OBJECTIVE: To construct recombination eukaryote expression plasmid for human parathyroid hormone (PTH) gene, assay PTH expression and biological activity after transfection in vitro and evaluate gene therapy effect on hypoparathyroidism (HPT). METHODS: (1) PTH gene was amplified from human embryonic parathyroid gland tissue, and plasmid pcDNA3.1-PTH-GFP (pcDPG) was constructed by TOPO recombination technique. Digestion, PCR and sequencing were used to identify the positive vectors. (2) pcDPG was transformed into 293 cells by Lipofectamine 2000(TM), fluorescent inverted microscope was used to observe GFP expression, and PTH gene expression was assayed by RT-PCR technique. (3) PTH protein in supernatant was purified and evaluated biological activity. (4) HPT rabbit models were developed and plasmid pcDPG was injected in skeletal muscles, respectively. Serum calcium, phosphonium and PTH were assayed and pathological changes observed. RESULTS: (1) The findings in digestion and PCR were accorded to anticipation and sequences in report were identified to reference at 99.30%. (2) 24 h after transfection GFP expression could be detected and enhanced with time prolonged arriving to 38.91% and 62.45% at 48 h. PTH gene expression could be detected by RT-PCR. (3) Purified PTH protein made the signs of HPT disappear. (4) Serum calcium and PTH levels were lower than those of pre-operation (P < 0.05) and serum phosphonium enhanced to normal standard 48 h after treatment at the plasmid pcDPG doses of 300 microg/kg and 500 microg/kg. CONCLUSION: Recombination plasmid pcDPG was transformed effectively in vitro and the transfected cells produced PTH protein with biological activity. Besides, the satisfactory therapeutic effect of HPT rabbits was attained by pcDPG plasmid, which provided a foundation for further study of HPT gene therapy.
OBJECTIVE: To construct recombination eukaryote expression plasmid for humanparathyroid hormone (PTH) gene, assay PTH expression and biological activity after transfection in vitro and evaluate gene therapy effect on hypoparathyroidism (HPT). METHODS: (1) PTH gene was amplified from humanembryonic parathyroid gland tissue, and plasmid pcDNA3.1-PTH-GFP (pcDPG) was constructed by TOPO recombination technique. Digestion, PCR and sequencing were used to identify the positive vectors. (2) pcDPG was transformed into 293 cells by Lipofectamine 2000(TM), fluorescent inverted microscope was used to observe GFP expression, and PTH gene expression was assayed by RT-PCR technique. (3) PTH protein in supernatant was purified and evaluated biological activity. (4) HPT rabbit models were developed and plasmid pcDPG was injected in skeletal muscles, respectively. Serum calcium, phosphonium and PTH were assayed and pathological changes observed. RESULTS: (1) The findings in digestion and PCR were accorded to anticipation and sequences in report were identified to reference at 99.30%. (2) 24 h after transfection GFP expression could be detected and enhanced with time prolonged arriving to 38.91% and 62.45% at 48 h. PTH gene expression could be detected by RT-PCR. (3) Purified PTH protein made the signs of HPT disappear. (4) Serum calcium and PTH levels were lower than those of pre-operation (P < 0.05) and serum phosphonium enhanced to normal standard 48 h after treatment at the plasmid pcDPG doses of 300 microg/kg and 500 microg/kg. CONCLUSION: Recombination plasmid pcDPG was transformed effectively in vitro and the transfected cells produced PTH protein with biological activity. Besides, the satisfactory therapeutic effect of HPT rabbits was attained by pcDPG plasmid, which provided a foundation for further study of HPT gene therapy.