LXRs激动剂T0901317对成人和SD大鼠骨骼肌细胞FAT/CD36基因mRNA表达的作用及FAT/CD36基因多态性与2型糖尿病关系

【作者】 曾蓉；

【导师】 宋滇平； 撒亚莲； 高建梅；

【作者基本信息】 昆明医学院 ， 内科学， 2008， 硕士

【摘要】 目的研究骨骼肌卫星细胞体外纯化、培养、鉴定的方法及其生物学特性。方法采用Ⅰ型胶原酶和胰蛋白酶两步消化法纯化出成人骨骼肌卫星细胞和大鼠骨骼肌卫星细胞,进行体外原代和传代培养,借此观察卫星细胞的增殖、分化特点并进行免疫细胞化学染色鉴定。结果两步消化法是获取卫星细胞可靠的方法。生长培养基促使细胞的增殖;延迟分瓶或分化培养基促使细胞分化形成骨骼肌细胞并最终融合形成肌管。骨骼肌结蛋白（desmin）单克隆抗体免疫细胞化学染色法是鉴定骨骼肌细胞的可靠方法。SD大鼠骨骼肌细胞生长曲线的每个时期较成人骨骼肌细胞的相应时期都缩短1-2天。结论体外培养的成人骨骼肌卫星细胞和大鼠骨骼肌卫星细胞在合适的培养条件和传代比例下能够增殖与分化并保持其生物学特性,为其在后续研究中的应用奠定了基础。目的探讨肝核受体LXRs激动剂T0901317对正常人骨骼肌细胞中FAT/CD36基因mRNA表达的影响。方法将原代培养的5例成人骨骼肌细胞分为用肝核受体LXRs激动剂T0901317（1μmol/L）作用组、T0901317（0.5μmol/L）作用组和未作用（阴性对照）组,采用SYBR GreenⅠ实时荧光定量PCR法检测各组成人骨骼肌细胞FAT/CD36基因mRNA表达水平,并进行比较分析。结果（1）以浓度为1μmol/L的T0901317作用组和0.5μmol/L的T090131作用组和未作用组样本的均数进行方差分析,差别有统计学意义（P＜0.05）。（2）进一步用2^-ΔΔCt公式法进行mRNA表达的倍数分析,浓度为1μmol/L的T0901317作用组和0.5μmol/L的T090131作用组成人骨骼肌细胞中FAT/CD36基因的mRNA表达分别是未作用组的3.03倍和2.91倍。结论肝核受体LXRs激动剂T0901317能够提高成人骨骼肌细胞中FAT/CD36基因mRNA的表达水平,提示T0901317可以加快骨骼肌细胞内脂肪酸的堆积,推测LXRs激动剂T0901317可能有增加糖尿病患者骨骼肌胰岛素抵抗的风险。目的探讨肝核受体LXRs激动剂T0901317对正常SD大鼠骨骼肌细胞中FAT/CD36基因mRNA表达的影响。方法将原代培养的5例SD大鼠骨骼肌细胞分为用肝核受体LXRs激动剂T0901317（1μmol/L）作用组、T0901317（0.5μmol/L）作用组和未作用（阴性对照）组,采用SYBR GreenⅠ实时荧光定量PCR法检测各组SD大鼠骨骼肌细胞FAT/CD36基因mRNA表达水平,并进行比较分析。结果肝核受体激动剂T0901317（1μmol/L）作用组与T0901317（0.5μmol/L）作用组和阴性对照组的Ct值样本均数进行方差分析,显示T0901317（1μmol/L）作用组和T0901317（0.5μmol/L）作用组与阴性对照组相比,差异没有统计学意义（P=0.116）,提示肝核受体LXRs的激动剂T0901317对于FAT/CD36基因mRNA在正常SD大鼠骨骼肌细胞中的表达没有明显作用。结论肝核受体LXRs激动剂T0901317对SD大鼠骨骼肌细胞中FAT/CD36基因mRNA的表达水平没有明显作用,提示T0901317在促进SD大鼠骨骼肌细胞内脂肪酸的堆积作用尚无确切的证据。目的探讨FAT/CD36脂肪酸转位酶基因启动子区-3489C/T和密码子区478C/T多态性与2型糖尿病（T2DM）的相关性。方法运用聚合酶链反应-限制性片段长度多态性技术对196例2型DM患者和120例正常对照者的-3489C/T与478C/T多态性进行检测,并比较各组间基因型频率和等位基因频率以及相关临床资料。结果（1）所有研究对象均无一例存在FAT/CD36基因启动子区478C→T突变,基因型均表现为478CC之纯合子,478CC和478TT等位基因频率分别为1和0。（2）T2DM组和正常对照组的-3489C/T多态性基因型和等位基因频率比较无显著性差异,P值分别为0.682和0.683。结论中国云南昆明地区的汉族人中FAT/CD36基因的启动子区-3489C/T和密码子区478 C/T多态性与昆明地区汉族人群2型糖尿病的发生无显著相关关系。更多还原

【Abstract】 Objective To establish the methods for purification,culture, identification and biological characteristics of adult human and rat skeletal muscle satellite cells（SCs）.Methods Human skeletal muscle cell and SD rat skeletal muscle cell were obtained by the two-steps method of collegenase-1 and trypsin,and were subjected to primary as well as secondary culture in vitro.Morphological characteristics,myotube formation and growth curve were observed to evaluate the proliferative and differentiation ability of SCs.The SCs were identified with cellular immuno-chemical stain.Results The two-steps method with collegenase-l and trypsin was reliable for collection of SCs.The cells showed high proliferative ability in the proliferative media and could form myotubes in differentiation media.The satellite cells of adult rat and human can be proliferated and differentiated in different serum concent ration,and can be identified by desmin stain.The growth periods in growth curve of rat was shorter than that of aldult human.Conclusion The SCs cultured in vitro from rat skeletal muscle or human skeletal muscle have high proliferative and differentiation ability and maintain their biological characteristics.They can be used to the continual study. Objective To investigate the Liver X receptors agonists T0901317’s effection on expression of FAT/CD36 gene mRNA in aldult human skeletal muscle cell.Methods Myotubes from humans were exposed to different T0901317 concentrations（0,0.5,and 1.0μmol/l）for 24 hours before experiments were performed.Then the expression of FAT/CD36 mRNA in skeletal muscle cell of each experimental human group was detected by SYBR Green I real-time quantitative polymerase chain reaction.The relative data were compared among groups by 2^-△△Ctmethod.Results（1）The Ct mean of control group,T0901317（0.5μmol/l）group,T0901317（1μmol/l）group was analyzed and the difference has significance（P＜0.05）.（2）The expression of FAT/CD36 mRNA with Liver X receptors agonists T0901317 in human skeletal muscle cell in the T0901317（0.5μmol/l）group and T0901317（1μmol/l）group was 2.91 times and 3.03 times than the control group.Conclusion The expression of FAT/CD36 mRNA in human skeletal muscle cell afer the treatment of Liver X receptors agonists T0901317 was increasing and the fatty acid in human skeletal muscle cell increased,so we may propose that T0901317 may increase the risk of resistance in aldult human skeletal muscle. Objective To investigate the Liver X receptors agonists T0901317’s effection on expression of FAT/CD36 gene mRNA in SD rat skeletal muscle cell.Metbods Myotubes from SD rats were exposed to different T0901317 concentrations（0,0.5,and 1.0μmol/l）for 24 hours before experiments were performed.Then the expression of FAT/CD36 gene mRNA in skeletal muscle cell of each experimental human group was detected by SYBR Green I real-time quantitative polymerase chain reaction.The relative data were compared among groups by 2^-△△Ctmethod.Results The Ct mean of control group,T0901317（0.5βmol/l）group,T0901317（1μmol/l）group was analyzed and the difference has no significance in the three groups.Conclusion The expression of FAT/CD36 gene in SD rat skeletal muscle cell after the treatment of Liver X receptors agonists T0901317 was the same as control group,so we propose that T0901317 may not increase the risk of skeletal muscle resistance of SD rats. Objective To study the correlation between fatty acid translocase （FAT/CD36）gene promoter region -3489C/T and code region 478C/T polymorphism and type 2 diabetes mellitus.Methods -3489C/T and 478C/T polymorphism were screened in 196 type 2 diabetic patients and 119 controls by polymerase chain reaction restriction fragment lenth polymorphism method（PCR-RFLP）.Results（1）All subjects proved to be homozygote wildtypes（CC）for the 478C→T substitution.（2）No significant differences were found in alleles and genotype frequencies of FAT/CD36 gene -3489C/T polymorphism between diabetic patients and non-diabetic contronl.Conclusion FAT/CD36 gene -3489C/T or 478C/T polymprphism was not significantly associated with type 2 diabetes mellitus in Han people of Yunnan.更多还原