TGF-β/Smad信号通路在Follistatin调节鸭骨骼肌卫星细胞增殖过程中的作用机制

【目的】卵泡抑素(Follistatin)能够调节骨骼肌肥大和脂肪沉积,可促进骨骼肌卫星细胞增殖。拟采用体外重组Follistatin处理增殖期的鸭骨骼肌卫星细胞,阐明TGF-β/Smad信号通路在Follistatin调节鸭骨骼肌卫星细胞增殖过程中的作用机制。【方法】以孵化14 d的鸭胚为试验材料,采用差速贴壁的方法分离骨骼肌卫星细胞,待细胞长到70%—80%时,将培养基换成含有浓度分别为0、1、10、100 ng·m L-1的Follistatin培养基,继续培养36 h后,采用CCK-8检测骨骼肌卫星细胞增殖情况;使用抗pax7抗体染色,DAPI染核,鉴定骨骼肌卫星细胞;采用real-time q PCR方法检测Follistatin对骨骼肌卫星细胞增殖过程中的标记基因PCNA、生肌因子基因Myo D和TGF-β信号通路中TGF-β、Smad2和Smad3的表达的影响。【结果】在倒置显微镜下观察,传代培养12 h鸭骨骼肌细胞一部分未贴壁呈圆形,一部分贴壁呈梭形。24 h后细胞全部贴壁,细胞略有变长。2 d后细胞继续增多,且呈长梭形。3 d后细胞数目增加,个别细胞融合。4 d后细胞数目进一步增加,细胞变粗,个别细胞融合。5 d后有少量细胞开始分化,细胞进一步融合。Pax7免疫荧光染色分析显示,95%以上的细胞中Pax7呈阳性表达;CCK-8检测细胞增殖分析表明,不同浓度的Follistatin处理鸭骨骼肌卫星细胞后,各处理组细胞增殖均显著高于对照组(P<0.01),且10 ng·m L-1 Follistatin处理鸭骨骼肌卫星细胞增殖效果最明显,为最佳处理浓度;与对照组相比,10 ng·m L-1 Follistatin处理组的Myo D基因表达量显著下降(P<0.05),PCNA基因表达量显著升高(P<0.05),Myf5基因表达量显著升高,TGF-β和Smad2基因表达显著升高(P<0.05),且Smad3基因表达量极限著升高(P<0.01);Western blotting检测蛋白表达水平结果表明,与对照组相比,TGF-β、Smad2和Smad3磷酸化水平也显著升高。【结论】10 ng·m L-1 Follistatin能显著促进鸭骨骼肌卫星细胞增殖,这一过程可通过TGF-β/Smad信号通路实现。使用最佳Follistatin处理浓度能够显著促进鸭骨骼肌卫星细胞增殖,该研究为鸭骨骼肌生长发育调控机理研究奠定分子基础。

猪Follistatin cDNA克隆及在大肠杆菌中的表达

提取猪卵巢总RNA,用RT-PCR方法克隆了猪FollistatincDNA的完整开放阅读框,长1038bp。将FollistatincDNA连接到原核表达载体pGEX-4T-3中,转化大肠杆菌BL21(DE3),以IPTG诱导,进行了GST-FS融合蛋白表达。用SDS-PAGE和Western杂交检测,结果显示在63kD处有特异性表达蛋白。

Follistatin-like 1(FSTL1)在单侧输尿管结扎诱导的小鼠肾间质纤维化中表达上调

Follistatin-like 1(FSTL1)是一个可以被TGF-β1诱导产生的分泌型胞外糖蛋白,它在输尿管和肾组织中都有较高表达.缺失FSTL1会导致小鼠先天性输尿管积水和肾盂积水.本研究拟建立单侧输尿管结扎(unilateral ureteral obstruction,UUO)诱导的小鼠肾间质纤维化模型,探讨FSTL1在肾间质纤维化中的表达及其在病理发生过程中的作用.小鼠右侧肾脏行UUO手术,左侧肾脏作为对照,UUO术后14d小鼠被处死.H&E和Masson染色检测肾间质纤维化程度;qRT-PCR和Western blot检测肾脏组织中纤维化特征分子,如α平滑肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原(typeⅠcollagen,COL1)和纤维连接蛋白(fibronectin,Fn),以及FSTL1的表达;免疫组化检测FSTL1表达和分布.UUO损伤的右肾组织表现出严重的间质纤维化,并且胞外基质(COL1,Fn)以及成纤维细胞活化标志物(α-SMA)的mRNA及蛋白表达水平也显著升高.同时发现,FSTL1的mRNA及蛋白表达水平在UUO损伤的右肾组织也显著升高,并且主要集中在肾小管上皮细胞中表达.因此推测,小鼠肾间质纤维化的病理过程可能与FSTL1表达上调有关,但其功能和确切作用机制还需进一步探讨.

肉兔Follistatin基因的表达研究

为了揭示肉兔卵泡抑素(Follistatin)基因对骨骼肌发育的作用,试验采用实时荧光定量PCR(qRT-PCR)方法,以加利福尼亚兔(U兔)和齐卡巨型白兔(G兔)为研究对象,检测了不同品种、不同性别、不同日龄肉兔Follistatin基因在股二头肌中的表达量,测定个体活体重和日增重。结果表明:300日龄时G兔Follistatin基因表达量极显著高于U兔(P<0.01),不同日龄公兔Follistatin基因表达量均高于母兔,35和56日龄公兔股二头肌中Follistatin基因表达量极显著高于母兔(P<0.01),并且Follistatin基因在两个品种肉兔股二头肌中的表达变化趋势较为一致,即随个体日龄增长呈现M型趋势。股二头肌中Follistatin基因发育变化与活体重呈负相关,与日增重的变化呈显著正相关(P<0.05)。说明Follistatin基因在肉兔的骨骼肌发育过程中具有重要的调控作用。

牙鲆Follistatin基因启动子克隆与分析

为研究海水鱼中Follsitatin基因的组织表达特异性,利用PCR方法获得牙鲆Follistatin基因启动子并进行序列分析,用RT-PCR和显微注射方法研究Follistatin基因与肌肉发育的关系.结果表明,所获得的启动子含有USF、E47、MyoD、NF-Y等多种转录因子结合位点;在牙鲆成体组织中,Follistatin基因在体肾、肠、心脏、头肾、脾中有表达,而在肌肉和肝脏中没有表达;显微注射证明Follistatin基因参与了肌肉的早期发育.

牙鲆Follistatin cDNA的克隆与序列分析

为研究海水鱼中Follistatin基因的序列特征,从受精后67 h的牙鲆受精卵中克隆获得牙鲆Follistatin基因cDNA序列,并对其进行序列分析.结果表明,在牙鲆中并没有发现Follistatin基因的可变式剪切现象;牙鲆Follistatin基因cDNA序列全长963 bp.序列分析结果显示:牙鲆与Takifugurubripes的亲缘关系最近.Follistatin蛋白中高度保守的半胱氨酸和甘氨酸残基的生物学功能可能是维持蛋白空间结构的稳定.

广西巴马小型猪卵泡抑素(Follistatin)cDNA的克隆和序列分析

从广西巴马小型猪的卵巢中提取总RNA,并以其为模板,应用逆转录-聚合酶链式反应(RT-PCR),在合成的特异性引物引导下,扩增得到巴马小型猪卵泡抑素(Follistatin)cDNA的全序列,长度为1035bp。该扩增片段经纯化后,连接到pMD18-T载体上扩增。经序列分析结果表明,本研究中克隆的猪卵泡抑素cDNA序列与GeneBank中已报道的家猪卵泡抑素同源性高达99.4%,与奶牛、家鼠、挪威鼠和马等其他物种的卵泡抑素cDNA序列间同源性也大于89%。

水牛Follistatin cDNA克隆及序列分析

卵泡抑素(Follistatin,FS)可通过旁分泌和自分泌的方式抑制促卵泡素(FSH)的分泌,从而影响动物的繁殖机能。本研究拟通过分析水牛的FS cDNA序列来分析其繁殖性能低的可能原因。试验参照GenBank公布的奶牛的FS cDNA序列设计引物,通过RT-PCR技术扩增目的基因,再将其亚克隆到pMD-18T克隆载体进行测序。测序结果显示本试验成功获得了水牛的FS cDNA序列。同源性及进化树分析提示FS在哺乳动物进化上高度保守。研究发现两处氨基酸的突变,这可能影响FS正常的生物学功能发挥,从而影响水牛的繁殖机能。

Transcriptomic analysis elucidates the enhanced skeletal muscle mass, reduced fat accumulation, and metabolically benign liver in human follistatin-344 transgenic pigs

Follistatin(FST) is an important regulator of skeletal muscle growth and adipose deposition through its ability to bind to several members of the transforming growth factor-β(TGF-β) superfamily, and thus may be a good candidate for future animal breeding programs. However, the molecular mechanisms underlying the phenotypic changes have yet to be clarified in pig. We generated transgenic(TG) pigs that express human FST specifically in skeletal muscle tissues and characterized the phenotypic changes compared with the same tissues in wild-type pigs. The TG pigs showed increased skeletal muscle growth, decreased adipose deposition, and improved metabolism status(P<0.05). Transcriptome analysis detected important roles of the PIK3–AKT signaling pathway, calcium-mediated signaling pathway, and amino acid metabolism pathway in FST-induced skeletal muscle hypertrophy, and depot-specific oxidative metabolism changes in psoas major muscle. Furthermore, the lipid metabolism-related process was changed in adipose tissue in the TG pigs. Gene set enrichment analysis revealed that genes related to lipid synthesis, lipid catabolism, and lipid storage were down-regulated(P<0.01) in the TG pigs for subcutaneous fat, whereas genes related to lipid catabolism were significantly up-regulated(P<0.05) in the TG pigs for retroperitoneal fat compared with their expression levels in wild-type pigs. In liver, genes related to the TGF-β signaling pathway were over-represented in the TG pigs, which is consistent with the inhibitory role of FST in regulating TGF-β signaling. Together, these results provide new insights into the molecular mechanisms underlying the phenotypic changes in pig.

Myeloid zinc finger 1(MZF1) is the most important transcriptional factor for porcine follistatin promoter

Follistatin （FS） is a secreted protein, which was originally isolated from porcine follicular fluid. Expression of follistatin is tightly regulated during porcine growth and development. To study the essential transcriptional regions of the porcine FS promoter, ten primer pairs were designed to amplify segments with different lengths of the FS promoter from -1 800 to ＋16 bp. The products were then inserted into the pGL3-basic vector to analyze the relative luciferase activity. The results showed that the most remarkable changes of promoter activity were observed between constructs （-302/＋16 bp）-FS and （-180/＋16 bp）-FS （P〈0.01）. Further research showed that the reconstructed reporter plasmid lacking myeloid zinc finger 1 （MZF1） binding sequence had significantly decreased luciferase activity （P〈0.05）. Furthermore, the FS protein expression was significantly increased in PK15 cells while the MZF1 was overexpressed, suggesting that the short sequence ＂TCCCCACC＂ （the recognition site of transcription factor MZF1） was the most important for FS transcription activation in the porcine.

广西巴马小型猪卵泡抑素(Follistatin)克隆及原核表达

目的通过RT-PCR扩增获得广西巴马小型猪Follistatin的cDNA全序列,经克隆测序分析其cDNA序列结构,进而构建pET-Fo原核表达质粒,使用IPTG诱导表达获得其融合蛋白,为将来进一步研究Follistatin对广西巴马小型猪肌肉生长和繁殖性能的影响奠定基础。方法从广西大学巴马小型猪卵巢中提取总RNA,并以其为模板,应用逆转录-聚合酶链式反应(RT-PCR),在合成的特异性引物引导下,扩增获得广西巴马小型猪卵泡抑素(Follistatin)cDNA的全序列,长度为1 035 bp。将PCR产物克隆到pMD18-T载体后进行序列测定及分析。结果广西巴马小型猪卵泡抑素cDNA序列与GenBank中已报道的家猪卵泡抑素同源性高达99.4%。同时,将目的基因插入到原核表达载体pET 32a+多克隆位点中,构建了Follistatin的原核表达载体,并转化于大肠杆菌BL21。转化菌经异丙基硫代半乳糖苷(IPTG)诱导,变性聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质免疫印迹(Western blotting)分析。结论实验已成功地表达了Follistatin的融合蛋白(约58.4 ku)。

Cloning and expression of follistatin gene in half-smooth tongue sole Cynoglossus semilaevis during the reproduction cycle

Follistatin(FST) is a monomeric glycoprotein highly enriched in cysteines and belongs to TGF-β superfamily. FST can suppress the secretion of follicle-stimulating hormone and plays a vital role in the reproduction of vertebrates. We used rapid amplification of cDNA ends technology to clone the FST gene of half-smooth tongue sole,Cynoglossus semilaevis. We characterized its phylogenetic context and expression patterns to elucidate its function in the breeding season. The full-length sequence of FST is 1 455 bp and encodes a protein of 321 amino acids. We investigated the expression pattern of FST in C. semilaevis at different stages of reproduction using reverse transcription-polymerase chain reaction(RTPCR). FST m RNA was expressed in all 13 tissues analyzed,and was expressed at high levels in gonad and at slightly lower levels in gill and brain. During the reproductive cycle of C. semilaevis,the transcript level of FST was the highest in the perinucleolus stage,decreased in the primary yolk stage,slightly increased in the tertiary yolk stage,and then reduced to a minimal level in the atretic follicles stage of the ovary. We concluded that FST suppressed follicle-stimulating hormone,which stimulated oocyte development. However,no significant variation was observed across all stages of testis development,although the expression level in the spermatogenesis stage was relatively low,which may result from the regulation of FST by aromatase.

Follistatin表达抑制促进BMP-2诱导人类骨髓间充质干细胞成骨分化的实验研究

目的探讨Follistatin表达抑制对人类骨髓间充质干细胞(BMSC)细胞活性及骨形态发生蛋白(BMP)-2诱导人类BMSC成骨分化的影响。方法采用Follistatin特异性siRNA转染人类BMSC,检测线粒体脱氢酶活性、细胞DNA含量及蛋白含量。采用定量逆转录-聚合酶链式反应(qRT-PCR)检测成骨相关基因表达,采用碱性磷酸酶(ALP)染色和茜素红染色检测Follistatin对BMP-2诱导人类BMSC成骨分化的影响。结果转染后3、7 d,人类BMSC代谢、DNA含量显著升高;转染后14 d,总蛋白含量增加。Follistatin表达抑制后成骨相关基因如ALP、OCN、OSX、OC、OPN、RUNX2等表达升高,且ALP活性及钙沉积量显著增加。结论抑制Follistatin表达可促进人类BMSC细胞活性和BMP-2诱导人类BMSC成骨分化能力,Follistatin可抑制人类BMSC成骨分化。

绵羊Follistatin基因不同功能区片段的原核表达与纯化

为了研究绵羊Follistatin基因的功能及与MSTN的结合能力,克隆了FS结构域1及结构域2,构建了405 bp插入序列的原核表达重组质粒pET-FS N+D1,以及630 bp插入序列的pET-FSN+D1+D2。通过SDS-PAGE及Western blot检验,在IPTG诱导表达。结果显示:分别获得了带有6×His的45 kDa和51 kDa融合蛋白,融合蛋白主要以包涵体形式存在。经His亲和层析分别纯化了pET-FSN+D1,pET-FSN+D1+D2,目的蛋白含量分别达2.58μg/mL和5.98μg/mL,为进一步研究Follistatin基因的功能及与MSTN的结合能力提供了基础。

Lentivirus-Mediated Short Hairpin RNA for Follistatin Downregulation Suppresses Tumor Progression in Hypopharyngeal Carcinoma

Objective:Follistatin(FST)inhibits the action of activin by interfering with the binding of activin to its receptor.Although the prognostic value of FST in various cancers has been investigated previously,studies rarely focused on hypopharyngeal carcinoma(HPC).In our study,the effect of FST expression on HPC tissues and cell lines was investigated.Methods:A total of 60 patients with HPC were recruited for this study.Levels of FST mRNA and protein were measured by quantitative polymerase chain reaction(PCR)and immunohistochemistry in HPC tissue samples and by qPCR in the HPC FaDu cells,as well as immortal nasopharyngeal epithelial cell line NP-69 cells.After silencing the FST expression in FaDu cells using lentivirus-mediated siRNA that was specific for FST mRNA,cell proliferation was determined by a Celigo assay.Tumor growth was monitored in nude mice and viability was determined by a methylthiazoletetrazolium assay.The ratio of cell cycle arrest and apoptosis was evaluated by flow cytometry.The colony formation ability was performed using Giemsa staining.In addition,wound healing and Transwell migration and invasion assays were performed for the analysis of cell motility.Results:FST expression was significantly higher in human HPC tissue and FaDu cells than in normal tissue and NP-69 cells.A higher expression of FST in HPC samples was positively correlated with advanced tumors.Moreover,FST knockdown by shRNA significantly decreased cell growth,colony formation,migration and invasion.Furthermore,FST silencing increased the cell apoptosis percentage,and arrested cell cycle in the S phase in FaDu cells.In addition,FST silencing suppressed tumor growth in vivo.Conclusions:Our results indicated that the FST gene was associated with HPC progression and may serve as a potential therapeutic target for the treatment of HPC.

Methylation Status of the Follistatin Gene at Different Development Stages of Japanese Flounder(Paralichthys olivaceus)

Follistatin （Fst） is a hyperplasia factor that plays a crucial role in muscle development. DNA methylation, a significant process, regulates gene expression. The aim of our study is to examine the DNA methylation and expression patterns ofFst gene at five different development stages of Japanese flounder （stage A, 7 dph; stage B, 90 dph; stage C, about 180 dph; stage D, about 24 months; stage E, about 36 months）. The muscle tissue of Japanese flounder was obtained at different development stages in dais experiment. DNA methylation levels in the promoter and exon 2 of Fst were determined by bisulfite sequencing, and the relative expression of the Fst gene at the five stages was measured by quantitative PCR. The results showed that the lowest methylation level was at stage A and the highest methylation level was at stage B. Moreover, the highest expression level of the Fst gene was observed at stage A. The mRNA abundance was negatively correlated with DNA methylation level. Three CpG islands in the promoter region and three CpG islands in exon 2 of Fst were found in the binding sequence of the putative transcription factor. These results offered a theoretical basis for the mechanism of Fst gene regulation to muscle development at different development stages.

Effects of follistatin on testosterone secretion of rat Leydig cell in vitro

Objective: To investigate the effects of follistatin(rhFS-288) on biosynthesis andsecretion of testosterone in rat Leydig cell in vitro.Methods: Leydig cells were isolated from Wistar rat testes by a discontinuous Per-coll gradient procedure. Purified cells were incubated in 24-well plate(105 cell/ml/well)and maintained for 24 h in a CO2 incubator. rhFS-288 and Ca2+ were added to the wellsindependently or jointly in both baseline (without hCG) and stimulation condition (1.0IU/ml of hCG) to observe the change of testosterone concentration in the media.Results: rhFS-288 showed a dose-dependent inhibiting effect on testosterone releasein baseline and stimulating condition. Ca2+ presented inhibitory effect either. Whereas,escape phenomenon emerged while Ca2+ concentration reached to 100 mmol/L. A com-bination of rhFS-288 with Ca2+ displayed a dose-dependent inhibition on testosterone se-cretion.Conclusion: rhFS-288 inhibits testosterone secretion in a dose-dependent manner.Calcium is thought to be the second messenger of FS action. The mechanism of escapephenomenon during high dose of Ca2+ along is unknown.

Follistatin, a Novel Biomarker for Malignant Gliomas

Molecular biomarkers are commonly used for the management of several types of malignant tumours in routine clinical practice. However, this is not the case for malignant gliomas. Cytokines and Angiogenesis factors are potential candidates due to their intrinsic role in tumourigenesis. Pre- and post-operative serum from 36 malignant glioma patients and 36 controls was analysed using the Bio-Plex Pro Angiogenesis and Cytokines Assay (Bio-Rad, USA). Amongst the molecules tested, the serum concentration of follistatin was significantly higher in patients than in controls. Moreover, the serum concentration of follistatin of the patients postoperatively was significantly reduced compared to that preoperatively. Factors such as age and gender did not affect the concentrations of follistatin measured in the serum of patients pre- and post-operatively as well as healthy controls. This is the first report of follistatin as potential biomarker for the detection of malignant gliomas.

Establishment of a Technique to PCR Detection about Follistatin Transgenic Sheep by Injection of Lentivirus

[Objective] The aim was to establish a stable detection method of lentivirus transgenic sheep at DNA level.[Method] The cotyledons,umbilical cord,tail tissue of newborn transgenic lambs and the body tissues of dead lambs were collected and used to extract DNA for PCR with primers designed for N＋D1 fragment of follistatin gene.At the same time,we detected the CMV promoter,5＇-LTR and so many other structure elements of lentiviral vector.The body tissues of dead lambs and muscle tissues of transgenic lambs in vivo were used to extract RNA for RT-PCR.[Result]The results showed that the DNA Extraction Kit was faster and more efficient than conventional method in extracting DNA and the DNA extracted with kit was easier to be amplified than that with conventional method.In order to avoid false positive caused by the interference of endogenous gene,the primers were designed for amplifying the combination of upstream of vector gene and downstream of target gene,increasing the specificity of detection.Tail tissue of newborn transgenic lambs could be used for detection and the detected results were reliable and accurate.The detection of CMV promoter,5＇-LTR and so many other structure elements of lentiviral vector provided a data support for the biological safety of transgenic animals and verify the detected results of target gene of transgenic lambs.The transcription products of RNA extracted from three of the lambs were not detected.[Conclusion] The PCR method established in our research for detecting transgenic sheep was efficient,fast and accurate.It would provide experimental basis for further detection at protein level,lay a foundation for the establishment of multi-level and systematic detection method of transgenic sheep and provide a stable technology platform for safety monitoring of transgenic sheep.

大黄鱼Follistatin基因克隆及表达分析

卵泡抑素(follistatin,FST)是转化生长因子β(Transforming growth factor-β)超家族成员之一,在动物肌肉生长中起重要作用。采用RT-PCR、RACE和常规PCR技术克隆了大黄鱼FST基因。获得的基因序列长3195 bp,其中5'非编码区96 bp,3'非编码区47 bp,含5个外显子及4个内含子。该基因开放阅读框972 bp,编码323个氨基酸,其中信号肽31个氨基酸,成熟肽292个氨基酸。Blast结果表明,大黄鱼FST基因与金鲷FST基因的核苷酸及蛋白序列同源性最高,分别达到96%和99%。FST基因在大黄鱼脑、眼、鳃、肾等多个组织中表达,其中鳃组织的表达量最高,脾组织中表达最低。检测水温19℃、25℃、30℃时大黄鱼不同组织FST基因表达量,眼和肌肉组织中FST基因表达量变化显著,推测FST基因可能在鱼类生长发育中发挥重要作用。