The changing AMPK expression profile in differentiating mouse skeletal muscle myoblast cells helps confer increasing resistance to apoptosis

AMP-activated protein kinase (AMPK) functions as a alpha/beta/gamma heterotrimer to preserve ATP levels and so cell viability during stressful conditions. However, its role in aiding survival of adult skeletal muscle precursor cells is unclear. Using the differentiating mouse C2C12 postnatal skeletal muscle myoblast cell line, we have determined that proteins for the AMPK subunit isoforms alpha2 and gamma2 are constitutively expressed, while those for alpha1, beta1 and beta2 are undetectable in undifferentiated myoblasts but increasingly expressed with differentiation to myotubes. Although the gamma3 subunit is expressed at a low level in myoblasts, it too is expressed increasingly with differentiation to myotubes. The p50 but not the p72 isoform of the embryonic alpha subunit homologue MELK is expressed only in proliferating myoblasts, while the ARK5 alpha subunit homologue is increasingly expressed with differentiation. Myotubes displayed higher basal and stimulated alpha1/alpha2 AMPK activation than myoblasts. Furthermore, serum starvation resulted in less apoptosis of differentiated myotubes than of undifferentiated myoblasts. This reflects, in part, the increased expression of functional AMPK in the myotubes, since specific inhibition of AMPK activity with 6-[4-(2-piperidin-1-ylethoxy)-phenyl]-3-pyridin-4-ylpyrazolo[1,5-alpha] pyrimidine (Compound C) exacerbated the apoptosis resulting from serum withdrawal. If these in vitro events can also occur in vivo, they could have implications for pathologies such as muscle wasting, in which undifferentiated satellite stem cells may be easier apoptotic targets than their differentiated counterparts. Furthermore, these results suggest that when interpreting results from in vitro or in vivo experiments on AMPK, the subunit expression profile should be taken into account.     

Phospho-tyrosine phosphatase inhibitor Bpv(Hopic) enhances C2C12 myoblast migration in vitro. Requirement of PI3K/AKT and MAPK/ERK pathways

Muscle progenitor cell migration is an important step in skeletal muscle myogenesis and regeneration. Migration is required for muscle precursors to reach the site of damage and for the alignment of myoblasts prior to their fusion, which ultimately contributes to muscle regeneration. Limited spreading and migration of donor myoblasts are reported problems of myoblast transfer therapy, a proposed therapeutic strategy for Duchenne Muscular Dystrophy, warranting further investigation into different approaches for improving the motility and homing of these cells. In this article, the effect of protein phospho-tyrosine phosphatase and PTEN inhibitor BpV(Hopic) on C2C12 myoblast migration and differentiation was investigated. Applying a wound healing migration model, it is reported that 1 μM BpV(Hopic) is capable of enhancing the migration of C2C12 myoblasts by approximately 40 % in the presence of myotube conditioned media, without significantly affecting their capacity to differentiate and fuse into multinucleated myotubes. Improved migration of myoblasts treated with 1 μM BpV(Hopic) was associated with activation of PI3K/AKT and MAPK/ERK pathways, while their inhibition with either LY294002 or UO126, respectively, resulted in a reduction of C2C12 migration back to control levels. These results propose that bisperoxovanadium compounds may be considered as potential tools for enhancing the migration of myoblasts, while not reducing their differentiation capacity and underpin the importance of PI3K/AKT and MAPK/ERK signalling for the process of myogenic progenitor migration.     

Expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in ischemic skeletal muscle and its regeneration

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible endothelial cell mitogen and survival factor. Its receptor VEGFR-2 (KDR/Flk-1) mediates these effects. We studied the expression of VEGF and VEGFR-2 in ischemic human and rabbit skeletal muscle by immunohistochemistry and in situ hybridization. Human samples were obtained from eight lower limb amputations because of acute or chronic critical ischemia. In chronically ischemic human skeletal muscle VEGF and VEGFR-2 expression was restricted to atrophic and regenerating skeletal myocytes, whereas in acutely ischemic limbs VEGF and VEGFR-2 were expressed diffusely in the affected muscle. Hypoxia-inducible factor-1alpha was associated with VEGF and VEGFR-2 expression both in acute and chronic ischemia but not in regeneration. Hindlimb ischemia was induced in 20 New Zealand White rabbits by excising the femoral artery. Magnetic resonance imaging and histological sections revealed extensive ischemic damage in the thigh and leg muscles of ischemic rabbit hindlimbs with VEGF expression similar to acute human lower limb ischemia. After 1 and 3 weeks of ischemia VEGF expression was restricted to regenerating myotubes and by 6 weeks regeneration and expression of VEGF was diminished. VEGFR-2 expression was co-localized with VEGF expression in regenerating myotubes. Macrophages and an increased number of capillaries were associated with areas of ischemic muscle expressing VEGF and VEGFR-2. In conclusion, two patterns of VEGF and VEGFR-2 expression in human and rabbit ischemic skeletal muscle are demonstrated. In acute skeletal muscle ischemia VEGF and VEGFR-2 are expressed diffusely in the affected muscle. In chronic skeletal muscle ischemia and in skeletal muscle recovering from ischemia VEGF and VEGFR-2 expression are restricted to atrophic and regenerating muscle cells suggesting the operation of an autocrine pathway that may promote survival and regeneration of myocytes.     

Transmural Replacement of Myocardium after Skeletal Myoblast Grafting into the Heart: Too Much of a Good Thing?

Skeletal myoblasts form stable grafts in the heart and may improve myocardial performance after infarction. The current study compared the ability of different immortalized myoblast lines, or primary myoblast isolates, to form grafts in the normal or cryoinjured heart. With a constant dose of 6 × 10⁶ cells, primary rat myoblasts and mouse C2C12 myoblasts formed similarly large skeletal muscle grafts in recipient rat hearts. Grafts often caused transmural replacement of the myocardium in normal hearts and almost completely replaced the damaged region in cryoinjured hearts. To test for possible artifacts due to immunosuppression and xenografting (mouse C2C12 cells into rat hearts), we grafted 1 × 10⁶ mouse MM14 or C2C12 myoblasts into normal hearts of nude mice. Again, the C2C12 grafts caused transmural replacement of the left ventricular wall and distorted the epi- and endocardial contours. The MM14 cells, however, formed relatively small grafts. The C2C12 grafts showed substantially higher BrdU incorporation rates at day 4 compared with MM14 cells, suggesting that ongoing proliferation was responsible for the increased graft size. None of the three skeletal muscle cell types expressed detectable amounts of the gap junction protein connexin43 after grafting. The intercellular adhesion protein N-cadherin was not expressed in primary skeletal muscle grafts, but was spotty or abundant in C2C12- and MM14-derived grafts, respectively. The absence of connexin43 precluded electrical coupling between graft and host muscle cells. Thus, when sufficient amounts of proliferation occur after grafting, skeletal muscle cells can effectively replace the volume of lost myocardium. Excess proliferation, however, can cause grafts to expand the ventricular wall and possibly impair pump function. Optimal cardiac repair strategies may need to incorporate methods to control graft cell proliferation.     

Genome-wide examination of myoblast cell cycle withdrawal during differentiation.

Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40% fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly-6A, whose expression specifically was up-regulated during cell cycle withdrawal coincident with early myoblast differentiation.     

Vascular endothelial growth factor-mediated autocrine stimulation of prostate tumor cells coincides with progression to a malignant phenotype

Vascular endothelial growth factor (VEGF), which is often produced at high levels by tumor cells, is a well-known mediator of tumor angiogenesis. VEGF receptor tyrosine kinases, KDR/Flk-1 and Flt-1, have been thought to be expressed exclusively by endothelial cells. In this study, we have used a prostate tumor progression series comprised of a differentiated rat prostate epithelial cell line, NbE-1, and its highly motile clonal derivative, FB2. Injection of NbE-1 cells into the inferior vena cava of syngeneic rats indicated that these cells are nontumorigenic. Using the same model, FB2 cells generated rapidly growing and well-vascularized tumors in the lungs. NbE-1 expressed marginal levels of VEGF, whereas high levels of VEGF protein were detected in FB2-conditioned medium and in FB2 tumors in vivo. Analysis of (125)I-VEGF(165) binding to NbE-1 and FB2 cells indicated that only motile FB2 cells expressed the VEGF receptor Flt-1. Consistent with this finding, physiological concentrations of VEGF induced chemotactic migration in FB2 but not in NbE-1 cells. This is the first documentation of a functional Flt-1 receptor in prostate tumor cells. Our results suggest two roles for VEGF in tumor progression: a paracrine role as an angiogenic factor and a previously undescribed role as an autocrine mediator of tumor cell motility.     

NDRG2, a novel regulator of myoblast proliferation, is regulated by anabolic and catabolic factors

Skeletal muscle tissue undergoes adaptive changes in response to stress and the genes that control these processes are incompletely characterised. NDRG2 (N-myc downstream-regulated gene 2), a stress- and growth-related gene, was investigated in skeletal muscle growth and adaption. While NDRG2 expression levels were found to be up-regulated in both differentiated human and mouse myotubes compared with undifferentiated myoblasts, the suppression of NDRG2 in C2C12 myoblasts resulted in slowed myoblast proliferation. The increased expression levels of the cell cycle inhibitors, p21 Waf1/Cip1 and p27 Kip1, and of various muscle differentiation markers in NDRG2-deficient myoblasts indicate that a lack of NDRG2 promoted cell cycle exiting and the onset of myogenesis. Furthermore, the analysis of NDRG2 regulation in C2C12 myotubes treated with catabolic and anabolic agents and in skeletal muscle from human subjects following resistance exercise training revealed NDRG2 gene expression to be down-regulated during hypertrophic conditions, and conversely, up-regulated during muscle atrophy. Together, these data demonstrate that NDRG2 expression is highly responsive to different stress conditions in skeletal muscle and suggest that the level of NDRG2 expression may be critical to myoblast growth and differentiation.     

大鼠骨骼肌卫星细胞的原代培养和鉴定

目的采用组织块培养技术探索大鼠骨骼肌卫星细胞的原代培养方法。方法以成年SPF级Sprague-Dawley大鼠为研究对象,采用组织块培养法获取大鼠骨骼肌卫星细胞,并与C2C12成肌细胞进行比较,对两种细胞进行形态学研究及采用免疫荧光和免疫组织化学法测定两种细胞α-actin蛋白和Desmin蛋白的表达及分布,从而对骨骼肌卫星细胞进行鉴定。结果通过组织块培养法获取的细胞增殖旺盛,分化良好。免疫细胞荧光和免疫组织化学实验结果显示,α-actin蛋白和Desmin蛋白在两种细胞胞浆中均有分布。结论用组织块培养法获取的骨骼肌卫星细胞具有良好的增殖与分化能力,用此种方法可培养出高纯度的骨骼肌卫星细胞。     

CC family chemokines directly regulate myoblast responses to skeletal muscle injury

Chemokines have been implicated in the promotion of leucocyte trafficking to diseased muscle. The purpose of this study was to determine whether a subset of inflammatory chemokines are able to directly drive myoblast proliferation, an essential early component of muscle regeneration, in a manner which is entirely independent of leucocytes. Cultured myoblasts (C2C12) were exposed to monocyte chemoattractant protein-1 (MCP-1; CCL2), macrophage inflammatory protein-1α(MIP-1α; CCL3) or MIP-1β (CCL4). All chemokines induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) and greatly increased myoblast proliferative responses. Chemokine-induced myoblast proliferation was abolished by pertussis toxin and the MEK1/2 inhibitor U0126, implicating both Gαi-coupled receptors and ERK1/2-dependent signalling. Myoblasts expressed receptors for all of the chemokines tested, and mitogenic responses were specifically inhibited by antibodies directed against CC family chemokine receptors 2 and 5 (CCR2 and CCR5). Within an in vitro myogenic wound healing assay devoid of leucocytes, all chemokines significantly accelerated the time course of myoblast wound closure after mechanical injury. Injections of MCP-1 into cardiotoxin-injured skeletal muscles in vivo also suppressed expression of the differentiation marker myogenin, consistent with a mitogenic effect. Taken together, our results indicate that CC chemokines have potent and direct effects on myoblast behaviour, thus indicating a novel role in muscle repair beyond leucocyte chemoattraction. Therefore, interventions aimed at modulating the balance between myoblast and leucocyte effects of CC chemokines in injured muscle could represent a novel strategy for the treatment of destructive muscle pathologies.     

Expression of vascular endothelial growth factor,placental growth factor,and their receptors Flt-1 and KDR in human placenta under pathologic conditions

The vascular endothelial growth factor (VEGF) family and its receptors have multifunctional activities besides angiogenesis, and some of these molecules are induced by hypoxia/ischemia. They are known to be expressed in human placenta, but little is known about their involvement in pathologic conditions. We have investigated the expression patterns of VEGF, placental growth factor (PlGF), and their receptors fms-like tyrosine kinase (Flt-1) and kinase insert domain-containing region (KDR) in placentas with histopathological changes. Forty-two placentas from normal and complicated pregnancies delivered in the second and third trimesters were fixed with paraformaldehyde and embedded in paraffin. In situ hybridization and immunohistochemistry were performed on serial sections. In the villi with characteristic hypoxic/ischemic changes (HIC), including increased syncytial knots, infarction, or hypercapillarization, intense immunostaining for VEGF was detected in the media of blood vessels, and increased staining for KDR was demonstrated in the endothelial cells. Strong PlGF immunoreactivity was localized to the degenerative trophoblasts around the infarctions. Marked Flt-1 mRNA expression in the syncytiotrophoblast layers of HIC villi was identified, but some samples did not show ligand expression in these regions. Positive immunostaining for VEGF, PlGF, and Flt-1 was observed in infiltrated neutrophils and macrophages in the placentas with chorioamnionitis (CAM). These findings suggested that in the hypoxic/ischemic regions, VEGF and KDR expression is increased within the villous vessels by paracrine regulation, whereas the expression of PlGF and Flt-1 is enhanced in villous trophoblasts by autocrine regulation. The Flt-1 gene may also be up-regulated directly by hypoxia/ischemia independently of ligand mediation. Furthermore, the results indicated that VEGF and PlGF stimulate inflammatory cell migration by autocrine regulation via the Flt-1 receptor in the CAM placenta. Thus, various functions of VEGF family members participate in the development of pathologic changes in the placenta.     

Flt-1、Flk-1在胶原诱导的关节炎小鼠关节组织中的表达

目的研究血管内皮生长因子(VEGF)受体F lt-1、F lk-1在Ⅱ型胶原诱导的关节炎(C IA)形成期的表达,探讨VEGF在类风湿性关节炎发病中的作用。方法于DBA/1 J小鼠皮下注射Ⅱ型胶原制作小鼠关节炎动物模型并进行关节指数评价,用ELISA法和免疫组织化学技术检测关节组织内VEGF及血管性假性血友病因子(vWF)含量,通过RT-PCR,Southern b lotting技术检测关节组织内VEGF及其特异性受体F lt-1、F lk-1mRNA表达。结果VEGF及vWF水平呈平行变化关系,均在关节炎发生后第四天达到最高水平,并与血管新生程度、关节炎严重程度呈正相关。关节组织内VEGF mRNA分别在279bp和304bp扩增片段有特异性表达、其特异性受体F lt-1、F lk-1 mRNA在377bp、402bp扩增片段有特异性表达。结论VEGF-F lt-F lk系统在关节炎形成早期起着重要作用,影响着实验诱导关节炎血管新生及病程。     

Expression of vascular endothelial growth factor and its receptors in the developing rat lung

We examined the regulation of the expression of vascular endothelial growth factor (VEGF) and its specific receptors, fetal liver kinase receptor (Flk-1), and fms-like tyrosine kinase receptor (Flt-1) during formation of the capillary network in the developing rat lung. An immunohistochemical study of lung tissue from 19- and 21-d-old fetuses and 1-, 3-, 5-, 7-, and 14-d-old animals revealed that the level of expression of both VEGF and Flk-1 is significantly higher before birth (p < 0.0001) than after. Increased expression of Flt-1 on the first day after birth (p < 0.0001) suggests that this receptor might play an important role in capillary growth in the perinatal period. Immunostaining also revealed the colocalization of VEGF, Flt-1, and Flk-1 in endothelial cells of the lung capillaries at the ultrastructural level. The present studies revealed that VEGF and its two receptors are upregulated during the development of capillaries in the fetal and newborn rat lung.     

TGF-�� isoforms inhibit IGF-1-induced migration and regulate terminal differentiation in a cell-specific manner

Following muscle injury, the damaged tissue and influx of inflammatory cells stimulate the secretion of growth factors and cytokines to initiate repair processes. This release of chemotactic signaling factors activates resident precursor cells and stimulates their mobilization and migration to the site of injury where terminal differentiation can occur. The three transforming growth factor-β (TGF-β) isoforms, and insulin-like growth factor-1 (IGF-1) are among the known regulatory factors released following muscle damage. We investigated the effect of recombinant active TGF-β1, -β2, -β3 and IGF-1 on C2C12 skeletal muscle satellite cell and P19 embryonal carcinoma cell terminal differentiation and migration. C2C12 myoblast fusion as well as P19 embryoid body formation and myogenic differentiation was assessed following 72?h TGF-β treatment (5?ng/ml), whereas the effect of the TGF-β isoforms on migration was determined following 7?h incubation. Our results showed that TGF-β decreases C2C12 myoblast fusion in an isoform-independent manner, whereas in the P19 cell lineage, results demonstrate that TGF-β1 specifically and significantly increased P19 embryoid body formation, but not expression of Connexin-43 or Myosin Heavy Chain. IGF-1 significantly increased migration compared to TGF-β isoforms, which, on their own, had no significant effect on the mobilization of either C2C12 or P19 cells. TGF-β isoforms decreased IGF-1-induced migration of both cell lineages. By distinguishing the factors involved in, and the molecular signals required for, myoblast recruitment during repair processes, strategies can be developed towards improved cell-mediated therapies for muscle injury.     

Therapeutic angiogenesis by a myoblast layer harvested by tissue transfer printing from cell-adhesive,thermosensitive hydrogels

Peripheral arterial disease (PAD) is characterized by the altered structure and function of arteries caused by accumulated plaque. There have been many studies on treating this disease by the direct injection of various types of therapeutic cells, however, the low cell engraftment efficiency and diffusion of the transplanted cells have been major problems. In this study, we developed an approach (transfer printing) to deliver monolayer of cells to the hindlimb ischemic tissue using thermosensitive hydrogels, and investigated its efficacy in long term retention upon transplantation and therapeutic angiogenesis. We first investigated the in vitro maintenance of robust cell–cell contacts and stable expression of the ECM proteins in myoblast layer following transfer printing process. In order to confirm the therapeutic effect of the myoblasts in vivo, we cultured a monolayer of C2C12 myoblasts on thermosensitive hydrogels, which was then transferred to the hindlimb ischemia tissue of athymic mice directly from the hydrogel by conformal contact. The transferred myoblast layer was retained for a longer period of time than an intramuscularly injected cell suspension. In addition, the morphology of the mice and laser Doppler perfusion (28 days after treatment) supported that the myoblast layer enhanced the therapeutic effects on the ischemic tissue. In summary, the transplantation of the C2C12 myoblast layer using a tissue transfer printing method could represent a new approach for the treatment of PAD by therapeutic angiogenesis.     

Flk-1+ adipose-derived mesenchymal stem cells differentiate into skeletal muscle satellite cells and ameliorate muscular dystrophy in mdx mice

Duchenne muscular dystrophy (DMD) is a severe hereditary disease characterized by the absence of dystrophin on the sarcolemma of muscle fiber. This absence results in widespread muscle damage and satellite cell activation. After depletion of the satellite cell pool, skeletal muscle is then invariably replaced by connective tissue, leading to progressive muscle weakness. Herein, we isolated Flk-1(+) mesenchymal stem cells (MSCs) from adult adipose tissue and induced them to differentiate into skeletal muscle cells in culture. Within mdx mice, an animal model of DMD, adipose tissue-derived Flk-1(+) MSCs (AD-MSCs) homed to and differentiated into cells that repaired injured muscle tissue. This repair correlated with reconstitution of dystrophin expression on the damaged fibers. Flk-1(+) AD-MSCs also differentiated into muscle satellite cells. This differentiation may have accounted for long-term reconstitution. These cells also differentiated into endothelial cells, thereby possibly improving fiber regeneration as a result of the induced angiogenesis. Therefore, Flk-1(+) AD-MSC transplants may repair muscular dystrophy.     

Expression of growth factors,growth inhibiting factors,and their receptors in invasive breast cancer. I: An inventory in search of autocrine and paracrine loops

The aim of the present study was to investigate which growth factors, receptors, and growth inhibiting factors are expressed in invasive breast cancer. Five (angiogenic) growth factors and their receptors: platelet-derived growth factor A chain (PDGF-AA) and PDGF receptor alpha (PDGFαR), PDGF-BB and PDGF beta receptor, transforming growth factor alpha (TGFα) and its receptor epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF) and its receptors vascular endothelial growth factor receptor I (Flt-1) and vascular endothelial growth factor receptor II (Flk-1/KDR); two growth inhibiting factors: transforming growth factor beta-1 (TGFβ1) and TGFβ2) and their receptor couple transforming growth factor beta receptor I (TGFβR-I) and TGFβR-II; and basic fibroblast growth factor (bFGF) were stained by standard immunohistochemistry on frozen sections in 45 cases of invasive carcinoma of the breast. Staining was scored as negative or positive in tumour epithelium, stroma, and blood vessels. TGFβ1 and TGFβ2 were expressed in the tumour cells in 67 per cent and 76 per cent of cases, respectively, whereas PDGFβR and TGFβR-II were expressed in 0 per cent and 2 per cent, respectively. The other factors showed variable expression in tumour cells. All factors were expressed in the stroma in most cases, except Flt-1, Flk-1/KDR, TGFβ2, and TGFβR-II, which showed variable expression, and EGFR, which showed no expression. The endothelium was in most cases positive for bFGF, PDGF-AA, PDGF-BB, VEGF, PDGFαR, PDGFβR, and TGFβ1 but TGFβ2 was negative in most cases and TGFα, EGFR, Flt-1, Flk-1/KDR, TGFβR-I, and TGFβR-II were variably expressed. The most interesting possible auto/paracrine loops, as demonstrated on serial sections and by fluorescence double staining, were the TGFα/EGFR, TGFβs/TGFβR, VEGF/Flt-1, and the VEGF/Flk-1 combinations. In conclusion, growth factors, growth inhibiting factors, and their receptors are frequently expressed in invasive breast cancer. Indications for some possible auto-and paracrine loops have been found, which should encourage further study on the role of these factors in breast cancer proliferation and angiogenesis. © 1998 John Wiley & Sons, Ltd.     

Control of attachment, morphology, and proliferation of skeletal myoblasts on silanized glass.

Generating skeletal muscle in vitro is an attractive approach to overcome problems associated with autologous transfer of muscle and donor site morbidity during plastic surgery. Such tissue engineering requires application of biomaterials that selectively control the attachment, morphology, and proliferation of muscle progenitor ("satellite") cells. This study examined the initial attachment, morphological characteristics, and proliferative behavior of murine C2C12 myoblasts on glass substrata derivatized with self-assembled monolayers (SAMs) of the organosiloxanes N-(2-aminoethyl)(3-aminopropyl)trimethoxysilane (EDA) and tridecafluoro-1,1,2,2-tetrahydrooctyl-1-dimethylchlorosil ane (13F). The fraction of myoblasts resisting detachment upon rinsing was greater on EDA than on 13F. Application of a quantitative moments-based analysis of cell morphology demonstrated that projected area and two size-invariant metrics of shape (extension and dispersion) for these cells were greater for EDA than for 13F. Myoblasts also proliferated faster on EDA than on 13F. These data indicate that EDA-derivatized glass provides a superior substratum for myoblast culture compared to 13F-derivatized glass. Understanding myoblast behavior on these biomaterials that promotes contrasting cellular responses is the first step toward using patterned SAMs to control myotube alignment for tissue engineering skeletal muscle.     

VEGF高表达的胶质瘤细胞C6对共培养微血管内皮细胞表达Flk-1及Flt-1的影响

目的:以正常鼠肝细胞系BRL 3A为对照,研究VEGF高表达的恶性胶质瘤细胞系C6对体外共培养的肺微血管内皮细胞表达Flt-1、Flk-1的影响。方法:建立体外C6,BRL 3A与肺微血管内皮细胞的共培养方法,利用免疫细胞化学方法检测共培养后的微血管内皮细胞上VEGF受体Flt-1、Flk-1蛋白的表达变化,进一步采用RT-PCR和Northernblot分析Flt-1、Flk-1mRNA表达的改变。结果:与胶质瘤细胞C6共培养的微血管内皮细胞Flk-1、Flt-1蛋白表达增加 (P <0.05),而与BRL 3A共培养的内皮细胞Flk-1、Flt-1蛋白表达下降 (P <0.01),RT-PCR和Northernblot检测发现与C6共培养后可上调微血管内皮细胞Flk-1、Flt-1mRNA的表达 (P <0.01),而与BRL 3A共培养下调了Flk-1、Flt-1mRNA的表达 (P <0.01)。结论:VEGF高表达的胶质瘤细胞C6对共培养的微血管内皮细胞表达Flk-1、Flt-1有明显的上调作用,这可能是体内胶质瘤血管新生的重要机制之一.