| Hypoparathyroidism is rare disease characterized by hypocalcemia and hyperphosphatemia due to low serum levels of parathyroid hormone. Most cases occur as a complication of thyroid or parathyroid surgery. Clinical features of hypoparathyroidism include symptoms of perioral numbness, paresthesias,carpal/pedal muscle spasms and tetany(when mean serum calcium was lower than2mmol/L). This study was designed to investigate PTH gene modified mesenchymal stem cells as treatment of hypoparathyroidism. We construct a PTH overexpression lentivirus vector, containing a mutant human prepro PTH gene that be identified and cut by Furin. The lentivirus infection to mesenchymal cells(MSCs) was followed by ELISA to detect the PTH expression. In the rat model of hypoparathyroidism, we found PTH gene modified MSCs by subcutaneously injection had a therapeutic effect in the treatment of hypoparathyroidism.Part Ⅰ Construction of carbon nanoparticles assisted hypoparathyroidism model in ratsObjective: To construct SD rat model of hypoparathyroidism. Methods:Parathyroidectomy was applied to rats with the help of carbon nanoparticles. The resected tissues were proved to be parathyoid glands using optical coherence tomography(OCT) and HE staining. Evaluation of the model was determined by serum calcium and PTH. Results: Rat model of hypoparathyroidism was successfully established through carbon nanoparticles assisted parathyroidectomy.Serum calcium levels on post-operation day 1, 5 and 10 were 1.79±0.09,1.63±0.07, 1.61±0.13 mmol/L and serum PTH leverls were 13.16±3.02,15.24±4.46, 15.91±5.76 ng/L, both of which were significantly lower than controls(P<0.05).PartⅡPTH expression of rat adipose derived stem cells transfected with lentivirusObjective: The aim of this part was to construct PTH overexpression lentivirus vector, and to determine the efficiency of lentivirus-mediated infection to rat MSCs. Methods: Human prepro PTH gene was amplified and integrated into lentivirus vector, and a Furin cutting site was introduced on the signal peptide using site-directed mutagenesis. Adipose-derived stem cells were isolated from the rat adipose tissue, and characterized by adipogenic and osteogenic differentiation. The MSCs were analyzed for the expression of Furin protease by RT-PCR. After be transfected by lentivirus, the expression of PTH was detected by ELISA. Results: Rats asipose-derived MSCs were successfully isolated;Lentivirus infected MSCs can secrete PTH.PartⅢ Experimental study on gene therapy of hypoparathyroidism with mesenchymal stem cellsObjective: To investigate the effects of gene therapy of hypoparathyroidism with MSCs. Methods: The model rats were administered subcutaneously with PTH-MSCs, wild type MSCs and normal saline subcutaneously immediately after surgery. The therapeutic effects were evaluated by general condition, survival and serum calcium and PTH levels. Results: Compare to controls, administration of PTH-MSCs improved survival rates and symptoms of hypocalcium; the serum calcium and PTH level on day 5 and 10 were significantly higher than those in control groups(P<0.05).Conclusions: Our results suggest that PTH gene modified MSCs by subcutaneously injection exert a therapeutic effect on aparathyroid rats, could be a novel and promising approach to treat hypoparathyroidism. |
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