浅论自发性高血压大鼠视网膜微血管稀少与细胞凋亡

1、浅论自发性高血压大鼠视网膜微血管稀少与细胞凋亡 作者：黄晓燕，陈碧新，高瞻，张明英，王赛斌，杨德业【摘要】 目的?：研究自发性高血压大鼠(SHR)视网膜毛细血管细胞凋亡的发生情况，探讨细胞凋亡与高血压视网膜微血管病变之间的关系，为研究高血压视网膜病变发病机制提供形态学依据。方法：10只雄性自发性高血压大鼠(SHR组)和10只雄性正常血压(WKY)大鼠(WKY组)分别在13周龄和18周龄取眼球做视网膜消化铺片，采用核苷酸末端转移酶介导dUTP缺口翻译法(TUNEL法)检测视网膜微血管细胞凋亡变化情况，HE染色及图像分析，动态观察视网膜微血管形态的改变及测定视网膜毛细血管面积及密度的改变。结果：T

2、UNEL法标记显示SHR各周龄大鼠视网膜微血管均出现阳性反应细胞，且18周龄大鼠阳性细胞与13周龄大鼠比较明显增多;WKY组大鼠视网膜微血管未见TUNEL阳性细胞。血管铺片HE染色显示SHR组部分毛细血管闭塞，内皮细胞和周细胞丢失，出现无细胞毛细血管，毛细血管密度较WKY组显着降低。结论：在高血压发展过程中，视网膜毛细血管内皮细胞和周细胞均可发生凋亡，同时伴有微血管稀疏，细胞凋亡可能在高血压视网膜病变的过程中扮演重要的角色。 【关键词】 高血压视网膜病;细胞凋亡;微血管稀少Abstract: ? Objective: To observe the apoptosis of retinal ca

3、pillary cells in spontaneously hypertensive rats and study the relationship between apoptosis and hypertensive retinopathy， in order to provide morphological evidence of hypertensive retinopathy. Methods: Experiments were performed on retinas from 13-week-old，18-week-old male SHR?(n?=10)?as experime

4、ntal group(SHR13 W，SHR18 W)and sex-and age-matched Wistar-Kyoto rats?(WKY)(n?=10)?as control group. Specimens were obtained using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling?(TUNEL)?methods to detect the apoptosis of retinal capillary cells， using hematoxylin and eosin?(HE)

5、?staining to observe the changes of retinal microvascular in morphology and determinate the retinal capillary area density. Results: Apoptotic cells labelled by TUNEL were emerged in SHR groups，including 13-week-old and 18-week-old. Apoptotic cells in 18-week-old SHR were significantly more than in

6、13-week-old SHR. No cell apoptosis was noted in each of WKY group. The level of apoptosis gradually increased as the weeks of age increasing in SHR， HE showed that some of capillary was occlusion， endothelial cells and pericytes lost，a large area of cell-free capillary was emerged. Compared with con

7、trol group，the retinal capillaries area density of SHR was significantly lower than that of the WKY groups. Conclusion: Apoptssis of the endothelial cells and pericytes of the retinal capillary all can occur in the development process of hypertension，at the same time microvascular rarefaction can ac

8、company. Apoptosis may play an important role in the development of hypertensive retinopathy.Key words: ? hypertensive retinopathy;apoptosis;capillary;microvascular rarefaction在我国高血压病人群中，高血压性视网膜病变的阳性率为70%左右，其所致眼底血管的改变基本上与高血压3个时期的变化相一致，在一定程度上能反映体内动脉的情况，对判断高血压病的严重程度和预后有一定的价值1。有关高血压性视网膜病变的发病机制尚未明了，其微血管

9、结构改变大多是通过检眼镜或眼底荧光素血管造影观察到的，如江时森等通过墨汁灌注法观察到自发性高血压大鼠(spontaneously hypertensive rats，SHR)视网膜微动脉存在微血管稀少现象，但在这一病理过程中，视网膜微血管病变是否与细胞凋亡有关鲜见报道。近年来，随着视网膜微血管消化铺片技术的应用，使直接观察视网膜微血管的形态学变化成为了可能。本研究应用视网膜消化铺片，HE染色直接观察视网膜微血管的形态学特征，并对微血管进行定量分析，同时采用TUNEL法特异性标记来观察13周龄和18周龄SHR视网膜毛细血管细胞凋亡情况，旨在探讨细胞凋亡与高血压视网膜微血管病变的关系。1材料和方法

10、1.1动物模型和试剂雄性SHR和正常血压大鼠(Wistar-Kyoto，WKY)各10只(其中13，18周龄各5只)，由上海中科院实验动物中心提供。大鼠饲养在温州医学院动物中心的标准化饲养房，饮自来水，标准饲料喂养，生活在昼夜节律为12 h、保持一定温度和湿度的空间。原位细胞凋亡检测试剂盒购于美国Roche公司。显色剂为DAB，购于福州迈新生物技术有限公司。1.3视网膜血管消化铺片的制备过量麻醉处死大鼠，用新鲜配制的4%多聚甲醛心脏灌流固定后摘除眼球，将眼球置于4%多聚甲醛中4 24 h。在显微镜下自角膜缘后0.5 mm处剪开眼球，去除眼前节和玻璃体，剥离视网膜，将其放入3%胰蛋白酶溶液内，3

11、7 孵育23 h，消化神经组织，然后在蒸馏水中轻轻吹打，仅留一薄层透明的视网膜血管网，将其贴于洁净的载玻片上，自然干燥，待染色。1.4视网膜血管形态学改变观察将制备好的消化铺片进行HE染色，光镜下观察视网膜血管形态学改变，各组大鼠选5张切片，每张切片计数5个高倍视野(400)，应用Image-Pro Plus 6.0软件分析图像，测定视网膜毛细血管面积密度，毛细血管面积密度(%)=毛细血管总面积/视网膜总面积。1.5TUNEL标记法将视网膜消化铺片按照ROCHE公司提供的TUNEL试剂盒说明书进行原位缺口末端标记检测。样本先用0.01 mol PBS缓冲液浸洗2次各5 min后再按照试剂盒说明

12、书进行。阴性对照：用50L指示液代替TUNEL反应液;阳性对照：实验前用Dnase-1消化组织(室温30 min)以打断所有细胞DNA链，余步骤同前;各步骤后均用0.01 mmol PBS缓冲液浸洗样本5 min3次。普通光镜下观察细胞凋亡，阳性染色随凋亡程度的不同呈浅棕至深棕褐色，定位于胞核，根据下列标准确定着色阳性细胞为凋亡细胞：单个散在分布;具有凋亡的核形态(核固缩或染色质浓聚附边或核碎裂);周围无炎症反应。对于缺乏凋亡核形态的阳性细胞，除非染色强度与背景有鲜明对比，且呈单个分布，否则不认为是凋亡细胞。非凋亡细胞呈紫蓝色，为阴性。1.5统计学处理方法两组间比较采用t检验。2 结果2.1各

13、组大鼠鼠尾动脉收缩压变化13周龄与18周龄SHR收缩压分别为(148.89.9)mmHg、(154.417.9)mmHg，WKY分别为(75.93.2)mmHg、(75.75.9)mmHg，相同周龄SHR组血压与WKY组比较差异均有显着性 (P?0.01)，两组组内不同周龄比较，差异均无显着性(P?0.05)。2.2眼底检查可见眼底动静脉由视乳头分出后呈放射状，与人眼底不同，无动静脉交叉，无黄斑结构。对照组眼底未见异常(见图1)，SHR组13周龄和18周龄均可见视网膜动脉变细，管径不规则，管壁反光增强，静脉迂曲扩张，视网膜水肿，反光增强，未见到片状出血点(见图2)。根据Reith- Wagen

14、er的4级分类法，相当于高血压眼底第I、第II级的改变。2.3视网膜血管形态学改变光镜下，WKY组毛细血管网管径粗细均匀一致，内皮细胞和周细胞均匀分布，内皮细胞核呈椭圆形，位于毛细血管中央或稍偏位，较周细胞略大，与血管平行;周细胞核呈短椭圆形、圆形或三角形，位于毛细血管壁的侧面，稍突出于血管壁(见图3)。SHR组血管管径粗细不均，周细胞和内皮细胞分布不均匀，部分毛细血管结构已破坏，13周龄 SHR部分毛细血管闭塞呈细线状(见图4)，18周龄SHR除了部分毛细血管表现为血管管腔闭塞外，其间无内皮细胞及周细胞影，血管腔完全闭锁，即无细胞毛细血管形成(见图5)。13周龄与18周龄SHR毛细血管面积密

15、度分别为(0.37200.0239)、(0.28400.0336)，WKY分别为(0.52400.0422)、(0.49400.0207)，SHR组毛细血管面积密度显着低于WKY组(P?0.01)，且随着周龄增加，SHR组毛细血管面积密度逐渐减少(P?0.05)。2.4视网膜微血管细胞凋亡变化SHR组大鼠视网膜微血管可见TUNEL阳性细胞，且18周龄阳性细胞与13周龄比较明显增多(见图6、8)，WKY组大鼠视网膜微血管未见TUNEL阳性细胞(见图7、9)。13周龄SHR组每个高倍视野凋亡细胞为(8.85.50)个，18周龄SHR组为(19.27.08)个，两组比较，差异有显着性(P?0.05)

16、。 【摘要】 目的?：研究自发性高血压大鼠(SHR)视网膜毛细血管细胞凋亡的发生情况，探讨细胞凋亡与高血压视网膜微血管病变之间的关系，为研究高血压视网膜病变发病机制提供形态学依据。方法：10只雄性自发性高血压大鼠(SHR组)和10只雄性正常血压(WKY)大鼠(WKY组)分别在13周龄和18周龄取眼球做视网膜消化铺片，采用核苷酸末端转移酶介导dUTP缺口翻译法(TUNEL法)检测视网膜微血管细胞凋亡变化情况，HE染色及图像分析，动态观察视网膜微血管形态的改变及测定视网膜毛细血管面积及密度的改变。结果：TUNEL法标记显示SHR各周龄大鼠视网膜微血管均出现阳性反应细胞，且18周龄大鼠阳性细胞与13

17、周龄大鼠比较明显增多;WKY组大鼠视网膜微血管未见TUNEL阳性细胞。血管铺片HE染色显示SHR组部分毛细血管闭塞，内皮细胞和周细胞丢失，出现无细胞毛细血管，毛细血管密度较WKY组显着降低。结论：在高血压发展过程中，视网膜毛细血管内皮细胞和周细胞均可发生凋亡，同时伴有微血管稀疏，细胞凋亡可能在高血压视网膜病变的过程中扮演重要的角色。 【关键词】 高血压视网膜病;细胞凋亡;微血管稀少Abstract: ? Objective: To observe the apoptosis of retinal capillary cells in spontaneously hypertensive rat

18、s and study the relationship between apoptosis and hypertensive retinopathy， in order to provide morphological evidence of hypertensive retinopathy. Methods: Experiments were performed on retinas from 13-week-old，18-week-old male SHR?(n?=10)?as experimental group(SHR13 W，SHR18 W)and sex-and age-matc

19、hed Wistar-Kyoto rats?(WKY)(n?=10)?as control group. Specimens were obtained using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling?(TUNEL)?methods to detect the apoptosis of retinal capillary cells， using hematoxylin and eosin?(HE)?staining to observe the changes of retinal mic

20、rovascular in morphology and determinate the retinal capillary area density. Results: Apoptotic cells labelled by TUNEL were emerged in SHR groups，including 13-week-old and 18-week-old. Apoptotic cells in 18-week-old SHR were significantly more than in 13-week-old SHR. No cell apoptosis was noted in

21、 each of WKY group. The level of apoptosis gradually increased as the weeks of age increasing in SHR， HE showed that some of capillary was occlusion， endothelial cells and pericytes lost，a large area of cell-free capillary was emerged. Compared with control group，the retinal capillaries area density

22、 of SHR was significantly lower than that of the WKY groups. Conclusion: Apoptssis of the endothelial cells and pericytes of the retinal capillary all can occur in the development process of hypertension，at the same time microvascular rarefaction can accompany. Apoptosis may play an important role in the development of hypertensive retinopathy.Key words: ? hypertensive retinopathy;apoptosis;capillary;microvascular rarefaction在我国高血压病人群中，高血压性视网膜病变的阳性率为70%左右，其所致眼底血管的改变基本上与高血压3个时期的变化相一致，在一定程度上能反映体内动脉的情况，对判断高血压病的