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Determination of Casepase3 Protein by Western Blotting Method The RPE cells were homogenized and solubilized in ice cold PBS containing protease inhibitors, phenylmethylsulfonyl fluoride(1mg/L), aprotinin(1mg/L), leupeptin (1mg/L), pepstatin A (1mg/L) and EDTA (1mmol/L).
The homogenate was centrifuged at 15 000r/min at 4℃ for 10 minutes. The protein content of the supernatants was determined by the Bradford method[10]. After sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) on 12% linear slab gel under reducing conditions, separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using a semidry electrophoretic transfer cell (Transblot; BioRad, Richmond, CA). Blot was stained at room temperature with a 1∶600 dilution of monoclonal mouse anticasepase3 antibody over night at 4℃. After washing and incubation with horseradish peroxidaseconjugated secondary antibody (1∶1000 dilution), blot was developed using the enhanced chemiluminescence Western blotting analysis detection system (ECL Plus; Amersham Pharmacia Biotech, Arlington Heights, IL).
Statistical Analysis All experiments were performed three times and results are shown as mean±SD. Statistical significance were determined by onefactor analysis of variance (ANOVA) followed by Fisher post hoc test for multiple comparisons. P<0.05 was considered significant.
RESULTS
The Expression of Growth Factors without RPE Cells and Medium There were upregulation of TNFα, VEGF, βFGF, TGFβ2 expression in presence of increasing FBS and no expression of TNFα, VEGF, βFGF, TGFβ2 among 10,20,30g/L ITS groups without RPE cells and medium.
Cell Cultures and Immunocytochemistry Primary cultures of RPE cells were successfully established. Clusters of flattened,polygonal cells in culture RPE cells (5×105 cells/well) for 5 days with 20,40,100mL/L FBS and 20,40,100mL/L FBS combined with 10g/L ITS as well as the cytokeratin positive RPE cells were observed with epithelial shape (Figure 1). There was no definite difference among the shape of RPE cells and number of cytokeratinpositive cells by cell counting in those groups. TNFα, VEGF, βFGF, TGFβ2 (but no IFNγ) and caspase3 activity were expressed and upregulated in increasing concentration of FBS (20,40,100mL/L), or increasing FBS combined with 10g/L ITS. There were no distinguishing differences in expression levels of different growth factors between these groups, but distinguishing differences were shown among different groups （P＜0.01,Table 1）. The lowest expression could be seen in RPE cells 20mL/L FBS and 20mL/L FBS combined with ITS medium. In contrast, RPE cells were shown in better growth condition in 20mL/L FBS combined with 10g/L ITS medium RPE.Table 1 Comparison with Concentrations of TNFα, VEGF, βFGF, TGFβ2 among various groups (略)
IFNγ in the RPE cells and supernatant. With increased concentrations of FBS, the expression of these growth factors showed a progressive increasing (Figure 2,3;P<0.01). We also confirmed that there was no compensatory expression of IFNγ in RPE cells and supernatant.
Western Blotting TNFα, VEGF, βFGF, TGFβ2 (but no expression of IFNγ) and caspase3 were expressed in RPE cells and expressions were upregulated in increasing concentration of FBS (20,40,100mL/L) or increasing FBS combined with 10g/L ITS. Western blotting analysis only showed a faint band of casepase3 in the control group. A weak to strong expression of casepase3 was observed in RPE cells in different concentration of FBS, the production of which are reduced greatly with the medium containing 20mL/L FBS and 20mL/L FBS combined with 10g/L ITS in DMEM (Figure 4). Computer photo analysis indicated that there were significant differences among three groups (P＜0.01, Figure 5; the experiment was performed in triplicate).
DISCUSSION
We found expressions of TNFα, VEGF, βFGF, TGFβ2, casepase3 but not IFNγ in the RPE cells and supernatants as previously reported. The key point for this study is to optimize cell culture medium that supports normal growth of RPE cells. TNFα is produced mainly by activated macrophages and T cells but may also be made by residual corneal cells[78]. In the eye, TNFα activity is associated with uveitis[910] and the corneal response to various type of injury and is related to corneal allograft survival[11]. Recently, researchers reported that exotic TNFα can induce apoptosis in human RPE cells[12] and play a role in regulation of proliferative vitreoretinopathy (PVR) and choroidal neovascular membranes(CNVMs), as well as regulation of RPE functions[12,13]. VEGF is a multifunctional cytokine that is related to angiogenesis and the accumulation of peritoneal fluid in a variety of physiologic and pathologic conditions. It is produced by a wide range of normal and neoplastic cells, including endothelial cells, smooth muscle cells, fibroblasts, inflammatory cells, and various cancer cells[14]. In the eye, the VEGF is known to be constitutively expressed in the normal corneal epithelium and to be upregulated after trauma. Several studies have demonstrated a role for VEGF in corneal neovascularization(NV) in various animal models of ocular surface inflammation [1517]. It is related to diabetic macular edema, agerelated macular degeneration (AMD)[18,19] and play an important role in the occurrence of PVR [20] and choroidal neovascularization (CNV)[21]. There is close relationship between βFGF and TGFβ2. TGFβ2 has been shown to have numerous diverse biologic effects on βFGF. TGFβ2 is the most potent growth inhibitory polypeptides known for a wide variety of cell types including most epithelial cells, endothelial cells, most lymphoid cells, and many myeloid cells[22]. In the eye, TGFβ2 and βFGF are elevated after laserinduced CNV in C57BL/6 mice [23]. Otherwise, βFGF has been identified as a factor capable of exacerbating the cataractogenic effects of TGFβ2. Thus, βFGF inhibitors, as well as TGFβ2 inhibitors, have the potential to protect the lens against TGFβ2induced cataractous changes[24] as well as stimulated connective tissue growth factor expression during corneal myofibroblast differentiation, etc[25] IFNγ is a lymphocyte cytokine with broad biologic effects[8]. Like wise, It is also produced in some part of the eyes[26,27], which is not observed in RPE cells and supertanants.
Realizing the detrimental role of most growth factors, more efforts have been made to look for the ideal medium for culturing these cells[28,29]. Our previous study[30] showed that the posterior part of the eye is not absolutely immunologically privileged and that rejection is a serious problem in human retinal transplantation. Many questions concern transplantation except technique. In present study, the expression of TNFα, VEGF, βFGF, TGFβ2 in RPE cells increases with the increased concentrations of FBS. Given that FBS was the origin of the complement, we can infer that complement in FBS played an important role in the experimental culture conditions critical for the expression of growth factors. Baraldet al [31] reported no neuronlike cells were found in liver, notocord, or neural tubeconditioned media if FBS was used. Douay et al [32] showed the same results. Tolnay et al[33] found that complement receptor 2 (CR2) participates in the regulation of B cell responses to antigen. The treatment of IM9 B lymphoblastoid cells or Raji Burkitts lymphoma cells with 10% heatinactivated fetal bovine serum for 24 hours increased both the CR2 mRNA level and CR2 surface protein expression more than twofold. However, no change in the CR2 expression level was observed when cells were cultured in serumfree medium. Leshem et al[34] tested various immune functions of lymphocytes growing in medium containing nontreated and heatinactivated FBS. The data clearly show that heat inactivation of the serum is not mandatory. In some cases, the addition of untreated FBS resulted in elevated response levels while maintaining immune function specialty. These data strongly support our results. ITS is a serum free media supplement and can substitute for FBS as a media supplement. Compared with 40mL/L and 100mL/L FBS in the DMEM, 20mL/L FBS in the medium showed decreased expression of growth factors. Therefore, the latter maybe the best choice for the RPE cell culture. Gruber et al[35] also showed that compared with the average 17.5% colony formation observed in controls, ITS, TGFbeta1 and ITS with IGFI significantly increased colony formation (28.4%, 30.4%, and 30.4%, respectively). It is of note that there is no expression of IFNγ in RPE cells and their supernatants. We speculate that the production of many damaged growth factors overwhelmed its expression. Casepase3 also played a role in RPE cells and showed the same result. As we all know, caspase3 is expressed in cells as an inactive 32kDa precursor from which the 17kDa and 11 kDa subunits are proteolytically generated during apoptosis[3638]. The upregulation of caspase3 activity was detected with different concentrations of FBS, and 20mL/L FBS showed lower apoptosis coinciding with the above four growth factors results.
In summary, we infer that 20mL/L FBS with 10g/L ITS medium could decrease the apoptosis during RPE cell culture, and maybe the ideal medium for the growth of normal RPE.
Acknowledgements:Dr.Kaplan and Prof. Bora from Kentucky Lions Eye Center of USA helped the reasearch.
【参考文献】
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5 McLaughlin BJ, Fan W, Zheng JJ, Cai H, Del Priore LV, Bora NS, Kaplan HJ. Novel role for a complement regulatory protein (CD46) in retinal pigment epithelial adhesion. Invest Ophthalmol Vis Sci 2003;44(8):36693674
6 Del Priore LV, Geng L, Tezel TH, Kaplan HJ. Extracellular matrix ligands promote RPE attachment to inner Bruchs membrane. Curr Eye Res 2002;25(2):7989
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8 Keadle TL, Usui N, Laycock KA, Miller JK, Pepose JS, Stuart PM. IL1 and TNFalpha are important factors in the pathogenesis of murine recurrent herpetic stromal keratitis. Invest Ophthalmol Vis Sci 2000;41(1):96102
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