豚鼠视网膜色素上皮细胞分泌TGF-β2的信号通路的研究

发布时间：2018-06-26 12:50

本文选题：豚鼠视网膜色素上皮细胞 + 视黄酸　；参考：《天津医科大学》2010年硕士论文

【摘要】： 目的在各种近视眼发病机制学说中,“视网膜局部调控学说”备受瞩目,该学说认为视网膜感知外界环境变化后产生一级近视信号因子(如视黄酸,RA)并作用于视网膜色素上皮(retinal pigment epithelium, RPE),使之产生二级信号因子(如TGF-β2),进而调控巩膜的生长,形成近视。本实验观察全反视黄酸(ATRA)对豚鼠RPE细胞的生长、分泌TGF-β2的影响及细胞内第二信使cAMP、IP3的变化。并研究磷脂酶C抑制剂U73122和腺苷酸环化酶抑制剂SQ22536分别对视网膜色素上皮细胞生长及分泌TGF-β2影响,分析RPE细胞分泌TGF-β2的相关胞内信号传导途径,进一步探讨RPE在近视发生发展中的呈递作用。方法选取健康3周龄花色豚鼠,麻醉处死后摘取眼球,采用组织消化法分离培养豚鼠RPE细胞,上皮细胞特异的角蛋白免疫组化染色鉴定,取第2-3代对数生长期的细胞用于后续实验。实验细胞于使用前均换无血清培养液培养24小时以使其同步化。 1. ATRA组：用MTT法检测不同浓度(5×10-6M,10×10-6M,40×10-6M)ATRA对豚鼠RPE细胞增殖的影响。选取浓度为10×10-6M的ATRA作用于RPE细胞,分别于2h、4h、6h、8h、16h取上清培养液用ELISA方法检测TGF-β2的分泌量；于0min、5min、30min、2h、6h收集细胞裂解液用ELISA和放射免疫的方法分别检测胞内cAMP和IP3的含量变化。 2.磷脂酶C抑制剂U73122和腺苷酸环化酶抑制剂SQ22536组：用MTT法检测不同浓度(5×10-6M,10×10-6M,15×106M,20×10-6M)U73122和(5×10-6M,10×10-6M,20x10-6M,50×10-6M)SQ22536对豚鼠RPE细胞增殖的影响。取浓度为1×10-5M的U73122、SQ22536作用于豚鼠RPE细胞,分别于2h、4h、6h、8h、16h用ELISA方法检测TGF-β2的分泌量。结果 1. RPE细胞原代培养及鉴定：原代培养的豚鼠RPE细胞48小时后已贴壁生长,细胞含有丰富的色素。传代后色素减少,细胞近融合时形态近六角形。免疫组化角蛋白染色RPE细胞为棕黄色阳性反应。 2. ATRA对豚鼠RPE细胞的影响 MTT检测：40×10-6M的ATRA作用RPE细胞24h后,细胞生长明显受到抑制。5×10"6M和10×10-6M的ATRA对RPE细胞的生长的作用与对照组相比差异无显著性(P0.05)。 TGF-β2的检测：加入10×10-6MATRA后,TGF-β2的分泌量与对照组相比,2h、4h、6h明显升高,8h无显著差异,16h降低(P0.05),呈随时间延长分泌量逐渐下降趋势。 cAMP及IP3的检测：加入含10×10-6MATRA的培养液后,RPE胞内cAMP含量在30min和2h时升高(p＜0.05),5min、6h时与对照组相比无明显差别。而胞内的IP3含量在各时间点均显著降低(p0.05),6h时降低最明显。 3.磷脂酶C抑制剂U73122和腺苷酸环化酶抑制剂SQ22536对豚鼠RPE细胞的影响 MTT检测：5×106M浓度U73122作用豚鼠RPE细胞24h后,MTT结果与对照组无显著差异(p0.05),浓度5×10-6M时均明显低于对照组(p0.01)。并随浓度的增OD值降低越明显。而SQ22536组RPE细胞生长无明显变化(p0.05)。 TGF-β2的检测：加入1×10-5M U73122后,TGF-β2的分泌量除2h无显著差异外,余各时间点均较对照组明显增加(P0.05)；而SQ22536组对RPE细胞分泌TGF-β2在2h,4h,6h,16h无明显影响(p0.05),8h时分泌量降低(P0.05)。结论 1.较高浓度的ATRA及U73122对豚鼠RPE细胞的增殖有抑制作用,而SQ22536对RPE细胞生长无明显影响。 2.10×10-6M的ATRA作用于RPE细胞后,TGF-β2的分泌量短期升高,随时间延长而降低。 3.10×10-6M的ATRA作用于RPE细胞后,IP3含量显著降低,cAMP含量在观察时间段中期有升高,后又降至对照组水平。 4.U73122可使RPE细胞分泌TGF-β2增加,而SQ22536对RPE TGF-β2的分泌无明显影响。研究结果提示RPE细胞的增殖与第二信使IP3有关。ATRA对RPE细胞分泌TGF-β2的影响可能与第二信使IP3的降低有关。而抑制磷脂酶C信号通路后,RPE细胞分泌TGF-β2增加,进一步证实了豚鼠RPE细胞分泌TGF-β2与磷脂酶C信号通路有关。
[Abstract]:objective
In the pathogenesis theory of myopia, the "retina local regulation theory" has attracted much attention. This theory suggests that the retina perceiving the external environment changes to produce the signal factor of the primary myopia (such as retinoic acid, RA) and acts on the retinal pigment epithelium (retinal pigment epithelium, RPE), making it produce two level signal factors (such as TGF- beta 2). It regulates the growth of the sclera and forms myopia.
The effect of total anti retinoic acid (ATRA) on the growth of RPE cells in guinea pigs, the effect of TGF- beta 2 and the changes in the second messenger cAMP and IP3 in the cells, and the effects of the phospholipase C inhibitor U73122 and adenylate cyclase inhibitor SQ22536 on the growth of retinal pigment epithelial cells and the secretion of TGF- beta 2 respectively, and the phase of TGF- beta 2 secreted by RPE cells were analyzed. Objective to further investigate the role of RPE in the development of myopia.
Method
A healthy 3 week old pigmented guinea pig was selected to extract the eyeball after anaesthesia. The guinea pig RPE cells were isolated and cultured by tissue digestion. The epithelial cell specific keratin immunohistochemical staining was used to identify the cells. The cells of the 2-3 generation logarithmic growth period were used for the follow-up experiment. The experimental cells were cultured for 24 hours before using the serum-free culture medium to synchronize them.
Group 1. ATRA: the effects of different concentrations (5 x 10-6M, 10 x 10-6M, 40 x 10-6M) ATRA on the proliferation of RPE cells in guinea pigs were detected by MTT method. The concentration of ATRA was selected as 10 x 10-6M in RPE cells. Radioimmunoassay was used to detect the changes of intracellular cAMP and IP3 levels.
2. phospholipase C inhibitor U73122 and adenylate cyclase inhibitor SQ22536 group: MTT method was used to detect the effects of different concentrations (5 x 10-6M, 10 x 10-6M, 15 x 106M, 20 x 10-6M) U73122 and (5 x 10-6M, 10 * 10-6M, 20x10-6M, 50). 4h, 6h, 8h and 16h detected the secretion of TGF- beta 2 by ELISA.
Result
Primary culture and identification of 1. RPE cells:
The primary cultured guinea pig RPE cells had been adhered to the wall after 48 hours. The cells were rich in pigments. The pigments were reduced and the cells were nearly hexagonal when the cells were near fusion. The immuno histochemical keratin staining RPE cells were brown and yellow positive.
Effect of 2. ATRA on RPE cells in guinea pigs
MTT detection: after 40 x 10-6M ATRA action RPE cell 24h, the cell growth was significantly affected by the inhibition of.5 * 10 "6M and 10 x 10-6M in the growth of RPE cells compared with the control group (P0.05).
Detection of TGF- beta 2: after adding 10 x 10-6MATRA, the secretion of TGF- beta 2 was significantly higher than the control group, 2h, 4h, 6h were significantly increased, 8h had no significant difference, 16h decreased (P0.05), and the secretion gradually decreased with time.
The detection of cAMP and IP3: after adding 10 x 10-6MATRA, the content of cAMP in RPE cells increased in 30min and 2H (P < 0.05), 5min and 6h, and there was no significant difference compared with the control group, but the IP3 content in the cell decreased significantly at every time point (P0.05), and the decrease was most obvious.
Effects of 3. phospholipase C inhibitor U73122 and adenylate cyclase inhibitor SQ22536 on guinea pig RPE cells
MTT detection: there was no significant difference in MTT results from the control group (P0.05) after 5 * 106M concentration of U73122 in the RPE cell of guinea pig (P0.05). The concentration of 5 x 10-6M was significantly lower than that of the control group (P0.01). The increase of the concentration increased obviously with the concentration increase, but there was no obvious change in RPE cell growth in SQ22536 group (P0.05).
The detection of TGF- beta 2: after adding 1 x 10-5M U73122, the secretion of TGF- beta 2 except 2H was significantly higher than that of the control group (P0.05), while the SQ22536 group secreted TGF- beta 2 in 2H, 4h, 6h, and decreased the secretion of RPE cells.
conclusion
1. the higher concentration of ATRA and U73122 inhibited the proliferation of RPE cells in guinea pigs, while SQ22536 had no significant effect on the growth of RPE cells.
After 2.10 * 10-6M ATRA acted on RPE cells, the secretion of TGF- beta 2 increased in short term and decreased with time.
After 3.10 * 10-6M ATRA acted on RPE cells, IP3 content decreased significantly, cAMP content increased in the middle of the observation period, and then decreased to the control group.
4.U73122 can increase the secretion of TGF- beta 2 in RPE cells, while SQ22536 has no significant effect on the secretion of RPE TGF- beta 2.
The results suggest that the proliferation of RPE cells with second messenger IP3 related to the effect of.ATRA on the secretion of TGF- beta 2 from RPE cells may be related to the decrease of second messenger IP3. But after the inhibition of phospholipase C signaling, the increase of TGF- beta 2 in RPE cells further confirms that TGF- beta 2 in guinea pig RPE cells is related to phospholipase C signaling pathway.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R778.11

【参考文献】