糖尿病大鼠视网膜组织CTGF、VEGF、TGF-β_2作用机制和CTGFsiRNA的干预作用
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摘要
研究背景和目的
糖尿病性视网膜病变(diabetic retinopathy,DR)是最严重和常见的糖尿病并发症之一,是50岁以上人群的重要致盲眼病。近年来糖尿病的发病率显著上升,其中在发展中国家的增长速度要远大于其他国家。其发病率高达38%～90%,现已引起人们广泛重视。糖尿病因糖代谢障碍致全身各组织器官的微血管异常,在眼部可引起视网膜缺血缺氧,新生血管形成,纤维增殖等以致视网膜脱离,甚至导致丧失视失。DR的发病机制十分复杂,至今尚未完全阐明。细胞因子在DR的发生、发展中起重要作用。其中新近发现的结缔组织生长因子(connective tissuegrowth factor,CTGF)引起了许多学者的关注,CTGF为CCN(CTGF/fisp12,Cef10/cry61,Nov,CCN)家族成员之一,为单体分泌性多肽。CTGF广泛存在于人类多种组织器官中,它具有促进血管生成、调节内皮细胞、诱导细胞凋亡,促进有丝分裂和细胞增生,趋化细胞等作用。近年来的研究显示CTGF参与了糖尿病视网膜病变的发病机制。与CTGF密切相关的细胞因子包括血管内皮生长因子(vascular endothelial growth factor VEGF)和转化生长因子(transforming growthfactor,TGF)等,它们在CTGF的作用过程中也发挥着重要的作用。研究显示,CTGF蛋白可与VEGF蛋白连接,相互作用影响新生血管形成;同时CTGF被认为是TGF-β_2的下游效应介质;TGF-β_2可诱导CTGF和VEGF表达增加;TGF-β_2可通过MAPK和PKC途径使VEGF表达增加。另外,细胞凋亡在糖尿病视网膜病变的发生发展中起了很重要的作用。细胞凋亡存在于糖尿病视网膜的神经节细胞层,光感受器细胞层和内核层。在糖尿病患者中,视网膜内细胞凋亡的程度明显高于非糖尿病者。多元醇通路的激活,山梨糖醇聚集可影响视网膜的功能,氧化损伤、糖基化终产物的形成可能对视网膜产生毒性作用,通过非酶糖基化途径诱导视网膜细胞凋亡。RNA干扰(RNA interference,RNAi)技术是一种转录后基因沉默的强大工具,已证实其为化学合成长21～23个核苷酸的小分子RNA(smallinterfered RNA,siRNA),能在哺乳动物体内外介导同源基因沉默。siRNA作为一种新方法可以用来研究在各种组织中基因产物的功能。总之,到目前为此,虽然取得了诸多的进展,但各因子间的关系并不十分清楚,DR还是严重威胁人们视力的疾病,也不能完全地预防及治疗,因此需要进一步对其进行研究。对三种细胞因子的深入研究,有利于破解糖尿病视网膜中微血管病变的发生机制。本实验以CTGF为主线,利用CTGF小干扰RNA技术的干预,通过检测糖尿病大鼠视网膜中CTGF、VEGF和TGF-β_2蛋白和基因的表达,细胞凋亡的发生情况,来分析三因子在糖尿病大鼠视网膜中的作用机制,以求寻找到防治DR的新策略。
方法
利用STZ诱导形成糖尿病大鼠模型。Wistar大鼠,体重为180-220g,240只。腹腔注射1%STZ溶液,剂量为60mg/kg。连续三天尿糖在+++以上,72小时后取大鼠尾静脉检测血糖,血糖值大于16.7mmol/L,为糖尿病大鼠造模成功。所有大鼠分为对照组,糖尿病4周、8周、12周、16周、24周组和干预组(糖尿病16周)。干预组为糖尿病16周时给予CTGFsiRNA玻璃体腔注射。
视网膜铺片ADP酶染色观察视网膜血管形态,HE染色光镜下观察视网膜各层结构。应用RT-PCR和Western-Blot方法检测视网膜内CTGF、VEGF和TGF-β_2基因和蛋白的表达;免疫组化观察以上三因子在视网膜内的分布情况。应用TUNEL方法检测视网膜内细胞凋亡的表达。
统计学分析:统计结果均以均数士标准差表示,应用SPSS10.0软件进行统计分析,包括两组资料之间比较的t检验和相关分析。
结果
成功建立了STZ诱导糖尿病大鼠模型。成功率为97.50%。在糖尿病16周以前,视网膜结构规则,血管走行良好与对照组无显著差异。到24周时,可见视网膜结构不规则,水肿,细胞排列紊乱,有部分细胞突出于内界膜;视网膜血管主干较为僵硬变窄,但未见缺血区及新生血管形成。细胞凋亡表达在糖尿病4周就已出现,并逐渐增强。CTGF,VEGF和TGF-β_2的表达水平在糖尿病视网膜内明显增强。CTGF开始于糖尿病8周,主要位于视网膜神经节细胞层;VEGF和TGF-β_2则在糖尿病12周时开始表达增强,CTGF的表达增强早于VEGF和TGF-β_2;差异具有统计学意义(P＜0.05),随着病程的延长,三因子的表达逐渐增强;到糖尿病24周时,三因子均达到表达均最强,在视网膜各层均可见免疫染色的蛋白。CTGFsiRNA在干预组大鼠视网膜内造成了CTGF基因的沉默,干扰效率为54.46%,CTGF蛋白的干扰效率为45.11%,同时也使VEGF、TGF-β_2 mRNA及其蛋白表达明显减弱,细胞凋亡表达也明显减少,统计学上差异具有显著性意义(P＜0.05)。我们发现在糖尿病早期大鼠视网膜内CTGF与VEGF(P＜0.05),CTGF与TGF-β_2(P＜0.05),VEGF与TGF-β_2(P＜0.01)之间,以及三种因子与细胞凋亡的表达(P＜0.01)之间呈明显的正相关性。
结论
本研究成功建立了糖尿病大鼠模型,在糖尿病早期视网膜内可见血管及网膜的结构无明显改变,但出现了逐渐增多的凋亡细胞,最早发生在视网膜神经节细胞层,之后出现于内核层及外核层,到糖尿病24周时出现视网膜血管变窄及僵硬,网膜水肿及细胞排列不规则等病变。这表明糖尿病早期视网膜细胞凋亡的发生要早于血管病变的发生。糖尿病可促使大鼠视网膜内的CTGF表达增强,同时也上调了视网膜内VEGF和TGF-β_2的表达,并参与了视网膜细胞凋亡的诱导。CTGF的表达增强的发生早于VEGF和TGF-β_2的表达。说明CTGF在三因子的相互作用中占了主要地位。CTGFsiRNA沉默了CTGF基因的表达,使VEGF、TGF-β_2蛋白和基因表达也明显下降,并使糖尿病视网膜内的细胞凋亡表达出现减弱趋势。总之,CTGF,VEGF和TGF-β_2三者在糖尿病早期视网膜中发挥了重要作用。CTGFsiRNA有可能为临床提供一个预防和治疗DR的新策略。
Background and Objective
Diabetic retinopathy is a leading cause of blindness,ultimately resulting in an advanced stage of proliferative retinopathy with neovascularization,fibrovascular proliferation,and retinal detachment.Recently the incidence of diabetes increased significantly,which is higher in developing countries than others as 38%～90%.It has been attracted extensive attention.The pathogenesis of DR is very complex and has not yet been fully clarified.Moreover cells are lost during this early stage of retinopathy in diabetes.Cytokines have been implicated in diabetic retinopathy,the most notable being vascular endothelial growth factor(VEGF) and transforming growth factor-beta2 (TGF-β2).Connective tissue growth factor(CTGF) is a cysteine-rich matricellular protein belonging to the CCN family of proteins,which have many diverse functions concerned with angiogenesis,fibrosis,and apoptosis etc.CTGF,accompanied by VEGF and TGF-β2 are increasingly recognized as playing important roles in the pathogenesis of diabetic retinopathy.Recently,a novel method of post-transcriptional silencing of gene expression,called RNA interference(RNAi),was discovered.RNAi is a conserved cellular mechanism that silences the expression of a protein in a specific and potent fashion.Thus small interference RNA(siRNA) is a potent method for investigating the function of gene products in tissues.Although much progress has been made recently,the interaction between these factors is not very clear and further research is needed,as the disease remains neither preventable nor curable.In this study, we measure the gene and protein expression and the distribution of CTGF,VEGF and TGF-β2 in the retina of diabetic and normal rats,and analyze the correlation between expression of these three factors and the relationship between the degree of apoptosis and cytokine production using siRNA targeting CTGF(CTGFsiRNA) to silence the CTGF gene.
Methods
Diabetes was induced in rats by the 1%β-cell toxin streptozotocin(STZ).The blood glucose above 16.7mmol/L and urine glaucose+++ is the standard of setting up the model.Animals were divided to control,diabetic rats in 4w,8w,12w,16w,24 weeks and similar animals diabetic 16 weeks treated with CTGFsiRNA by intravitreal injection.The vessels of retina were detected by ADPase stained mRNA level and protein expression of CTGF,VEGF and TGF-β_2 was measured by RT-PCR and Western-blotting,and located by immunohistochemistry.Retinal apoptosis was detected by TUNEL staining.
The results were statistically tested by SPSS10.0 with t test and correlations analysis.
Results
The model of diabetes in rat was set up successfully.At early stage of diabetic retina before 16 weeks,there were similar to the control.Till 24 weeks diabetes,the vessels became straight and narrow,and the layers of the retina became unregular and the inner layer membrane edema.The levels of CTGF,VEGF and TGF-β_2 and TUNEL-positive nuclei number in the diabetic retinas were significantly higher than the control(P＜0.05).CTGF rose at 8 weeks,earlier than VEGF and TGF-β_2 at 12 weeks after the onset of diabetes.The difference was significant(P＜0.05). siRNA-mediated inhibition of CTGF mRNA inhibited retinal VEGF and TGF-β_2 and also resulted in a significant decrease in apoptosis.Significant correlations were found between CTGF and VEGF(P=0.04),CTGF and TGF-β_2(P=0.05),VEGF and TGF-β_2 (P＜0.01),apoptosis and these three cytokines(P＜0.01) in the rat retina early in diabetes.
Conclusion
These results suggest that the diabetes-mediated increase in CTGF up-regulates VEGF and TGF-β_2 expression and induces apoptosis in retina.And the elevation could be inhibited by treatment with CTGFsiRNA.CTGF,VEGF and TGF-β_2 play critical roles in the early stage of diabetic retina.CTGF could serve as a potential target for the prevention and treatment of diabetic retinopathy.
糖尿病性视网膜病变(diabetic retinopathy,DR)是最严重和常见的糖尿病并发症之一,是50岁以上人群的重要致盲眼病。近年来糖尿病的发病率显著上升,其中在发展中国家的增长速度要远大于其他国家。其发病率高达38%～90%,现已引起人们广泛重视。糖尿病因糖代谢障碍致全身各组织器官的微血管异常,在眼部可引起视网膜缺血缺氧,新生血管形成,纤维增殖等以致视网膜脱离,甚至导致丧失视失。DR的发病机制十分复杂,至今尚未完全阐明。细胞因子在DR的发生、发展中起重要作用。其中新近发现的结缔组织生长因子(connective tissuegrowth factor,CTGF)引起了许多学者的关注,CTGF为CCN(CTGF/fisp12,Cef10/cry61,Nov,CCN)家族成员之一,为单体分泌性多肽。CTGF广泛存在于人类多种组织器官中,它具有促进血管生成、调节内皮细胞、诱导细胞凋亡,促进有丝分裂和细胞增生,趋化细胞等作用。近年来的研究显示CTGF参与了糖尿病视网膜病变的发病机制。与CTGF密切相关的细胞因子包括血管内皮生长因子(vascular endothelial growth factor VEGF)和转化生长因子(transforming growthfactor,TGF)等,它们在CTGF的作用过程中也发挥着重要的作用。研究显示,CTGF蛋白可与VEGF蛋白连接,相互作用影响新生血管形成;同时CTGF被认为是TGF-β_2的下游效应介质;TGF-β_2可诱导CTGF和VEGF表达增加;TGF-β_2可通过MAPK和PKC途径使VEGF表达增加。另外,细胞凋亡在糖尿病视网膜病变的发生发展中起了很重要的作用。细胞凋亡存在于糖尿病视网膜的神经节细胞层,光感受器细胞层和内核层。在糖尿病患者中,视网膜内细胞凋亡的程度明显高于非糖尿病者。多元醇通路的激活,山梨糖醇聚集可影响视网膜的功能,氧化损伤、糖基化终产物的形成可能对视网膜产生毒性作用,通过非酶糖基化途径诱导视网膜细胞凋亡。RNA干扰(RNA interference,RNAi)技术是一种转录后基因沉默的强大工具,已证实其为化学合成长21～23个核苷酸的小分子RNA(smallinterfered RNA,siRNA),能在哺乳动物体内外介导同源基因沉默。siRNA作为一种新方法可以用来研究在各种组织中基因产物的功能。总之,到目前为此,虽然取得了诸多的进展,但各因子间的关系并不十分清楚,DR还是严重威胁人们视力的疾病,也不能完全地预防及治疗,因此需要进一步对其进行研究。对三种细胞因子的深入研究,有利于破解糖尿病视网膜中微血管病变的发生机制。本实验以CTGF为主线,利用CTGF小干扰RNA技术的干预,通过检测糖尿病大鼠视网膜中CTGF、VEGF和TGF-β_2蛋白和基因的表达,细胞凋亡的发生情况,来分析三因子在糖尿病大鼠视网膜中的作用机制,以求寻找到防治DR的新策略。
方法
利用STZ诱导形成糖尿病大鼠模型。Wistar大鼠,体重为180-220g,240只。腹腔注射1%STZ溶液,剂量为60mg/kg。连续三天尿糖在+++以上,72小时后取大鼠尾静脉检测血糖,血糖值大于16.7mmol/L,为糖尿病大鼠造模成功。所有大鼠分为对照组,糖尿病4周、8周、12周、16周、24周组和干预组(糖尿病16周)。干预组为糖尿病16周时给予CTGFsiRNA玻璃体腔注射。
视网膜铺片ADP酶染色观察视网膜血管形态,HE染色光镜下观察视网膜各层结构。应用RT-PCR和Western-Blot方法检测视网膜内CTGF、VEGF和TGF-β_2基因和蛋白的表达;免疫组化观察以上三因子在视网膜内的分布情况。应用TUNEL方法检测视网膜内细胞凋亡的表达。
统计学分析:统计结果均以均数士标准差表示,应用SPSS10.0软件进行统计分析,包括两组资料之间比较的t检验和相关分析。
结果
成功建立了STZ诱导糖尿病大鼠模型。成功率为97.50%。在糖尿病16周以前,视网膜结构规则,血管走行良好与对照组无显著差异。到24周时,可见视网膜结构不规则,水肿,细胞排列紊乱,有部分细胞突出于内界膜;视网膜血管主干较为僵硬变窄,但未见缺血区及新生血管形成。细胞凋亡表达在糖尿病4周就已出现,并逐渐增强。CTGF,VEGF和TGF-β_2的表达水平在糖尿病视网膜内明显增强。CTGF开始于糖尿病8周,主要位于视网膜神经节细胞层;VEGF和TGF-β_2则在糖尿病12周时开始表达增强,CTGF的表达增强早于VEGF和TGF-β_2;差异具有统计学意义(P＜0.05),随着病程的延长,三因子的表达逐渐增强;到糖尿病24周时,三因子均达到表达均最强,在视网膜各层均可见免疫染色的蛋白。CTGFsiRNA在干预组大鼠视网膜内造成了CTGF基因的沉默,干扰效率为54.46%,CTGF蛋白的干扰效率为45.11%,同时也使VEGF、TGF-β_2 mRNA及其蛋白表达明显减弱,细胞凋亡表达也明显减少,统计学上差异具有显著性意义(P＜0.05)。我们发现在糖尿病早期大鼠视网膜内CTGF与VEGF(P＜0.05),CTGF与TGF-β_2(P＜0.05),VEGF与TGF-β_2(P＜0.01)之间,以及三种因子与细胞凋亡的表达(P＜0.01)之间呈明显的正相关性。
结论
本研究成功建立了糖尿病大鼠模型,在糖尿病早期视网膜内可见血管及网膜的结构无明显改变,但出现了逐渐增多的凋亡细胞,最早发生在视网膜神经节细胞层,之后出现于内核层及外核层,到糖尿病24周时出现视网膜血管变窄及僵硬,网膜水肿及细胞排列不规则等病变。这表明糖尿病早期视网膜细胞凋亡的发生要早于血管病变的发生。糖尿病可促使大鼠视网膜内的CTGF表达增强,同时也上调了视网膜内VEGF和TGF-β_2的表达,并参与了视网膜细胞凋亡的诱导。CTGF的表达增强的发生早于VEGF和TGF-β_2的表达。说明CTGF在三因子的相互作用中占了主要地位。CTGFsiRNA沉默了CTGF基因的表达,使VEGF、TGF-β_2蛋白和基因表达也明显下降,并使糖尿病视网膜内的细胞凋亡表达出现减弱趋势。总之,CTGF,VEGF和TGF-β_2三者在糖尿病早期视网膜中发挥了重要作用。CTGFsiRNA有可能为临床提供一个预防和治疗DR的新策略。
Background and Objective
Diabetic retinopathy is a leading cause of blindness,ultimately resulting in an advanced stage of proliferative retinopathy with neovascularization,fibrovascular proliferation,and retinal detachment.Recently the incidence of diabetes increased significantly,which is higher in developing countries than others as 38%～90%.It has been attracted extensive attention.The pathogenesis of DR is very complex and has not yet been fully clarified.Moreover cells are lost during this early stage of retinopathy in diabetes.Cytokines have been implicated in diabetic retinopathy,the most notable being vascular endothelial growth factor(VEGF) and transforming growth factor-beta2 (TGF-β2).Connective tissue growth factor(CTGF) is a cysteine-rich matricellular protein belonging to the CCN family of proteins,which have many diverse functions concerned with angiogenesis,fibrosis,and apoptosis etc.CTGF,accompanied by VEGF and TGF-β2 are increasingly recognized as playing important roles in the pathogenesis of diabetic retinopathy.Recently,a novel method of post-transcriptional silencing of gene expression,called RNA interference(RNAi),was discovered.RNAi is a conserved cellular mechanism that silences the expression of a protein in a specific and potent fashion.Thus small interference RNA(siRNA) is a potent method for investigating the function of gene products in tissues.Although much progress has been made recently,the interaction between these factors is not very clear and further research is needed,as the disease remains neither preventable nor curable.In this study, we measure the gene and protein expression and the distribution of CTGF,VEGF and TGF-β2 in the retina of diabetic and normal rats,and analyze the correlation between expression of these three factors and the relationship between the degree of apoptosis and cytokine production using siRNA targeting CTGF(CTGFsiRNA) to silence the CTGF gene.
Methods
Diabetes was induced in rats by the 1%β-cell toxin streptozotocin(STZ).The blood glucose above 16.7mmol/L and urine glaucose+++ is the standard of setting up the model.Animals were divided to control,diabetic rats in 4w,8w,12w,16w,24 weeks and similar animals diabetic 16 weeks treated with CTGFsiRNA by intravitreal injection.The vessels of retina were detected by ADPase stained mRNA level and protein expression of CTGF,VEGF and TGF-β_2 was measured by RT-PCR and Western-blotting,and located by immunohistochemistry.Retinal apoptosis was detected by TUNEL staining.
The results were statistically tested by SPSS10.0 with t test and correlations analysis.
Results
The model of diabetes in rat was set up successfully.At early stage of diabetic retina before 16 weeks,there were similar to the control.Till 24 weeks diabetes,the vessels became straight and narrow,and the layers of the retina became unregular and the inner layer membrane edema.The levels of CTGF,VEGF and TGF-β_2 and TUNEL-positive nuclei number in the diabetic retinas were significantly higher than the control(P＜0.05).CTGF rose at 8 weeks,earlier than VEGF and TGF-β_2 at 12 weeks after the onset of diabetes.The difference was significant(P＜0.05). siRNA-mediated inhibition of CTGF mRNA inhibited retinal VEGF and TGF-β_2 and also resulted in a significant decrease in apoptosis.Significant correlations were found between CTGF and VEGF(P=0.04),CTGF and TGF-β_2(P=0.05),VEGF and TGF-β_2 (P＜0.01),apoptosis and these three cytokines(P＜0.01) in the rat retina early in diabetes.
Conclusion
These results suggest that the diabetes-mediated increase in CTGF up-regulates VEGF and TGF-β_2 expression and induces apoptosis in retina.And the elevation could be inhibited by treatment with CTGFsiRNA.CTGF,VEGF and TGF-β_2 play critical roles in the early stage of diabetic retina.CTGF could serve as a potential target for the prevention and treatment of diabetic retinopathy.
引文
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