| Myogenesis and maintenance of muscle is a complicated and highly regulated process, which is regulated by hormone, nutrition, growth factors, myogenic regulatory factors (MRFs) as well as exercise. These factors interact with each other, and then a regulatory network forms. This thesis would focus on two most important regulatory axes in the network:the positive GH/IGF-1axis and the negative myostatin axis. Immuno-neutralization techniques tagetting these two axes were used to explore their effects on the myogenesis and growth of mice. And the novel immune regulatory functions of myostatin were investigated as well. Here are the results of the thesis:1. Effects of myostatin on the expression of inflammatory cytokine genes and pathways involvedExpression of myostatin was detected in skeletal muscle, fat and spleen of high-fat-diet fed mice, and high expression levels of murine macrophage marker gene, F4/80and CD68, were also detected in fat and spleen of high-fat-diet fed mice.Expression of myostatin was further detected in splenic lymphocyte, and its expression could be induced by LPS, after being treated with LPS, myostatin mRNA increased by259.37%(P=0.082). Further study showed that myostatin could induce the expression of inflammatory cytokine genes from macrophage. Myostatin treatment could increase the expression of TNFa by545.71%(P=0.0011), IL1β by14.97fold (0.039) and1L6by225.48%(0.012). while there was no effect on those genes from dendritic cells. RNAi targeting the ActRIIB receptor could greatly block the expression of those inflammatory cytokines induced by myostatin, the expression of TNFa and IL1β decreased by50.43%(P<0.01) and79.18%(P<0.05). respectively; while RNAi targeting the ActRIIA receptor and the negative control RNAi could not inhibit the expression of these genes. Further exploration of the potential pathways involved in the above mentioned process showed that Smad and JNK pathway inhibitors could greatly block the expression of TNFa induced by myostatin by51.72%(P=0.016) and69.24%(P=0.0023) respectively, while Smad, p38and Erk pathway inhibitors could greatly inhibit the expression of IL1|3induced by myostatin by58.21%(P<0.01),55.64%(P=0.002) and37.8%(P<0.05), respectively. Thus we conclude that myostatin induce the expression of TNFa, IL1β and IL6through the ActRIIB receptor, and it might induce the expression of TNFa through the Smad and JNK pathway and induce the expression of IL1β through Smad, p38and Erk pathway.2. Immno-neutralization of somatostatinFusion genes of mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) fused with somatostatin (SS)(GM-CSF/SS) and cholera toxin B subunit (CTB) fused with SS were constructed, expressed in Escherichia coli and purified as fusion proteins. And female BALB/c mice were actively immunized subcutaneously with these purified fusion proteins, GM-CSF/SS treatment increased the body weight of mice by4.62%compared with the control (P<0.05), together with the induction of detectable serum antibodies against SS. The level of serum growth hormone (GH) elevated by44.54%(P<0.05) and the mRNA expression of muscular IGF-1increased by94%for the GM-CSF/SS-treated mice. Though CTB/SS treatment didn’t increase the body weight of mice as expected, the purified fusion protein, CTB/SS. could polymerize into a biologically active pentamer required for CTB binding to GM1receptors. The recombinant protein CTB/SS was efficient in inducing specific immunity against CTB and SS in mice. The serum growth hormone of CTB/SS-treated mice increased by28.69%(P<0.05) compared with the control. The results indicated that the recombinant protein GM-CSF/SS was efficient in inducing specific immunity against SS, subsequently leading to the increase of the GH level by SS neutralization, and ultimately improving the growth of mice, and this protein might be promising to be used in livestocks. Although CTB/SS could also induce the antibody against SS, the high level of anti-CTB antibody induced at the same time might indirectly inhibit the growth and weight gain of mice.3. Single chain antibody fragment against myostatinIn this work, we first reported the construction, expression and characterization of a single chain variable fragment (scFv) antibody against myostatin. A759bp whole-length scFv was constructed using the splicing overlap extension PCR technique after codon-optimization according to the Escherichia coli favored codons and expressed in Escherichia coli. ELISA detection showed that the scFv could bind to myostatin, and the scFv N-terminal fusion significantly decreased its binding affinity (P<0.05). The scFv could rescue the inhibitory effects of myostatin to myoblast proliferation and viability.8μ.g/ml scFv enhanced the cell viability by31.11%(P<0.01) and55.48%(P<0.01) compared with the pBS and myostatin group, respectively. Cell morphology showed obvious increase in the cell number for the scFv-treated cells. Myostatin increased the expression level of the cyclin-dependent kinase inhibitor p21, and the scFv could antagonize these functions of myostatin. The p21mRNA expression increased by52.4%(P<0.05) when treated with0.4μg/ml myostatin, and pretreatment with8μg/ml scFv significantly decreased the elevated level of p21by22.48%(P<0.05). The scFv could also decrease the expression of E3ubiquitin ligase atrogin-1(MAFbx) and muscle ring finger1(MuRFl) induced by hydrogen dioxide in C2C12myoblast by80.41%(P=0.00013) and57.94%(P=0.049), respectively. It could also decrease the expression of MuRF1induced by hydrogen dioxide in C2C12myotube by33.35%(P=0.014). Finally, the SBE-luciferase activity decreased by57.4%when co-treated with50ng/ml scFv compared with the myostatin (10ng/ml)-treated cells (P<0.05). These results indicated that the scFv might act through the Smad and p21pathway to rescue the myostatin-mediated inhibition of myogenesis and alleviate muscle atrophy through inhibiting the expression of E3ubiquitin ligases. |
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