糖尿病視網(wǎng)膜病變對(duì)鼠循環(huán)外周血內(nèi)皮祖細(xì)胞數(shù)量的影響
發(fā)布時(shí)間：2018-08-24 08:45
【摘要】： 目的：本實(shí)驗(yàn)旨在建立Wistar大鼠糖尿病模型、糖尿病視網(wǎng)膜病變(diabetic retinopathy, DR)模型,分析糖尿病視網(wǎng)膜病變對(duì)循環(huán)外周血的內(nèi)皮祖細(xì)胞(endothelial progenitor cells, EPCs)的影響。探討內(nèi)皮祖細(xì)胞在糖尿病視網(wǎng)膜病變發(fā)病機(jī)制中的作用,為治療糖尿病視網(wǎng)膜病變開拓思路、提供理論依據(jù)。 方法：選取體重250-350g的2月齡雄性成年Wistar大鼠60只隨機(jī)分為正常對(duì)照組,腹腔注射鏈脲佐菌素(streptozotocin, STZ)大鼠1周、1、3、6個(gè)月組。1周、1、3、6個(gè)月組按50mg/kg腹腔快速注射2%STZ溶液,對(duì)照組注射等量的枸櫞酸-枸櫞酸鈉緩沖液。進(jìn)行血糖、體重,流式細(xì)胞儀計(jì)數(shù)幾組大鼠循環(huán)外周內(nèi)皮祖細(xì)胞數(shù)量,HE染色,視網(wǎng)膜血管鋪片PAS染色、透射電鏡檢測并行統(tǒng)計(jì)分析。 結(jié)果：1.大鼠腹腔注射鏈脲佐菌素72 h后,血糖(空腹血糖大于16.65mmol/L)達(dá)成模標(biāo)準(zhǔn)。成模后1周即出現(xiàn)多飲、多食、多尿等糖尿病癥狀,體質(zhì)量減小。對(duì)照組大鼠每日飲水量少,尿量少,毛色光滑,體質(zhì)量隨周齡增大而穩(wěn)步增長。腹腔注射STZ后1周、1、3、6個(gè)月組和對(duì)照組大鼠的血糖比較有統(tǒng)計(jì)學(xué)差異(P0.05),它們的體重比較有統(tǒng)計(jì)學(xué)差異(P0.05)。 2. EPCs流式細(xì)胞儀計(jì)數(shù)：1周、1個(gè)月、3個(gè)月、6個(gè)月、對(duì)照組循環(huán)血內(nèi)皮祖細(xì)胞計(jì)數(shù)分別為25±7,28±8,39±7,43±7,45±4。與對(duì)照組相比,注射STZ后EPCs計(jì)數(shù)均降低。注射STZ 1周、1、3、6個(gè)月組EPCs計(jì)數(shù)逐漸升高。幾組外周血EPCs計(jì)數(shù)比較有統(tǒng)計(jì)學(xué)差異(P0.05)。 3.HE染色：和正常對(duì)照組相比,一周組和一個(gè)月組的視網(wǎng)膜各層未見明顯變化。在三個(gè)月組中,視網(wǎng)膜各層開始較明顯的變薄,排列紊亂,部分血管擴(kuò)張。在六個(gè)月組中,視網(wǎng)膜各層變薄、排列紊亂更加明顯,部分血管擴(kuò)張更加明顯,甚至有向外突出、破潰的趨勢。總體趨勢是實(shí)驗(yàn)組大鼠視網(wǎng)膜組織較正常對(duì)照組大鼠視網(wǎng)膜組織各層厚度變薄,視細(xì)胞數(shù)目隨月齡增加而逐漸減少。 4.視網(wǎng)膜血管消化鋪片：一周組可見視網(wǎng)膜毛細(xì)血管網(wǎng)管徑粗細(xì)均勻一致,內(nèi)皮細(xì)胞核長呈橢圓形、染色淡,周細(xì)胞核呈圓形、染色深；大鼠糖尿病3個(gè)月時(shí),視網(wǎng)膜毛細(xì)血管周細(xì)胞核多數(shù)與正常組相似,僅個(gè)別細(xì)胞核體積稍大,染色稍淺,毛細(xì)血管管腔通暢。6個(gè)月時(shí),視網(wǎng)膜毛細(xì)血管常見周細(xì)胞核腫脹,體積增大,染色變淺,毛細(xì)血管可見局灶性多形核白細(xì)胞阻塞管腔,偶見閉鎖的毛細(xì)血管。 5.透射電鏡檢測：一個(gè)月組毛細(xì)血管內(nèi)皮細(xì)胞及周細(xì)胞核膜輕微凹陷,異染色質(zhì)聚集靠邊,管腔未變形；線粒體脊清晰,部分腫脹的線粒體均質(zhì)溶解。3個(gè)月組內(nèi)皮細(xì)胞水腫,核染色質(zhì)聚集、靠邊,管腔變形不顯著；內(nèi)網(wǎng)層細(xì)胞內(nèi)大量空泡,線粒體腫脹,嵴消失,毛細(xì)血管基底膜增厚,電子密度明顯增大。6個(gè)月組內(nèi)皮細(xì)胞腫脹變形,核染色質(zhì)凝聚靠邊,核變形,線粒體明顯腫脹,并見空泡變性；基底膜增厚顯著,電子密度增加；內(nèi)皮細(xì)胞的胞質(zhì)向管腔內(nèi)指狀突起,管腔明顯狹窄,甚至閉塞。節(jié)細(xì)胞層可見節(jié)細(xì)胞胞核凹陷,形成假包環(huán)體,高爾基囊泡擴(kuò)張。 結(jié)論： 1.腹腔注射STZ 1周后即可形成穩(wěn)定的糖尿病大鼠模型。 2.腹腔注射STZ后1、3、6個(gè)月組出現(xiàn)DR的改變,且隨月齡增加而逐漸加重。 3.注射STZ后EPCs計(jì)數(shù)均降低。注射STZ 1周、1、3、6個(gè)月組EPCs計(jì)數(shù)逐漸升高。 4.推測EPC在糖尿病視網(wǎng)膜病變發(fā)病機(jī)制中起著非常重要的作用。EPCs動(dòng)員及生存時(shí)間,導(dǎo)致血管的再內(nèi)皮化的過程受損,促進(jìn)缺血性疾病的發(fā)生發(fā)展,EPCs參與了DR過程。 5.探討內(nèi)皮祖細(xì)胞在糖尿病視網(wǎng)膜病變發(fā)病機(jī)制中的作用,為治療糖尿病視網(wǎng)膜病變開拓思路、提供理論依據(jù)。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R774.1
本文編號(hào)：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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R774.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 吳波;盧正茂;王堯;羅天航;薛緒潮;畢建威;康俊升;方國恩;;兔骨髓源性血管內(nèi)皮祖細(xì)胞的分離、培養(yǎng)及鑒定[J];中國實(shí)驗(yàn)血液學(xué)雜志;2010年02期
，本文編號(hào)：2200251
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