骨骼肌细胞的介电弛豫特性

在100Hz～100MHz频率范围内,利用非线性数值计算和曲线拟合分析,验证了蛙离体骨骼肌细胞的介电弛豫特性满足Cole-Cole公式(误差≤3.45%),通过频域介电谱、Cole-Cole图、介电损耗因子和介电损耗角正切频率谱的曲线拟合分析,确定了蛙骨骼肌细胞的Cole-Cole介电参数:高频段相对介电常数εh=78,第一相对介电增量△ε1=113000,第二相对介电增量△ε2=45000,第一特征频率fc1=9 kHz,第二特征频率fc2=158 kHz,第一相位角β1=0.881,第二相位角β2=0.984,低频段电导率κL=0.55mS/cm,常数A=35,常数m=1.08.     

Characteristics of the Localization of Connexin 43 in Satellite Cells during Skeletal Muscle Regeneration In Vivo

For myogenesis, new myotubes are formed by the fusion of differentiated myoblasts. In the sequence of events for myotube formation, intercellular communication through gap junctions composed of connexin 43 (Cx43) plays critical roles in regulating the alignment and fusion of myoblasts in advances of myotube formation in vitro. On the other hand, the relationship between the expression patterns of Cx43 and the process of myotube formation in satellite cells during muscle regeneration in vivo remains poorly understood. The present study investigated the relationship between Cx43 and satellite cells in muscle regeneration in vivo. The expression of Cx43 was detected in skeletal muscles on day 1 post-muscle injury, but not in control muscles. Interestingly, the expression of Cx43 was not localized on the inside of the basement membrane of myofibers in the regenerating muscles. Moreover, although the clusters of differentiated satellite cells, which represent a more advanced stage of myotube formation, were observed on the inside of the basement membrane of myofibers in regenerating muscles, the expression of Cx43 was not localized in the clusters of these satellite cells. Therefore, in the present study, it was suggested that Cx43 may not directly contribute to muscle regeneration via satellite cells.     

APOA-1 is a Novel Marker of Erythroid Cell Maturation from Hematopoietic Stem Cells in Mice and Humans

The mechanism that regulates the terminal maturation of hematopoietic stem cells into erythroid cells is poorly understood. Therefore, identifying genes and surface markers that are restricted to specific stages of erythroid maturation will further our understanding of erythropoiesis. To identify genes expressed at discrete stages of erythroid development, we screened for genes that contributed to the proliferation and maturation of erythropoietin (EPO)-dependent UT-7/EPO cells. After transducing erythroid cells with a human fetal liver (FL)-derived lentiviral cDNA library and culturing the cells in the absence of EPO, we identified 17 candidate genes that supported erythroid colony formation. In addition, the mouse homologues of these candidate genes were identified and their expression was examined in E12.5 erythroid populations by qRT-PCR. The expression of candidate erythroid marker was also assessed at the protein level by immunohistochemistry and ELISA. Our study demonstrated that expression of the Apoa-1 gene, an apolipoprotein family member, significantly increased as hematopoietic stem cells differentiated into mature erythroid cells in the mouse FL. The Apoa-1 protein was more abundant in mature erythroid cells than hematopoietic stem and progenitor cells in the mouse FL by ELISA. Moreover, APOA-1 gene expression was detected in mature erythroid cells from human peripheral blood. We conclude that APOA-1 is a novel marker of the terminal erythroid maturation of hematopoietic stem cells in both mice and humans.     

骨骼肌卫星细胞的培养及其在骨骼肌组织工程研究中的应用

近年来,随着组织工程技术的不断发展,生命科学、材料科学及制造科学的相互渗透,使得体外构建人体组织和器官的功能性替代物成为可能.骨骼肌卫星细胞作为组织工程的种子细胞在体外扩增至一定数量后与生物可降解三维支架材料结合,植入患者体内来修复缺损及恢复生理功能.目前对肌卫星细胞的体外培养已进行了较多研究,而对于肌卫星细胞的体外生长、增殖、鉴定,以及与组织工程三维支架的相互作用和生物功能的研究尚处于发展阶段,文中将对这些方面的研究进展作一综述.     

负荷渐增式训练对老年小鼠骨骼肌卫星细胞AMPK磷酸化的影响

目的 探讨负荷渐增式训练对老年小鼠骨骼肌卫星细胞腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)磷酸化的影响.方法 实验小鼠分为3组:青年对照组(YC组,n=12)、老年对照组(OC组,n=12)与老年运动组(OT组,n=12).OT组进行负荷渐增式训练,流式细胞分选技术分离CD...     

The Impact of CB1 Receptor on Nuclear Receptors in Skeletal Muscle Cells

Cannabinoids are abundant signaling compounds; their influence predominantly arises via engagement with the principal two G-protein-coupled cannabinoid receptors, CB1 and CB2. One suggested theory is that cannabinoids regulate a variety of physiological processes within the cells of skeletal muscle. Earlier publications have indicated that expression of CB1 receptor mRNA and protein has been recognized within myotubes and tissues of skeletal muscle from both murines and humans, thus representing a potentially significant pathway which plays a role in the control of skeletal muscular activities. The part played by CB1 receptor activation or inhibition with respect to these functions and relevant to targets in the periphery, especially skeletal muscle, is not fully delineated. Thus, the aim of the current research was to explore the influence of CB1 receptor stimulation and inhibition on downstream signaling of the nuclear receptor, NR4A, which regulates the immediate impacts of arachidonyl-2′-chloroethylamide (ACEA) and/or rimonabant in the cells of skeletal muscle. Murine L6 skeletal muscle cells were used in order to clarify additional possible molecular signaling pathways which contribute to alterations in the CB1 receptor. Skeletal muscle cells have often been used; it is well-documented that they express cannabinoid receptors. Quantitative real-time probe-based polymerase chain reaction (qRT-PCR) assays are deployed in order to assess the gene expression characteristics of CB1 receptor signaling. In the current work, it is demonstrated that skeletal muscle cells exhibit functional expression of CB1 receptors. This can be deduced from the qRT-PCR assays; triggering CB1 receptors amplifies both NR4A1 and NR4A3 mRNA gene expression. The impact of ACEA is inhibited by the selective CB1 receptor antagonist, rimonabant. The present research demonstrated that 10 nM of ACEA notably amplified mRNA gene expression of NR4A1 and NR4A3; this effect was suppressed by the addition of 100 nM rimonabant. Furthermore, the CB1 receptor antagonist led to the downregulation of mRNA gene expression of NR4A1, NR4A2 and NR4A3. In conclusion, in skeletal muscle, CB1 receptors were recognized to be important moderators of NR4A1 and NR4A3 mRNA gene expression; these actions may have possible clinical benefits. Thus, in skeletal muscle cells, a possible physiological expression of CB1 receptors was identified. It is as yet unknown whether these CB1 receptors contribute to pathways underlying skeletal muscle biological function and disease processes. Further research is required to fully delineate their role(s).     

The Origin and Fate of Muscle Satellite Cells

Satellite cells represent the primary population of stem cells resident in skeletal muscle. These adult muscle stem cells facilitate the postnatal growth, remodeling, and regeneration of skeletal muscle. Given the remarkable regenerative potential of satellite cells, there is great promise for treatment of muscle pathologies such as the muscular dystrophies with this cell population. Various protocols have been developed which allow for isolation, enrichment, and expansion of satellite cell derived muscle stem cells. However, isolated satellite cells have yet to translate into effective modalities for therapeutic intervention. Broadening our understanding of satellite cells and their niche requirements should improve our in vivo and ex vivo manipulation of these cells to expedite their use for regeneration of diseased muscle. This review explores the fates of satellite cells as determined by their molecular signatures, ontogeny, and niche dependent programming.     

运动对骨骼肌卫星细胞的影响及作用机制

骨骼肌卫星细胞在骨骼肌生长发育、损伤修复以及骨骼肌重塑等生理病理过程中具有重要的作用。适宜的运动训练可活化卫星细胞,促进卫星细胞增殖并向成肌细胞分化。本文就骨骼肌卫星细胞的起源、形态特征和特异性的标记以及运动训练调控骨骼肌卫星细胞活化、增殖、分化的作用机制进行综述。     

Isolation and Differentiation of Chondrocytic Cells Derived from Human Embryonic Stem Cells Using dlk1/FA1 as a Novel Surface Marker

Few surface markers are available to monitor lineage differentiation during chondrogenesis. Recently, delta-like1/fetal antigen1 (dlk1/FA1), a transmembrane protein of the Notch/Delta/Serrata family, was shown to be essential for inducing early chondrogenesis. Thus, we investigated the possible use of dlk1/FA1 as a novel surface marker for chondroprogenitor cells during hESC differentiation. We found that, Dlk1/FA1 is expressed specifically in cells undergoing transition from proliferating to prehypertrophic chondrocytes during endochondral ossification of the mouse limb. In hESC cells, dlk1/FA1 was not expressed by undifferentiated hESC, but expressed during in vitro embryoid bodies (hEBs) formation upon down-regulation of undifferentiated markers e.g. Oct 3/4. Similarly, dlk1/FA1 was expressed in chondrocytic cells during in vivo teratoma formation. Interestingly, treatment of hEBs with Activin B, a member of TGF-ß family, markedly increased Dlk1 expression in association with up-regulation of the mesoderm-specific markers (e.g. FOXF1, KDR and VE-cadherin) and SOX9. dlk1/FA1⁺ cells isolated by fluorescence activated cell sorting (FACS) were capable of differentiating into chondrocytic cells when cultured as micromass pellets in a xeno-free system containing TGFβ1. In conclusion, we identified dlk1/FA1 as a novel marker of chondroprogenitor cells that undergo embryonic lineage progression from proliferation to the prehypertrophic stage. Tracking dlk1/FA1 expression as a mesoderm/chondroprogenitor surface marker provides a novel strategy for designing clinically relevant protocols to direct the differentiation of hESC into chondrocytes.     

Chitinase 3-Like-1 Expression in Colonic Epithelial Cells as a Potentially Novel Marker for Colitis-Associated Neoplasia

Chitinase 3-like-1 (CHI3L1/YKL-40) is a protein secreted from restricted cell types including colonic epithelial cells (CECs) and macrophages. CHI3L1 is an inflammation-associated molecule, and its expression is enhanced in persons with colitis and colon cancer. The biological function of CHI3L1 on CECs is unclear. In this study, we investigated the role of CHI3L1 on CECs during the development of colitis-associated neoplasia. We analyzed colonic samples obtained from healthy persons and from persons with ulcerative colitis with or without premalignant or malignant changes. DNA microarray and RT-PCR analyses significantly increased CHI3L1 expression in non-dysplastic mucosa from patients with inflammatory bowel disease (IBD) who had dysplasia/adenocarcinoma compared with that in healthy persons and in patients with IBD who did not have dysplasia. As determined by IHC, CHI3L1 was expressed in specific cell types in the crypts of colonic biopsies obtained from patients with ulcerative colitis who have remote dysplasia. Purified CHI3L1 efficiently activated the NF-κB signaling pathway and enhanced the secretion of IL-8 and TNF-α in SW480 human colon cancer cells. In addition, colon cancer cell proliferation and migration were significantly promoted in response to CHI3L1 in these cells. In summary, CHI3L1 may contribute to the proliferation, migration, and neoplastic progression of CECs under inflammatory conditions and could be a useful biomarker for neoplastic changes in patients with IBD.Chitinase 3-like-1 (CHI3L1, also known as YKL-40 or HC-gp39) is classified in the glycosyl hydrolases 18 family based on the structural similarity with other chitinases.^1,2 However, functionally, CHI3L1 lacks enzymatic activity and belongs to the family of chi-lectins (chitinase-like lectins) that includes Ym-1³ and stabilin-1-interacting chitinase-like protein.⁴ CHI3L1 is a 40 kDa protein and is produced by restricted cell types, including colonic epithelial cells (CECs) and macrophages.^5–7 CHI3L1 can be detected in the Golgi apparatus and the endoplasmic reticulum,⁸ but its major sites of action seems to be extracellular as a secreted protein.⁹ The secreted form of CHI3L1 has growth-stimulating effects in connective tissue cells, including synoviocytes and chondrocytes.¹⁰ In addition, CHI3L1 shows dose-dependent growth-stimulating effects in human fibroblasts and shows similar and synergistic effects with well-characterized mitogen, insulin-like growth factor 1 (IGF-1).¹¹ However, the exact biological function of CHI3L1 in CECs remains uncertain.It is well documented that elevated levels of CHI3L1 can be detected in the sera of persons with rheumatoid arthritis, bronchial asthma, or inflammatory bowel disease (IBD).^12–15 Serum CHI3L1 is significantly increased in active but not quiescent IBD.^5,9,15 In agreement with this observation, approximately 64% of persons with Crohn''s disease (CD) who have extra-intestinal manifestations such as erythema nodosum and fistulas showed significantly increased serum levels of CHI3L1.^15,16 In addition, patients with CD who had stenotic disease had higher serum CHI3L1 than did patients with non-stenotic disease.¹⁷ Of note, the colonic CHI3L1 mRNA level was increased in persons with active ulcerative colitis (UC) and CD but was in the normal range in persons with quiescent UC and the uninvolved regions of CD.⁵ In addition, CHI3L1 serum concentrations seem to be not only highly up-regulated in persons with active CD and UC but also correlated with poor prognosis of solid tumors, including breast cancer and colon cancer.⁹Patients with chronic IBD have an increased risk of developing colitis-associated cancer, which increases by 0.5% to 1% annually after 10 years of chronic inflammation.¹⁸ A growing amount of evidence indicates that various soluble factors produced by epithelial cells and immune cells play a pathogenic role in the carcinogenic change of CECs.^19,20 CHI3L1 seems to be one of the soluble factors that play a pivotal role in protecting cancer cells from undergoing apoptosis, as well as promoting tissue remodeling by interacting with the extracellular matrix and by stimulating angiogenesis.²¹ However, little is know about the role of CHI3L1 in IBD-associated colon cancer.In this study we show that CHI3L1 expression in CECs is significantly and specifically increased in non-dysplastic mucosa of patients with UC who have dysplasia that is away from the non-dysplastic mucosa (remote dysplasia) as well as colorectal adenocarcinoma; we also show that it may be a reliable biological marker of neoplasia in high-risk individuals. In addition, we demonstrate a new mechanism by which CHI3L1 may contribute to IBD-associated neoplasia through a growth-stimulating effect on enhancing the production of NF-κB–induced IL-8 and tumor necrosis factor (TNF)-α, which presumably are associated with chronic inflammation-mediated malignant transformation in CECs.     

细胞外灌流对骨骼肌细胞介电谱的影响

通过灌流的方法,探讨细胞外液对骨骼肌细胞介电谱的影响,应用非线性Co le-Co le公式的曲线拟合与数值计算,比较灌流后骨骼肌细胞介电谱参数,结果表明:(1)低频段介电常数Lε和高频段电导率hκ随着细胞外灌流液电导率的降低而减少;(2)细胞外灌流对骨骼肌细胞介电谱的特征频率(fC 1,fC 2)影响不大;(3)细胞外灌流主要影响骨骼肌细胞Δε″和tgδ的峰值。     

AMPK调节骨骼肌细胞GLUT4基因表达的机制研究

腺苷酸活化蛋白激酶(AMPK)能调节运动/肌肉收缩所引起的骨骼肌细胞葡萄糖转运蛋白4(GLUT4)基因的表达,但至今它的调节机制不清.研究显示在非运动刺激引起的细胞信号事件中由组蛋白去乙酰化酶(HDACs)以及组蛋白乙酰化酶(HATs)控制的组蛋白乙酰化状态是调节基因表达的重要机制,所以我们假设AMPK信号途径是通过征用HDACs中的HDAC5(在骨骼肌细胞内高表达)来实现对运动/肌肉收缩引起的GLUT4基因表达控制.细胞分为正常浓度葡萄糖对照组(NGLU组)、正常浓度AICAR组(NGLU AICAR组)、高浓度对照组(HGLU组)、高浓度AICAR组(HGLU AICAR组).用5 mmol/L和20 mmol/L葡萄糖浓度培养骨骼肌细胞后,NGLU AICAR组和HGLU AICAR组与肌肉收缩模拟信号刺激5-氨基-4-甲酰胺咪唑核糖核苷酸(AICAR)孵育.AICAR能激活NGLU组骨骼肌细胞AMPKα2、减少骨骼肌细胞核HDAC5蛋白、促使HDAC5与骨骼肌细胞加强因子(MEF2)蛋白分离和上调GLUT4基因的表达;相反,高浓度葡萄糖延迟由AICAR引起的AMPKα2磷酸化、AMPKα2向细胞核转入、HDAC5向细胞核转出和GLUT4基因的表达.实验结果说明在不同葡萄糖浓度下的骨骼肌细胞GLUT4基因表达变化都对应着上游AMPK蛋白和下游HDAC5蛋白的变化,AMPK可能是征用转录抑制子HDAC5来调节MEF2的活性而达到控制肌肉收缩所引起的GLUT4基因表达.     

Delivery of Factor VIII Gene into Skeletal Muscle Cells Using Lentiviral Vector

Purpose

This study was designed to investigate whether transduction of lentiviral vectors (LV) carrying human coagulation factor VIII (hFVIII) cDNA into skeletal muscle could increase circulating hFVIII concentrations.

Materials and Methods

A LV containing bacterial LacZ gene as a control or human FVIII gene was intramuscularly administered into the thigh muscle of 5 weeks old Sparague-Dawley rats. The plasma human FVIII concentration and neutralizing anti-FVIII antibodies were measured for up to 12 weeks in these experimental animals.

Results

The plasma human FVIII levels in the rats injected with LV carrying FVIII cDNA peaked at post-injection 1st week (5.19 ± 0.14 ng/mL vs. 0.21 ± 0.05 ng/mL in control rats , p < 0.05). Elevated hFVIII concentrations were maintained for 4 weeks (2.52 ± 0.83 ng/mL vs. 0.17 ± 0.08 ng/mL in control rats, p < 0.05) after a single intramuscular injection. In the Bethesda assay, neutralizing antibodies for FVIII protein were detected only in FVIII-LV injected rats by the 10th week, but not in control rats.

Conclusion

This study suggested that a single administration of an advanced generation LV carrying the human FVIII cDNA resulted in elevation of FVIII level in immune competent rats, and that this gene transfer approach to the skeletal muscle could be an effective tool in treatment of hemophilia A.     

吡格列酮对大鼠骨骼肌细胞脂联素受体基因表达的影响

为研究吡格列酮对大鼠骨骼肌细胞脂联素受体表达的影响,应用体外原代培养骨骼肌细胞和SYBR Green Ⅰ实时定量PCR,观察不同浓度吡格列酮对鼠骨骼肌细胞脂联素受体表达水平的影响.结果表明,不同浓度的吡格列酮作用于大鼠骨骼肌细胞20h后对脂联素的受体R1表达没有显示出任何有意义的变化.大鼠骨骼肌细胞脂联素受体基因表达可能与胰岛素增敏剂噻唑烷二酮类吡格列酮无关.     

Adrenergic and Cholinergic Nerve Terminals in Skeletal Muscle Vessels1

By the use of histochemical methods the enzyme acetylcholinesterase (AcChE) was visualized in nerve terminal appearing structures in the adventitia of small intramuscular arteries (30–100 μ) of cat and dog. These AcChE-rich structures may represent cholinergir nerve terminals. Following chronic sympathectomy in dogs no AcChE-rich nerve terminals were found in muscles on the operated side, indicating that these nerves represented sympathetic cholinergic innerva-tion of skeletal muscle vessels, presumably the vasodilator nerves. In adjacent tissue sections of the muscle, adrenergic nerve terminals were histochemically visualized. Adrenergic nerve terminals were. seen in the same layer of the vessel wall as the cholinergic ones, i.e. in the adventitia surrounding the media. Adrenergic nerve terminals were found to innervate both large and small arteries. In muscle samples from monkey and human subjects no AcChE-rich nerve terminals were observed around the vessels. However, adrenergic nerve terminals were found in these species with the same appearance as in cat and dog. This finding supports previous physiological experiments indicating that skeletal muscle of monkey lack sympathetic cholinergic vasodilator innervation.     

Mast Cells Appearing in Long‐term Skeletal Muscle Cell Cultures of Rat

Mast cells are known to be involved in type I allergy and to be localized in almost all tissues in the body. However, they have slightly different properties depending on their tissue of residence. Although mast cells are found in skeletal muscle tissue, there have been no reports of their appearance in cultured skeletal muscles. We report here that mast cells appear in long‐term cultures of skeletal muscles from neonatal rats and rat fetuses. When muscle cells were disseminated and cultured in minimum essential medium with 10% fetal calf serum and 10% horse serum, oval cells containing large granules started to appear on myotube sheets at 5 days of culture. These oval cells continued to proliferate for 2–3 months, and showed immunoreactivity for histamine, tryptase, Fc_εRI, and c‐kit. They showed metachromatic staining with 0.5% toluidine blue at pH 0.5 and were stained with both Alcian blue and safranin. Biochemically measured histamine content per dish was significantly higher in 2‐month than in 5‐day culture. From these results, we concluded that these oval cells were mast cells. Because proteases from mast cells have been reported previously to affect myoblast proliferation, the present findings suggest that there may be some interaction between mast cells and muscle cell proliferation or differentiation. The present finding that mast cells are easily obtained from ordinary skeletal muscle cultures provides a useful method for the study of the diverse functions of mast cells. Anat Rec, 1424‐1430, 2007. © 2007 Wiley‐Liss, Inc.