a Department of Ophthalmology , Pusan National University Hospital , Busan , South Korea.
b Biomedical Research Institute , Pusan National University Hospital , Busan , South Korea.
Curr Eye Res. 2018 Dec;43(12):1484-1488. doi: 10.1080/02713683.2018.1501075. Epub 2018 Sep 5.
To isolate and culture human extraocular muscle (EOM) myoblasts and facilitate their differentiation to myotubes in vitro, and to determine whether these myoblasts express thyroid-stimulating hormone receptor (TSHR).
Human EOM myoblasts were isolated from EOM samples, and identified by immunostaining for PAX7 and MYOD1 (markers of human skeletal myoblasts), and western blot for desmin (muscle marker). In addition, we investigated the expressions of SHOX2 (a genetic marker of EOM myoblasts) and HOXC10 (an exclusive marker of hind-limb muscle-derived myoblasts) by RT-PCR. Fusion index and myotube area were measured to quantify myotube differentiation. TSHR immunostaining and western blot were used to determine the presence of TSHR on human EOM myoblasts and investigate its expression during myogenesis.
Human EOM myoblasts were immunopositive for PAX7 and MYOD1 staining, and had desmin expression during myogenesis. The EOM-specific gene SHOX2 was detected by RT-PCR, but HOXC10 was not detected. The significant change in both fusion index and myotubes were shown at 8 days after induction of differentiation myotubes. Immunostaining revealed TSHR was expressed on human EOM myoblasts and western blot demonstrated the presence of TSHR protein and highest TSHR protein expression was shown at 10 days after myogenic differentiation.
Human EOM myoblasts were cultured and underwent myogenic differentiation in vitro. TSHR protein was detected on human EOM myoblasts and increasing TSHR expression during myogenic differentiation.
分离和培养人眼外肌（EOM）成肌细胞，并促进其在体外分化为肌管，并确定这些成肌细胞是否表达促甲状腺激素受体（TSHR）。
从 EOM 样本中分离出人眼外肌成肌细胞，并通过 PAX7 和 MYOD1（人骨骼肌成肌细胞标志物）的免疫染色以及结蛋白（肌肉标志物）的 Western blot 进行鉴定。此外，我们通过 RT-PCR 研究 SHOX2（EOM 成肌细胞的遗传标志物）和 HOXC10（后肢肌肉衍生成肌细胞的特有标志物）的表达。融合指数和肌管面积用于量化肌管分化。TSHR 免疫染色和 Western blot 用于确定人眼外肌成肌细胞上 TSHR 的存在，并研究其在肌发生过程中的表达。
人眼外肌成肌细胞对 PAX7 和 MYOD1 染色呈免疫阳性，在肌发生过程中有结蛋白表达。通过 RT-PCR 检测到 EOM 特异性基因 SHOX2，但未检测到 HOXC10。分化为肌管后 8 天，融合指数和肌管均发生显著变化。免疫染色显示 TSHR 表达于人眼外肌成肌细胞，Western blot 显示 TSHR 蛋白的存在，在肌生成分化后 10 天表达最高。
人眼外肌成肌细胞在体外培养并进行肌生成分化。人眼外肌成肌细胞上检测到 TSHR 蛋白，并且在肌生成分化过程中 TSHR 表达增加。
