【摘要】 目的：通过研究肿瘤坏死因子(TNFα),血管内皮生长因子（VEGF）、β成纤维细胞生长因子(βFGF)、转移生长因子β2 (TGFβ2)、干扰素γ(IFNγ)及半胱氨酸天冬氨酸蛋白酶3（Casepase3）在不同浓度胎牛血清（fetal bovine serum，FBS）与胰岛素转铁蛋白亚硒酸钠（insulintransferrinsodium selenite，ITS)混合培养基中的表达及其对正常视网膜色素上皮（retinal pigment epithelium，RPE)细胞生长的影响以探索维持正常RPE细胞生长的理想培养基。方法：首先分别在不含RPE细胞和DMEM培养基的20,40,100mL/L FBS和10,20,30g/L ITS中检测TNFα, VEGF、βFGF、TGFβ2、IFNγ及Casepase3是否存在。然后取四十只C57 BL/6系小鼠眼的RPE细胞分别培养于含20,40,100mL/L FBS以及20,40,100mL/L FBS与10g/L ITS分别组合的DMEM培养基中。免疫组织化学染色以及细胞计数鉴定、评估RPE细胞的存在与生长状况。采用原代培养48h的第三代、第四代RPE细胞，分别用反转录聚合酶链反应（RTPCR）以及酶联免疫吸附（ELISA）法检测RPE细胞和上清液中TNFα, VEGF、βFGF、TGFβ2、IFNγ及casepase3的表达强弱；用蛋白印记（Western blotting）法检测RPE细胞中Casepase3的表达。结果：在20,40,100mL/L FBS中检测到了TNFα, VEGF、βFGF、TGFβ2及casepase3 （IFNγ没有表达）并且随浓度增加而表达上调。在10,20,30g/L ITS中没有检测到上述生长因子。RPE细胞培养成功。在含20,40,100mL/L不同浓度的FBS以及20,40,100mL/L FBS与10g/L ITS分别组合的DMEM培养基中随浓度增加，TNFα, VEGF、βFGF、TGFβ2及casepase3的表达呈上调趋势，各对应组组间的表达强度没有明显差异，但不同浓度组组内的表达强度有明显差异（P＜0.01）。IFNγ没有表达。上述因子在含20mL/L FBS与20mL/L FBS +10g/L ITS的培养基中表达最低，但20mL/L FBS +10g/L ITS的培养基中RPE细胞生长良好。结论：在RPE细胞和上清液中有TNFα, VEGF、βFGF、TGFβ2及casepase3的表达。20mL/L FBS +10g/L ITS的混合培养基可能是维持正常RPE细胞生长的一种理想培养基。
【关键词】 视网膜色素上皮细胞；生长因子；半胱氨酸天冬氨酸蛋白酶3；培养
INTRODUCTION
Retinal pigment epithelial (RPE) cells play a fundamental role in many normal biological processes including absorption of light adjacent to the outer segments of the photoreceptors, storage and conversion of vitamin A esters, formation of an acid mucopolysaccharide complex that surrounds the outer segments of rods and cones, as well as phagocytosis of rod and, to a lesser extent, cone outer segments. There are a number of retinal disorders in which dysfunction of the RPE is the key to the pathogenetic process[1]. Recent researches on transplantation RPE cells into subretinal space have made great progresses[26]. However, problems, such as how to supply healthy RPE cells, are the key to successful transplantation which need to be solved. Recent study has shown that vascular endothelial growth factor (VEGF) is responsible for many ocular pathologies[7], which indicates that growth factors could be one main reason that affects ocular pathophysiological proporties. Therefore, we established primary cultures of RPE cells to investigate if growth factors critical for RPE cell function, such as tumor necrosis factor (TNFα), vascular endothelial growth factor(VEGF), β fibroblast growth factor (βFGF), transforming growth factor β2 (TGFβ2) and interferonγ (IFNγ) as well as casepase3, are expressed in RPE cells,and the ideal medium for the growth of normal RPEs.
METHODS
Detecting the Expression of Growth Factors The expression of TNFα, VEGF, βFGF, TGFβ2 and IFNγ as well as caspase3 in groups of 20,40,100mL/L fetal bovine serum (FBS) and 10,20,30g/L insulintransferrinsodium selenite media supplement (ITS) without RPE cells and DulbeccoS modified Eagles medium (DMEM) were tested in order to exclude the influence of the RPE cells and medium. Saline were taken as control groups. Then, the medium were divided into control (only with DMEM) , concentration of 20, 40, 100mL/L groups for the experiment.
Animals and Cell Cultures Forty pathogenfree C57BL/6 mice, aged 23 weeks, were used in this study. All animals were treated in accordance with ARVO Statement for the use of Animals in Ophthalmic and Vision Reaseach in U.S. and China. Donor C57BL/6 mice were decapitated and their eyes were immediately enucleated and placed in icecold DMEM with 2mmol/L glutamine. The whole eyeballs were incubated for 20 minutes at 37℃ in solutions of 20mL/L disperse concentrations. The eyes were rinsed twice in DMEM and placed in a Petri dish containing fresh DMEM. Under a dissecting microscope, the eyeballs were cut open along the edge of the cornea. After the lenses and irises were removed, the RPE and their attached neural retina were gently separated from the eye cup and transferred to a new Petri dish containing fresh DMEM. After two changes to fresh medium and incubation for 15 minutes at room temperature, the entire RPE sheets were easily separated from the neural retina. Separated RPE sheets were cut into pieces then transferred into culture flasks precoated with 1mg/L fibronectin and grew at 37℃ in a 50mL/L CO2 atmosphere in DMEM containing 20,40,100mL/L FBS and 10g/L ITS. It is the same experiment condition of the FBS and ITS during the whole cell cultures course. Cell counting was used to evaluate RPE cell growth. Passage 3 to 4 RPE cells and their supernatants after being cultured for 48 hours were used and all these cells were routinely stained with antibodies against a broad range of epidermal keratins (AE1/AE3; RDI, Flanders,NJ07836) for determination of their epithelial origins. All cell culture material and chemicals were purchased from Invitrogen(Carlsbad,CA).
Characterization of the Primary Culture by Immunocytochemistry RPE cell cultures were washed in PBS and fixed in 4% paraformaldehyde (PFA) at room temperature (RT) for 15 minutes. Subsequently, nonspecific binding was blocked with normal sheep serum (Serotec, Raleigh, NC) at RT for 30 minutes. The primary antibody (mouse anticytokeratin: RDI, PRO 61835: 1∶100) used to characterize the cells was diluted in PBS with 0.1% Tween 20 and incubated for 60 minutes at RT. After the cells were washed three times for 5 minutes each in PBS with 0.1% Tween 20, the secondary antibody (sheep antimouseCy3; Sigma, C2181; 1∶400) was applied for 30 minutes. Subsequent to repeat washing, numbers of cells in four random visual fields were counted and numbers of positive cells were used to calculate the percent positive. Universal negative control mice (Dako, NP015) were use for negative control: Specific binding were visualized under fluorescence optics (Olympus, Melville, NY). Cell morphologies were characterized by phasecontrast microscopy.
Determination of TNFα, VEGF, βFGF, TGFβ2 and IFNγ Expression by RTPCR Total RNA (1μg) which was isolated from RPE cells in different concentration of FBS using SV. Total RNA Isolation System kits ( Promega, Madison, WI, USA) and PCR kit ( Applied Biosystems, CA) were used for RTPCR to detect the mRNA levels of βactin, TNFα, VEGF, βFGF, TGFβ2 and IFNγ. The negative controls consisted omission of RNA or reverse transcripts from the reaction mixture. PCR products were quantified by densitometery, using the Molecular Analyst/PC program (BioRad, Hercules, California). RTPCR was carried out with the following primers: βactin (F5′GCC ACC AGT TCG CCA TGG ATG A 3′, R5′GTC AGG CAG CTC ATA GCT CTT C3′);TNFα(F5′TCT CAT CAG TTC TAT GGC CC3′, R5′GGG AGT AGA CAA GGT ACA AC3′) [8];VEGF(F5′GCG GGC TGC CTC GCA GTC3′, R5′TCA CCG CCT TGG CTT GTC AC3′); βFGF(F5′AGC GGC TCT ACT GCA AGA AC3′, R5′TCG TTT CAG TGC CAC ATA CC3′); TGFβ2(F5′CCA AAG ACT TAA CAT CTC CCA CC3′, R5′GTT CGA TCT TGGGCGTATTTC3′);IFNγ(F5′GCATCTTGGCTTTGCAGCTC3′,R5′CGACTCCTTTTCCGCTTCCT3′) [9]; For TNFα: The amplification consisted of 50 cycles of denaturation for 1 minute 45 seconds at 95°C, annealing for 45 seconds at 61°C, and extension for 7 minutes at 72°C: For VEGF: annealing for 45 seconds at 60°C, 32 cycles; For βFGF: annealing for 45 seconds at 60°C, 30 cycles; For TGFβ2: annealing for 45 seconds at 60°C, 25 cycles; For IFNγ: annealing for 45 seconds at 60°C, tried 2560 cycles. Other amplification conditions were the same as TNFα.
Determination of TNFα, VEGF, βFGF, TGFβ2 and IFNγ Protein by Enzymelinked Immunosorbent Assay Levels of TNFα, VEGF, βFGF, TGFβ2 and IFNγ were measured in supernatant of RPE cells under different concentration of FBS with ITS media supplement using enzymelinked immunosorbent assay (ELISA) according to manufacturer instructions as follows: The supernatant of RPE cells were collected at passage 3 to 4 primary RPE cell cultures and centrifuged for 10 minutes to remove cells and other large debris, aliquoted, and immediately stored at 20°C until assay for TNFα, VEGF, βFGF, TGFβ2 and IFNγ production by ELISA (Quantikine Kits MTA00 for TNFα, MMV00 for VEGF, DFB50 for βFGF, DB250 for TGFβ2, MIF00 for IFNγ respectively, from R&D Systems). Frozen cell culture medium (background for assay) was used as negative control for ELISA measurements. In addition, unspecific color production by plastic was quantified by a control provided by the supplier (control kit component).
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