Research Of The Effects On T/B Lymphocytes Of Regenerated Bombyx Mori Silk Fibroin And Its Degradation Rate In The Wound

Objective:studying the effects on lymphocytes of regenerated Bombyx mori silk fibroin and its degradation rate in vivo.Mothods:1. The activation on lymphocytes of regenerated Bombyx mori silk fibroin which is soluble in vivo and in vitro1.1 The activation on T cell of regenerated Bombyx mori silk fibroin in vitroThe regenerated Bombyx mori silk fibroin film was prepared with RMPI1640 into the silk fibroin solution. Then, human PBMC and rat lymph node cell suspension were cultured with the silk fibroin solution of the final concentration of 0.01% and 0.1%. The activation was observed and the proliferation was detected by MTT test after 72 hours.1.2 The activation on B cell of regenerated Bombyx mori silk fibroin in vivoThe soluble regenerated Bombyx mori silk fibroin that was used to immunize New Zealand rabbits mixed with Freund's adjuvant and the dose was 15 mg/kg weight. After the immunization was strengthened for 4-5 times, the specific antibody was detected by double immunodiffusion test and the globulin was detected by ultraviolet spectrophotometry.2. The degradation of porous regenerated Bombyx mori silk fibroin membrane that is not soluble in the wound and its effect on immune cell2.1 Research of the degradation rate of porous regenerated Bombyx mori silk fibroin membranePorous regenerated Bombyx mori silk fibroin membrane was labeled by 125I and the labeling efficiency and stability was measured. SD rats were selected to create wound surface by removing its skin of back. Then, the wound surface was covered with 125I-porous regenerated silk fibroin membrane and the epidermis was layed on the porous regenerated silk fibroin membrane. At last, the wound was sutured. After operation, single photon emission computed tomography (SPECT) was used for imaging at different time and ROI technology was used to analyse. It was stopped when the isotope signal disappeared and the rats were sacrificed at this time. Various organs were taken to detect the distribution.2.2 The effect on immune cellsAfter rats were sacrificed, inflammatory cells of the wound surface were analysed through HE staining. PBMC were obtained at 3 d,14 d,28 d,55 d and splenic cells were obtained at 55 d. Then, two-color immunofluorescence and flow cytometry technology were used to detect the ratio of CD3+CD25+T/CD3+T cells.Results:1. The activation on lymphocytes of regenerated Bombyx mori silk fibroin which is soluble in vivo and in vitro1.1 The activation on T cell of regenerated Bombyx mori silk fibroin in vitroAfter human PBMC stimulation by two kinds of concentration of regenerated Bombyx mori silk fibroin solution, the OD values of MTT test were consistent with negative controls and significantly different from positive controls. The results of rat lymph node cells stimulation were consistant with human PBMC.1.2 The activation on B cell of regenerated Bombyx mori silk fibroin in vivoIn double immunodiffusion test, the precipitation lines were not observed in the immune serum of soluble regenerated Bombyx mori silk fibroin, but it was observed in the serum of positive control. The globulin content of the immune serum of silk fibroin were consistent with negative controls and significantly different from positive controls.2. The degradation of porous regenerated Bombyx mori silk fibroin membrane that is not soluble in the wound and its effect on immune cell2.1 Research of the degradation rate of porous regenerated Bombyx mori silk fibroin membraneThe labeling efficiency was (21.1±1.11)% and the stability was well. After operation, the isotope signal intensity decreased with time. After 55 days of implanting, the isotope signal disappeared. The residual rate decreased gradually with time and it was (3.39±2.59)% at the time of the 55th day through ROI analysis. The intensity of radioactivity was low in various orgens. In comparison, the kidney obtained high distribution and its %ID/g was 0.29±0.06. The distribution of immune orgens were low.2.2 The effect on immune cellsA small amount of silk fibroin composition were obtained and its proportion was similar with the results of ROI analysis. After 3 days of implanting, the ratio of CD3+CD25+/CD3+T cells of PBMC was higher than normal. After 14 days of implanting, the ratio was consistent with normal. After 28 days and 55 days, the ratio was lower than normal. After 55 days, the ratio of splenic CD3+CD25+/CD3+T cells was consistent with normal.Conclusion:The ability to stimulate lymphocytes of regenerated Bombyx mori silk fibroin is weak. The rate of degradation is cpordinate with the rate of tissue repair.