Background Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human growth hormone （hGH） has been shown to promote angiogensis and attenuate apoptosis in the experimental an...Background Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human growth hormone （hGH） has been shown to promote angiogensis and attenuate apoptosis in the experimental animal. This study was conducted to explore the effects of myoblast-based hGH gene therapy on heart function restoration and angiogenesis after myocardial infarction, and to compare the differences between myoblast-based hGH gene therapy and myoblast therapy.Methods Myoblasts were isolated from several SD rats, cultured, purified, and transfected with plasmid pLghGHSN and pLgGFPSN. Radioimmunoassay （RIA） was used to detect the expression of hGH in these myoblasts. SD rats underwent the ligation of the left anterior descending coronary artery so as to establish a heart ischemia model. Thirty surviving rats that underwent ligation were randomly divided into 3 equal groups 2 weeks after left coronary artery occlusion： pLghGHSN group received myoblast infected with hGH gene transplantation; pLgGFPSN group received myoblast infected with GFP gene transplantation; control group： received cultured medium only. Four weeks after the injection the surviving rat underwent evaluation of cardiac function by echocardiography. The rats were killed and ventricular samples were undergone immunohistochemistry with hematoxylin-eosin and factor Ⅷ. Cryosection was analyzed by fluorescence microscopy to examine the expression of green fluorescent protein. Reverse transcriptase-polymerase chain reaction （RT-PCR） was used to examine the mRNA expression of vascular endothelial growth factor （VEGF）, bax and Bcl-2. hGH expression in myocardium was examined by Western blot.Results Myoblast can be successfully isolated, cultured and transfected. The expression of hGH in transfected myoblast was demonstrated with RIA. Four weeks after therapy, the cardiac function was improved significantly in pLghGHSN group and pLgGFPSN group. Fractional shortening （FS） and ejection fraction （EF） in pLghGHSN group were 展开更多
Complete skeletal muscle repair and regeneration due to severe large injury or disease is still a challenge.Biochemical cues are critical to control myoblast cell function and can be utilized to develop smart biomater...Complete skeletal muscle repair and regeneration due to severe large injury or disease is still a challenge.Biochemical cues are critical to control myoblast cell function and can be utilized to develop smart biomaterials for skeletal muscle engineering.Citric acid-based biodegradable polymers have received much attention on tissue engineering,however,their regulation on myoblast cell differentiation and mechanism was few investigated.Here,we find that citrate-based polycitrate-polyethylene glycol-polyethylenimine(POCG-PEI600)nanoclusters can significantly enhance the in vitro myoblast proliferation by probably reinforcing the mitochondrial number,promote the myotube formation and full-thickness skeletal muscle regeneration in vivo by activating the myogenic biomarker genes expression of Myod and Mhc.POCG-PEI600 nanoclusters could also promote the phosphorylation of p38 in MAP kinases(MAPK)signaling pathway,which led to the promotion of the myoblast differentiation.The in vivo skeletal muscle loss rat model also confirmed that POCG-PEI600 nanoclusters could significantly improve the angiogenesis,myofibers formation and complete skeletal muscle regeneration.POCG-PEI600 nanocluster could be also biodegraded into small molecules and eliminated in vivo,suggesting their high biocompatibility and biosafety.This study could provide a bioactive biomaterial-based strategy to repair and regenerate skeletal muscle tissue.展开更多
N6-methyladenosine(m6A)represents the most abundantly occurring m RNA modification and is involved in the regulation of skeletal muscle development.However,the status and function of m6A methylation in prenatal myogen...N6-methyladenosine(m6A)represents the most abundantly occurring m RNA modification and is involved in the regulation of skeletal muscle development.However,the status and function of m6A methylation in prenatal myogenesis remains unclear.In this study,we first demonstrated that knockdown of METTL14,an m6A methyltransferase,inhibited the differentiation and promoted the proliferation of C2 C12 myoblast cells.Then,using a refined m6A-specific methylated RNA immunoprecipitation(RIP)with next generation sequencing(Me RIP-seq)method that is optimal for use with samples containing small amounts of RNA,we performed transcriptome-wide m6A profiling for six prenatal skeletal muscle developmental stages spanning two important waves of porcine myogenesis.The results revealed that,along with a continuous decrease in the m RNA expression of the m6A reader protein insulin-like growth factor 2 m RNA-binding protein 1(IGF2 BP1),the m6A methylome underwent highly dynamic changes across different development stages,with most of the affected genes being enriched in pathways related to skeletal muscle development.RNA immunoprecipitation confirmed that IGF2 BP1 targets 76 genes involved in pathways associated with muscle development,including the key marker genes MYH2 and Myo G.Moreover,small interfering RNA(si RNA)-mediated knockdown of IGF2 BP1 induced phenotypic changes in C2C12 myoblasts similar to those observed with knockdown of METTL14.In conclusion,we clarified the dynamics of m6A methylation and identified key genes involved in the regulatory network of porcine skeletal muscle development.展开更多
基金This work was supported by a grant from the National Natural Science Foundation of China （No. 30470457）.
文摘Background Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human growth hormone （hGH） has been shown to promote angiogensis and attenuate apoptosis in the experimental animal. This study was conducted to explore the effects of myoblast-based hGH gene therapy on heart function restoration and angiogenesis after myocardial infarction, and to compare the differences between myoblast-based hGH gene therapy and myoblast therapy.Methods Myoblasts were isolated from several SD rats, cultured, purified, and transfected with plasmid pLghGHSN and pLgGFPSN. Radioimmunoassay （RIA） was used to detect the expression of hGH in these myoblasts. SD rats underwent the ligation of the left anterior descending coronary artery so as to establish a heart ischemia model. Thirty surviving rats that underwent ligation were randomly divided into 3 equal groups 2 weeks after left coronary artery occlusion： pLghGHSN group received myoblast infected with hGH gene transplantation; pLgGFPSN group received myoblast infected with GFP gene transplantation; control group： received cultured medium only. Four weeks after the injection the surviving rat underwent evaluation of cardiac function by echocardiography. The rats were killed and ventricular samples were undergone immunohistochemistry with hematoxylin-eosin and factor Ⅷ. Cryosection was analyzed by fluorescence microscopy to examine the expression of green fluorescent protein. Reverse transcriptase-polymerase chain reaction （RT-PCR） was used to examine the mRNA expression of vascular endothelial growth factor （VEGF）, bax and Bcl-2. hGH expression in myocardium was examined by Western blot.Results Myoblast can be successfully isolated, cultured and transfected. The expression of hGH in transfected myoblast was demonstrated with RIA. Four weeks after therapy, the cardiac function was improved significantly in pLghGHSN group and pLgGFPSN group. Fractional shortening （FS） and ejection fraction （EF） in pLghGHSN group were
基金supported by National Natural Science Foundation of China(Grant No.51872224)Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research,College of Stomatology,Xi’an Jiaotong University(Grant No.2018LHMKFKT004)+1 种基金Special Guidance Funds for the Construction of World-class Universities(disciplines)and Characteristic Development in Central Universities(grant No.PY3A078)the Fundamental Research Funds for the Central Universities(grant No.xzy022019050)。
文摘Complete skeletal muscle repair and regeneration due to severe large injury or disease is still a challenge.Biochemical cues are critical to control myoblast cell function and can be utilized to develop smart biomaterials for skeletal muscle engineering.Citric acid-based biodegradable polymers have received much attention on tissue engineering,however,their regulation on myoblast cell differentiation and mechanism was few investigated.Here,we find that citrate-based polycitrate-polyethylene glycol-polyethylenimine(POCG-PEI600)nanoclusters can significantly enhance the in vitro myoblast proliferation by probably reinforcing the mitochondrial number,promote the myotube formation and full-thickness skeletal muscle regeneration in vivo by activating the myogenic biomarker genes expression of Myod and Mhc.POCG-PEI600 nanoclusters could also promote the phosphorylation of p38 in MAP kinases(MAPK)signaling pathway,which led to the promotion of the myoblast differentiation.The in vivo skeletal muscle loss rat model also confirmed that POCG-PEI600 nanoclusters could significantly improve the angiogenesis,myofibers formation and complete skeletal muscle regeneration.POCG-PEI600 nanocluster could be also biodegraded into small molecules and eliminated in vivo,suggesting their high biocompatibility and biosafety.This study could provide a bioactive biomaterial-based strategy to repair and regenerate skeletal muscle tissue.
基金supported by the Agricultural Science and Technology Innovation ProgramThe Elite Young Scientists Program of CAAS+3 种基金supported by the National Natural Science Foundation of China(31830090)the National Key Project(2016ZX08009003-006)the Shenzhen Dapeng New District Special Fund for Industry Development(KY20180114)the Agricultural Science and Technology Innovation Program(ASTIP-AGIS5)
文摘N6-methyladenosine(m6A)represents the most abundantly occurring m RNA modification and is involved in the regulation of skeletal muscle development.However,the status and function of m6A methylation in prenatal myogenesis remains unclear.In this study,we first demonstrated that knockdown of METTL14,an m6A methyltransferase,inhibited the differentiation and promoted the proliferation of C2 C12 myoblast cells.Then,using a refined m6A-specific methylated RNA immunoprecipitation(RIP)with next generation sequencing(Me RIP-seq)method that is optimal for use with samples containing small amounts of RNA,we performed transcriptome-wide m6A profiling for six prenatal skeletal muscle developmental stages spanning two important waves of porcine myogenesis.The results revealed that,along with a continuous decrease in the m RNA expression of the m6A reader protein insulin-like growth factor 2 m RNA-binding protein 1(IGF2 BP1),the m6A methylome underwent highly dynamic changes across different development stages,with most of the affected genes being enriched in pathways related to skeletal muscle development.RNA immunoprecipitation confirmed that IGF2 BP1 targets 76 genes involved in pathways associated with muscle development,including the key marker genes MYH2 and Myo G.Moreover,small interfering RNA(si RNA)-mediated knockdown of IGF2 BP1 induced phenotypic changes in C2C12 myoblasts similar to those observed with knockdown of METTL14.In conclusion,we clarified the dynamics of m6A methylation and identified key genes involved in the regulatory network of porcine skeletal muscle development.
