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Title: Permanent myopathy caused by mutation of SCN4A Metl592Val: Observation on myogenesis in vitro and on effect of basic fibroblast growth factor on the muscle.

Authors: Yu Feng, Hong Wang, Xiao-Guang Luo, Yan Ren

Journal, date & volume: , 2009 Apr , 25, 61-6

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/19290024

Abstract
The present study is to observe in vitro the proliferation ability of the muscle cells from permanent myopathy (PM) patients of nomokalaemic periodic paralysis (normKPP), which is caused by mutations of Met1592Val in the skeletal muscle voltage gated sodium channel (SCN4A) gene on chromosome 17q23.1. We also evaluate the possible effect of the foreign basic fibroblast growth factor (bFGF) in preventing and curing PM.The gastrocnemius muscle cells were taken from two male patients with PM of the same Chinese family with Met1592Val mutation of SCN4A, determined by gene screening. Four male patients suffering from the skeletal injury without PM were taken as control. All preparations were protogenerationally cultured in vitro. Proliferation of the cultured preparations was measured by MTT. Activities of the lactic dehydrogenase (LDH), creatine kinase (CK), and protein content in these cells were also detected. The effects of bFGF with different doses (10 ng/mL, 20 ng/mL, 40 ng/mL, 80 ng/mL, 120 ng/mL and 160 ng/mL) on the above mentioned parameters were also evaluated.Cells from both PM and control subjects were successfully cultured in vitro. The cultivation of the muscle cells from PM patients in vitro was not yet seen. Results indicated the obvious stimulation of bFGF on cell proliferation, activities of LDH and CK, protein synthesis, in a dose dependent manner. The optimal dose of bFGF was 120 ng/mL (P<0.05), beyond which greater dose caused a less effect. The effect of bFGF on 160 ng /mL was stronger than that on 80 ng/mL, but there was no significant difference (P>0.05).Myoblastic cells from patients with PM had a weaker ability of developing into the myotubules, thus they were unable to perform effective regeneration, which resulted in a progressive necrosis. The exogenous bFGF could promote the division and proliferation of the muscle cells in vitro. These results shield a light on bFGFos potential role in preventing and treating PM.正常血钾性周期麻痹是由位于17号染色体上编码人类骨骼肌钠通道基因(SCN4A)发生突变所致, 以肢体无力短暂、 反复发作并完全恢复为特点, 部分患者可发展为永久性肌病(permanent myopathy, PM),该病的发病机制尚不清楚, 至今也无有效的治疗方法。 本文观察SCN4A Met1592Val突变所致PM患者骨骼肌细胞体外生长过 程, 同时探讨外源性碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)对PM防治作用的可能性。对两名来自一个由基因诊断确诊为SCN4A Met1592Val突变家系的患者和四名正常对照者的腓肠肌细胞进行体外培养, 观察其生长过程; 同时给予6个浓度(10 ng/mL, 20 ng/mL, 40 ng/mL, 80 ng/mL, 120 ng/mL和160 ng/mL)的bFGF刺激, 用MTT 法、 考马斯亮蓝比色法、 反复冻融细胞, 对骨骼肌细胞增殖进行检测。腓肠肌细胞可体外培养, 与在体发生过程相似,但PM组肌细胞发育迟滞; bFGF 对肌细胞生长的增殖作用、 促LDH、 CK活性及蛋白质合成作用呈明显的浓度依赖性, 浓度为120 ng/mL 时作用最显著(P<0.05), 此时浓度再增大其效应反而下降, 在浓 度为160 ng/mL 时的作用大于80 ng/mL, 但无统计学意义(P>0.05) 。PM 患者受损腓肠肌在体外有再生能力, 但融合成肌맜能力极弱, 存在再生障碍, 不能对变性、 坏死的肌纤维形成有效再生, 故呈进行性肌纤维耗竭; 外源性bFGF 可促进PM患者体外培养骨骼肌细胞增殖分裂, 并可促进肌맜成熟, 对进展性肌病应具有防治作用。