SLC39A5在人巩膜成纤维细胞中的表达及其与TGF-β2、MMP-2关系的实验研究
发布时间：2018-11-23 19:28
【摘要】：目的:研究近视相关因子SLC39A5在人胚胎眼巩膜成纤维细胞(Human fetal sclera fibroblasts,HFSFs)中的表达及近视调控因子转化生长因子-β2(Transforming growth factorβ2,TGF-β2)对其表达的影响,并观察干预(上调/下调)SLC39A5表达后基质金属蛋白酶2(Matrix metalloproteinase 2,MMP-2)的表达变化,分析SLC39A5与TGF-β2信号通路的关系,初步探讨SLC39A5在巩膜重塑及近视发病机制中的作用。方法:1.传代培养人胚胎眼巩膜成纤维细胞,选取5-7代生长状态良好的HFSFs用于实验。2.通过定量反转录聚合酶连锁反应(Quantificational real-time polymerase chain reaction,QRT-PCR)、免疫印迹法(Western-blot,WB)以及细胞免疫荧光等方法检测正常人巩膜成纤维细胞中SLC39A5的表达,以THP-1细胞为阳性对照,研究SLC39A5在HFSFs中m RNA、蛋白水平的表达及在细胞内的定位。3.予入液终浓度分别为2.5μmol/L、5μmol/L、10μmol/L、20μmol/L的重组人TGF-β2蛋白刺激HFSFs,刺激时间分别为4h、8h、16h,采用QRT-PCR、Western-blot检测TGF-β2刺激后的人巩膜成纤维细胞中SLC39A5的表达。4.利用RNA干扰和重组蛋白技术分别下调和上调SLC39A5的表达,采用Western-blot方法检测HFSFs中SLC39A5、MMP-2蛋白的表达,观察改变SLC39A5的表达对MMP-2的影响。结果:1、成功传代培养HFSFs。2、采用QRT-PCR和细胞免疫荧光定位检测后发现正常HFSFs中存在SLC39A5的表达。3、当刺激HFSFs的TGF-β2浓度从2.5μg/ml逐渐增加到20μg/ml,刺激时间由4h增加到16h时,HFSFs中MMP-2 m RNA的表达量明显增加,且在TGF-β2浓度为5μg/ml、作用时间为8h时MMP-2 m RNA的表达量达到峰值,此时细胞中SLC39A5 m RNA表达量也较正常对照组明显增加,差异均有统计学意义(P0.05)。Western-blot检测TGF-β2刺激后的HFSFs,TGF-β2增加时,MMP-2的表达也随之增加,证明在HFSFs中TGF-β2对MMP-2有上调作用。4、选取被TGF-β25ug/ml刺激8h的HFSFs,通过RNA干扰和重组蛋白分别下调/上调SLC39A5后,western-blot检测发现两组细胞中SLC39A5蛋白的表达量存在差异,且差异具有统计学意义(P0.05),但在成功干预SLC39A5后MMP-2的表达并未发生明显改变。结论:1.在人胚胎眼巩膜成纤维细胞中存在SLC39A5 m RNA及蛋白的表达。2.TGF-β2刺激可导致HFSFs中SLC39A5的表达量明显增加,SLC39A5可能与TGF-β2共同参与近视的形成。3.通过si RNA下调和重组蛋白上调SLC39A5表达成功后,人巩膜成纤维细胞中MMP-2的表达无显著性变化,提示SLC39A5参与近视的作用机制可能与MMP-2无关。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R778.11
本文编号：2352482
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R778.11
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