研究背景：细胞外基质（ECM）是一个由非细胞成分组成的大型网络结构，包裹、支撑并维持体内组织和器官的相应结构。ECM不仅为组织和器官提供结构支撑，还通过协助细胞附着和与邻近细胞交流，为组织形态发生、分化和同源性提供生化和生物力学支持。然而，ECM的病理性积累可以引起细胞纤维化，进而导致器官和组织的功能衰竭。有很多种TGF-β超家族成员都参与了各种器官和组织的细胞外基质的调控，比如TGF-βs家族（TGF-β 1,2,3）， BMPs 家族（BMP2, 4,6和7）和GDFs 家族（GDF2, 8和11）。其中，TGF-β家族是调节成纤维细胞活性和胶原蛋白表达的重要细胞因子。近年来，由于肌生长抑制素（MSTN）与TGF-β共享相同的跨膜受体和胞内信号通路，人们对其在调节成纤维细胞中的作用越来越感兴趣。作为TGF-β超家族的成员之一，肌生长抑制素（MSTN）可以负调控肌肉的生长发育。此外，最近的研究表明，肌生成抑制素可以直接调节骨骼肌成纤维细胞的胶原蛋白表达。然而，在跟腱和心脏组织中，肌生成抑制素的是否具有促纤维化的作用仍有待阐明。随着基因修饰的发展，利用猪作为大型动物模型，科学家们在生物医学研究方面取得了许多令人瞩目的成就。由于在器官大小、器官解剖学和同源性方面与人类相似，猪被认为是首选的非啮齿动物模型。因此，本研究以MSTN基因缺失猪作为动物模型，探索MSTN基因缺失对肌腱和心脏的影响。

1、PCR-RFLP方法在MSTN基因缺失猪后代的基因型鉴定中的应用

在之前的研究中，我们已经使用转录激活因子效应物核酸酶(TALEN)和体细胞核转移(SCNT)技术来生产MSTN 基因敲除猪。在目前的研究中，我们试图通过这些MSTN敲除猪与野生型（WT）猪交配生产MSTN突变猪。基因修饰动物的子代一般通过基因测序的方式进行基因型鉴定，这种鉴定方式价格昂贵且耗时。为了克服这个问题，我们开发了限制性内切酶介导的PCR-RFLP检测MSTN 基因缺失猪基因分型的方法。在此过程中，根据MSTN基因缺失猪的突变序列推导出保守寡核苷酸引物和酶切位点。利用PCR技术扩增纯合子MSTN突变体（MSTN^-/-）、杂合子MSTN突变体（MSTN^+/-）和WT猪的MSTN基因，产生一条长度为167 bp片段。经过BstNI酶处理后，MSTN^+/-样品产生两个片段，分别在167bp和86 bp位置，WT样品仅在86 bp的位置上有一个条带。由于MSTN^-/-样品中MSTN基因转录本不受BstNI酶的作用，仅在167 bp的位置上形成条带。因此，我们可以使用BstNI限制性内切酶介导的PCR-RFLP方法确定所有猪的基因型。总而言之，本研究为建立了一种简单、快速的PCR-RFLP基因分型方法，用于MSTN 基因缺失猪育种过程中仔猪基因型的鉴定。

2、MSTN基因缺失降低肌腱中的胶原蛋白表达，导致猪踮脚站立

本研究选取2日龄雄性36头MSTN^-/-和30头WT猪，用于研究MSTN基因在肌腱生长发育中的作用。研究发现MSTN^-/-猪中有69.4%的猪出现一种与肌腱胶原蛋白相关的踮脚站立的病理特征，而WT猪未发现这种病理特征。与WT猪相比，MSTN^-/-猪的肌腱与体重比值显著降低（p<0.05）。MSTN^-/-肌腱的卷曲长度显著长于野生型猪（p<0.05）。在肌腱中检测了MSTN和受体（ACVRIIB）的表达。MSTN^-/-猪体肌腱中I型胶原（Col1A）（p<0.05）和巩膜炎因子（Scleraxis, Scx）（p<0.05）表达水平明显低于WT猪，而cyclin依赖性激酶抑制剂1（p21）表达水平较高（p<0.05）。且MSTN^-/-肌腱中Smad2/3的磷酸化水平明显低于WT（p<0.05）。体外实验结果显示：MSTN处理显著增加了肌腱成纤维细胞中Smad2/3的磷酸化（p<0.05）；此外，MSTN处理增加Col1A（p<0.05）和Scx的表达（p<0.01），降低p21的表达（p<0.001）；并且MSTN处理后成纤维细胞增殖能力显著提升（p<0.05）。结果表明，MSTN可调节肌腱成纤维细胞中胶原蛋白的表达和细胞增殖；且MSTN基因缺失导致MSTN^-/-猪发生胶原相关的病理性踮脚站立。

3、MSTN基因缺失降低猪心脏胶原蛋白表达

本研究选取6月龄的雄性MSTN^-/-和WT猪（各3头）用于研究MSTN基因缺失对心脏ECM的影响及其潜在机制。并通过MSTN活性蛋白体外处理心脏成纤维细胞，研究MSTN对胶原蛋白表达的影响。在WT心脏中检测到MSTN蛋白，而在MSTN^-/-心脏中未检测到MSTN蛋白的表达。与WT猪相比，MSTN^-/-猪的心重比显著降低（p<0.05）。天狼猩红染色（p<0.01）、免疫荧光染色（p<0.01）和超显微结构检查（p<0.001）等形态学分析结果显示，与WT相比，MSTN^-/-心脏结缔组织含量显著减少。与WT猪相比，MSTN^-/-猪的心脏中羟脯氨酸含量（p<0.01），I型胶原蛋白（Col1A）含量（p<0.05）及p-Smad3/Smad3水平（p<0.05）显著降低。外源性MSTN蛋白处理可显著提高心脏成纤维细胞中Col1A的表达量（p<0.05），并激活细胞Smad（p<0.05）和AKT（p<0.05）信号通路，与体外研究结果相一致。

结论：本研究首次建立了MSTN 基因缺失猪的PCR-RFLP鉴定方法，并将其应用于MSTN 基因缺失猪的育种中。借助MSTN基因缺失猪的动物模型，本研究证实了MSTN可以通过调节肌腱和心脏的Smad信号直接调节ECM含量。

Background: The extracellular matrix (ECM) is a large network of non-cellular components that surround, support, and give the structure of all tissues and organs in the body. The ECM provides not only structural support for tissues and organs but also provides biochemical and biomechanical support for tissue morphogenesis, differentiation and homeostasis by assisting cells to attach to and communicate with nearby cells. However, pathologic accumulation of the ECM causes fibrosis, which leads to a functional failure of the organs and tissues. Variety of TGF-β superfamily members such as TGF-βs (TGF-β1, 2, and 3), BMPs (BMP2, 4, 6, and 7), and GDFs (GDF2, 8, and 11) are known to participate in regulating extracellular matrix in various organs and tissues. Among them, TGF-β is the well-known cytokine that regulates fibroblast activity and collagen expression. Recently, there is growing interest in the role of myostatin (MSTN) in regulating fibroblast since it shares the same transmembrane receptors and intracellular signaling pathway with TGF-β. MSTN is a member of the TGF-β superfamily and negatively regulates muscle growth. Moreover, recent studies revealed that MSTN directly regulates the collagen expression of skeletal muscle fibroblasts. However, the profibrotic role of MSTN in other organs and tissues is unclear and requires more study.

With the development of genome editing technology, a growing number of efforts are being made to employ pigs as a large animal model for biomedical research. Pigs are considered the preferred nonrodent model because of their similarities in size, organ anatomy, and homology to humans. Thus, the present study produced MSTN mutant pigs and applied them in tendon and heart research.

Application of PCR-RFLP method in MSTN mutant pig production

In the previous study, we already produced MSTN knockout pigs using transcription activator-like effector nucleases (TALEN) mediated gene editing and somatic cell nuclear transfer (SCNT) mediated pig production. In the present study, we were trying to produce MSTN mutant pigs by mating these MSTN knockout pigs with WT pigs. However, the identification of gene mutated animals is labor-intensive and costly in mutated animal breeding. The identification of these mutations is more technically demanding and time-consuming than the generation of the animals themselves. To overcome this problem, we developed the restriction enzyme-mediated PCR-RFLP assay for MSTN mutant pig genotyping. To accomplish this, conserved oligonucleotide primer and restriction site were deduced according to the mutated sequence of the MSTN mutant pigs. PCR amplification yielded a 167 bp band for all homozygous MSTN mutant (MSTN^-/-), heterozygous MSTN mutant (MSTN^+/-), and wild-type (WT) pigs. However, MSTN^+/- samples produced two fragments with 167bp and 86 bp, and WT samples produced one fragment with 86 bp after being digested by BstNI. MSTN^-/- samples were not digested by BstNI and yielded a 167 bp band. Thus, we were able to determine the genotype of all pigs using BstNI restriction enzyme-mediated PCR-RFLP method. Overall, the present study established a simple and fast PCR-RFLP genotyping method and applied it in MSTN mutant pig production.

MSTN deficiency decreases collagen expression in tendon and causes tippy-toe standing in pigs

In order to investigate the role of MSTN in tendon, 36 MSTN^-/- and 30 WT male pigs were used in tendon research at 2-day-old age, respectively. 69.4% of the MSTN^-/- pigs showed the tippy-toe standing, which is known as a collagen related pathological feature in tendon, while no tippy-toe standing was detected in WT pigs. The tendon to body weight ratio was significantly decreased in MSTN^-/- pigs compared to WT pigs (p<0.05). The crimp length of the MSTN^-/- tendon was significantly longer than that of WT pigs (p<0.05). The expression of MSTN and the activin receptor type IIB (ACVRIIB) was detected in the tendon. Type I collagen (Col1A) and Scleraxis (Scx) expression levels in tendon were significantly lower than those in WT in vivo (p<0.05, respectively), whereas cyclin-dependent kinase inhibitor 1 (p21) expression level was higher (p<0.05). Phosphorylation of Smad2/3 was significantly lower in MSTN^-/- tendon compared with that of WT tendon in vivo (p<0.05). MSTN treatment significantly increased the phosphorylation of Smad2/3 in tendon fibroblasts (p<0.05). Moreover, the MSTN treatment increased Col1A and Scx (p<0.05, p<0.01, respectively) and decreased p21 expression in vitro (p<0.001). There was a significant increase in fibroblast proliferation after MSTN treatment (p<0.05). The results indicated that MSTN regulates collagen expression and proliferation in tendon fibroblasts; thus, MSTN deficiency causes collagen related pathological tippy-toe standing in MSTN^-/- pigs.

MSTN deficiency decreases collagen expression in pig heart

We investigated whether loss of MSTN affects the cardiac ECM in pigs. Three male MSTN^-/- and WT pigs were used in heart research at 6-month-old age, respectively. Cardiac ECM and underlying mechanisms were determined post-mortem. The role of MSTN on collagen expression was investigated by treating cardiac fibroblasts with active MSTN protein in vitro. MSTN protein was detected in WT hearts, while no expression was detected in MSTN^-/- hearts. The heart-to-body weight ratio was significantly decreased in MSTN^-/-pigs (p<0.05). The morphometric analysis, including picrosirius red staining (p<0.01), immunofluorescent staining (p<0.01), and ultra-structural thickness examination of the endomysium (p<0.001), revealed a significant reduction of connective tissue content in MSTN^-/- hearts compared to WT. Hydroxyproline (p<0.01), Col1A (p<0.05), and p-Smad3/Smad3 levels (p<0.05) were significantly lower in MSTN^-/- hearts in vivo. Consistently, cardiac fibroblasts treated with exogenous MSTN protein overexpressed Col1A (p<0.05) and activated Smad (p<0.05) and AKT signaling pathways (p<0.05) in vitro. The present study suggests that inhibition of MSTN decreases cardiac ECM.

Conclusion: The present study firstly established PCR-RFLP method to identify MSTN mutant pigs and applied it in MSTN mutant pig production. Using these MSTN pigs as a large animal model, we revealed that MSTN directly regulate the ECM content through modulating Smad signaling in tendon and heart.

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