聚集蛋白基因在原代培养成肌细胞中的表达

目的研究神经源性聚集蛋白（agrin）基因在原代培养成肌细胞中的表达。方法原代培养成年大鼠的成肌细胞，免疫细胞化学方法进行鉴定，将agrin—Y428基因cDNA片段亚克隆入pCDNA3真核表达载体，重组子经脂质体介导转染成肌细胞，G418筛选，获得具有抗性的克隆，增殖后以逆转录一聚合酶链反应（RT—PCR）和免疫荧光的方法检测基因的转录和蛋白表达，并初步测定其生物学活性。结果原代培养8周后，90％以上仍为成肌细胞，转染神经源性agrin基因后，成肌细胞可以表达相应的mRNA和有功能活性的蛋白。结论原代培养的成肌细胞可以作为agrin基因转移的有效载体，可用于进一步肌肉功能减退的基因治疗中。     

Stable hybrid myotubes: A new model for studying re-expression of enzymatic activities in vitro

Heterokaryons represent a stable and reproducible model system for the study of biochemical and molecular aspects responsible for muscle gene activation. Previous experiments have used this fusion system to demonstrate human gene activation in hybrids formed between human and non-human cells. The aim of this research was to apply this experimental model to the correction of a cytoplasmic activity, namely glucose-6-phosphate dehydrogenase (G6PD), in vitro, in hybrid myotubes formed between G6PD-negative and positive myoblasts. Different identification methods were used (Hoechst stain and Fluorescent Latex Microspheres, FLMs) to identify hybrid myotubes formed. We demonstrated the restoration of G6PD activity in all hybrid myotubes formed; we then tried to elucidate the mechanisms underlying the restoration of this specific activity and apply the results obtained to the understanding of more complex mechanisms involved in muscle gene activation. Paper presented at the National Congress at Sorrento in 1991 and selected by the Editorial Board of the Journal     

Promotion of muscle regeneration by myoblast transplantation combined with the controlled and sustained release of bFGFcpr

Although myoblast transplantation is an attractive method for muscle regeneration, its efficiency remains limited. The efficacy of myoblast transplantation in combination with the controlled and sustained delivery of basic fibroblast growth factor (bFGF) was investigated. Defects of thigh muscle in Sprague–Dawley (SD) rats were created, and GFP‐positive myoblasts were subsequently transplanted. The rats were divided into three groups. In control group 1 (C1) only myoblasts were transplanted, while in control group 2 (C2) myoblasts were introduced along with empty gelatin hydrogel microspheres. In the experimental group (Ex), myoblasts were transplanted along with bFGF incorporated into gelatin hydrogel microspheres. Four weeks after transplantation, GFP‐positive myoblasts were found to be integrated into the recipient muscle and to contribute to muscle fibre regeneration in all groups. A significantly higher expression level of GFP in the Ex group demonstrated that the survival rate of transplanted myoblasts in Ex was remarkably improved compared with that in C1 and C2. Furthermore, myofibre regeneration, characterized by centralization of the nuclei, was markedly accelerated in Ex. The expression level of CD31 in Ex was higher than that in both C1 and C2, but the differences were not statistically significant. A significantly higher expression level of Myogenin and a lower expression level of MyoD1 were both observed in Ex after 4 weeks, suggesting the promotion of differentiation to myotubes. Our findings suggest that the controlled and sustained release of bFGF from gelatin hydrogel microspheres improves the survival rate of transplanted myoblasts and promotes muscle regeneration by facilitating myogenesis rather than angiogenesis. Copyright © 2013 John Wiley & Sons, Ltd.     

周期性牵张应力下成肌细胞面积、周长的变化研究

目的基于成熟的细胞力学加载和细胞图像处理、分析方法,从细胞层次上定量观察成肌细胞周期性牵张应力加载后的面积、周长的时间改建效应。方法通过4点加力装置给成肌细胞施与各种时间段的生理性牵张应力(0.1Hz,2000μstrain),在相差显微镜下观察并记录细胞形态变化;借助计算机细胞图像处理和分析系统对成肌细胞形态进行定量分析。结果加力组和对照组的成肌细胞面积、周长测量值在加力0.5、1.0、2.0h后,两者差异不大,加力4h后两者的差异开始出现,加力8h后两者的差异变得明显;随着加力时间的延长,加力组和对照组间细胞形态参数测量的差别越来越明显。而去除细胞力学刺激后,成肌细胞形态都出现回复趋势。结论连续加力时细胞形态面积、周长变化明显,而停止加力后细胞面积、周长有回复的趋势。     

connexin43基因体外转染大鼠L6骨骼肌成肌细胞的研究

目的将含有connexin43基因的质粒在脂质体的介导下转染进入大鼠L6骨骼肌成肌细胞,使用G418根据有限稀释法筛选单克隆细胞株L6 myoblast-Cx43。方法通过免疫细胞化学技术、Western blot技术,对整合有connexin43基因的阳性细胞克隆进行检测。结果两个阳性克隆组Cx43蛋白表达均明显高于对照组。结论获得的两个持续高表达connexin43蛋白的细胞株可用于进行移植细胞之间及移植细胞与宿主细胞之间能否形成良好的电偶联的研究。     

CPUHY002对大鼠心肌细胞瞬时外向钾电流的影响

目的观察一种新合成的磷酸二酯酶Ⅲ(PDEⅢ)抑制剂CPUHY002对大鼠心肌细胞瞬时外向钾电流(Ito)的影响。方法采用全细胞膜片钳技术考察CPUHY002对急性分离大鼠心肌细胞和H9c2细胞系Ito的作用。结果在+50 mV下,CPUHY002对急性分离细胞和H9c2细胞系的Ito呈浓度依赖性抑制,其半数有效抑制浓度(IC50)分别为0.98μmol/L和0.91μmol/L;1μmol/L CPUHY002可使I-V曲线明显下移,Ito峰值分别下降(39.10±2.30)%和(47.32±2.06)%,激活曲线右移,失活曲线左移(未见于H9c2),恢复曲线无显著变化。结论 CPUHY002可浓度依赖性地抑制大鼠心肌细胞Ito。     

静电纺丝纳米纤维膜作为骨骼肌组织工程支架材料的细胞相容性

摘要 背景：有报道以生物可降解的胶原盘或聚L-乳酸、聚羟基乙酸、聚L-乳酸/聚羟基乙酸共聚物等作为骨骼肌组织工程的支架材料,各有优缺点,不能完全满足骨骼肌组织工程的需要。 目的：探讨静电纺丝纳米纤维膜作为骨骼肌组织工程支架材料的可行性。 方法：制备7种不同组分的静电纺丝纳米纤维膜,以其浸提液为培养基培养第3代SD乳鼠成肌细胞,以含体积分数20%新生小牛血清的F12培养基培养的为对照。采用MTT法和扫描电镜检测成肌细胞在各组材料的黏附及生长情况。 结果与结论：各组分静电纺丝纳米纤维膜吸光度值与对照组间差异无显著性意义(P > 0.05)。各组分静电纺丝纳米纤维膜组成肌细胞黏附率差异有显著性意义(P < 0.05)。扫描电镜与上述结果一致。含70%聚乳酸+20%蚕丝蛋白+10%胶原组成电纺丝纳米纤维膜组可见大量成肌细胞黏附,呈梭形,两极伸展,排列规律,效果最好。其他各组细胞少,形态不规则,似衰退期成肌细胞。提示静电纺丝纳米纤维膜无细胞毒性,对成肌细胞的增殖无影响,成肌细胞能良好地黏附;以70%聚乳酸+ 20%蚕丝蛋白+10%胶原组分效果最佳。 关键词：聚乳酸;蚕丝蛋白;胶原;成肌细胞;静电纺丝技术;纳米纤维膜;组织工程 doi:10.3969/j.issn.1673-8225.2011.12.020     

17β-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts

The skeletal muscle is one of the important target tissues for the actions of estrogen via both nuclear and extranuclear (non-genomic) pathways. However, there is a paucity of information about the receptor (ER) involved. The aim of this study was thus to explore the ER expression in skeletal muscle, and the influence of estrogen on it, by using C2C12 myoblasts derived from mouse skeletal muscle. Significant expression of a ~66-kD protein immunoreactive to ER type α (ERα) monoclonal antibody, which was comparable to that in ovary, was detected in the whole-cell (total) and nucleus-free (nonnuclear) fractions of C2C12 myoblasts. The expression level of these ER proteins increased in several hours with treatment with 17β-estradiol (E2), which was preceded by the elevation of the ER mRNA level. This increase appeared to reflect the acceleration of de novo synthesis of ER protein, as proved by the ³⁵S-methionine immunoprecipitation method. A similar extent of fast increase in ER expression was also induced by a membrane-impermeable, BSA-conjugated estradiol (E2-BSA). Unexpectedly, the E2-induced increases in total and nonnuclear ER were further enhanced by the classic ER antagonists tamoxifen and ICI182,780 in a wide concentration range, implying some structural difference of the involved ER from the classical one. Treatment with the ERK1/2 inhibitor, PD98059 (10 μM), or the p38 MAPK-specific inhibitor, SB203580 (10 μM), greatly inhibited the E2-induced ER increase, while the protein kinase C (PKC) activator TPA (1 μM) enhanced it. These results collectively suggest that C2C12 skeletal myoblasts express a high level of ER, a considerable part of which is extranuclear. Further, the expression of ER in these cells may be significantly upregulated by estrogen itself via increased biosynthesis linked to membrane-bound ER and downstream MAPK-mediated signaling pathways. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Modulation of the microenvironment by growth factors regulates the in vivo growth of skeletal myoblasts

OBJECTIVES

To investigate the optimal microenvironment for efficient myoblast transplantation in vivo.

MATERIALS AND METHODS

The effects of co‐culture with growth factors, including basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin‐like growth factor‐I) and platelet‐derived growth factor (PDGF), on in vitro growth, migration and proteolytic activity of mouse skeletal myoblasts were investigated. Myoblasts were co‐injected with growth factors into the subcutis and bladder wall of nude mice, and its impact on the growth patterns of myoblasts in vivo assessed.

RESULTS

There was dose‐dependent stimulation of in vitro myoblast growth after treatment with each of the four growth factors, but bFGF induced the most marked increase in the growth of myoblasts. Treatment of myoblasts with all types of growth factors also resulted in a dose‐dependent increase in the in vitro migration of myoblasts, and PDGF had the most prominent effect on myoblast migration. Increased secretion of matrix metalloproteinase‐9 (MMP‐9) in myoblasts induced by growth factors was proportional to their increased migration capacity, which was partly inhibited by SB‐3CT, an inhibitor of MMP‐9. The in vivo growth of myoblasts was significantly enhanced by co‐injection with all types of growth factor into both the subcutis and bladder wall, but this effect was most marked 1 and 2 weeks after co‐injection with bFGF and PDGF, respectively. Furthermore, there was synergistic in vivo growth of myoblasts by co‐injection of both bFGF and PDGF compared with that achieved with either agent alone.

CONCLUSIONS

These findings suggest that modulation of the microenvironment using growth factors, particularly bFGF and PDGF, could provide the optimum condition for effective myoblast transplantation in vivo.