| Acute and chronic rejection have constantly been two obstacles which puyzzled organ transplantation since long time ago. There are many obviously toxic and ill effects when using immuno-suppressive agent. At present, it is widely accepted that inducing recipient specific immunologic tolerance to donor tissue or organs is the key point to solving these problems. Immune exemption has been put forward for more than 100 years, and it has been discovered that only few organs, such as cornea, testicle and placenta, are the location of immune exemption in organism. Available researches have confirmed that it was one of the important reasons that certain cells expressed FasL in the immune exemption places. Identification of this mechanism has important significance, because it provides new ways to approach the aims of inducing immunologic tolerance.Testicular supporting cells (Sertoli cells) expressing plenty of FasL is one of the important reasons that testicle become immune exemption organ. We were to establish chimera of liver and testicular Sertoli cells to make the liver richly express FasL, and to observe the efficacy of this method to induce rat liver transplantation immunologic tolerance.Experiment 1: the preparation of testicular Sertoli cells and their effect on lymphocytesObjective: To investigate the isolation and purification method for testicular Sertoli cells and their effect on immunologic function of lymphocytes. Methods: (1) Testicular Sertoli cells purification: 32 male SD rats were randomly divide into 4 groups, each group has 8 rats. 2 methods of digestion and sieve filtration were employed to separate Sertoli cells from testicle as below: Group A:2.5g/L of collagenase V digestion and twice sieve filtrating; Group B: 2.5g/L of collagenase V digestion and once sieve filtrating; Group C: 2.0g/L of collagenase V instead of 2.5g/L of collagenase V digestion and twice sieve filtrating; Group D: 2.0g/L of collagenase V digestion and once sieve filtrating. Counted the number of isolated and purified Sertoli cells, and judged the activity of Sertoli cells. (2) Cytotoxic test: Obtained Wistar rats splenocytes(106/mL), added 200uL splenocyte and 100uL phytohaemagglutinin (PHA, 20ug/ml) in each well and cultured in the 37℃ 5% CO2 constant temperature oven for 48 hours. Irradiated the Sertoli cells from donor SD rats with 15Gy for 1 hour, then added the cells into culture blade containing splenocytes in different concentrations as following: 1 × 105 (Group A), 1 × 106 (Group B), 1 × 107 (Group C) and Group D added 1 × 107/ml Sertoli cells that co-cultured with FasL-mAb for 30 minutes in advance. Then added in 10ul MTT ( 1ug/ul), after cultured for another 4-6 hours, added in 0.1ml acidulated isoamyl alcohol. Measureed the OD litres by MTT chromatometry and judged the cytotoxicity of Sertoli cells to activated lymphocytes. Results: (1) The harvested testicular Sertoli cells from each fetal rat was: (0.95 ±0.06) ×106 in Group A, (0.60 ± 0.06) × 106 in Group B, (1.03± 0.09)×106 in Group C and (0.50±0.23)× 106 in Group D, respectively; the survival rate (%) was: 88.8 ±2.5, 89.7 ± 2.7,89.8 ± 2.3 and 88.3 ±3.2, respectively. The isolated and purified Sertoli cells of Group A and Group C were much more than that of Group B and Group D. Among the four groups, the cell number of Group C was highest and it was (1.03 ±0.09) × 106. The survival rate of the four groups was similar, and all above 88%. (2) Testicular Sertoli cells had obviously cytotoxicity on lymphocytes in vitro, and had significant difference between every two groups(P<0.01). The larger Sertoli cells number, the stronger the killing or inhibition effect on lymphocytes was. When the Sertoli cells number increased to 1 × 106/ml, cocultered for 3 days, the killing effect reached its peak, afterward it didn't increase any more and maintained at a high level. Conclusions:The method of digesting testicular tissue with collagenase V(2.0g/L) and filtrating with sieve twice can obtain more Sertoli cells and save 25% colla... |
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