实验性糖尿病大鼠视网膜细胞凋亡和微血管改变及结缔组织生长因子的表达

目的 研究糖尿病大鼠视网膜凋亡细胞和结缔组织生长因子(CTGF)表达情况及其在微循环改变中的作用.方法 实验研究.55只成年雄性Wistar大鼠,以随机数字表法,随机分为正常对照(CON)组(10只鼠)和糖尿病(DM)组(45只鼠).根据病程不同,将存活的30只DM组大鼠再分为2个月组(DM2)、4个月组(DM4)及6个月组(DM6),每组各10只鼠.用末端脱氧核糖核酸转移酶介导的dUTP缺口末端标记(TUNEL)法检测视网膜凋亡细胞,用免疫组织化学法测定CTGF表达水平,以视网膜消化铺片过碘酸雪夫(PAS)染色法观察视网膜血管改变情况.观察并比较不同病程的DM大鼠视网膜凋亡细胞及CTGF表达情况、视网膜微血管形态变化及周细胞数量变化.对各组大鼠视网膜的细胞凋亡指数、CTGF表达阳性率及血管周细胞数量进行比较采用LSD-t检验法,两变量间的相关性分析采用线性相关法.结果 CON组大鼠视网膜细胞凋亡和CTGF表达均阴性.DM2组、DM4组、DM6组大鼠视网膜细胞凋亡指数分别为0.05±0.01、0.25±0.03及0.52±0.02;组间两两比较,差异均有统计学意义(DM2与DM4组比较t=21.432,DM2与DM6组比较t=50.843,DM4与DM6组比较t=29.410;P<0.05),表明随DM病程延长,视网膜凋亡指数逐渐增加.DM2组、DM4组、DM6组大鼠视网膜CTGF表达阳性率分别为(22.79±2.99)%、(41.73±2.59)%、(55.27±2.68)%,组间两两比较,差异均有统计学意义(DM2与DM4组比较t=15.345,DM2与DM6组比较t=26.316,DM4与DM6组比较t=10.971;P<0.05),表明随DM病程延长,视网膜CTGF表达水平逐渐增高.CON及DM2组大鼠视网膜血管走形良好,DM4组大鼠视网膜部分血管渐变僵硬、狭窄;DM6组大鼠视网膜血管主干僵硬,部分微血管狭窄明显.与CON组视网膜血管周细胞数量对比,DM2组未见明显变化;随病程延长,DM4组和DM6组大鼠视网膜血管周细胞数量较CON组逐渐减少,差异均有统计学意义(t=3.367,6.667;P<0.05).DM大鼠视网膜细胞凋亡指数与CTGF阳性表达水平呈显著正相关(r=0.958,P<0.05),凋亡指数和CTGF阳性表达水平与视网膜血管周细胞数量均呈显著负相关(r=-0.540,-0.595;P<0.05).结论 在DM大鼠视网膜组织中,凋亡细胞和致纤维化因子CTGF的产生均早于微循环血管走形及周细胞的改变.(中华眼科杂志,2011,47:521-526)

Abstract：

Objective To study the cell apoptosis and the expression of connective tissue growth factor (CTGF) in the retina of diabetic rats and to explore their contributions to the changes of microcirculation. Methods It was a experiment study. Fifty-five adult male Wistar rats were divided into two groups, normal control group (CON,10 rats) and diabetes mellitus group (DM, 45 rats). The 30 surviving rats in the DM group were further divided into 3 groups based on the time of observation, 2 month (DM2), 4 month (DM4) and 6 month (DM6) groups, with 10 rats in each group. Cell apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL). Expression of CTGF was determined by immunohistochemical study. Retinal vessels were observed by retinal digest stretched periodic acid Schiff (PAS) staining. Results Immunohistochemical staining and TUNEL staining revealed negative results in normal control group. Retinal cell apoptosis index increased gradually from DM2 to DM6, the differences between any two groups were statistically significant (t2-4,2-6,4-6=21.432, 50.843, 29.410;P<0.05). Expression of CTGF in the retina increased from DM2-DM6, the differences between any two groups were statistically significant (t2-4,2-6,4-6=15.345, 26.316, 10.971;P<0.05). PAS staining of retinal blood vessels obtained negative results in the CON and DM2 groups. Part of retinal capillaries were slightly stiff and narrow in DM4 group. Retinal capillaries in DM6 group were trunk stiff and were narrowed obviously. The number of pericytes was reduced in DM4, and progressed following the course of diabetes. The number of pericytes in the DM2 group did not different from that in the CON group (t=0.875,P=0.387). The number of pericytes in the DM4 and DM6 group were significantly decreased as compared to the CON group (t=3.367,6.667;P<0.05). Retinal cellular apoptosis index had a significant positive correlation to the expression of CTGF (r=0.958,P<0.05). Number of pericytes was significantly correlated (negative correlation) with retinal cellular apoptosis index and the expression of CTGF (r=-0.540, -0.595; P<0.05). Conclusions The appearances of cellular apoptosis and fibrosing factor CTGF in the retina of diabetic rats occurred earlier than the changes of microcirculation and the number of capillary pericytes.

Retinal cell apoptosis, microvascular changes and expression of connective tissue growth factor in experimental diabetic rats

Objective To study the cell apoptosis and the expression of connective tissue growth factor (CTGF) in the retina of diabetic rats and to explore their contributions to the changes of microcirculation. Methods It was a experiment study. Fifty-five adult male Wistar rats were divided into two groups, normal control group (CON,10 rats) and diabetes mellitus group (DM, 45 rats). The 30 surviving rats in the DM group were further divided into 3 groups based on the time of observation, 2 month (DM2), 4 month (DM4) and 6 month (DM6) groups, with 10 rats in each group. Cell apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL). Expression of CTGF was determined by immunohistochemical study. Retinal vessels were observed by retinal digest stretched periodic acid Schiff (PAS) staining. Results Immunohistochemical staining and TUNEL staining revealed negative results in normal control group. Retinal cell apoptosis index increased gradually from DM2 to DM6, the differences between any two groups were statistically significant (t2-4,2-6,4-6=21.432, 50.843, 29.410;P<0.05). Expression of CTGF in the retina increased from DM2-DM6, the differences between any two groups were statistically significant (t2-4,2-6,4-6=15.345, 26.316, 10.971;P<0.05). PAS staining of retinal blood vessels obtained negative results in the CON and DM2 groups. Part of retinal capillaries were slightly stiff and narrow in DM4 group. Retinal capillaries in DM6 group were trunk stiff and were narrowed obviously. The number of pericytes was reduced in DM4, and progressed following the course of diabetes. The number of pericytes in the DM2 group did not different from that in the CON group (t=0.875,P=0.387). The number of pericytes in the DM4 and DM6 group were significantly decreased as compared to the CON group (t=3.367,6.667;P<0.05). Retinal cellular apoptosis index had a significant positive correlation to the expression of CTGF (r=0.958,P<0.05). Number of pericytes was significantly correlated (negative correlation) with retinal cellular apoptosis index and the expression of CTGF (r=-0.540, -0.595; P<0.05). Conclusions The appearances of cellular apoptosis and fibrosing factor CTGF in the retina of diabetic rats occurred earlier than the changes of microcirculation and the number of capillary pericytes.