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 elevated significantly compared with pLgGFPSN group and control group after therapy （FS： 36.9±5.3 vs 29.5±3.5, 21.8±2.9; EF： 56.9±4.3 vs 47.1±3.6, 38.4±4.8, P〈0.05）. Left ventricular end-diastolic dimension （LVEDD） and heart infracted size in pLghGHSN group were decreased significantly compared with pLgGFPSN group and control group after therapy （LVEDD： 5.9±0.3 vs 6.8±0.2, 8.6±0.3; heart infracted size： （34.5±4.2）% vs （40.0±3.9）%, （46.1±3.8）%, P〈0.05）; Green fluorescence was detected in cryosection of pLgGFPSN group. The capillary density of the pLgGFPSN group was significantly greater than those of the pLghGHSN group and control group （P〈0.05）. The mRNA expression of VEGF and Bcl-2/bax in pLghGHSN group was higher than in pLgGFPSN group or control group （P〈0.05）. The expression of hGH clene in myocardium tissue can be detected by Westem blot assay in pLghGHSN group.Conclusions Transplantation of heart cells transfected with hGH induced greater angiogenesis and effect of antiapoptosis than transplantation of cells transfected with GFP. Combined GH gene transfer and cell transplantation provided an effective strategy for improving postinfarction ventricular function.展开更多
Background This study transferred a recombinant gene encoding human insulin like growth factor-1 （hIGF-1） into modified primary skeletal myoblasts with a retroviral vector （pLgXSN） and determined whether the hIGF-...Background This study transferred a recombinant gene encoding human insulin like growth factor-1 （hIGF-1） into modified primary skeletal myoblasts with a retroviral vector （pLgXSN） and determined whether the hIGF-1 promoted growth of skeletal muscle in rat.Methods hlGF-lcDNA was amplified in vitro from normal human liver cells by using RT-PCR and cloned into plasmid vector pLgXSN. The recombinant vector pLghIGF-1SN and control vector pLgGFPSN were transfected into packaging cell PT67 and G418 was used to select positive colony. Myoblasts were infected with a high titre viral supernatant and transduction efficiency was evaluated as GFP expression. The expression of hIGF-1 mRNA in myoblasts was investigated by immunocytochernistry and RT-PCR. MTT assays detected the growth of myoblasts in vitro. Myoblasts transduced with pLghlGF-1SN were injected into hind limb muscles of 10-12 week male SD rats. Formed tissues were harvested 4 weeks later. Myocyte diameter, mean weight of hind limb and body were measured to evaluate the skeletal muscle growth. Results Recombinant retroviral plasmid vector pLghlGF-1SN was constructed successfully. The titre of the packaged recombinant retrovirus was 1 × 106 cfu/ml. The transfection rate of PT67 cells reached 100% after G418 screening, hIGF-1 expression was positive in myoblast-IGF-1. The proliferation rate of myoblast-IGF-1 in vitro was higher than GFP-myoblast or myoblast （P〈 0.05）. The mean weights of hind limb and body of rats injected myoblast-IGF-1 were higher than those of the rats injected with myoblast-GFP or myoblast （P〈 0.05）. Myocyte diameter had a significant increase in IGF-1 group compared to GFP group and myobiast group （P〈 0.05）. Conclusions The transfection of the human IGF- 1 gene mediated by a retroviral vector can promote the growth of skeletal muscle in rats. Genetically modified primary skeletal myoblasts provide a possibly effective approach to treat some skeletal muscle diseases.展开更多
Background Experimental studies and preliminary clinical studies have suggested that growth hormone （GH） treatment may improve cardiovascular parameters in chronic heart failure （CHF）. Recombinant human GH （rhGH...Background Experimental studies and preliminary clinical studies have suggested that growth hormone （GH） treatment may improve cardiovascular parameters in chronic heart failure （CHF）. Recombinant human GH （rhGH） has been delivered by a recombinant protein, by plasmid DNA, and by genetically engineered cells with different pharmacokinetic and physiological properties. The present study aimed to examine a new method for delivery of rhGH using genetically modified bioartificial muscles （BAMs）, and investigate whether the rhGH delivered by this technique improves left ventricular （LV） function in rats with CHE Methods Primary skeletal myoblasts were isolated from several Sprague-Dawley （SD） rats, cultured, purified, and retrovirally transduced to synthesize and secrete human rhGH, and tissue-engineered into implantable BAMs. Ligation of the left coronary artery or sham operation was performed. The rats that underwent ligation were randomly assigned to 2 groups： CHF control group （n=6） and CHF treatment group （n=6）. The CHF control group received non-rhGH-secreting BAM （GFP-BAMs） transplantation, and the CHF treatment group received rhGH-secreting BAM （GH-BAMs） transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups： sham control group （n=6） and sham treatment group （n=6）. The sham control group underwent GFP-BAM transplantation, and the sham treatment group underwent GH-BAM transplantation. GH-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats with ligation of the left coronary artery or sham operation was performed. Eight weeks after the treatment, echocardiography was performed, hGH, insulin-like growth factor-1 （IGF-1） and TNF-a levels in rat serum were measured by radioimmunoassay and ELISA, and then the rats were killed and ventricular samples were subjected to immunohistochemistry. Results Primary rat myoblasts were retrovirally transduced to secrete rhGH and tissue-engineered into implantable BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted 1 to 2 lug of bioactive rhGH per day. When implanted into syngeneic rat, GH-BAMs secreted and delivered rhGH. Eight weeks after therapy, LV ejection fraction （EF） and fractional shortening （FS） were significantly higher in CHF rats treated with GH-BAMs than in those treated with GFP-BAMs （（65.0i-6.5）% vs （48.1±6.8）%, P 〈0.05）, （（41.3±7.4）% VS （26.5±7.1）%, P 〈0.05）. LV end-diastolic dimension （LVEDD） was significantly lower in CHF rats treated with GH-BAM than in CHF rats treated with GFP-BAM （P 〈0.05）. The levels of serum GH and IGF-1 were increased significantly in both CHF and sham rats treated with GH-BAM. The level of serum TNF-α decreased more significantly in the CHF treatment group than in the CHF control group.Conclusions rhGH significantly improves LV function and prevents cardiac remodeling in rats with CHF. Genetically modified tissue-engineered bioartificial muscle provides a method delivering recombinant protein for the treatment of heart failure.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 elevated significantly compared with pLgGFPSN group and control group after therapy （FS： 36.9±5.3 vs 29.5±3.5, 21.8±2.9; EF： 56.9±4.3 vs 47.1±3.6, 38.4±4.8, P〈0.05）. Left ventricular end-diastolic dimension （LVEDD） and heart infracted size in pLghGHSN group were decreased significantly compared with pLgGFPSN group and control group after therapy （LVEDD： 5.9±0.3 vs 6.8±0.2, 8.6±0.3; heart infracted size： （34.5±4.2）% vs （40.0±3.9）%, （46.1±3.8）%, P〈0.05）; Green fluorescence was detected in cryosection of pLgGFPSN group. The capillary density of the pLgGFPSN group was significantly greater than those of the pLghGHSN group and control group （P〈0.05）. The mRNA expression of VEGF and Bcl-2/bax in pLghGHSN group was higher than in pLgGFPSN group or control group （P〈0.05）. The expression of hGH clene in myocardium tissue can be detected by Westem blot assay in pLghGHSN group.Conclusions Transplantation of heart cells transfected with hGH induced greater angiogenesis and effect of antiapoptosis than transplantation of cells transfected with GFP. Combined GH gene transfer and cell transplantation provided an effective strategy for improving postinfarction ventricular function.
基金This work was supported by a grant from the National Natural Science Foundation of China (No.30470457).
文摘Background This study transferred a recombinant gene encoding human insulin like growth factor-1 （hIGF-1） into modified primary skeletal myoblasts with a retroviral vector （pLgXSN） and determined whether the hIGF-1 promoted growth of skeletal muscle in rat.Methods hlGF-lcDNA was amplified in vitro from normal human liver cells by using RT-PCR and cloned into plasmid vector pLgXSN. The recombinant vector pLghIGF-1SN and control vector pLgGFPSN were transfected into packaging cell PT67 and G418 was used to select positive colony. Myoblasts were infected with a high titre viral supernatant and transduction efficiency was evaluated as GFP expression. The expression of hIGF-1 mRNA in myoblasts was investigated by immunocytochernistry and RT-PCR. MTT assays detected the growth of myoblasts in vitro. Myoblasts transduced with pLghlGF-1SN were injected into hind limb muscles of 10-12 week male SD rats. Formed tissues were harvested 4 weeks later. Myocyte diameter, mean weight of hind limb and body were measured to evaluate the skeletal muscle growth. Results Recombinant retroviral plasmid vector pLghlGF-1SN was constructed successfully. The titre of the packaged recombinant retrovirus was 1 × 106 cfu/ml. The transfection rate of PT67 cells reached 100% after G418 screening, hIGF-1 expression was positive in myoblast-IGF-1. The proliferation rate of myoblast-IGF-1 in vitro was higher than GFP-myoblast or myoblast （P〈 0.05）. The mean weights of hind limb and body of rats injected myoblast-IGF-1 were higher than those of the rats injected with myoblast-GFP or myoblast （P〈 0.05）. Myocyte diameter had a significant increase in IGF-1 group compared to GFP group and myobiast group （P〈 0.05）. Conclusions The transfection of the human IGF- 1 gene mediated by a retroviral vector can promote the growth of skeletal muscle in rats. Genetically modified primary skeletal myoblasts provide a possibly effective approach to treat some skeletal muscle diseases.
基金This work was supported by grants from the National High-Technology Research and Development Program of China （863 Program） （No. 2002AA745070）, Shanxi Province Natural Science Foundation （No. 2009011055-4）, and Scientific Research Foundation of High Education Institutions of Shanxi Province, China （No. 200811034）.
文摘Background Experimental studies and preliminary clinical studies have suggested that growth hormone （GH） treatment may improve cardiovascular parameters in chronic heart failure （CHF）. Recombinant human GH （rhGH） has been delivered by a recombinant protein, by plasmid DNA, and by genetically engineered cells with different pharmacokinetic and physiological properties. The present study aimed to examine a new method for delivery of rhGH using genetically modified bioartificial muscles （BAMs）, and investigate whether the rhGH delivered by this technique improves left ventricular （LV） function in rats with CHE Methods Primary skeletal myoblasts were isolated from several Sprague-Dawley （SD） rats, cultured, purified, and retrovirally transduced to synthesize and secrete human rhGH, and tissue-engineered into implantable BAMs. Ligation of the left coronary artery or sham operation was performed. The rats that underwent ligation were randomly assigned to 2 groups： CHF control group （n=6） and CHF treatment group （n=6）. The CHF control group received non-rhGH-secreting BAM （GFP-BAMs） transplantation, and the CHF treatment group received rhGH-secreting BAM （GH-BAMs） transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups： sham control group （n=6） and sham treatment group （n=6）. The sham control group underwent GFP-BAM transplantation, and the sham treatment group underwent GH-BAM transplantation. GH-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats with ligation of the left coronary artery or sham operation was performed. Eight weeks after the treatment, echocardiography was performed, hGH, insulin-like growth factor-1 （IGF-1） and TNF-a levels in rat serum were measured by radioimmunoassay and ELISA, and then the rats were killed and ventricular samples were subjected to immunohistochemistry. Results Primary rat myoblasts were retrovirally transduced to secrete rhGH and tissue-engineered into implantable BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted 1 to 2 lug of bioactive rhGH per day. When implanted into syngeneic rat, GH-BAMs secreted and delivered rhGH. Eight weeks after therapy, LV ejection fraction （EF） and fractional shortening （FS） were significantly higher in CHF rats treated with GH-BAMs than in those treated with GFP-BAMs （（65.0i-6.5）% vs （48.1±6.8）%, P 〈0.05）, （（41.3±7.4）% VS （26.5±7.1）%, P 〈0.05）. LV end-diastolic dimension （LVEDD） was significantly lower in CHF rats treated with GH-BAM than in CHF rats treated with GFP-BAM （P 〈0.05）. The levels of serum GH and IGF-1 were increased significantly in both CHF and sham rats treated with GH-BAM. The level of serum TNF-α decreased more significantly in the CHF treatment group than in the CHF control group.Conclusions rhGH significantly improves LV function and prevents cardiac remodeling in rats with CHF. Genetically modified tissue-engineered bioartificial muscle provides a method delivering recombinant protein for the treatment of heart failure.
