Identification of two SNPs in myostatin (MSTN) gene of Takifugu rubripes and their association with growth traits

The myostatin (MSTN) is a member of transforming growth factor-β superfamily which inhibits muscle growth. In this study, the genomic DNA sequence of MSTN gene was cloned from Takifugu rubripes (T. rubripes). Two polymorphisms of the MSTN gene were detected by polymerase chain reaction-single strand conformation polymorphism (PCR–SSCP) and DNA sequencing methods in 296 T. rubripes. One A748G locates in exon 2 and the other, C1197T, in intron 2. Analysis showed that the A748G mutation caused an amino acid change from Thr to Ala (Ala166Glu). These two SNPs showed a low degree of linkage disequilibrium and four haplotypes were identified. The most frequent haplotype was AC, which occurred at a frequency of 44.3%. Association analyses between these two SNPs and growth traits showed that the individuals with genotype CT and TT of the mutation C1197T had significantly higher body mass, body length and body height than those with genotype CC (P < 0.05). These results show that MSTN gene can be utilized as a candidate gene for molecular marker-assisted breeding of T. rubripes.     

黄河裸裂尻鱼MSTN基因RNA干扰研究

目的 利用反义寡合甘酸技术抑制肌肉生长抑制素（myostatin, MSTN）的表达,评估MSTN基因的沉默效果,并检测RNA干扰后对下游基因的影响。方法 通过构建黄河裸裂尻鱼（Schizopygopsis pylzovi）MSTN基因RNA干扰（RNAi）重组腺病毒载体1P3（DSP MSTN 273+250+1737）和1P2（DSP MSTN 195+1670）,并将其注射黄河裸裂尻鱼肌肉组织,进行活体RNA干扰;利用Real-time PCR和Western-blotting评估MSTN基因的沉默效果,并检测MSTN基因RNA干扰后肌肉肌酸激酶（muscle-type creatine kinase, M-CK）基因转录水平的调控作用。结果 Real-time PCR分析结果表明,与HK组（病毒通用阴性对照组）和N组（空白对照组）相比,重组腺病毒载体1P3对黄河裸裂尻鱼肌肉MSTN基因的转录具有明显的干扰作用（P<0.05）,抑制率达53.5%;而重组腺病毒载体1P2对MSTN基因的转录无明显干扰作用（P>0.05）。Western-blotting分析结果与Real-time PCR结果相一致。同时,经1P3干扰后随着MSTN基因转录水平的下降,其肌肉肌酸激酶M-CK基因表达水平显著上升。结论 通过RNAi技术能够有效的抑制MSTN基因的表达,并能够上调M-CK基因的表达量。因此,证明了在黄河裸裂尻鱼中MSTN能够抑制M-CK的转录。揭示高原土著鱼类MSTN 基因的对肌肉的生长发育起到调控作用。     

生长抑制素14肽治疗肝硬化门脉高压并发食管胃底静脉曲张破裂出血的临床研究

目的探讨生长抑制素14肽治疗肝硬化门脉高压合并食管胃底静脉曲张破裂出血的临床效果。方法选取2010年4月至2013年2月在我院诊治的肝硬化门脉高压合并食管胃底静脉曲张破裂出血患者76例,按照随机数字表法随机分为对照组(n=38)和观察组(n=38)。两组患者进行常规基础治疗,对照组给予垂体后叶素治疗,观察组给予生长抑制素14肽治疗,比较两组的临床疗效及不良反应。结果观察组的总有效率(92.11%)显著高于对照组(73.68%),两组比较差异具有统计学意义(P<0.05),两组不良反应比较差异无统计学意义。结论生长素抑制素14肽治疗肝硬化门脉高压并发食管胃底静脉曲张破裂出血疗效显著,并且不良反应少,在临床上值得推广运用。     

大鼠Myostatin蛋白成熟肽的原核表达及纯化

目的:利用谷胱甘肽S-转移酶(GST)融合蛋白表达系统在大肠杆菌中表达纯化Myostatin成熟肽。方法:应用RT-PCR从大鼠的腓肠肌mRNA中逆转录扩增Myostatin成熟肽编码序列,克隆入原核表达载体pGEX-4T1中,IPTG诱导其在大肠杆菌BL21(DE3)中表达,采用Western-blot用抗-GST抗体验证融合蛋白表达。融合蛋白经Glutathione-agarose色谱纯化。结果:PCR扩增的Myostatin基因序列与GenBank数据库中的序列一致;30°C,0.1mmol/LIPTG诱导16小时,GST-Myostatin融合蛋白获得优化表达,表达效率为25%;Western-blot证实融合蛋白的特异性表达。结论:成功构建融合蛋白GST-Myostatin重组基因,并在大肠杆菌中获得有效表达,为Myostatin抗体的制备及进一步研究该蛋白的功能奠定基础。     

Endoplasmic reticulum stress induces myostatin precursor protein and NF-kappaB in cultured human muscle fibers: relevance to inclusion body myositis

Sporadic-inclusion body myositis (s-IBM) is the most common progressive muscle disease of older persons. It leads to pronounced muscle fiber atrophy and weakness, and there is no successful treatment. We have previously shown that myostatin precursor protein (MstnPP) and myostatin (Mstn) dimer are increased in biopsied s-IBM muscle fibers, and proposed that MstnPP/Mstn increase may contribute to muscle fiber atrophy and weakness in s-IBM patients. Mstn is known to be a negative regulator of muscle fiber mass. It is synthesized as MstnPP, which undergoes posttranslational processing in the muscle fiber to produce mature, active Mstn. To explore possible mechanisms involved in Mstn abnormalities in s-IBM, in the present study we utilized primary cultures of normal human muscle fibers and experimentally modified the intracellular micro-environment to induce endoplasmic-reticulum (ER)-stress, thereby mimicking an important aspect of the s-IBM muscle fiber milieu. ER stress was induced by treating well-differentiated cultured muscle fibers with either tunicamycin or thapsigargin, both well-established ER stress inducers. Our results indicate for the first time that the ER stress significantly increased MstnPP mRNA and protein. The results also suggest that in our system ER stress activates NF-kappaB, and we suggest that MstnPP increase occurred through the ER-stress-activated NF-kappaB. We therefore propose a novel mechanism leading to the Mstn increase in s-IBM. Accordingly, interfering with pathways inducing ER stress, NF-kappaB activation or its action on the MstnPP gene promoter might prevent Mstn increase and provide a new therapeutic approach for s-IBM and, possibly, for muscle atrophy in other neuromuscular diseases.     

A soluble activin type IIB receptor improves function in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurologic disease characterized by progressive weakness that results in death within a few years of onset by respiratory failure. Myostatin is a member of the TGF-β superfamily that is predominantly expressed in muscle and acts as a negative regulator of muscle growth. Attenuating myostatin has previously been shown to produce increased muscle mass and strength in normal and disease animal models. In this study, a mouse model of ALS (SOD1^G93A transgenic mice) was treated with a soluble activin receptor, type IIB (ActRIIB.mFc) which is a putative endogenous signaling receptor for myostatin in addition to other ligands of the TGF-β superfamily. ActRIIB.mFc treatment produces a delay in the onset of weakness, an increase in body weight and grip strength, and an enlargement of muscle size whether initiated pre-symptomatically or after symptom onset. Treatment with ActRIIB.mFc did not increase survival or neuromuscular junction innervation in SOD1^G93A transgenic mice. Pharmacologic treatment with ActRIIB.mFc was superior in all measurements to genetic deletion of myostatin in SOD1^G93A transgenic mice. The improved function of SOD1^G93A transgenic mice following treatment with ActRIIB.mFc is encouraging for the development of TGF-β pathway inhibitors to increase muscle strength in patients with ALS.     

Impact of repeated bouts of eccentric exercise on myogenic gene expression

Evidence indicates that repeated-bouts of eccentric exercise (EE) do not exacerbate the extent of muscle damage indices, as compared to a single-bout. We hypothesized that molecular adaptations, under repeated-bouts of EE, would include suppression of muscle repair inhibitory factors such as myostatin and up-regulation of muscle repair positive regulatory factors such as myogenic regulatory factors (MRFs). Fifteen males were recruited for this study. The exercise group (n = 9) successfully completed six sets of 15 reps of maximum voluntary eccentric contractions, for six consecutive days, using a dynamometer (Multicont-II). Blood and muscle biopsy samples were obtained from each subject 1 week prior to exercise, 2 days post the first training session, and 24 h after the last training session. Gene expression levels were determined using real-time RT-PCR. Blood samples were analyzed for creatine kinase (CK) and lactate-dehydrogenase (LDH) activity. Repeated-bouts of EE induced a large down-regulation of myostatin mRNA (−73%) which persisted throughout the study. The responses of MRFs were mild. At day 3 only myogenin increased significantly (1.9 fold) while MyoD decreased by 45%. Surprisingly, at day 7, despite the presence of muscle damage indices, all MRFs returned to the pre-exercise levels. The results of the present study showed that repeated-bouts of EE, for six consecutive days, dramatically decreased Myostatin mRNA expression but impaired the expression patterns of MRFs such that, with the exception of myogenin that showed a moderate non-sustained increase, MyoD and MYf5 response was minimal. Grants: Funding was provided by the Ministry of Health of the Hungarian Government. Grant number: ETT 388/2003.