糖尿病视网膜病变对鼠循环外周血内皮祖细胞数量的影响
发布时间：2018-08-24 08:45
【摘要】： 目的：本实验旨在建立Wistar大鼠糖尿病模型、糖尿病视网膜病变(diabetic retinopathy, DR)模型,分析糖尿病视网膜病变对循环外周血的内皮祖细胞(endothelial progenitor cells, EPCs)的影响。探讨内皮祖细胞在糖尿病视网膜病变发病机制中的作用,为治疗糖尿病视网膜病变开拓思路、提供理论依据。 方法：选取体重250-350g的2月龄雄性成年Wistar大鼠60只随机分为正常对照组,腹腔注射链脲佐菌素(streptozotocin, STZ)大鼠1周、1、3、6个月组。1周、1、3、6个月组按50mg/kg腹腔快速注射2%STZ溶液,对照组注射等量的枸橼酸-枸橼酸钠缓冲液。进行血糖、体重,流式细胞仪计数几组大鼠循环外周内皮祖细胞数量,HE染色,视网膜血管铺片PAS染色、透射电镜检测并行统计分析。 结果：1.大鼠腹腔注射链脲佐菌素72 h后,血糖(空腹血糖大于16.65mmol/L)达成模标准。成模后1周即出现多饮、多食、多尿等糖尿病症状,体质量减小。对照组大鼠每日饮水量少,尿量少,毛色光滑,体质量随周龄增大而稳步增长。腹腔注射STZ后1周、1、3、6个月组和对照组大鼠的血糖比较有统计学差异(P0.05),它们的体重比较有统计学差异(P0.05)。 2. EPCs流式细胞仪计数：1周、1个月、3个月、6个月、对照组循环血内皮祖细胞计数分别为25±7,28±8,39±7,43±7,45±4。与对照组相比,注射STZ后EPCs计数均降低。注射STZ 1周、1、3、6个月组EPCs计数逐渐升高。几组外周血EPCs计数比较有统计学差异(P0.05)。 3.HE染色：和正常对照组相比,一周组和一个月组的视网膜各层未见明显变化。在三个月组中,视网膜各层开始较明显的变薄,排列紊乱,部分血管扩张。在六个月组中,视网膜各层变薄、排列紊乱更加明显,部分血管扩张更加明显,甚至有向外突出、破溃的趋势。总体趋势是实验组大鼠视网膜组织较正常对照组大鼠视网膜组织各层厚度变薄,视细胞数目随月龄增加而逐渐减少。 4.视网膜血管消化铺片：一周组可见视网膜毛细血管网管径粗细均匀一致,内皮细胞核长呈椭圆形、染色淡,周细胞核呈圆形、染色深；大鼠糖尿病3个月时,视网膜毛细血管周细胞核多数与正常组相似,仅个别细胞核体积稍大,染色稍浅,毛细血管管腔通畅。6个月时,视网膜毛细血管常见周细胞核肿胀,体积增大,染色变浅,毛细血管可见局灶性多形核白细胞阻塞管腔,偶见闭锁的毛细血管。 5.透射电镜检测：一个月组毛细血管内皮细胞及周细胞核膜轻微凹陷,异染色质聚集靠边,管腔未变形；线粒体脊清晰,部分肿胀的线粒体均质溶解。3个月组内皮细胞水肿,核染色质聚集、靠边,管腔变形不显著；内网层细胞内大量空泡,线粒体肿胀,嵴消失,毛细血管基底膜增厚,电子密度明显增大。6个月组内皮细胞肿胀变形,核染色质凝聚靠边,核变形,线粒体明显肿胀,并见空泡变性；基底膜增厚显著,电子密度增加；内皮细胞的胞质向管腔内指状突起,管腔明显狭窄,甚至闭塞。节细胞层可见节细胞胞核凹陷,形成假包环体,高尔基囊泡扩张。 结论： 1.腹腔注射STZ 1周后即可形成稳定的糖尿病大鼠模型。 2.腹腔注射STZ后1、3、6个月组出现DR的改变,且随月龄增加而逐渐加重。 3.注射STZ后EPCs计数均降低。注射STZ 1周、1、3、6个月组EPCs计数逐渐升高。 4.推测EPC在糖尿病视网膜病变发病机制中起着非常重要的作用。EPCs动员及生存时间,导致血管的再内皮化的过程受损,促进缺血性疾病的发生发展,EPCs参与了DR过程。 5.探讨内皮祖细胞在糖尿病视网膜病变发病机制中的作用,为治疗糖尿病视网膜病变开拓思路、提供理论依据。
[Abstract]:AIM: To establish diabetic retinopathy (DR) and diabetic retinopathy (DM) models in Wistar rats, and to investigate the effects of DM on endothelial progenitor cells (EPCs) in peripheral blood. To provide a theoretical basis for the development of diabetic retinopathy.
METHODS: Sixty 2-month-old male Wistar rats weighing 250-350 g were randomly divided into normal control group and intraperitoneally injected with streptozotocin (STZ) for 1 week, 1,3,6 months. Weight, the number of circulating EPCs, HE staining, PAS staining of retinal blood vessels, transmission electron microscopy and statistical analysis were performed.
After 72 hours of intraperitoneal injection of streptozotocin, the blood glucose (fasting blood glucose > 16.65 mmol/L) reached the model standard. One week after the injection, diabetic symptoms such as drinking more, eating more, urinating less, body weight decreased. The control group rats drank less water, urine less, hair color smooth, and body weight increased steadily with age. There were significant differences in blood glucose (P 0.05) and body weight (P 0.05) between the week, 1,3,6 months group and the control group.
2. EPCs counting by flow cytometry: 1 week, 1 month, 3 months and 6 months, the EPCs counting of circulating blood in control group were 25 (+) 7, 28 (+) 8, 39 (+) 7, 43 (+) 7, 45 (+) 4. Compared with control group, the EPCs counting decreased after STZ injection.
3. HE staining: Compared with the normal control group, there was no significant change in the retinal layers of the week group and the month group. In the three-month group, the retinal layers began to become thinner, arranged disorderly, and some blood vessels dilated. In the six-month group, the retinal layers became thinner, arranged disorderly, and some blood vessels dilated more obviously, even directed. The general trend is that the retinal tissue of the experimental group is thinner than that of the normal control group, and the number of optic cells decreases with the increase of age.
4. Retinal blood vessel digestion: the diameter of retinal capillary network was uniform in one week group, the length of endothelial cell nucleus was elliptic, the staining was light, the peripheral cell nucleus was round and the staining was deep. At 3 months of diabetes, the nucleus of pericapillary cells of retina was mostly similar to the normal group, only a few nuclei were slightly larger and slightly lighter stained. At 6 months, the pericyte nuclei of retinal capillaries were swollen, enlarged and stained lightly. Focal polymorphonuclear leukocytes obstructed the lumen and occasionally atresia of capillaries could be seen in the capillaries.
5. Transmission electron microscopy: in one month group, the endothelial cells and nuclear membrane of peripheral cells were slightly depressed, heterochromatin aggregated near the edge, and the lumen was not deformed; mitochondrial ridge was clear, and some of the swollen mitochondria were homogeneously dissolved. Mitochondria swelled, cristae disappeared, basement membrane of capillaries thickened, electron density increased significantly. Endothelial cells swelled and deformed, nuclear chromatin coagulated near the edge, nuclear deformation, mitochondria swelled and vacuolar degeneration were observed in 6 months group; basement membrane thickened significantly, electron density increased; endothelial cell cytoplasm toward the lumen finger process, lumen narrowed significantly. In the ganglion cell layer, the nuclei of the ganglion cells are depressed, forming pseudo inclusion rings, and dilated Golgi vesicles.
Conclusion:
1. after 1 weeks of intraperitoneal injection of STZ, a stable diabetic rat model can be formed.
2. after intraperitoneal injection of STZ, the change of DR appeared in 1,3,6 months group, and gradually increased with the increase of age.
3. the EPCs count decreased after injection of STZ. After injection of STZ for 1 weeks, the EPCs count increased gradually in 1,3,6 months.
4. It is speculated that EPCs play an important role in the pathogenesis of diabetic retinopathy. Mobilization and survival time of EPCs lead to impaired endothelialization of blood vessels and promote the occurrence and development of ischemic diseases. EPCs participate in DR process.
5. To explore the role of endothelial progenitor cells in the pathogenesis of diabetic retinopathy and provide theoretical basis for the treatment of diabetic retinopathy.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.1
本文编号：2200251
[Abstract]:AIM: To establish diabetic retinopathy (DR) and diabetic retinopathy (DM) models in Wistar rats, and to investigate the effects of DM on endothelial progenitor cells (EPCs) in peripheral blood. To provide a theoretical basis for the development of diabetic retinopathy.
METHODS: Sixty 2-month-old male Wistar rats weighing 250-350 g were randomly divided into normal control group and intraperitoneally injected with streptozotocin (STZ) for 1 week, 1,3,6 months. Weight, the number of circulating EPCs, HE staining, PAS staining of retinal blood vessels, transmission electron microscopy and statistical analysis were performed.
After 72 hours of intraperitoneal injection of streptozotocin, the blood glucose (fasting blood glucose > 16.65 mmol/L) reached the model standard. One week after the injection, diabetic symptoms such as drinking more, eating more, urinating less, body weight decreased. The control group rats drank less water, urine less, hair color smooth, and body weight increased steadily with age. There were significant differences in blood glucose (P 0.05) and body weight (P 0.05) between the week, 1,3,6 months group and the control group.
2. EPCs counting by flow cytometry: 1 week, 1 month, 3 months and 6 months, the EPCs counting of circulating blood in control group were 25 (+) 7, 28 (+) 8, 39 (+) 7, 43 (+) 7, 45 (+) 4. Compared with control group, the EPCs counting decreased after STZ injection.
3. HE staining: Compared with the normal control group, there was no significant change in the retinal layers of the week group and the month group. In the three-month group, the retinal layers began to become thinner, arranged disorderly, and some blood vessels dilated. In the six-month group, the retinal layers became thinner, arranged disorderly, and some blood vessels dilated more obviously, even directed. The general trend is that the retinal tissue of the experimental group is thinner than that of the normal control group, and the number of optic cells decreases with the increase of age.
4. Retinal blood vessel digestion: the diameter of retinal capillary network was uniform in one week group, the length of endothelial cell nucleus was elliptic, the staining was light, the peripheral cell nucleus was round and the staining was deep. At 3 months of diabetes, the nucleus of pericapillary cells of retina was mostly similar to the normal group, only a few nuclei were slightly larger and slightly lighter stained. At 6 months, the pericyte nuclei of retinal capillaries were swollen, enlarged and stained lightly. Focal polymorphonuclear leukocytes obstructed the lumen and occasionally atresia of capillaries could be seen in the capillaries.
5. Transmission electron microscopy: in one month group, the endothelial cells and nuclear membrane of peripheral cells were slightly depressed, heterochromatin aggregated near the edge, and the lumen was not deformed; mitochondrial ridge was clear, and some of the swollen mitochondria were homogeneously dissolved. Mitochondria swelled, cristae disappeared, basement membrane of capillaries thickened, electron density increased significantly. Endothelial cells swelled and deformed, nuclear chromatin coagulated near the edge, nuclear deformation, mitochondria swelled and vacuolar degeneration were observed in 6 months group; basement membrane thickened significantly, electron density increased; endothelial cell cytoplasm toward the lumen finger process, lumen narrowed significantly. In the ganglion cell layer, the nuclei of the ganglion cells are depressed, forming pseudo inclusion rings, and dilated Golgi vesicles.
Conclusion:
1. after 1 weeks of intraperitoneal injection of STZ, a stable diabetic rat model can be formed.
2. after intraperitoneal injection of STZ, the change of DR appeared in 1,3,6 months group, and gradually increased with the increase of age.
3. the EPCs count decreased after injection of STZ. After injection of STZ for 1 weeks, the EPCs count increased gradually in 1,3,6 months.
4. It is speculated that EPCs play an important role in the pathogenesis of diabetic retinopathy. Mobilization and survival time of EPCs lead to impaired endothelialization of blood vessels and promote the occurrence and development of ischemic diseases. EPCs participate in DR process.
5. To explore the role of endothelial progenitor cells in the pathogenesis of diabetic retinopathy and provide theoretical basis for the treatment of diabetic retinopathy.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.1
【参考文献】
相关期刊论文 前1条
1 吴波;卢正茂;王尧;罗天航;薛绪潮;毕建威;康俊升;方国恩;;兔骨髓源性血管内皮祖细胞的分离、培养及鉴定[J];中国实验血液学杂志;2010年02期
，本文编号：2200251
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