| Objective:To obtain corneal epithelial cells and endothelial cells of SD rats by primary culture.To determine cytotoxic effects of small molecule compounds J2 on the corneal epithelial cells and endothelial cells by MTT. To determine the effect of small molecule compounds J2 on CD80 of corneal epithelial cells and endothelial cells by FCM and membrane ultrastructure of them by AFM. Corneal allograft models were established to determine the effection of J2 on transplant rejection, to observe the changes on corneal endothelium by J2 with AFM.Methods:1. The eyes were enucleated from 5 SPF SD rats anaesthetized intraperitoneal with 2% sodium pentobarbital according to 0.2ml/100g. Corneal epithelium and endothelium were isolated from corneas, incubated with DMEM/F12 at 37℃and identified by immunohistochemistry.2. According to MTT test, cells were incubated into 96 well-plates for 24h and cocultured with J2 for selected observation time (10min,20min,30min, Ih,2h,4h,8h,16h, 32h,48h). Then add MTT in maintenance medium and incubated for 4 hours at 37℃. 96 well-plates were placed on an orbital shaker for 10 minutes after which the absorbance of the extracts was measured at 490 nm. To observe the effect of J2 on morphology of corneal epithelial and endothelial cells by TEM.3. Peripheral blood was isolated from mesenteric venous blood and separated by density gradient centrifugation to obtain MNCs. Corneal eptithelial cells were cultured with MNCs, added with 20000U/L IL-2,500000U/L IFN-y cytokines. There were three groups:group A=MNCs+corneal epithelial cells+J2, group B=MNCs+corneal epithelial cells, group C=corneal epithelial cells.4. To determine the expression of CD80 on corneal epithelial cells by FCM. First, adjust the cell density to 1×106/mL. Second, label CD80 by adding goat anti-mouse monoclonal antibody 0.1ml, and incubate for 30min at 37℃. Third, add rabbit anti-goat FITC-IgG secondary antibody 100μL, and incubate for 30min at 37℃. Last, the supernatant was discarded by wash to remove unbound fluorescent secondary antibody.5. Preparation and observation of AFM sample:cultured cells of three groups were washed by PBS and immersed in distilled water for 30min in order to wash off residual on cells'surface. AFM samples were fixed with 2.5% glutaraldehyde for 20min and dried. To observe the changes on membrane surface of corneal epithelial cells by AFM.6. To determine the effect of J2 on corneal endothelial cells with the same methods (MTT,FCM and AFM).7. The Wistar and SD rats were used as donors and recipients to establish corneal allograft model in vivo. There were three groups:(A) J2 trial group, (B) CsA control group, (C) placebo control group. Drop the same times of drug per day,4 times/day. Grafts were checked with slit lamp each day. According to corneal neovascularization score and corneal opacity score to record survival time of graft. Expression of CD80 was determined by immunohistochemistry. Ultrastructued changes on corneal endothelial layer of graft were observed by AFM.Results:1. Corneal epithelial and endothelial cells were proliferated by primary culture. Morphology of corneal epithelial cells was fusiform, that of corneal endothelial cells was polygonal.2. IC50 of J2 for corneal epithelial cells were:10min (4.43±0.59mmol/L),20min(4.40±0.51mmol/L),30min(4.31±0.48mmol/L),1h(4.17±0.45mmol/L),2h(4.15±0.46mmol/L),4h(4.14±0.48mmol/L),8h(4.12±0.43mmol/L),16h( 4.09±0.45mmol/L),32h(4.04±0.44mmol/L),48h(4.03±0.47mmol/L). TEM scanned: morphology of corneal epithelial cells became fusiform into swollen round. Microvilli were drop off, a large number of vacuole were seen, mitochondrial was swollen with disappearance of the ridge and particles of rough endoplasmic reticulum were drop off.3. Fluorescence index of CD80 for corneal epithelial cells was 1.944±0.237 in group A, 3.128±0.175 in group B,1.082±0.120 in group C by FCM. For ANOVA analysis, there was a significant difference on three groups (F=156.307, P＜0.00). For LSD-t test, there was significant difference between each group (P<0.05).4. IC50 of J2 for corneal endothelial cells were:10min (3.20±0.33mmol/L),20min(2.97±0.28mmol/L),30min(2.88±0.26mmol/L),1h(2.68±0.25 mmol/L),2h(2.59±0.26mmol/L),4h(2.58±0.14mmol/L),8h(2.53±0.17mmol/L),16h(2.45±0.11mmol/L),32h(2.44±0.21mmol/L),48h(2.43±0.28mmol/L). TEM scanned: morphology of corneal endothelial cells became polygonal into swollen round. Microvilli were also dropped off, a large number of vacuoles were seen, and no nucleus was seen.5. Fluorescence index of CD80 for corneal endothelial cells was 2.132±0.173 in group A, 4.023±0.164 in group B,0.985±0.118 in group C by FCM. For ANOVA analysis, there was a significant difference on three groups (F=4989.799,P< 0.00). For LSD-t test, there was significant difference between each group (P<0.05).6. Corneal epithelial cells were observed by AFM, its morphology was fusiform, the size of the nucleus was about 14.07μm×20.04μm, the length of the cell was about 54.65μm and height was about 580-610nm. Morphology of corneal endothelial cells was polygonal, the size of the nucleus was about 17.28μm×20.43μm, the length of the cell was about 52.45μm and height was about 356nm. The membrane surfaces of both two kinds of cells were general smooth, but uneven ultrastructure could be observed by AFM. For ANOVA analysis, there were significant differences on Ra,Rq,Rvm,Rt for three groups.7. Survival time of graft in three groups were:(A)28.73±0.37d, (B) 31.47±0.57d, (C) 16.60±0.42d. For Log-Rank analysis, there were significant differences on three groups(F=60.788,P<0.00); for the pairwise comparison after controling of confounding factors between the level, it was found there was no significant difference between group A and group B. Ultrastructure changes on corneal endothelium of graft could be observed by AFM. Conclusion:1. Cytotoxic effects of small molecule compounds J2 on the corneal epithelial cells and endothelial cells are not linear relationships with time, at the first 1h, IC50 values decrease obviously. And IC50 values of corneal epithelial cells and endothelial cells can provide reliable reference data for further research.2. Corneal epithelial cells can be activated by MNCs and its expression of CD80 is highly expressed, these phenomena can be affected by J2 and expression of CD80 can be reduced. The same changes are also found in corneal endothelial cells. It suggests J2 can affect the expression of costimulatory molecules CD80 by compounds with CDR3-CC'on the surface of CD4+T cell in order to delay or inhibit the occurrence of corneal graft rejection.3. AFM can be applied to observe the effect of J2 on membrane surface of corneal epithelial and endothelial cells.4. The evidence of effection of J2 on corneal transplant rejection was further confirmed by animal models in vivo. However, compared with the traditional drugs CsA, there was no obvious advantagement for J2 on transplant rejection due to its formulation or pharmacokinetics. These problems should be solved on the next step. The changes on corneal endothelium of graft could be observed by AFM.5. AFM represents a new powerful tool for corneal imaging, and its application in this field needs further investigation.
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