Modified methods for culturing myoblasts of rats: Combination of multi-enzymatic digestion and double purification

BACKGROUND： With developments of tissue engineering and genetic engineering, we aim to culture myoblasts, which are characterized by high purity, high quality and high production, for wide application in neural regeneration researches. OBJECTIVE： To modify traditional dissociation method in order to obtain myoblasts, which are characterized by high purity, high quality and high production, and explore the biological properties under in vitro culture. DESIGN： Observational study. SETTING： Basic Institute of Academy of Military Medical Sciences of Chinese PLA. MATERIALS： Four neonatal Wistar rats of 5 days old, both genders and mean body mass of 10 g were selected in this study. The main reagents and devices were detailed as follows： DMEM medium （Gibco Company）, fetus bovine serum （FBS, Hycolne Company）, collagenase Ⅱ （Sigma Company）, trypsin （Sigma Company）, dispase Ⅱ （Sigma Company）, desmin antibody （Fuzhou Maixin Company）, antibody Ⅱ and ABC kit （Wuhan Baster Biotechnology Company）, desk centrifuge （KUBATO, Japan）, and inverted phase contrast microscope （LEICA DMIRB, Germany）. METHODS： The experiment was carried out in the Basic Institute of Academy of Military Medical Sciences of Chinese PLA from June to October 2006. Neonatal rats were sacrificed under sterile condition to obtain skeletal muscles of limbs, which were washed with cold PBS （containing benzylpenicillin and estreptomicina）, and muscular tissue was sheared into pieces. Then, those muscular pieces were added with mixed digestive enzyme （containing 2 g/L collagenase Ⅱ ＋ 5 g/L dispase Ⅱ ＋ 0.28 g/L CaCl2） as twice volume as pieces, dealt with mechanical pipetting for 5 minutes and cultured in CO2 incubator for 10 minutes. The operation was done for three times and the muscular pieces were digested for 45 minutes in total. Moreover, cells were suspended again in order to obtain myoblasts from skeletal muscle of neonatal rats. In addition, myoblasts were purified with differential attachment technique and enzyme digestion so as to observe morphological characteristics and growth, draw growth curve, analyze surface structure under scanning electron microscope, and evaluate with Desmin immunohistochemical staining. MAIN OUTCOME MEASURES： Morphological characteristics and growth ofmyoblasts cultured in vitro. RESULTS： ①Growth of myoblasts of skeletal muscle： Primary cells had well growth, mature and differentiation. The positive rate of Desmin was 94% and purification of cells was ideal. Growth curve of cells demonstrated that myoblasts which were characterized by high purification started proliferation plentiful through transient growth lag phase （about at one or two days after inoculation）. If myoblasts were not dealt with any interventions, they might become sarcotubule gradually at 3 - 5 days after proliferative phase. During this period, myoblasts maintained a monocaryon-bipolarity state under inverted phase contrast microscope. Furthermore, the growth of cells was the strongest and reproductive activity was the most powerful. This suggested that myotube started to form; in addition, muscle fiber of contractility might form under a well culturing condition. ②Immunocytochemical stain with desmin antibody： Interzonal fiber of desmin from myoblasts showed strongly positive reaction. Positive staining existed in cytoplasm had a high nucleus-cytoplasm ratio. However, myoblasts showed negative or mildly positive reaction. CONCLUSION： It is ideal for modified multi-enzymatic digestion and double purification method to dissociate and purify myoblasts of skeletal muscle; meanwhile, these two methods are both the effective ways to provide convenient conditions to obtain seed cells for neural regeneration researches.     

脂肪干细胞原代培养及诱导成肌细胞的实验研究

目的 探讨大鼠脂肪干细胞(ADSC)的培养及诱导分化为成肌细胞的方法 .方法 取SD大鼠脂肪组织,酶消化法分离、培养ADSC,免疫荧光和流式细胞仪检测相关表面抗原CD90、CD105和CD34的表达.取培养至第2代处于对数生长期细胞,分别用含5-氮杂胞苷(5-aza)的诱导培养液(诱导组)和基础培养液(对照组)进行培养.诱导时间为7、14、21、28和35 d,倒置相差显微镜观察细胞的生长情况和形态变化,免疫荧光和流式细胞仪检测成肌细胞特异性抗原desmin和myosin的表达.结果 成功从大鼠脂肪组织分离、培养出ADSC,相关表面抗原表达检测证实其干细胞特性.诱导组细胞诱导28 d后呈现成肌细胞特有的"漩涡"样生长形态,单个细胞表现出多核化;免疫荧光和流式细胞仪检测显示,desmin和myosin的表达率在诱导28 d时达最高,分别为52.57%和50.04%,而诱导前和对照组细胞均呈阴性表达.结论 成功从大鼠脂肪组织分离、培养ADSC,含5-aza的诱导培养液可将其诱导分化为成肌细胞,诱导 28 d成肌细胞特异性抗原表达率最高.     

高强度周期性牵张应力促进体外培养成肌细胞凋亡的机制探讨

目的：探讨高强度应力作用下体外培养成肌细胞凋亡的分子机制。方法：采用Flexercell Strain Unit系统对体外培养成肌细胞施加牵张应力,通过DNA ladder实验观察细胞凋亡情况。Western Blot检测磷酸化和总体JNK1的水平,通过NFkappa B报告系统检测其活性。通过JNK1的RNAi实验观察抑制JNK1后,是否补救被抑制的NFkappa B活性。结果：10%表面拉伸幅度的牵张应力作用24 h后,细胞形态梭形稍变长,且排列方向有一致性的趋势,DNA ladder实验证实15%以上较高强度应力引起C2C12细胞凋亡,高强度应力条件下成肌细胞凋亡过程中,JNK1通路被激活,抑制JNK1的活化,补救被抑制的NFkappa B活性。结论：高强度周期性牵张应力促进体外培养成肌细胞凋亡是通过激活JNK1通路而抑制NFkappa B活性实现的。     

番石榴叶提取物调节白色脂肪组织棕色化作用机制研究

目的:探究番石榴叶提取物调节脂肪细胞分化的作用机制.方法:6周龄雄性SPF级db/db小鼠24只、同龄C57BL/6J正常组小鼠6只,将符合纳入标准的db/db小鼠根据体重、血糖随机分为模型组和番石榴叶提取物高剂量组、番石榴叶提取物中剂量组、番石榴叶提取物低剂量组,并将番石榴叶提取物按高、中、低剂量进行灌胃,于4周后空...     

绞股蓝皂苷对C2C12细胞成肌分化的影响

目的 研究发现，绞股蓝皂苷不仅具有抗炎、抗衰老等多重作用，而且还能促进成骨细胞增殖分化，保护其氧化应激损伤；本研究拟探讨绞股蓝皂苷对C2C12细胞成肌分化的影响。方法 完全培养基培养C2C12细胞，细胞增殖贴壁后用含有不同浓度绞股蓝皂苷的分化培养基诱导细胞分化，每个浓度均设置3个复孔；第5天观察细胞分化的形态、计算细胞的分化活力，Western blot检测相关成肌标志物MYF5和MyoD1的表达，免疫荧光检测MyoD1的表达，Image J软件计算肌管融合指数。结果 诱导C2C12细胞分化的第5天可见细胞分化为典型的肌管形态，10 mg/L和5 mg/L的绞股蓝皂苷浓度干预下的MYF5和MyoD1的表达较为显著，且肌管融合指数更高，相较于对照组差异均具有统计学意义（P＜0.05）。结论 绞股蓝皂苷可促进C2C12细胞的成肌分化，以10 mg/L和5 mg/L的浓度较好。