Tissue-Engineered Bioartificial Muscles Expressing A Foreign Recombinant Protein For Gene Therapy

Part one Study on construction of recombinant vector carrying human growth hormone geneObjective: To construct a recombinant vector carrying human growth hormone (hGH) gene. To set the stage for achieving efficient and stable expression of hGH gene in primary rat skeletal myoblasts.Methods: (1)The plasmid containing hGH fragment was cleaved by restriction enzyme digestion,and the resultant fragment hGHcDNA was inserted directionally into pcDNA3.0.(2)A pair of primers were designed and synthesized according to human hGH cDNA sequences. pZ12l-hGH-2 was used as a template of PCR reaction. (3)hGH cDNA was subcloned into the vector pLenti6/v5-D-TOPO and pLgXSN.(4)The recombinant vector was identified by restriction endonuclease analysis and DNA sequence analysis.Results Restriction enzyme and DNA sequencing analysis revealed that hGH gene was correctly inserted into the blank vector pcDNA3.0,pLenti6/v5-D-TOP and pLgXSN.Conclusions The recombinant vector pcDNA-hGH,PLghGHSN and pLenti6/V5 -hGH were constructed successfully. Part two Tissue-Engineered Bioartificial muscles Secrete and delivere recombinant human growth hormoneObjective To achieve efficient and stable expression of human growth hormone ( hGH) gene in primary rat skeletal myoblasts. Genetically modified myoblast with hGH will be tissue-engineered into BAM which delivere recombinant protein.Methods (1)To establish a practical method of isolation and culture of primary rat skeletal myoblasts. (2)Using a cationic polymer Sofast, primary rat myoblasts were transfected by pcDNA-hGH. hGH protein levels in culture medium from the myoblasts were measured with radioimmunoassay.(3)The plasmid pLenti6/V5-hGH was transfected into the human embryonic kideny 293T cells with Lipofectamine 2000,and blasticidin select positive colony, a high-titer retrovirus were obtained as a result.(4)pLghGHSN was packaged by PT67 and E86 packaging cells. Retroviral producer cell line were generated for pLghGHSN after a 2-step transfection. G418 select positive colony, a high-titer retrovirus were obtained.(5)Myoblast were infected by a high-titer lentiviral and retroviral supernatant. The expression of the hGH was detected with radioimmunoassay. To compare the expression of hGH in culture medium from pcDNA-hGH-myoblast,pLenti6/V5-hGH-myoblast,pLghGHSN-myoblast. (6)Myoblast expressed high level hGH were tissue-engineered into BAMs, and rhGH levels in culture medium from BAMs were measured by a radioimmunoassay technique that does not cross-react with rat GH. The expression of protein in culture medium from BAMs was detected by westerblot.Results (1)The method of isolating and culture of primary rat skeletal myoblasts was established. The levels of the hGH in culture medium from pcDNA-hGH-myoblast,pLenti6/V5-hGH-myoblast,pLghGHSN-myoblast were 40.41±0.1ng/ml; 58.58±3.31 ng/ml; 680.58±3.31ng/ml, respectively. The hGH levels of culture medium were higher in pLghGHSN-myoblast than in pLenti6/V5–hGH-myoblast and pcDNA–hGH -myoblast. Proliferating rat skeletal myoblasts stably transduced with the pLghGHSN were tissue engineered in vitro into bioartificial muscles containing organized postmitotic myofibers secreting 1-2μg of rhGH/day in vitro.Conclusions The primary rat skeletal muscle cells could be transfected efficiently with pcDNA-hGH,pLenti6/V5-hGH and pLghGHSN,and secreted hGH proteins.hGH gene modifing BAM could secrete and delivere recombinant protein.Part three Recombinant human growth hormone Secreted From Tissue-Engineered Bioartificial muscle improves Ventricular function in AMI RatObjective Heart failure is characterized by a number of nurohormonal abnormalities, including derangements in the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) signaling axis. Studies showed that patients with heart failure exhibit a low serum GH and IGF-1 levels. GH administration might improve ventricular function. In the present study, we will examine a new method for delivery of rhGH using genetically modified bioartificial muscles (BAM). To investigate whether the rhGH delivered by this technique improved LV function in rats with CHF.Methods (1)SD male rats underwent left anterior descending coronary artery so as to establish acute myocardial infarction (AMI) models. Similar surgery was performed in sham-operated rats but without coronary artery ligation.(2)Twelve rats that underwent ligation were randomly divided into 2 equal groups: MI group and MI-GH: MI group received GFP-BAM transplantation; MI-GH group received GH-BAM transplantation; Another 12 rats were used as sham operation group(S group). S group were also randomly divided into 2 equal groups: S group and S-GH, S group received GFP-BAM transplantation; S -GH group received GH-BAM transplantation; (3)Ligation of the left anterior descending branch or sham operation was performed with subcutane implantation of GFP-BAM or GH-BAM. (4)hGH and IGF-1 levels in rat serum were measured by a radioimmunoassay 4 and 8 weeks after treatment. Echocardiography was performed 4 and 8 weeks after treatment.Results Serum GH and IGF-1 level were significantly higher in both CHF and sham rats treated with GH-BAMs than in those treated with GFP-BAMs. ( P<0.05 for both). There were significantly higher in LV ejection fraction (EF), fractional shortening (FS) in CHF rats treated with GH-BAM compared with CHF rats treated with GFP-BAM (65.0±6.5% verse 48.1±6.8%, 41.3±7.4% verse 26.5±7.1%. P<0.05). There were significantly lower in LV end-diastolic dimension (LVEDD) in CHF rats treated with GH-BAM compared with CHF rats treated with GFP-BAM (7.2±0.42 versus 8.25±0.31, P<0.05). There were no significant differences in EF,FS and LVEDD between S group and S-GH.Conclusions(1)When implanted subcutaneously into syngeneic rat, genetically modified BAMs delivered a sustained physiologic dose of rhGH and improved LV myocardial function in AMI rat. (2)Genetically modified bioartificial muscles provides a new method delivering recombinant protein for gene therapy.