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     

血管紧张素II通过PI3K/Akt/FOXO1信号通路诱导骨骼肌细胞萎缩

目的探讨血管紧张素II(Ang II)对骨骼肌细胞的调控作用及其作用机制。方法使用Ang II及其1型受体(AT1R)拮抗剂(ARB)奥美沙坦干预C2C12成肌细胞,分为Control、Ang II和Ang II+ARB三组。使用2%马血清诱导成肌细胞分化为肌管细胞。免疫荧光染色检测肌管细胞的平均面积改变,Western blot检测肌管细胞泛素连接酶、生肌调节因子(MRFs)和PI3K/Akt/FOXO1信号通路蛋白表达的变化。结果免疫荧光染色显示,Ang II干预后,肌管细胞的平均直径和面积减小(P0.001);使用奥美沙坦干预后,肌管细胞平均直径和面积明显增大(P 0.01)。Western blot显示,Ang II干预后,肌管细胞pPI3K/PI3K(P0.01),p-Akt/Akt (P 0.001)和p-FOXO1/FOXO1 (P 0.01)的比率降低; MURF1 (P 0.05)和MAFbx (P 0.001)蛋白表达明显升高,MHC(P0.01)和MyoD(P 0.05)蛋白表达明显降低;使用奥美沙坦处理后,能够减轻Ang II对肌管细胞的干预作用。结论 Ang II能够通过抑制PI3K/Akt/FOXO1信号通路的磷酸化,促进蛋白的降解,抑制蛋白的合成,诱导骨骼肌细胞萎缩。     

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.     

L6细胞复合培养中平行结构支架材料的细胞黏附性观察

目的：检测在L6细胞培养过程中，具有平行结构支架材料的生物相容性。方法：将医用可吸收缝线平行折叠并制备成类似圆柱体样，与大鼠L6成肌细胞株体外复合培养。利用扫描电镜观察平行结构支架材料吸附的L6细胞的形态学结构，观察L6细胞在材料上的表面相容性。结果l天后，细胞在支架材料表面黏附、伸展，随时间的延长，显示出沿支架材料纵轴排列趋势，第6天的时候，可以见到类似肌小管的结构。结论：所制备的具有平行结构的支架材料具有良好的生物相容性。     

Effects of leukotriene C4 on macrophage association with and intracellular fate of Trypanosoma cruzi

Leukotriene C4 (LTC4) enhanced the association of mouse peritoneal macrophages (MPM) with Trypanosoma cruzi, increasing the proportion of MPM associating with parasites and the number of trypanosomes per MPM. LTC4 affected both cells since pretreatment of either one increased the association. LTC4 also enhanced MPM uptake of killed T. cruzi or latex beads, denoting stimulation of phagocytosis. However, since LTC4 pretreatment of rat heart myoblasts--nonphagocytic cells--also increased the association, host cell membrane alterations induced by LTC4 may also facilitate parasite invasion. Inhibition of MPM guanylate cyclase abrogated the LTC4 effect, suggesting a role for elevated levels of cyclic GMP. LTC4 also increased the rate of intracellular parasite killing by MPM. These results suggest that LTC4, occurring in inflammation such as develops in T. cruzi infection, regulates parasite clearance by MPM by increasing uptake and intracellular destruction.     

p38丝裂素活化蛋白激酶信号通路在应力调控成肌细胞分化中的作用

目的研究在C2C12细胞成肌分化过程中应力对丝裂原活化蛋白激酶（MAPKs）信号通路中p38丝裂素活化蛋白激酶（p38MAPK）信号通路的影响。方法将6孔Bio Flex培养板上贴壁培养的C2C12细胞,以0.5 Hz的加载频率和10%的细胞拉伸变形幅度,分别进行拉伸培养2、6、12、24 h,应用Western blot免疫印迹检测总p38和磷酸化p-p38（Thr180/Tyr182）蛋白的表达情况。结果周期性机械拉伸在调控C2C12成肌细胞分化过程中,p38MAPK信号通路被激活。p38MAPK信号通路蛋白磷酸化水平在较高水平;而p38MAPK通路总蛋白表达维持在一基线水平,各组之间差异无统计学意义。加入p38MAPK信号通路特异性抑制剂SB203580后再加力,Myogenin的表达明显降低。结论p38MAPK信号通路在应力介导的C2C12成肌细胞分化过程中发挥重要作用,但不是这一调控过程的唯一通路。     

携人胰岛素样生长因子-1基因的成肌细胞移植后在体存活及产物表达的研究

目的:观察并探讨携人胰岛素样生长因子-1(hIGF-1)基因的成肌细胞移植入雄性C3H小鼠体内后的存活、转归以及移植后hIGF-1基因的表达。方法:84只雄性C3H小鼠随机(20~30g,7~11w)分为A组(正常小鼠转基因细胞移植组)、B组(正常小鼠空白成肌细胞移植组)、C组(受伤小鼠转基因细胞移植组)和D组(受伤小鼠空白成肌细胞移植组),每组20只,另4只作正常对照。A、B两组分别于右侧腓肠肌中段注射转基因成肌细胞或空白成肌细胞;C、D两组以重力打击造成小鼠右侧腓肠肌中段钝挫伤,伤后第3天分别于致伤局部注射转基因成肌细胞或空白成肌细胞。注射细胞后第2、5、10、20、30天,各组随机抽取4只小鼠处死,取右侧腓肠肌中段检测,Br-dU免疫组化染色检测外源细胞在体内的存活情况;4组另行hIGF-1免疫组化染色及实时PCR检测外源转基因细胞在体内表达hIGF-1情况。结果:各组小鼠均有BrdU免疫组化阳性染色,A、C两组均有hIGF-1mRNA表达及hIGF-1分泌,B、D两组未检测到hIGF-1mRNA表达及hIGF-1分泌。结论:携hIGF-1基因的成肌细胞移植入正常及钝挫伤小鼠体内后,可存活一段时间,并能稳定地分泌hIGF-1因子。