小环肽CHSDGIC保护大鼠视网膜神经节细胞株RGC-5的实验研究

发布时间：2018-12-23 12:28

【摘要】：目的：通过体外实验，观察垂体腺苷酸环化酶激活多肽(pituitary adenylate cyclaseactivating polypeptide，PACAP)衍生的小环肽CHSDGIC对紫外光B照射诱导凋亡的大鼠视网膜神经节细胞株RGC-5的保护作用，为CHSDGIC预防视网膜神经节细胞病变提供实验依据。方法：用高糖DMEM常规培养RGC-5，然后将RGC-5置于紫外光B（Ultraviolet,UVB;中波312nm）照射下进行细胞凋亡诱导处理。(1)采用Western-blot法检测正常条件下和UVB照射1min、3min、5min后RGC-5体内PAC1受体的表达情况。(2)常规培养RGC-5，然后分为4个实验组和1个对照组，各实验组加入不同质量浓度的CHSDGIC(1μmol/L、10μmol/L、100μmol/L、1000μmol/L)，对照组加入0.9%NaCl，培养1h，各组细胞同时接受UVB照射3min，24h后采用CCK-8试剂盒酶标仪检测各组RGC-5存活情况；流式细胞仪Annexin-V/PI双染法检测RGC-5凋亡情况，主要分析10μmol/L CHSDGIC对细胞凋亡率的影响。结果：(1) Western-Blot检测发现，正常条件及UVB照射下RGC-5体内均有PAC1受体的表达；(2) CCK-8法检测显示1μmol/L、10μmol/L、100μmol/L、1000μmol/LCHSDGIC的细胞存活率分别为(34.00±1.07)%、(55.20±2.23)%、(36.69±1.96)%、(42.93±1.91)%，，对照组的存活率为(24.62±1.72)%，差异有统计学意义(P值均0.05)，其中10μmol/L组的细胞存活率最高；(3)流式Annexin-V/PI双染法凋亡检测发现，UVB作用于RGC-5的对照组细胞凋亡率(37.40±2.40)%较正常生长的RGC-5细胞凋亡率(5.43±1.02)%高，差异有统计学意义(P值0.05)；10μmol/LCHSDGIC实验组细胞凋亡率为(15.30±1.10)%，较对照组相比差异有统计学意义(P值0.05)。结论：RGC-5存在PACAP的特异性受体PAC1，CHSDGIC对UVB诱导凋亡条件下的RGC-5有明显的保护作用。
[Abstract]:Aim: to observe the protective effect of pituitary adenylate cyclase activated polypeptide (pituitary adenylate cyclaseactivating polypeptide,PACAP) -derived small cyclic peptide CHSDGIC on rat retinal ganglion cell line RGC-5 induced by ultraviolet B irradiation in vitro. To provide experimental evidence for the prevention of retinal ganglion cytopathia by CHSDGIC. Methods: RGC-5, was cultured with high glucose DMEM and then RGC-5 was placed in ultraviolet B (Ultraviolet,UVB;). (1) the expression of PAC1 receptor in RGC-5 was detected by Western-blot method in normal condition and 3 mins 5 min after UVB irradiation. (2) the expression of PAC1 receptor in RGC-5 was detected by routine culture of RGC-5,. Then they were divided into four experimental groups and one control group. Each group was treated with different concentration of CHSDGIC (1 渭 mol/L,10 渭 mol/L,100 渭 mol/L,1000 渭 mol/L), the control group was treated with 0.9 NaCl1 for 1 hour, and the cells in each group were irradiated with UVB for 3 min at the same time. After 24 hours, the survival of RGC-5 in each group was detected by CCK-8 kit enzyme labeling instrument. The apoptosis of RGC-5 was detected by flow cytometry (Annexin-V/PI) double staining, and the effect of 10 渭 mol/L CHSDGIC on the apoptosis rate was analyzed. Results: (1) the expression of PAC1 receptor in RGC-5 was found in normal condition and UVB irradiation by Western-Blot. (2) the cell survival rate of 1 渭 mol/L,10 渭 mol/L,100 渭 mol/L,1000 渭 mol/LCHSDGIC was (34.00 卤1.07)%, (55.20 卤2.23)%, (36.69 卤1.96)%, (42.93 卤1.91)%, respectively. The survival rate of the control group was (24.62 卤1.72)%, and the difference was statistically significant (P < 0. 05). The cell survival rate of the 10 渭 mol/L group was the highest. (3) the apoptosis rate of the control group treated with UVB was (37.40 卤2.40)% higher than that of the normal growth RGC-5 cell (5.43 卤1.02)%, and the apoptosis rate of the control group was (37.40 卤2.40)% higher than that of the normal growth group (5.43 卤1.02)%). The difference was statistically significant (P 0.05). The apoptotic rate of the 10 渭 mol/LCHSDGIC experimental group was (15.30 卤1.10)%, which was significantly higher than that of the control group (P < 0. 05). Conclusion: PAC1,CHSDGIC, a specific receptor for PACAP in RGC-5, has an obvious protective effect on RGC-5 induced by UVB.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1

【参考文献】