈长梭形,数量达(5.32±0.03)×10⁷ L^-1并趋于稳定;脂肪基质细胞的CD44、CD29阳性表达率为(84.35±9.73)%,(86.37± 8.45)%,CD34为阴性表达;透射电镜观察到处于幼稚状态的细胞器等超微结构。脂肪基质细胞作为一种多能干细胞,传至第3-6代8-12 d时的细胞数量多且形态稳定,具有细胞分化和合成所需蛋白的超微结构,此时是开始诱导分化反应的最佳时期。     

尾悬吊大鼠骨髓基质干细胞体外分化能力及相关因子的表达

背景：尾悬吊模拟失重可造成骨丢失,骨髓基质干细胞增殖能力与分化方向也直接影响着骨髓腔内细胞水平骨代谢的平衡并最终改变骨的质量。 目的：观察尾悬吊大鼠骨髓基质干细胞体外向成骨细胞和脂肪细胞分化的能力及相关因子的表达。 方法：SD大鼠随机分为正常对照组、尾悬吊组。取大鼠骨髓细胞体外培养,传1代后分别向成骨细胞和脂肪细胞两个方向诱导分化,成骨组第16天行碱性磷酸酶染色,第28天检测细胞外基质矿化能力,real-time PCR法检测第28天骨形态发生蛋白2 mRNA的表达,成脂组第21天检测脂蛋白酯酶的比活性及mRNA的表达,第30天油红O法检测脂滴形成能力。 结果及结论：尾悬吊组大鼠骨髓基质干细胞成骨分化早期碱性磷酸酶阳性细胞表达率显著高于对照组,但分化晚期骨形态发生蛋白2 mRNA的表达水平及细胞外基质矿化能力较对照组显著降低;尾悬吊大鼠骨髓基质干细胞成脂分化过程中脂蛋白酯酶的表达、比活性及脂滴形成能力均显著高于对照组。说明尾悬吊可潜在刺激骨髓基质干细胞早期成骨分化能力,但显著抑制其矿化能力,从而抑制成骨,其机制可能与抑制骨形态发生蛋白2的表达有关;可显著促进骨髓基质干细胞向脂肪细胞分化。尾悬吊造成大鼠骨量的丢失可能与其对骨髓基质干细胞分化方向的影响有关。     

成人骨髓间充质干细胞体外培养、鉴定与成骨诱导

背景：骨髓间充质干细胞具有自我增殖和多向分化潜能,然而间充质干细胞在骨髓中含量极少,体外纯化、扩增与成骨诱导是进行骨组织工程研究的关键。 目的：拟建立一套简便可靠的成人骨髓间充质干细胞体外培养及鉴定体系,并定向诱导其向成骨细胞分化。 方法：抽取髂前上棘处骨髓,采用密度梯度离心法与贴壁筛选法相结合,对骨髓间充质干细胞分离、纯化、培养并扩增。将地塞米松、β-甘油磷酸钠、维生素Ｃ配制成骨诱导液,将细胞分为成骨诱导组和空白对照组进行观察。 结果与结论：成人骨髓间充质干细胞呈典型的长梭形、纺锤状。8-11 d细胞进入快速增殖期,生长曲线呈S型,CD44、CD90均呈阳性表达,而CD34、CD45呈阴性表达。碱性磷酸酶活性随培养时间而增高,并在第12天达到高峰,成骨诱导组各时间点碱性磷酸酶活性均高于空白对照组。说明实验建立的成人骨髓间充质干细胞体外培养、鉴定及诱导成骨细胞分化体系可以获得纯度较高、成骨分化能力较好的间充质干细胞。     

In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells

Although the existence of mammary stem cells has been suggested by serial transplantation studies in mice, their identification has been hindered by the lack of specific surface markers, and by the absence of suitable in vitro assays for testing stem cell properties: self-renewal and ability to generate differentiated progeny. We have developed an in vitro cultivation system that allows for propagation of human mammary epithelial cells (HMECs) in an undifferentiated state, based on their ability to proliferate in suspension, as nonadherent mammospheres. We demonstrate that nonadherent mammospheres are enriched in early progenitor/stem cells and able to differentiate along all three mammary epithelial lineages and to clonally generate complex functional structures in reconstituted 3D culture systems. Gene expression analysis of cells isolated from nonadherent mammospheres revealed overlapping genetic programs with other stem and progenitor cells and identified new markers that may be useful in the identification of mammary stem cells. The isolation and characterization of these stem cells should help elucidate the molecular pathways that govern normal mammary development and carcinogenesis.