胰高血糖素对豚鼠巩膜成纤维细胞增生的影响及其机制

Effect of glucagon on proliferation of scleral fibroblast cells of guinea pig in vitro and its mechanism

背景胰高血糖素与近视的发生密切相关，在一定浓度范围内可抑制近视的发展，但其确切的作用机制尚不明确。目的研究胰高血糖素对豚鼠巩膜成纤维细胞（GSFCs）生长的影响及作用机制。方法获取出生15d的健康纯种豚鼠的巩膜组织，采用组织块培养法进行GSFCs的培养和传代，采用免疫组织化学法检测培养的细胞中波形蛋白抗体、细胞角蛋白抗体及S-100抗体的表达。用完全随机法将培养的细胞分组，分别在不同组的培养孔中加入5、10、50、100、200μg／L胰高血糖素培养GSFCs24h，不加胰高血糖素培养的为对照组。利用MTT比色法观察各质量浓度胰高血糖素作用后及50btg／L胰高血糖素作用1、2、3、5、7dGSFCs的生长情况，酶联免疫检测仪测量波长490nm处各孔细胞的吸光度（A490）值，并用ELISA法检测培养72h不同质量浓度胰高血糖素对GSFCs中基质金属蛋白酶2／组织金属蛋白酶抑制剂2（MMP-2／TIMP-2）表达水平的影响，以酶标仪测定450nm波长处各孔细胞的A450值。结果GSFCs传代培养后密度较低时呈枝状排列，密度较高时呈束状或漩涡状排列，波形蛋白抗体反应阳性。随着胰高血糖素质量浓度的增加，细胞的A490值逐渐升高，差异有统计学意义（F=32．340，P=0．013），胰高血糖素质量浓度〉10μg／L时，细胞的A490值较对照组明显升高，差异均有统计学意义（t=5．575、6．627、16．074、12．003，P〈0．05）。培养孔中加入50μg／L胰高血糖素后随着培养时间的延长，细胞的A450值升高，差异有统计学意义（F时间=10．610，P=0．024），且与对照组比较细胞的A490值升高，差异有统计学意义（F。=9．068，P=0．039）。胰高血糖素质量浓度在5～200μg／L时，随胰高血糖素质量浓度的增加，MMP-2含量逐渐减少，差异有统计学意义（F=153．639，P=0．036），但TIMP-2含量的变化差异无统计学意义（F=24．770，P=3．250）。结论胰高血糖素可促进体外培养的GSFCs的增生，其作用呈剂量和时间依赖性，其作用机制可能是下调MMP-2在GSFCs中的表达，MMP-2／TIMP-2失衡所致。

Background The occurrence of experimental myopia may be related to glucagon,and within the range of certain concentration,glucagon may inhibit the development of myopia,but its exact action mechanism is not completely clear. Objective Purpose of this study was to evaluate the effects of glucagon on the proliferation of guinea pig scleral fibroblast cells（GSFCs） and the possible role of glueagon in myopic scleral remodeling. Methods The seleral tissue was obtained from the clean blooded guinea pig aged 15 days. GSFCs were cultured and identified with vimentin antibody, cytokeratin antibody and S-100 antibody. 0,5,10,50,100,200 p,g/L glucagon was added into the different cultured hole for 24 hours respectively, and the growth and proliferation （A4~~ value ）of GSFCs was detected by MTT eolorimetric assay. Then the A^90 value of GSFCs was assayed in 1 day,2,3,5,7 days under the 50 Ixg/L glucagon action. Matrix metalloproteinase-2 （ MMP-2 ） and tissue inhibitor of metalloproteinase-2 （ TIMP-2 ） levels（A450 value）in GSFCs were detected by enzyme-linked immuno sorbent assay（ELISA） 72 hours after the cells cultured. Results Passaged GSFCs showed the dcndric array at lower density and gyrate array at the higher density with the positive response for vimentin. A49o values of GSFCs were gradually increased with the rise of glueagon concentration（ F= 32. 340 ,P= 0. 013 ）. When the glucagon concentration was 10-200 μg/L, the A490 value of GSFCs was higher than that without glucagon group, showing signifitant differences between them（ t = 5. 575,6. 627,16. 074,12. 003 ,P〈0.05 ）. Under the 50 p,g/L glucagon action,A490 values were significantly accented with the time lapse （Ftime = 10. 610,P=0. 024） ,and the A490values also were significantly higher than the parallel control groups without glueagon（F rout, =9. 068 ,P=0. 039）. MMP-2 level was gradually declined with the enhance of glucagon within range of 5-200 μg/L（F= 153. 639,P=0. 036） ,but no significant difference was found in TIMP-2 expression（F=24. 770, P=3. 250）. Conclusions Glucagon ean promote the proliferation of GSFCs in vitro,and the synthesis of MMP-2 shows a concentration- and time-dependent manner.