CAJ | 학술논문

目的:通过研究肿瘤坏死因子(TNF-α),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(IFN-γ)及半胱氨酸天冬氨酸蛋白酶-3(Casepase-3)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(insulin-transferrin-sodium 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TNF-α, VEGF、βFGF、TGFβ2、IFN-γ及Casepase-3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(RT-PCR)以及酶联免疫吸附(ELISA)法检测RPE细胞和上清液中TNF-α, VEGF、βFGF、TGFβ2、IFN-γ及casepase-3的表达强弱;用蛋白印记(Western blotting)法检测RPE细胞中Casepase-3的表达.结果:在20,40,100mL/L FBS中检测到了TNF-α, VEGF、βFGF、TGFβ2及casepase-3 (IFN-γ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,TNF-α, VEGF、βFGF、TGFβ2及casepase-3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).IFN-γ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TNF-α, VEGF、βFGF、TGFβ2及casepase-3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. 目的:通過研究腫瘤壞死因子(TNF-α),血管內皮生長因子(VEGF)、β成纖維細胞生長因子(βFGF)、轉移生長因子β2 (TGFβ2)、榦擾素-γ(IFN-γ)及半胱氨痠天鼕氨痠蛋白酶-3(Casepase-3)在不同濃度胎牛血清(fetal bovine serum,FBS)與胰島素轉鐵蛋白亞硒痠鈉(insulin-transferrin-sodium selenite,ITS)混閤培養基中的錶達及其對正常視網膜色素上皮(retinal pigment epithelium,RPE)細胞生長的影響以探索維持正常RPE細胞生長的理想培養基.方法:首先分彆在不含RPE細胞和DMEM培養基的20,40,100mL/L FBS和10,20,30g/L ITS中檢測TNF-α, VEGF、βFGF、TGFβ2、IFN-γ及Casepase-3是否存在.然後取四十隻C57 BL/6繫小鼠眼的RPE細胞分彆培養于含20,40,100mL/L FBS以及20,40,100mL/L FBS與10g/L ITS分彆組閤的DMEM培養基中.免疫組織化學染色以及細胞計數鑒定、評估RPE細胞的存在與生長狀況.採用原代培養48h的第三代、第四代RPE細胞,分彆用反轉錄聚閤酶鏈反應(RT-PCR)以及酶聯免疫吸附(ELISA)法檢測RPE細胞和上清液中TNF-α, VEGF、βFGF、TGFβ2、IFN-γ及casepase-3的錶達彊弱;用蛋白印記(Western blotting)法檢測RPE細胞中Casepase-3的錶達.結果:在20,40,100mL/L FBS中檢測到瞭TNF-α, VEGF、βFGF、TGFβ2及casepase-3 (IFN-γ沒有錶達)併且隨濃度增加而錶達上調.在10,20,30g/L ITS中沒有檢測到上述生長因子.RPE細胞培養成功.在含20,40,100mL/L不同濃度的FBS以及20,40,100mL/L FBS與10g/L ITS分彆組閤的DMEM培養基中隨濃度增加,TNF-α, VEGF、βFGF、TGFβ2及casepase-3的錶達呈上調趨勢,各對應組組間的錶達彊度沒有明顯差異,但不同濃度組組內的錶達彊度有明顯差異(P＜0.01).IFN-γ沒有錶達.上述因子在含20mL/L FBS與20mL/L FBS +10g/L ITS的培養基中錶達最低,但20mL/L FBS +10g/L ITS的培養基中RPE細胞生長良好.結論:在RPE細胞和上清液中有TNF-α, VEGF、βFGF、TGFβ2及casepase-3的錶達.20mL/L FBS +10g/L ITS的混閤培養基可能是維持正常RPE細胞生長的一種理想培養基.
목적:통과연구종류배사인자(TNF-α),혈관내피생장인자(VEGF)、β성섬유세포생장인자(βFGF)、전이생장인자β2 (TGFβ2)、간우소-γ(IFN-γ)급반광안산천동안산단백매-3(Casepase-3)재불동농도태우혈청(fetal bovine serum,FBS)여이도소전철단백아서산납(insulin-transferrin-sodium selenite,ITS)혼합배양기중적표체급기대정상시망막색소상피(retinal pigment epithelium,RPE)세포생장적영향이탐색유지정상RPE세포생장적이상배양기.방법:수선분별재불함RPE세포화DMEM배양기적20,40,100mL/L FBS화10,20,30g/L ITS중검측TNF-α, VEGF、βFGF、TGFβ2、IFN-γ급Casepase-3시부존재.연후취사십지C57 BL/6계소서안적RPE세포분별배양우함20,40,100mL/L FBS이급20,40,100mL/L FBS여10g/L ITS분별조합적DMEM배양기중.면역조직화학염색이급세포계수감정、평고RPE세포적존재여생장상황.채용원대배양48h적제삼대、제사대RPE세포,분별용반전록취합매련반응(RT-PCR)이급매련면역흡부(ELISA)법검측RPE세포화상청액중TNF-α, VEGF、βFGF、TGFβ2、IFN-γ급casepase-3적표체강약;용단백인기(Western blotting)법검측RPE세포중Casepase-3적표체.결과:재20,40,100mL/L FBS중검측도료TNF-α, VEGF、βFGF、TGFβ2급casepase-3 (IFN-γ몰유표체)병차수농도증가이표체상조.재10,20,30g/L ITS중몰유검측도상술생장인자.RPE세포배양성공.재함20,40,100mL/L불동농도적FBS이급20,40,100mL/L FBS여10g/L ITS분별조합적DMEM배양기중수농도증가,TNF-α, VEGF、βFGF、TGFβ2급casepase-3적표체정상조추세,각대응조조간적표체강도몰유명현차이,단불동농도조조내적표체강도유명현차이(P＜0.01).IFN-γ몰유표체.상술인자재함20mL/L FBS여20mL/L FBS +10g/L ITS적배양기중표체최저,단20mL/L FBS +10g/L ITS적배양기중RPE세포생장량호.결론:재RPE세포화상청액중유TNF-α, VEGF、βFGF、TGFβ2급casepase-3적표체.20mL/L FBS +10g/L ITS적혼합배양기가능시유지정상RPE세포생장적일충이상배양기.
AIM: To optimize the conditions for in vitro culture of retinal pigment epithelium (RPE) cells, we characterized expressions of various growth factors in RPE cells, including tumor necrosis factor (TNF-α), vascular endothelial growth factor (VEGF), β fibroblast growth factor (βFGF), transforming growth factor β2 (TGFβ2), and interferon-γ (IFN-γ). We also studied expressions of caspase-3 under different concentrations of fetal bovine serum (FBS) with insulin-transferrin-sodium selenite (ITS) supplement. METHODS: First, we investigated if expressions of TNF-α, VEGF, βFGF, TGFβ2, IFN-γ, and caspase-3 in FBS and ITS with of concentration. Second, we cultured primary RPE cells from eyes of forty C57 BL/6 mice in standard dulbecco's modified eagle's medium (DMEM) containing 20,40,100mL/L FBS and 20,40,100mL/L FBS together with 10g/L ITS. Immunohisto-chemical staining and cell counting were performed to verify the existence and growth condition of RPE cells. Expressions of TNF-α, VEGF, βFGF, TGFβ2 and IFN-γ were determined using cells and supernatant from passage-3 to -4 primary RPE cell after 48 hours of culture with RT-PCR and enzyme-linked immunosorbent assays (ELISA). The expression of casepase-3 was determined via Western blotting. The major outcome measurement is the expression level of growth factors in cultured RPE cells and the experiment design is to expose the RPE cells to different culture medium. RESULTS: TNF-α, VEGF, βFGF, TGFβ2, but not IFN-γ, were expressed and the expressions increased with concentration. No expression of the aforementioned genes was detected in presence of ITS. The primary cultures of RPE cells were successfully established. TNF-α, VEGF, βFGF, TGFβ2 (but no IFN-γ) and the active caspase-3 were detected in 20,40,100mL/L FBS or 20,40,100mL/L FBS combined with 10g/L ITS; the expressions were upregulated with increasing concentration of FBS. There is no significant difference in the expression of growth factors between these groups. However, significant differences were shown among different concentration of FBS (P＜0.01）. The lowest expression was observed in 20mL/L FBS or 20mL/L FBS combined with 10g/L ITS medium with RPE cells. But RPE cells were shown in better growth condition in 20mL/L FBS combined with 10g/L ITS.CONCLUSION: TNF-α, VEGF, βFGF, TGFβ2 and caspase-3 were expressed in RPE cells and supernatants. The production of above 20mL/L FBS combined with 10g/L ITS in DMEM may be the ideal cell culture medium that supports the normal growth of RPE cells.