Toxicity Effect Of Immunosuppressive Drugs On Human Adult Islets In Vitro And The Evaluation Of Protective Strategies

Objectives: Human islet transplantation is one of the effective treatments for T1DM. Up to date, the toxicity effect of immunosuppressive drugs on human adult islets negatively influents the islet allo-transplantation in clinics. This research focuses on the exploration of the toxicity mechanism of immunosuppressive drugs on human islets in vitro, and the evaluation of protective effects of nicotinamide (NAA), pentoxifylline (PTX), or PDTC on islets injury.Methods: 1. Using the refined Ricordi chamber, we isolated the human islets from the cadaveric pancreas, then evaluated its quality by the staining of dithizone (DTZ) and acridine orange-propidium iodide (AO-PI). 2. In vitro, after the 24h incubation of purified human islets with immunosuppressive drugs (MP, CsA, MPA, FK506, Rapa, FTY720) in different concentrations, the morphological changes were observed using microscope, the viability was justified by MTT methods, and the endocrine function was evaluated using the insulin-release assay. 3. The analysis of TNFa in culture supernates, the insulin-release assay, and the TUNEL detective method for apoptosis were used to evaluate the protective effect of NAA or PTX on human islets after 3 days co-culture with the drugs (FK506, MPA, Rapa, FTY720). 4. Pyrrolidine dithiocarbamate (PDTC), which is an inhibitor of the nuclear factor kB (NF-kB), was added into the incubation of human islets with drugs (FK506, MPA, Rapa, FTY720). After 3 days, the islets function and apoptosis was detected using methods as above. At the same time, a semi-quantitative analysis, named electrophoretic mobility shift assay (EMSA), was ensued to identify the changes of expression of NF-kB in the nuclei.Results: 1. In this study, the human islets was well isolated from the adult pancreas with good quality (viability rate is about 98%) and quantity (purification rate is 95%). 2. Under the microscope, significant apoptosis of islets was detected. And except for FTY720, all the other drugs induced the deduction of islet viability when analyzed by MTT (p<0.01). MP, CsA, FK506, and MPA dramatically decreased islet secreting...