Investigation Of T Cellular Immunologic Tolerance Induced By MIDO Gene-transfected Tissue Engineering Chondrocytes

Current clinical practices to treat damaged cartilage is a hang-up question. Because the resources of autoallergic cartilage transplantation are limited, tissue engineering presents an alternative to cartilage replacements. However, tissue engineering cartilage's clinical application is greatly limited for its immunogenicity. This study intends to change local immunological respone of T cells around transplanted cartilage cells through indoleamine 2,3 dioxygenase(IDO) gene transfection, to induce T cells' immunity tolerance to allograft tissue engineering cartilage transplantation by lower adjusting T cell immunological response, and to develop a transgenic method for preventing autoimmunity rejection, which allow engineering seed cells to be used for patients who have aniso-immune genetic background. By this way, immunologic barrier formed by MHC from polymorphism and polygene should be overcomed, and tissue engineering seed cells be generalized. This study will break a new path for solving immunologic rejection from local reimplantation. Isolation and culture of mouse chondrocyte:Harvesting of C57 mouse articular cartilage: the cartilage was digested by 2% II type collagenase in vitro at 37℃for 3～4 hours, and then centrifuged. Isolated cells were then collected for experiment. chondrocyte viability determined by tryptan blue stain was above 95%.Primary cultured chondrocyte were detected by MTT which could describe cell growth curve. And we found that cartilage cell entered exponential growth phase at the second or fifth day after adherence and reach a confluence for about 7 days. but along with the increasing of cell passage times, the growth cycle will obviously become longer than before .Extracellular matrix (ECM) secreted by chondrocyte show a markedly response to toluidine blue staining. We stained the chondrocyte by toluidine blue and found that the purity is above 85%. But along with the increasing of cell passage times, the capability of secretion in cartilage cell was decreasing. Therefore two or three passages are the best time for cells to be used in the experiment.pEGFP-N1-IDO plasmid tranfection and gene expression in chondrocyte:Plasmid pEGFP-N1-IDO was transfcted into primary chondrocyte by lipidsome at the best rate 2:1 between lipidsome and plasmid. and it showed that cartilage cell could adherence and grow well after been transfected.The EGFP expression after transfection in chondrocyte was monitored by fluorescence microscope and laser confocal microscopy, and it was shown that green fluorescence distributed equably on the whole cell which began to express after 12 hours of transfection and reach a higher level after 48 hours. EGFP expressed at both endocymia and nuclus but it detected higher in kytoplasm than in nucleus.Chondrocytes were collected at different times after tranfection, fixed by paraform, and then detected EGFP expression in chondrocyte by flow cytometry.along with the time prolongation, transfection efficency presented a trend of ascending firstly and then descending at the 24 hours after tranfection, the EGFP expression was at a climax. the transfection efficency could achieve at the rate of 37% after 48 hours.RT-PCR could detect mIDO expression at RNA level. compared toβ-actin optical density value, the value of chondrocyte gene tranfection was relatively stable.The impact of chondrocyte transfected by mIDO on T lymphocyte proliferation:We analyzed the contents of tryptophan in the medium of mIDO transfected chondrocyte by means of HPLC. 24 hours later, we found that the contents of purified chondrocyte group was between 4.93～5.71 mg/L, averaged at 5.3±0.39 mg/L whereas gene tranfected chondrocyte group couldn't detected any tryptophan. The degradation of tryptophan in the cell medium was accompanied with increasing of kynurenine metabolic products. We studied the elimination kinetics of tryptophan existed in the supernatant lipid and found that the degradation rate of tryptophan in the gene tranfected chondrocyte group is obviously quicker than that in the purified chondrocyte.Transgenic chondrcyte mixed lymphocyte reaction:We treated 2×105 Splenic lymph cells with 3 000 radγradiation exposure and supposed them as antigen presenting cells(APC), and then we supposed 3×105 lymph node lymphocytes as immunologic response cells(IRC). After mixed lymphcyte reaction, we detected the situation of proliferation by MTT at the 5th,6th,7th and 8th day. Results showed that compared with purified chondrocyte group, the transgenic group and the non-transgenic group presented proliferation (P<0.02), however, the proliferation rate of mIDO transgenic group was obviously slower than that of the transgenic group(P<0.05).Immunologic response cells were marked by CFSE in the mixed lymphocyte reaction, cocultured for 96 hours, and then T lymphocyte was deteced by flow cytometry. we found that the transgenic group and the non-transgenic group presented proliferation (P<0.01) and formed 9 generation cells. The total proliferation index of mIDO transgenic group was at 1.026±0.016 after 96 hours. the index of positive purified chondrocyte group was at 1.201±0.026, therefore, the allogeneic T lymphocyte proliferation of mIDO transgenic group was inhabited markedly compared with positive control group.Conclusion: digested method can obtained a great number of primary cultured chondocytes and two or three passages are the best time for cells to be used in the experiment; lipidosome is suitable to become the gene transduction vector in the primary chondrocyte culture; EGFP expressed stably at both endocymia and nuclus but it mainly in kytoplasm after mIDO was transducted into the primary cultured chondrocyte; The concentration of tryptophan in the culture medium of transgenic cell decrease obviously which supply the evidence that IDO can suppress allogeneic T lymphocyte proliferation through degrading tryptophan concentration in the local microenvironment. In the mixed lymphocyte reaction in vitro, expressed mIDO transfected chondocytes suppress allogeneic T lymphocyte proliferation which supply a new hypothesis that transfected chondrocyte can suppress allogeneic T lymphocyte proliferation through degrading tryptophan concentration in the local microenvironment and prolong the survival time of graft .