摘要
大量的研究表明,骨骼肌同其它分泌型器官一样也能产生具有生物学活性的蛋白因子,调节机体的脂代谢和糖代谢。如当机体运动时,骨骼肌收缩能够产生白细胞介素6以调节肝糖元合成,促进脂肪组织脂解。最近人们利用信号捕获(singal sequence trap)技术从1812个克隆中筛选出一种几乎仅在小鼠骨骼肌中表达的具有单一信号序列未知蛋白,被命名为“Musclin”。胰岛素可诱导Musclin mRNA表达而转染Musclin的腺病毒可引起小鼠脂肪组织体积明显下降,且脂质代谢的一个重要转录因子Foxo1可显著下调Musclin mRNA表达,推测Musclin基因在脂代谢中也具有重要的调节作用,提示骨骼肌分泌的Musclin可能是糖、脂肪代谢中的又一个活性因子。为初步揭示Musclin调控生脂的分子机制及最终为瘦肉型猪选育提供理论依据。本试验以猪为研究对象,克隆了猪Musclin cDNA,利用用生物信息学分析探讨了Musclin蛋白的结构和特性,RT-PCR技术研究了猪Musclin mRNA在不同组织、不同月龄、不同品种、以及猪皮下前体脂肪细胞、骨骼肌肌卫星细胞、联合培养细胞的表达规律,并探讨了猪Musclin基因与生脂基因LPL、FAS、PPARγ、TGH以及骨骼肌分化特异性基因Myf5与MyoG mRNA表达的相关性,为初步揭示Musclin调控脂代谢的分子机制及最终为瘦肉型猪选育提供理论依据。结果如下:
1.克隆了374 bp的猪Musclin基因片段,通过对预测的氨基酸序列分析发现,猪Musclin基因序列和小鼠骨骼肌获得Musclin cDNA结构一致都有一个丝氨酸蛋白酶切割位点——KKKR,同源序列比对发现,猪Musclin基因碱基序列与家兔、牛、绵羊和人的同源性达85%以上;而与鸡的同源性只有59%,说明Musclin在进化过程保守性较差。
2.Musclin蛋白属于亲水性不稳定蛋白,以α螺旋为主,中间夹杂一些Extended strand可确定Musclin蛋白属于混合型蛋白。主要集中在分泌途径上,是分泌型蛋白且第1—26位氨基酸序列是一段信号肽,26与27之间存在一个酶切位点。羟基端含有较多的转角结构,同时该区域的抗原指数较高,其亲水性指数和呈现在蛋白表面的可能性也比较大,因此该区段的抗原表位应该是优势抗原表位所在。这为研究Musclin蛋白的免疫功能的研究提供理论依据和一定的指导意义。
3.猪Musclin mRNA在内脏脂肪和皮下脂肪表达丰度最高,脾脏表达量最低(P<0.05);随月龄增长猪肌肉和皮下脂肪组织中Musclin mRNA表达量呈显著下降(P<0.05);瘦肉型猪肌肉和皮下组织Musclin mRNA表达显著高于脂肪型猪(P<0.05)。皮下脂肪组织中该基因mRNA表达分别与FAS、PPARγ呈显著负相关和正相关(P<0.05),与TGH相关性不显著(P>0.05)。因此,猪Musclin基因除在肌肉中表达外,在脂肪等其他组织也能表达;该基因的表达与猪月龄、品种有关,且与生脂基因FAS、PPARγ密切相关,推测该基因可能在脂代谢中起到一定的作用。
4 Musclin在原代脂肪细胞中的表达呈时间依赖性减少;联合培养细胞中Musclin表达显著高于同期单独培养原代脂肪细胞(P < 0.01)。而低于同期单独培养的骨骼肌卫星细胞,第10天的表达量达到显著水平(P >0.01),细胞Musclin mRNA表达与LPL、PPARγ、Myf5、MyoG mRNA表达都有显著相关。推测Musclin参与了脂肪和肌代谢过程,但具体机制较为复杂,有待我们深入的研究。
Accumulating evidences exist that skeletal muscle acts as an endocrine organ modulating hepatic glucose production and white adipose tissue lipolysis. such as production of interleukin-6 (IL-6) during exercise. Recently the people using an efficient signal sequence trap (SST) method from the 1812 cloning technology in almost a screened only in the expression of skeletal muscle in mice with a single signal sequence without cross-domain model of the unknown protein, was named "Musclin". Insulin can induce Musclin mRNA expression and the transfer Musclin Adenovirus can cause mice fat tissue volume decreased significantly, and lipid metabolism in an important transcription factor Foxo1 can significantly lower Musclin mRNA expression, to speculate Musclin gene in lipid metabolism also has important The regulation of, suggesting that skeletal muscle could be Musclin secretion of sugar and fat metabolism in the activity of another factor. Musclin for control of Health revealed the molecular mechanism of fat and lean pig breeding eventually provide a theoretical basis. The present study examined the distrution of pig,we cloned the pig Musclin gene and analyzed structure and characteristics of Musclin by bioinformatics. Levels of Musclin mRNA in different tissues, at different ages ,in adipose muscle tissues of different breeds, primary adipocytes and co-culture cells (adipocyte and muscle satellite cell), muscle satellite cell were measured by Semi Quantitative Reverse Transcription-Polymerase Chain Reaction ( SQ RT-PCR). Correlation were studied between mRNA level of musclin and that of lipogenetic genes including lipoprotein lipase(LPL), fatty acid synthetase (FAS), peroxisome proliferator-activated receptorγ(PPARγ), triacylglycerol hydrolase(TGH).and specificity genes of skeletal muscle including Myf5 and MyoG. It may plays an important role in lipid metabolism. The main results were summarized as following:
1.Successfully cloned 374bp of the pig Musclin gene, Musclin pigs and mice muscle gene sequences were consistent Musclin cDNA structure has a serine protease cleavage point - KKKR, homologous sequences than on that pig Musclin gene sequence and rabbits, cattle, sheep and The identity of more than 85 percent, while the identity of chicken and only 59 percent of that in the course of evolution Musclin conservative poor.
2.Musclin hydrophilic proteins are unstable protein, to the Rulesαspiral and non-curly-based structure, inclusion of some middle Extended strand of protein can be identified Musclin hybrid protein. Mainly concentrated in the secretion of ways, the mitochondria in the small, is secreted protein and 1-26 in the first amino acid sequence is a signal peptide, between 26 and 27 there is a restriction site. The N-terminal No. 3-44 , 81-93 and 111-131 maybe the predominant epitopes of the B cell. This study would be helpful for research of mmunological function of Musclin.
3.The highest of Musclin expression levels were found in visceral adipose tissues and subcutaneous adipose tissues and the lowest expression in spleen among all other tissues(P<0.05); the expression of musclin mRNA in muscle and subcutaneous adipose tissue were decreased significantly during aging (P<0.05); in the same ages, the levels of Musclin mRNA in subcutaneous adipose and muscle of lean-type pig was significantly higher than that of obesity-type pig ( P<0.05); and the express of Musclin was negative correlated and positively correlated with FAS and PPARγrespectively (P<0.05). However,it had no significant correlation with TGH ( P>0.05). So, we concluded that pig Musclin mRNA was expressed in the muscle as well as other tissues such as adipose tissue. The express of porcine Musclin gene was related to age, breed and FAS, PPARγ. it may plays an important role in lipid metabolism
4.Musclin in the primary fat cells of a time-dependent reduction in the cultured cells Musclin expression was significantly higher than the same period in a separate training of primary fat cells (P <0.01). But lower than the same period a separate culture of skeletal muscle satellite cells, the expression of 10 days to achieve significant levels (P> 0.01). Expression of Musclin mRNA in cell had significant correlation with and LPL, PPARγ, Myf5, MyoG. Musclin speculate may be involved in regulating fat synthesis, but we have to further study this mechanism.
引文
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