Adipose Tissue-Derived Mesenchymal Stem Cells Transfected with pcDNA3-Pth (1-34) Gene for the Treatment of Hypoparathyroidism---A Rat Model

1st year project: Although permanent hypoparathyroidism after the thyroid or parathyroid surgery rarely ours in general practice (1%). It is a trouble problem. Long-term vit. D3 and calcium therapy are inconvenient and cause gastric hyper-secretion and constipation. Long-term parathyroid hormone therapy is rarely applied nowadays because of its short half life. We have tried to treat hypoparathyroidism with oral gene therapy and have some progress.. However, we have found in our previous experiments that oral gene therapy can hardly achieve the goal of normocalcemia. In this study we will try to use mesenchymal stem cells, which are transfected with human PTH (1-34) gene to treat hypoparathyroidism. Adult male Sprague-Dawley (300gm) rats will be used in this study. Four groups will be divided after parathyroidectomy. Group I (N=10) will accept implantation of TheraCyte containing 4 x 106 cells, Group II (N=10) will accept implantation of TheraCyte containing 4 x 107 live cells, Group III will not accept cells as a control group, and Group IV (N=10) will accept sham operation. The C3H10T1/2 cell line was established in 1973 from C3H embryos. These cells display fibroblastic morphology and are functionally similar to mesenchymal stem cells. The plasmid containing full-length human PTH (1-34) (179 bps) driven under a human crytomegalovirus promoter (pcDNA3) is used with cloing site of Hind III, Bam HI, and E-Coli competent cell are used to amplify cDNA. Lipofectamine is used to contransfect plasmid DNA PTH (1-34) into C3H10T1/2 cells. Live cells number of 4 x 106 or 4 x 107 are loaded in TheraCyte using the centrifuging loading method. The TheraCyte encapsulated with 4 x 106 or 4 x 107will be implanted to the back of parathyrectomized rats. After 0 week, 2 weeks, 4 weeks, 8 weeks ,12 weeks and 16 weeks after implantation, blood will be drawn from rats’tail for the determination of Ca, P, rat’s PTH, PTH (1-34) levels. Sixteen weeks after implantation, the TheraCyte at implantation site will be removed for histological examination with immunohistochemical stain with PTH (1-34) antibodies. 

Project ID:PC9902-1701 
External Project ID:NSC98-2314-B182-028-MY2