| Objective1. Two specific media (ECM and ACF) were compared with the routinely used basic medium MEM and DMEM for their potentials in serum free culture of rabbit and rat corneas in the first part study.2. To investigate the feasibility to use a new dilation agent, hydroxyethyl starch(HES), as an alternative to the standard deswelling additive dextran T500 in organ culture preservation media.3. To modulate alloimmunity prior to keratoplasty so as to prevent corneal endothelial cell death,we optimized adenovirus-mediated gene transfer to organ-cultured donor corneal endothelium in vitro and to delineate the kinetics of reporter gene EGFP expression in rabbit corneal endothelial cells.4. To eatablish several rat corneal transplantion models using organ-cultured rat or rabbit corneas so ao to investigate the roles of several related cytokines and lymphocyte subsets in the corneal transplantions rejection after organ culture storage.Methods1. Eighty rabbit and eighty rat corneas were divided into 5 groups and cultured in five test organ culture media with or without serum content for 3 weeks and viability evaluation of corneal endothelial cells was performed. The evaluation parameters included: Endothelial cells densitie(ECD)before and after preservation and trypan blue vital staining of the endothelium after preservation; The mean central thickness of rabbit cornea before and after preservation;The expression of rabbit F-actin and rat ZO-1 in corneal endothelium using IHC,WB and RT-PCR;The transmission electron micronscopy observation of rabbit corneal endothelial cells.2. A half of 18 pairs of rabbit corneas were cultured in serum free ACF culture medium for 21d and then were dehydrated in ACF with a concentration of 5% dextran T500 as acontrol.The other corneas were cultured in ACF medium with a supplement of HES 130/0.4 in concentrations of 10% from the beginning.The evaluation parameters included: The endothelial cells viability before and after storage; The mean central thickness of rabbit cornea before and after preservation;The mean water content after storage; The transparency and folding of rabbit corneas of two groups using two-level system; The expression of F-actin in corneal endothelium using IHC and WB;The electron micronscopy observation of rabbit corneal endothelial cells.3. Twenty-four Newtherland rabbit corneas which had been stored in ECM+2%FBS mediun for 2~3weeks were incubated with replication-deficient adenovirus encoding enhanced green fluorescent protein (EGFP) or empty vector ex vivo at a dose of 5×106 to 5×109vp/ul at temperatures of 37°C. The epithelium had been removed before transduction.After ex vivo infection, and EGFP expression in the grafts was visualized in vitvo by inverted fluorescence microscope over 2 extended weeks.Finally,vital staining was performed to count endothelial cells desity in all grafts.4. A total of 24 Wistar rats, 54 SD rats and 12 Newtheland rabbits were selected. The 54 SD rats were randomly divided into five groups:①normal control group(n=6),②allograft transplantions group with fresh rat cornea (n=12),③allograft transplantions group with fresh rat cornea without descemet membrane combined with organ-cultured rabbit endothelium(n=12),④allograft transplantions group with organ-cultured rat cornea (n=12),and⑤corneal transplantion group with fresh rat isograft without descemet membrane and organ-cultured rabbit endothelium.The former four groups received Wistar→SD keratoplasty and the latter 5 group SD→SD. After transplantion, immunological rejections were observed with a slit lamp microscope. Serum and aqueous humor levels of IL-2,IFN-γ,IL-4 and TNF-αwere measured by ELISA. IHC was performed to examine CD25+T expressions in grafts. Levels of TNF-αmRNA, IFN-γmRNA, IL-4mRNA and CD25mRNA in grafts were detected by using RT-PCR. The expression levels of CD25 and CD28 lymphocyte subsets in peripheral blood were also determined by Flow Cytometry(FCM).Results1. After storage,only corneas cultured in MEM medium with low serum content exhibited a higher endothelial cells loss and the highest densities were found in corneas cultured in ECM and ACF.F-actin and ZO-1 could be seen in all corneal endothelialiums using IHC.Further WB showed that F-actin protein expression of rabbit corneal endotheliums achieved the lowest level in MEM medium and the highest level in ECM and ACF medium.So did ZO-1mRNA expression levels using RT-PCR in rat corneas.2. After 3 weeks storage, ECD of rabbit corneas cultured in ACF medium with 10% HES130/0.4 was slightly lower than that of the control group without dehydration. The experiment group also exhibited a thinner corneal thickness, better transparency and less folding compared with the control group. After dehydration for 48h using dextran T500, the control corneas became thin and transparent, however, the endothelial cell density decreased greatly at the same time. WB showed that F-actin protein expression in rabbit corneal endothelium achieved higher level in ACF medium with 10% HES130/0.4.3. EGFP expression was found to be restricted to the corneal endothelium.Transduction of organ-cultured rat corneas with high doses (5×107~5×108 vp/μL) of AdV caused EGFP expression in 67～84% of corneal endothelial cells. Very high AdV dose (5×109 vp/μL) reduced endothelial cell densities to 1978(SD=281) nuclei/mm2.In vitro expression of EGFP in organ-cultured corneal grafts was demonstrated in 2 weeks observation period and weakened gradually in warm storage.4.①The graft survival time were longer in the fourth and fifth groups than in the second and third groups which reached the longest 16.3d in the fifth group.②The serum and aqueous humor levels of IL-2,IFN-γ,IL-4 and TNF-αwere higher in all surgical groups than in the normal control group at days 6 ,13 and 24d after surgeries (P < 0.05),and the second and third groups had the highest expression level.③At day 13, CD25 was weakly expressed in all surgery groups.④The expressions of IFN-γmRNA,TNF-αmRNA,IL-4 mRNA and CD25mRNA in grafts were markedly increased (P < 0.05) at day 13 after surgeries,whereas there was no expression of these genes in normal corneas.⑤At day 13, the percentages of CD25 in lymphocytes of all groups didn't fluctuate greatly.However, the percentages of CD28 in lymphocytes of the second and third groups increased significantly and weakly expressed in the other two surgery groups.Conclusion1. ECM and ACF medium showed superiority over standard FCS medium in the ability to preserve endothelial cells viability.They have great potentials comparing with conventional MEM and DMEM medium and may play an important part in serum free organ cultured method in the future.2. HES 130/0.4 keeps the corneas thin and transparent, is well torelated for endothelial cells and can be used as a continuous supplement during organ culture. It also simplifies the processing steps of corneas,makes further dehydration before transplantation unnecessary and reduces infection risk in a certain extent. HES 130/0.4 appears to be an alternative to the use of dextran as a deswelling additive in cornea organ culture media.3. Organ-cultured rabbit corneal endothelial cells can be selectively and efficiently transduced by AdV vector following extended periods of warm storage, and reporter gene EGFP expression is retained in vitro during extended 2 weeks organ culture preservation. EGFP expression in fresh corneal grafts is more extensive than in organ-cultured grafts.4. The cytokines involved in Th1/Th2-type immune response (IL-2,IFN-γ,IL-4 and TNF-α) and lymphocyte subsets CD28 play important roles during corneal allograft rejection. Monitoring the expression of them after transplantion surgeries can help to indicate the degree of local immune reaction and predict the corneal allograft rejection.It's good for prolonging survival time to make use of organ-cultured corneal grafts.Increasing the expression of Th2 cytokines and decreasing the expression of Th1 cytokines and CD28 may help to inhibit corneal allograft rejection. |
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