骨形成蛋白对骨骼肌卫星细胞增殖与胶原蛋白合成的影响

目的　探讨骨形成蛋白 (BMP)对骨骼肌卫星细胞增殖与胶原蛋白合成的影响。　方法　体外获取与培养 Wistar大鼠骨骼肌卫星细胞 ,分别用含 BMP浓度为 0、50、1 0 0、50 0和 1 0 0 0 ng/ml的诱导培养基培养 72小时。通过 MTT法测定细胞的增殖 ,光镜观察细胞融合率 ,3 H-脯氨酸掺入法测定细胞胶原蛋白合成量。　结果　 BMP可促进骨骼肌卫星细胞的增殖 ,降低其细胞融合率 ,同时增加胶原蛋白的合成量。这种作用在 BMP浓度为 50 0 ng/ml即可表现出来 ,并随着浓度的增加越明显。　结论　 BMP可促进骨骼肌卫星细胞的增殖 ,抑制成肌表型促进向成骨细胞分化     

Laser skin rejuvenation: epidermal changes and collagen remodeling evaluated by in vivo confocal microscopy

Fractionated carbon dioxide (CO₂) laser resurfacing is an effective treatment of skin aging. Several studies investigated the morphologic changes due to this laser treatment by using skin biopsies or animal model. Recently, reflectance confocal microscopy (RCM) has emerged as a new tool that can “optically” scan the skin in vivo with a nearly histologic resolution and in a totally noninvasive modality. Our study aims to analyze the skin changes following the ablative fractional CO₂ laser sessions by using RCM. Ten patients were subjected to ablative fractional CO₂ laser sessions for skin aging. Confocal microscopic images were acquired at baseline (w0), 3 weeks (w3), 6 weeks (w6), and 12 weeks (w12) after laser session. Previously identified confocal parameters were used to assess the skin aging at baseline and after treatment. At w3, the epidermis showed a complete disappearance of the mottled pigmentation upon RCM along with the presence of few Langherans’ cells. The collagen type as seen upon RCM observed at baseline was replaced by a newly formed collagen type of long, bright and straight fibers (collagen remodeling). These fibers were parallel arranged and observed throughout the entire RCM mosaic. At w6 and w12 the confocal aspects of the skin was unchanged compared to w3. RCM confirmed the presence of an intense collagen remodeling following laser resurfacing. In line with previous studies, this collagen showed a peculiar arrangement and distribution. The collagen remodeling was still present after 3 months and confirms the long-term effect of the treatment. This is the first time that the skin can be analyzed in vivo at patient’s bedside. In the near future, RCM can be an essential adjunct for Clinicians to measure the effects of laser treatment and possibly to gain new insights into the development of side effects.     

Selection of cell source for ligament tissue engineering

Use of appropriate types of cells could potentially improve the functionality and structure of tissue engineered constructs, but little is known about the optimal cell source for ligament tissue engineering. The object of this study was to determine the optimal cell source for anterior cruciate ligament (ACL) tissue engineering. Fibroblasts isolated from anterior cruciate ligament, medial collateral ligament (MCL), as well as bone marrow mesenchymal stem cells (MSC) were compared using the following parameters: proliferation rate, collagen excretion, expression of collagen type I, II, and III, as well as alpha-smooth muscle actin. Green fluorescent protein (GFP) transfected MSCs were used to trace their fate in the knee joints. MSC, ACL, and MCL fibroblasts were all highly stained with antibodies for collagen types I and III and alpha-smooth muscle actin while negatively stained with collagen type II. Proliferation rate and collagen excretion of MSCs were higher than ACL and MCL fibroblasts (p < 0.05), and MSCs could survive for at least 6 weeks in knee joints. In summary, MSC is potentially a better cell source than ACL and MCL fibroblasts for anterior cruciate ligament tissue engineering.     

Focused In Vivo Genetic Analysis of Implanted Engineered Myofascial Constructs

Successfully engineering functional muscle tissue either in vitro or in vivo to treat muscle defects rather than using the host muscle transfer would be revolutionary. Tissue engineering is on the cutting edge of biomedical research, bridging a gap between the clinic and the bench top. A new focus on skeletal muscle tissue engineering has led investigators to explore the application of satellite cells (autologous muscle precursor cells) as a vehicle for engineering tissues either in vitro or in vivo. However, few skeletal muscle tissue-engineering studies have reported on successful generation of living tissue substitutes for functional skeletal muscle replacement. Our model system combines a novel aligned collagen tube and autologous skeletal muscle satellite cells to create an engineered tissue repair for a surgically created ventral hernia as previously reported [SA Fann, L Terracio, W Yan, et al., A model of tissue-engineered ventral hernia repair, J Invest Surg. 2006;19(3):193–205]. Several key features we specifically observe are the significant persistence of transplanted skeletal muscle cell mass within the engineered repair, the integration of new tissue with adjacent native muscle, and the presence of significant neovascularization. In this study, we report on our experience investigating the genetic signals important to the integration of neoskeletal muscle tissue. The knowledge gained from our model system applies to the repair of severely injured extremities, maxillofacial reconstructions, and restorative procedures following tumor excision in other areas of the body.     

Ureteral Cell Cultures II: Collagen Production and Response to Pharmacologic Agents

Purpose

To investigate the in vitro response of ureteral cells to potentially anti-fibrotic agents.

Materials and Methods

Cultured human uroepithelial cells, smooth muscle cells, and myofibroblasts were assayed for proliferation and production of collagen types I and III, with and without the presence of hydrocortisone, colchicine, retinol, verapamil, and D-penicillamine.

Results

Hydrocortisone stimulated the proliferation of all three cell types and reduced the type I and type III collagen production by myofibroblasts and smooth muscle cells, respectively. Verapamil enhanced the growth of uroepithelial cells and decreased collagen III production by both uroepithelial and smooth muscle cells. D-penicillamine increased the proliferation of uroepithelial and smooth muscle cells, and inhibited collagen type III production by all three cell types.

Conclusions

In vitro evidence suggests that hydrocortisone, verapamil, and D-penicillamine have effects that could favorably alter the healing of endoscopic ureteral incisions.     

Intracardiac transplantation of skeletal myoblasts yields two populations of striated cells in situ

BACKGROUND: Adult heart lacks stem cells and cannot effectively regenerate. In contrast, skeletal muscle is constantly undergoing repair. We proposed to transplant immature skeletal myoblasts into injured myocardium. METHODS: Approximately 7x10(6) soleus skeletal myoblasts were expanded in vitro from adult New Zealand White rabbits (n = 23) whose posterior left ventricle was cryoinjured to create a transmural lesion. Autologous myoblasts (n = 18) or saline (n = 5) was transplanted into the central cryolesion at the time of injury (n = 6) or 1 week later (n = 12). Hearts were harvested 2 weeks after injection. RESULTS: Myoblast transfer did not incur further morbidity. After cryolesion, grossly, a 1.6-cm epicardial hemorrhagic lesion could be seen. Histologically, the transmural lesion contained inflammatory cells and active scarring but no viable cardiomyocytes. Electron microscopy demonstrated a predominance of collagen and fibroblasts. Nine hearts contained multinucleated cells within the cryolesion that covered approximately 75% of the central cryolesion in 17% of animals. Immunohistochemical analysis confirmed their skeletal muscle origin. At the periphery of the lesion, isolated clusters of nonskeletal muscle cells could be visualized (n = 12) that resembled immature cardiocytes. CONCLUSIONS: Autologous skeletal myoblasts can regenerate viable striated tissue within damaged myocardium. Myoblast transfer warrants further investigation as a new method for improving myocardial performance within infarcted myocardium.     

Transplanted fetal cardiomyocytes as cardiac pacemaker.

BACKGROUND: While morphologic integration of transplanted fetal cardiomyocytes into the ventricular myocardium is a well-known fact, no studies have yet shown transplanted cells to coherently contribute to contraction and electrical excitation of the host myocardium. The aim of this study was to prove the hypothesis that by transplanting cardiomyocytes with a higher intrinsic rhythmic rate into the myocardium of the left ventricle, these cells could act as an ectopic pacemaker by functional coupling with host cardiomyocytes. METHODS AND RESULTS: Dissociated fetal canine atrial cardiomyocytes including sinus nodal cells were delivered into the free wall of the left ventricle of adult canine X-linked muscular dystrophy dogs (n=2). These dogs fail to express Dystrophin in both cardiac and skeletal muscle. In the control group (n=2) fetal skin fibroblasts were used for grafting. A total of 3-4 weeks after transplantation the dogs underwent catheter ablation of the atrioventricular node (AV-node) and subsequent electrophysiological mapping studies. Transplanted cells were identified by Dystrophin immunoreactivity, indicating survival and morphological integration in the recipient heart. The expression of Connexin 43 between donor and recipient cells suggested formation of gap junctions between injected and host cardiomyocytes. After catheter ablation of the AV-node, a ventricular escape rhythm emerged driving the pace of the heart and originating from the labeled transplantation site. This effect could not be observed in the control group (n=2). CONCLUSIONS: The results constitute the first observation of phenomena indicating electrical and mechanical coupling between allogeneic donor cardiomyocytes and recipient myocardium in-vivo. Further experiments are necessary to evaluate the technique as a potential therapy for atrioventricular block.     

Construction of a bioengineered cardiac graft

OBJECTIVES: Currently available graft materials for repair of congenital heart defects cause significant morbidity and mortality because of their lack of growth potential. An autologous cell-seeded graft may improve patient outcomes. We report our initial experience with the construction of a biodegradable graft seeded with cultured rat or human cells and identify their 3-dimensional growth characteristics. METHODS: Fetal rat ventricular cardiomyocytes, stomach smooth muscle cells, skin fibroblasts, and adult human atrial and ventricular cardiomyocytes were isolated and cultured in vitro. These cells were injected into or laid onto biodegradable gelatin meshes, and their rate of proliferation and spatial location within the mesh was evaluated by using a cell counter and histologic analysis. RESULTS: Rat cardiomyocytes, smooth muscle cells, and fibroblasts demonstrated steady proliferation over 3 to 4 weeks. The gelatin mesh was slowly degraded, but this process was most rapid after seeding with fibroblasts. Human atrial cardiomyocytes proliferated within the gelatin meshes but at a slower rate than that of fetal rat cardiomyocytes. Human ventricular cardiomyocytes survived within the gelatin mesh matrix but did not increase in number during the 2-week duration of evaluation. Grafts seeded with rat ventricular cells exhibited spontaneous rhythmic contractility. All cell types preferentially migrated to the uppermost surface of each graft and formed a 300- to 500-microm thick layer. CONCLUSIONS: Fetal rat ventricular cardiomyocytes, gastric smooth muscle cells, skin fibroblasts, and adult human atrial cardiomyocytes can grow in a 3-dimensional pattern within a biodegradable gelatin mesh. Similar autologous cell-seeded constructs may eventually be applied to repair congenital heart defects.     

氨哮素对失神经骨肌内胶原代谢的影响

为探讨氨哮素对人体失神经骨骨各肌内胶原代谢的影响，采用安慰剂对照的随机双盲研究法，对７１例因臂丛神经损伤而致肌皮神经功能完全丧失的患者，给予氨哮素或安慰剂６０μｇ，每天２次，治疗３个月。实验前、后均行肱二头肌活检，活检肌肉作Ⅰ型、Ⅳ型胶原的免疫组织化学染色和图像分析。结果表明，氨哮素治疗组肌内膜中Ⅰ型胶原明显低于安慰剂组，Ⅳ型胶原纤维的增生明显少于安慰剂组（Ｐ＜０．０５）。证明，氨哮素能抑制失神经骨骨各肌内胶原的增生     

A model of tissue-engineered ventral hernia repair.

We have developed a tissue-engineered ventral hernia repair system using our novel aligned collagen tube and autologous skeletal muscle satellite cells. In this model system, skeletal muscle satellite cells were isolated from a biopsy, expanded in culture, and incorporated into our collagen tube scaffold, forming the tissue-engineered construct. We characterized the results of the repaired hernias on both the gross and microscopic scales and compared them to an unrepaired control, an autologous muscle repair control, and a collagen-tube-only repair. Untreated animals developed a classic hernia sac, devoid of abdominal muscle and covered only with a thin layer of mesothelial tissue. Significant muscle, small-diameter blood vessels, and connective tissue were apparent in both the autologous control and the engineered muscle repairs. The engineered muscle repairs became cellularized, vascularized, and integrated with the native tissue, hence becoming a "living" repair. A tissue-engineered construct repair of ventral hernias with subsequent incorporation and vascularization could provide the ultimate in anterior wall myofascial defect repair and would further the understanding of striated muscle engineering. The knowledge gained from our model system would have immediate application to mangled extremities, maxillofacial reconstructions, and restorative procedures following tumor excision in other areas of the body.     

Regenerative potential of mesodermal stem cells for the heart

There has been increasing interest in recent years in the phenomenon of "regeneration," especially in the function of the bone marrow stromal cell system in the support of hematopoiesis. The stromal cell system has been proposed to consist of mesodermal stem cells that are capable of self-renewal and differentiation into a variety of mesodermal tissues, including bone, cartilage, tendon, fat, endothelium, skeletal muscle, and cardiomyocytes. These findings raise the possibility that bone marrow-derived cells may provide an alternative source of cardiomyocytes in patients with severe cardiac failure due to loss of muscle cells. Some studies have indicated that locally or systemically delivered mesodermal stem cells can generate de novo cardiomyocytes. Despite their potential clinical utility for cellular and gene therapy, the mechanism of differentiation in mesodermal stem cells and characterization of stem cells in terms of surface antigen expression remain to be resolved. Although some clinical trials have been initiated using crude bone marrow-derived stromal cells, we need more knowledge of stem cells to establish a standard protocol for cellular therapy.     

The SNARE Protein SNAP23 and the SNARE-Interacting Protein Munc18c in Human Skeletal Muscle Are Implicated in Insulin Resistance/Type 2 Diabetes

OBJECTIVE

Our previous studies suggest that the SNARE protein synaptosomal-associated protein of 23 kDa (SNAP23) is involved in the link between increased lipid levels and insulin resistance in cardiomyocytes. The objective was to determine whether SNAP23 may also be involved in the known association between lipid accumulation in skeletal muscle and insulin resistance/type 2 diabetes in humans, as well as to identify a potential regulator of SNAP23.

RESEARCH DESIGN AND METHODS

We analyzed skeletal muscle biopsies from patients with type 2 diabetes and healthy, insulin-sensitive control subjects for expression (mRNA and protein) and intracellular localization (subcellular fractionation and immunohistochemistry) of SNAP23, and for expression of proteins known to interact with SNARE proteins. Insulin resistance was determined by a euglycemic hyperinsulinemic clamp. Potential mechanisms for regulation of SNAP23 were also investigated in the skeletal muscle cell line L6.

RESULTS

We showed increased SNAP23 levels in skeletal muscle from patients with type 2 diabetes compared with that from lean control subjects. Moreover, SNAP23 was redistributed from the plasma membrane to the microsomal/cytosolic compartment in the patients with the type 2 diabetes. Expression of the SNARE-interacting protein Munc18c was higher in skeletal muscle from patients with type 2 diabetes. Studies in L6 cells showed that Munc18c promoted the expression of SNAP23.

CONCLUSIONS

We have translated our previous in vitro results into humans by showing that there is a change in the distribution of SNAP23 to the interior of the cell in skeletal muscle from patients with type 2 diabetes. We also showed that Munc18c is a potential regulator of SNAP23.Insulin resistance plays a major role in the development of type 2 diabetes and is highly related to the accumulation of triglycerides in skeletal muscle (1,2). Triglycerides are stored in the cell in cytosolic lipid droplets, which consist of a core of neutral lipids surrounded by a monolayer of amphipathic lipids (3,4). It is now recognized that lipid droplets are dynamic organelles with a complex surface that contains a number of different proteins, including the structural PAT proteins (5), lipid metabolic enzymes, and proteins involved in processing and sorting of the droplets (6,7).Lipid droplets are formed as primordial droplets and increase in size by a fusion process that requires the SNARE proteins synaptosomal-associated protein of 23 kDa (SNAP23), syntaxin-5, and vesicle-associated membrane protein four (VAMP4) (8). SNAP23 is also required for the insulin-stimulated translocation of GLUT4 to the plasma membrane (9,10), and we previously demonstrated that SNAP23 may play a role in the development of insulin resistance (8). Specifically, we showed that accumulation of lipid droplets in cardiomyocytes after fatty acid treatment results in a redistribution of SNAP23 to the interior of the cell, which coincides with the development of cellular insulin resistance (8). However, this treatment does not affect the total amount of SNAP23 (8). We also showed that the fatty acid–induced increase in SNAP23 in the interior of cardiomyocytes is at least partly explained by increased levels of SNAP23 on lipid droplets (8). However, the major amount of immunoreactive SNAP23 is spread diffusely in the interior of the cell (8), and the mechanism behind the redistribution has not been clarified.Here, we tested if our in vitro observations in fatty acid–treated cardiomyocytes (8) could be extrapolated to the situation in vivo by comparing skeletal muscle biopsies from patients with type 2 diabetes and matched lean and obese control subjects. We showed that skeletal muscle from patients with type 2 diabetes (and thus insulin resistance) had increased lipid accumulation and redistribution of SNAP23 to the microsomal/cytosolic fraction, observations that were comparable with our findings in fatty acid–treated cardiomyocytes (8). However, contrary to the observation in cardiomyocytes, there was an increase in total SNAP23 protein in the skeletal muscle biopsies from patients with type 2 diabetes. We also found that the SNARE-regulating protein Munc18c was increased in skeletal muscle biopsies from patients with type 2 diabetes and participates in the regulation of SNAP23 expression.     

From dynamic to cellular cardiomyoplasty

Dynamic Cardiomyoplasty. Latissimus dorsi dynamic cardiomyoplasty has been used in our institution for heart failure patients refractory to medical therapy; 113 cases were operated at Broussais and Pompidou Hospitals and 75 patients by our team abroad, in the scope of an international cooperative program. Cardiomyoplasty has been associated with better results due to technical improvements, the most significant mini-invasive techniques, the latest the use of growth factors to enhance muscle vascularization. Risk factors have been identified, resulting in more precise indications, a lower hospital mortality, and a wider use of this operation. There has been a new tendency to associate cardiomyoplasty with electrophysiological therapies: implantation of ventricular defibrillators and multisite cardiac pacing (for atrioventricular and interventricular resynchronization). Cellular Cardiomyoplasty. Adult myocardium cannot repair after infarction due to the absence of stem cells. Cell transplantation strategies for heart failure have been designed to replace damaged cells with cells that can perform cardiac work. Current possibilities in cell therapy for heart failure is the transplantation into the infarcted myocardium of autologous myoblasts (satellite cells originated from skeletal muscle), fetal cardiomyocytes, autologous heart cells, cells derived from bone marrow stem cells, and smooth muscle cells. Experimental studies demonstrated that cell transplantation into the myocardium was associated with the recovery of myocardial contractility and compliance, as well as the diastolic pressure-strain relationship in animal models (infarctlike myocardial lesions and dilated cardiomyopathy models). Healthy myoblasts and myotubes were observed 2 months after myocardial implantation. Clinical studies are now in progress.     

骨骼肌无细胞基质的制备及其生物相容性研究

目的细胞外基质是脂肪组织工程材料的研究热点之一。通过探讨骨骼肌无细胞基质的制作方法及生物相容性,为其在脂肪组织工程中的应用奠定基础。方法取健康成年小香猪新鲜骨骼肌组织,横切成厚2～3 mm的组织块,采用低渗-去垢剂法脱细胞处理。处理后采用HE染色、Masson三色染色、免疫组织化学染色及扫描电镜检测骨骼肌无细胞基质是否有细胞成分残留,并观察其基本结构;应用MTT法检测骨骼肌无细胞基质细胞毒性。取乳腺癌患者自愿捐赠脂肪组织,分离培养人脂肪干细胞(human adipose-derived stem cells,hADSCs),从形态学、流式细胞学和成脂、成骨分化能力方面进行鉴定。将骨骼肌无细胞基质与第3代hADSCs共培养,于培养后第1、3、5、7天通过细胞活性检测材料上细胞黏附、扩散和增殖情况,了解其与细胞之间的相互作用。结果 HE、Masson、免疫组织化学染色及扫描电镜观察显示骨骼肌无细胞基质肌纤维去除完全,无细胞核残留,基质结构保留完整;大量连接成网状的胶原纤维呈多孔隙样结构,规则排列。MTT检测示骨骼肌无细胞基质细胞毒性为1级,细胞相容性好。细胞活性检测示hADSCs在骨骼肌无细胞基质上能很好地伸展,且能与周围基质黏附,进入基质内部并相互交织。结论经脱细胞处理的骨骼肌无细胞基质具有良好生物相容性,可能作为脂肪组织工程的支架材料。     

Differential effects of nicotine and smoke condensate on bone cell metabolic activity

OBJECTIVE: Delayed or impaired healing of skeletal trauma in patients who smoke has been attributed to vascular responses of nicotine absorption and/or a direct effect of nicotine or other smoke components on bone cells. In vivo studies indicate variability in osteosynthetic response to nicotine versus smoke inhalation. We tested the hypothesis that components of cigarette smoke other than nicotine may be responsible for the adverse skeletal effects of smoking. DESIGN: In vitro cultures of MC3T3-E1 osteoblastlike cells were exposed to varying doses of nicotine or condensates of cigarette smoke. Metabolic assays included alkaline phosphatase activity, collagen synthesis, and total protein synthesis as well as cell proliferation. RESULTS: Variations in the degree of response were noted between bone cell preparations. Nicotine elicited a significant dose-dependent stimulation of bone cell metabolism in all studies. This was detected as increases in alkaline phosphatase activity and increases in total protein and collagen synthesis. Responses were noted with nicotine doses as low as 12.5 ng/mL (half the nicotine level circulating in smokers). In one study, maximum stimulation occurred at 250 ng/mL with levels reaching 74% (total protein) and 104% (collagen) greater than control cultures. In a second study, 222% and 627% stimulation of protein and collagen synthesis over controls was noted using 100 ng/mL. Addition of the nicotine receptor antagonist mecamylamine reduced the nicotine stimulation. Preparations of smoke condensate with equivalent nicotine concentrations reduced all indices of metabolic activity. Cell proliferation was stimulated by both nicotine (20-25%) and smoke condensate (38-46%). CONCLUSION: The data suggest that nicotine acts as a direct stimulant of bone cell metabolic activity. Smoke condensate containing equivalent levels of nicotine elicits an inhibitory effect. A probable speculation is that the delay in clinical healing of skeletal trauma in smoking patients may in part be a result of absorption of components of smoke other than nicotine.     

Vascular endothelial growth factor transgene expression in cell-transplanted hearts

OBJECTIVE: We evaluated the effect of transplanted cell type, time, and region of the heart on transgene expression to determine the potential of combined gene and cell delivery for myocardial repair. METHODS: Lewis rats underwent myocardial cryoinjury 3 weeks before transplantation with heart cells (a mixed culture of cardiomyocytes, smooth muscle cells, endothelial cells and fibroblasts, n = 13), vascular endothelial growth factor-transfected heart cells (n = 13), skeletal myoblasts (n = 13), vascular endothelial growth factor-transfected skeletal myoblasts (n = 13), or medium (control, n = 12). Vascular endothelial growth factor expression in the scar, border zone, and normal myocardium was evaluated at 3 days and at 1, 2, and 4 weeks by means of quantitative polymerase chain reaction. Transplanted cells and vascular endothelial growth factor protein were identified immunohistologically on myocardial sections. RESULTS: Vascular endothelial growth factor levels were very low in control scars but increased transiently after medium injection. Transplantation with heart cells and skeletal myoblasts significantly increased vascular endothelial growth factor expression in the scar and border zone. Transplantation of vascular endothelial growth factor-transfected heart cells and vascular endothelial growth factor-transfected skeletal myoblasts further augmented vascular endothelial growth factor expression, resulting in 4- to 5-fold greater expression of vascular endothelial growth factor in the scar at 1 week. Peak vascular endothelial growth factor expression was greater and earlier in vascular endothelial growth factor-transfected heart cells than in vascular endothelial growth factor-transfected skeletal myoblasts. Vascular endothelial growth factor was primarily expressed by the transplanted cells. Some of the transplanted heart cells and vascular endothelial growth factor-transfected heart cells were identified in the endothelial layer of blood vessels in the scar. CONCLUSIONS: Transplantation of heart cells and skeletal myoblasts induces vascular endothelial growth factor expression in myocardial scars and is greatly augmented by prior transfection with a vascular endothelial growth factor transgene. Vascular endothelial growth factor expression is limited to the scar and border zone for 4 weeks. Both heart cells and skeletal myoblasts may be excellent delivery vehicles for cell-based myocardial gene therapy.     

自噬在骨骼肌质量维持中的作用

作为人体重要的运动和能量代谢器官,骨骼肌质量的正常维持对于机体发挥正常的生理功能至关重要。自噬-溶酶体(autophagy-lysosome,AL)途径是一种在正常和病态细胞中普遍存在的生理或病理机制,对于维持细胞内蛋白质平衡,清除细胞内受损的细胞器,及维持内环境稳定起到关键作用。自噬过程的顺利进行需要经历多个步骤,在多重因子的协调作用下完成。自噬通过清除受损的肌原纤维和隔离的胞浆蛋白等细胞成分来维持健康机体的肌肉内稳态。自噬还可以提供细胞增殖所需的初始能量,促进损伤后肌肉的再生和重塑。同时,自噬失调也是导致年龄相关性骨骼肌萎缩的一个重要诱因。自噬可以影响骨骼肌对运动的反应,增加基础自噬水平有利于提高骨骼肌对运动的适应性。本文将自噬在骨骼肌质量维持中的作用与通路进行总结,以便为临床预防和治疗肌萎缩提供有效的康复策略。     

The effect of a cyclic uniaxial strain on urinary bladder cells

Purpose

Pre-conditioning of a cell seeded construct may improve the functional outcome of a tissue engineered construct for augmentation cystoplasty. The precise effects of mechanical stimulation on urinary bladder cells in vitro are not clear. In this study we investigate the effect of a cyclic uniaxial strain culture on urinary bladder cells which were seeded on a type I collagen scaffold.

Methods

Isolated porcine smooth muscle cells or urothelial cells were seeded on a type I collagen scaffolds and cultured under static and dynamic conditions. A uniform cyclic uniaxial strain was applied to the seeded scaffold using a Bose Electroforce Bio-Dynamic bioreactor. Cell proliferation rate and phenotype were investigated, including SEM analysis, RT-PCR and immunohistochemistry for α-Smooth muscle actin, calponin-1, desmin and RCK103 expression to determine the effects of mechanical stimulation on both cell types.

Results

Dynamic stimulation of smooth muscle cell seeded constructs resulted in cell alignment and enhanced proliferation rate. Additionally, expression of α-Smooth muscle actin and calponin-1 was increased suggesting differentiation of smooth muscle cells to a more mature phenotype.

Conclusions

Mechanical stimuli did not enhance the proliferation and differentiation of urothelial cells. Mechanical stimulation, i.e., preconditioning may improve the functional in vivo outcome of smooth muscle cell seeded constructs for flexible organs such as the bladder.

     

连续传代人胚骨骼肌成肌细胞生物学特性研究

目的 探讨成肌细胞连续传代能力,选择适宜肌组织工程研究的成肌细胞。方法 常规传代培养人胚骨骼肌细胞,以生长曲线、融合率分别观察细胞增列、分化能力,探讨成纤维细胞沾染对传代细胞的影响。结果 第６代以内细胞成纤维细胞沾染轻,主要表现出成肌细胞的生长特性,增殖较旺盛,分化能力高。第８代￣第１６代细胞成纤维细胞沾染重,表现出成纤维细胞的生长特性,增殖速度明显加快但分化能力低。第２０代细胞退变明显,细胞增殖     

MT1‐MMP and Type II Collagen Specify Skeletal Stem Cells and Their Bone and Cartilage Progeny

Skeletal formation is dependent on timely recruitment of skeletal stem cells and their ensuing synthesis and remodeling of the major fibrillar collagens, type I collagen and type II collagen, in bone and cartilage tissues during development and postnatal growth. Loss of the major collagenolytic activity associated with the membrane‐type 1 matrix metalloproteinase (MT1‐MMP) results in disrupted skeletal development and growth in both cartilage and bone, where MT1‐MMP is required for pericellular collagen dissolution. We show here that reconstitution of MT1‐MMP activity in the type II collagen‐expressing cells of the skeleton rescues not only diminished chondrocyte proliferation, but surprisingly, also results in amelioration of the severe skeletal dysplasia associated with MT1‐MMP deficiency through enhanced bone formation. Consistent with this increased bone formation, type II collagen was identified in bone cells and skeletal stem/progenitor cells of wildtype mice. Moreover, bone marrow stromal cells isolated from mice expressing MT1‐MMP under the control of the type II collagen promoter in an MT1‐MMP‐deficient background showed enhanced bone formation in vitro and in vivo compared with cells derived from nontransgenic MT1‐MMP‐deficient littermates. These observations show that type II collagen is not stringently confined to the chondrocyte but is expressed in skeletal stem/progenitor cells (able to regenerate bone, cartilage, myelosupportive stroma, marrow adipocytes) and in the chondrogenic and osteogenic lineage progeny where collagenolytic activity is a requisite for proper cell and tissue function.