本文選題：豚鼠視網(wǎng)膜色素上皮細(xì)胞 + 視黃酸　； 參考：《天津醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的 在各種近視眼發(fā)病機(jī)制學(xué)說(shuō)中,“視網(wǎng)膜局部調(diào)控學(xué)說(shuō)”備受矚目,該學(xué)說(shuō)認(rèn)為視網(wǎng)膜感知外界環(huán)境變化后產(chǎn)生一級(jí)近視信號(hào)因子(如視黃酸,RA)并作用于視網(wǎng)膜色素上皮(retinal pigment epithelium, RPE),使之產(chǎn)生二級(jí)信號(hào)因子(如TGF-β2),進(jìn)而調(diào)控鞏膜的生長(zhǎng),形成近視。 本實(shí)驗(yàn)觀察全反視黃酸(ATRA)對(duì)豚鼠RPE細(xì)胞的生長(zhǎng)、分泌TGF-β2的影響及細(xì)胞內(nèi)第二信使cAMP、IP3的變化。并研究磷脂酶C抑制劑U73122和腺苷酸環(huán)化酶抑制劑SQ22536分別對(duì)視網(wǎng)膜色素上皮細(xì)胞生長(zhǎng)及分泌TGF-β2影響,分析RPE細(xì)胞分泌TGF-β2的相關(guān)胞內(nèi)信號(hào)傳導(dǎo)途徑,進(jìn)一步探討RPE在近視發(fā)生發(fā)展中的呈遞作用。 方法 選取健康3周齡花色豚鼠,麻醉處死后摘取眼球,采用組織消化法分離培養(yǎng)豚鼠RPE細(xì)胞,上皮細(xì)胞特異的角蛋白免疫組化染色鑒定,取第2-3代對(duì)數(shù)生長(zhǎng)期的細(xì)胞用于后續(xù)實(shí)驗(yàn)。實(shí)驗(yàn)細(xì)胞于使用前均換無(wú)血清培養(yǎng)液培養(yǎng)24小時(shí)以使其同步化。 1. ATRA組：用MTT法檢測(cè)不同濃度(5×10-6M,10×10-6M,40×10-6M)ATRA對(duì)豚鼠RPE細(xì)胞增殖的影響。選取濃度為10×10-6M的ATRA作用于RPE細(xì)胞,分別于2h、4h、6h、8h、16h取上清培養(yǎng)液用ELISA方法檢測(cè)TGF-β2的分泌量；于0min、5min、30min、2h、6h收集細(xì)胞裂解液用ELISA和放射免疫的方法分別檢測(cè)胞內(nèi)cAMP和IP3的含量變化。 2.磷脂酶C抑制劑U73122和腺苷酸環(huán)化酶抑制劑SQ22536組：用MTT法檢測(cè)不同濃度(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對(duì)豚鼠RPE細(xì)胞增殖的影響。取濃度為1×10-5M的U73122、SQ22536作用于豚鼠RPE細(xì)胞,分別于2h、4h、6h、8h、16h用ELISA方法檢測(cè)TGF-β2的分泌量。 結(jié)果 1. RPE細(xì)胞原代培養(yǎng)及鑒定： 原代培養(yǎng)的豚鼠RPE細(xì)胞48小時(shí)后已貼壁生長(zhǎng),細(xì)胞含有豐富的色素。傳代后色素減少,細(xì)胞近融合時(shí)形態(tài)近六角形。免疫組化角蛋白染色RPE細(xì)胞為棕黃色陽(yáng)性反應(yīng)。 2. ATRA對(duì)豚鼠RPE細(xì)胞的影響 MTT檢測(cè)：40×10-6M的ATRA作用RPE細(xì)胞24h后,細(xì)胞生長(zhǎng)明顯受到抑制。5×10"6M和10×10-6M的ATRA對(duì)RPE細(xì)胞的生長(zhǎng)的作用與對(duì)照組相比差異無(wú)顯著性(P0.05)。 TGF-β2的檢測(cè)：加入10×10-6MATRA后,TGF-β2的分泌量與對(duì)照組相比,2h、4h、6h明顯升高,8h無(wú)顯著差異,16h降低(P0.05),呈隨時(shí)間延長(zhǎng)分泌量逐漸下降趨勢(shì)。 cAMP及IP3的檢測(cè)：加入含10×10-6MATRA的培養(yǎng)液后,RPE胞內(nèi)cAMP含量在30min和2h時(shí)升高(p＜0.05),5min、6h時(shí)與對(duì)照組相比無(wú)明顯差別。而胞內(nèi)的IP3含量在各時(shí)間點(diǎn)均顯著降低(p0.05),6h時(shí)降低最明顯。 3.磷脂酶C抑制劑U73122和腺苷酸環(huán)化酶抑制劑SQ22536對(duì)豚鼠RPE細(xì)胞的影響 MTT檢測(cè)：5×106M濃度U73122作用豚鼠RPE細(xì)胞24h后,MTT結(jié)果與對(duì)照組無(wú)顯著差異(p0.05),濃度5×10-6M時(shí)均明顯低于對(duì)照組(p0.01)。并隨濃度的增OD值降低越明顯。而SQ22536組RPE細(xì)胞生長(zhǎng)無(wú)明顯變化(p0.05)。 TGF-β2的檢測(cè)：加入1×10-5M U73122后,TGF-β2的分泌量除2h無(wú)顯著差異外,余各時(shí)間點(diǎn)均較對(duì)照組明顯增加(P0.05)；而SQ22536組對(duì)RPE細(xì)胞分泌TGF-β2在2h,4h,6h,16h無(wú)明顯影響(p0.05),8h時(shí)分泌量降低(P0.05)。 結(jié)論 1.較高濃度的ATRA及U73122對(duì)豚鼠RPE細(xì)胞的增殖有抑制作用,而SQ22536對(duì)RPE細(xì)胞生長(zhǎng)無(wú)明顯影響。 2.10×10-6M的ATRA作用于RPE細(xì)胞后,TGF-β2的分泌量短期升高,隨時(shí)間延長(zhǎng)而降低。 3.10×10-6M的ATRA作用于RPE細(xì)胞后,IP3含量顯著降低,cAMP含量在觀察時(shí)間段中期有升高,后又降至對(duì)照組水平。 4.U73122可使RPE細(xì)胞分泌TGF-β2增加,而SQ22536對(duì)RPE TGF-β2的分泌無(wú)明顯影響。 研究結(jié)果提示RPE細(xì)胞的增殖與第二信使IP3有關(guān)。ATRA對(duì)RPE細(xì)胞分泌TGF-β2的影響可能與第二信使IP3的降低有關(guān)。而抑制磷脂酶C信號(hào)通路后,RPE細(xì)胞分泌TGF-β2增加,進(jìn)一步證實(shí)了豚鼠RPE細(xì)胞分泌TGF-β2與磷脂酶C信號(hào)通路有關(guān)。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R778.11

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