【摘要】：目的:研究近視相關(guān)因子SLC39A5在人胚胎眼鞏膜成纖維細(xì)胞(Human fetal sclera fibroblasts,HFSFs)中的表達(dá)及近視調(diào)控因子轉(zhuǎn)化生長因子-β2(Transforming growth factorβ2,TGF-β2)對其表達(dá)的影響,并觀察干預(yù)(上調(diào)/下調(diào))SLC39A5表達(dá)后基質(zhì)金屬蛋白酶2(Matrix metalloproteinase 2,MMP-2)的表達(dá)變化,分析SLC39A5與TGF-β2信號通路的關(guān)系,初步探討SLC39A5在鞏膜重塑及近視發(fā)病機(jī)制中的作用。方法:1.傳代培養(yǎng)人胚胎眼鞏膜成纖維細(xì)胞,選取5-7代生長狀態(tài)良好的HFSFs用于實驗。2.通過定量反轉(zhuǎn)錄聚合酶連鎖反應(yīng)(Quantificational real-time polymerase chain reaction,QRT-PCR)、免疫印跡法(Western-blot,WB)以及細(xì)胞免疫熒光等方法檢測正常人鞏膜成纖維細(xì)胞中SLC39A5的表達(dá),以THP-1細(xì)胞為陽性對照,研究SLC39A5在HFSFs中m RNA、蛋白水平的表達(dá)及在細(xì)胞內(nèi)的定位。3.予入液終濃度分別為2.5μmol/L、5μmol/L、10μmol/L、20μmol/L的重組人TGF-β2蛋白刺激HFSFs,刺激時間分別為4h、8h、16h,采用QRT-PCR、Western-blot檢測TGF-β2刺激后的人鞏膜成纖維細(xì)胞中SLC39A5的表達(dá)。4.利用RNA干擾和重組蛋白技術(shù)分別下調(diào)和上調(diào)SLC39A5的表達(dá),采用Western-blot方法檢測HFSFs中SLC39A5、MMP-2蛋白的表達(dá),觀察改變SLC39A5的表達(dá)對MMP-2的影響。結(jié)果:1、成功傳代培養(yǎng)HFSFs。2、采用QRT-PCR和細(xì)胞免疫熒光定位檢測后發(fā)現(xiàn)正常HFSFs中存在SLC39A5的表達(dá)。3、當(dāng)刺激HFSFs的TGF-β2濃度從2.5μg/ml逐漸增加到20μg/ml,刺激時間由4h增加到16h時,HFSFs中MMP-2 m RNA的表達(dá)量明顯增加,且在TGF-β2濃度為5μg/ml、作用時間為8h時MMP-2 m RNA的表達(dá)量達(dá)到峰值,此時細(xì)胞中SLC39A5 m RNA表達(dá)量也較正常對照組明顯增加,差異均有統(tǒng)計學(xué)意義(P0.05)。Western-blot檢測TGF-β2刺激后的HFSFs,TGF-β2增加時,MMP-2的表達(dá)也隨之增加,證明在HFSFs中TGF-β2對MMP-2有上調(diào)作用。4、選取被TGF-β25ug/ml刺激8h的HFSFs,通過RNA干擾和重組蛋白分別下調(diào)/上調(diào)SLC39A5后,western-blot檢測發(fā)現(xiàn)兩組細(xì)胞中SLC39A5蛋白的表達(dá)量存在差異,且差異具有統(tǒng)計學(xué)意義(P0.05),但在成功干預(yù)SLC39A5后MMP-2的表達(dá)并未發(fā)生明顯改變。結(jié)論:1.在人胚胎眼鞏膜成纖維細(xì)胞中存在SLC39A5 m RNA及蛋白的表達(dá)。2.TGF-β2刺激可導(dǎo)致HFSFs中SLC39A5的表達(dá)量明顯增加,SLC39A5可能與TGF-β2共同參與近視的形成。3.通過si RNA下調(diào)和重組蛋白上調(diào)SLC39A5表達(dá)成功后,人鞏膜成纖維細(xì)胞中MMP-2的表達(dá)無顯著性變化,提示SLC39A5參與近視的作用機(jī)制可能與MMP-2無關(guān)。
[Abstract]:Objective: to investigate the expression of myopia related factor SLC39A5 in human fetal scleral fibroblasts (Human fetal sclera fibroblasts,HFSFs) and the effect of transforming growth factor 尾 2 (Transforming growth factor 尾 2 TGF- 尾 2 on myopia. The changes of matrix metalloproteinase 2 (Matrix metalloproteinase 2 (MMP-2) expression after intervention (upregulation / down-regulation) were observed, the relationship between SLC39A5 and TGF- 尾 2 signaling pathway was analyzed, and the role of SLC39A5 in scleral remodeling and myopia was preliminarily investigated. Methods: 1. Human embryonic ocular scleral fibroblasts were subcultured, and 5-7 passages of HFSFs with good growth state were selected for the experiment. 2. The expression of SLC39A5 in scleral fibroblasts was detected by quantitative reverse transcription polymerase chain reaction (Quantificational real-time polymerase chain reaction,QRT-PCR), Western blotting (Western-blot,WB) and cellular immunofluorescence. Using THP-1 cells as positive control, the expression and localization of m RNA, protein in HFSFs were studied. Recombinant human TGF- 尾 2 protein with final concentration of 2.5 渭 mol/L,5 渭 mol/L,10 渭 mol/L,20 渭 mol/L stimulated HFSFs, for 4 h and 8 h for 16 h, respectively. QRT-PCR, was used. Western-blot was used to detect the expression of SLC39A5 in human scleral fibroblasts stimulated by TGF- 尾 2. 4. RNA interference and recombinant protein techniques were used to down-regulate and up-regulate the expression of SLC39A5, and Western-blot method was used to detect the expression of SLC39A5,MMP-2 protein in HFSFs. The effect of SLC39A5 expression on MMP-2 was observed. Results: 1. The expression of SLC39A5 was found in normal HFSFs by QRT-PCR and immunofluorescence localization assay. 3. When the TGF- 尾 2 concentration of HFSFs was stimulated from 2.5 渭 g/ml to 20 渭 g / ml, the expression of TGF- 尾 2 in normal HFSFs was increased gradually from 2.5 渭 g/ml to 20 渭 g / ml. When the stimulation time was increased from 4 h to 16 h, the expression of MMP-2 m RNA in HFSFs increased significantly, and the expression of MMP-2 m RNA reached its peak when the concentration of TGF- 尾 2 was 5 渭 g / ml and the exposure time was 8 h. At the same time, the expression of SLC39A5 m RNA in the cells was significantly higher than that in the normal control group (P0.05). The expression of MMP-2 was also increased when the HFSFs,TGF- 尾 2 stimulated by TGF- 尾 2 was detected by Western-blot. The results showed that TGF- 尾 2 could up-regulate MMP-2 in HFSFs. 4. HFSFs, stimulated by TGF- 尾 25ug/ml for 8 h was down-regulated / upregulated SLC39A5 by RNA interference and recombinant protein, respectively. Western-blot analysis showed that there was a significant difference in the expression of SLC39A5 protein between the two groups (P0.05), but the expression of MMP-2 did not change significantly after the successful intervention of SLC39A5. Conclusion: 1. The expression of SLC39A5 m RNA and protein was found in human embryonic scleral fibroblasts. 2. TGF- 尾 2 stimulated the expression of SLC39A5 in HFSFs. SLC39A5 and TGF- 尾 2 may be involved in the formation of myopia. After down-regulation of si RNA and up-regulation of SLC39A5 expression by recombinant protein, there was no significant change in MMP-2 expression in human scleral fibroblasts, suggesting that the mechanism of SLC39A5 involvement in myopia may not be related to MMP-2.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R778.11

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