【摘要】：目的探討中藥蘇木(Caesalpinia Sapan L)及其主要有效成分巴西蘇木素(Brazilin)對(duì)高糖誘導(dǎo)血管內(nèi)皮細(xì)胞損傷的保護(hù)作用及其機(jī)制,為其治療糖尿病大血管病變提供實(shí)驗(yàn)依據(jù)。方法動(dòng)物實(shí)驗(yàn):30只健康雄性SD大鼠隨機(jī)分為正常組(n=10)與造模組(n=20),正常組正常飼養(yǎng),造模組高脂高糖飼料連續(xù)喂養(yǎng)4周后,一次性尾靜脈注射鏈脲佐菌素(STZ)溶液造成糖尿病模型,成模后的大鼠隨機(jī)分為對(duì)照組與蘇木組,蘇木組每天按人等效劑量810mg/kg體重生藥量,灌服溶于2ml蒸餾水中的蘇木顆粒劑,正常組與對(duì)照組每天灌服等量的生理鹽水,以上大鼠4周后測(cè)血糖及體重,取主動(dòng)脈,運(yùn)用HE染色觀察糖尿病大鼠主動(dòng)脈形態(tài)結(jié)構(gòu);透射電鏡(tem)觀察糖尿病大鼠血管內(nèi)皮細(xì)胞中的自噬結(jié)構(gòu),探討蘇木對(duì)糖尿病大鼠血管內(nèi)皮細(xì)胞自噬的影響。細(xì)胞實(shí)驗(yàn):培養(yǎng)人臍靜脈內(nèi)皮細(xì)胞(human umbilical vein endothelial cells,HUVEC),采用MTT法確定巴西蘇木素濃度;加入濃度為33.3 mmol/l的葡萄糖溶液,建立高糖誘導(dǎo)HUVEC損傷體外模型,給予濃度為15 ug/ml巴西蘇木素,Western-blot檢測(cè)自噬相關(guān)基因Beclin1、LC3-Ⅱ、p62蛋白的表達(dá)。結(jié)果動(dòng)物實(shí)驗(yàn):糖尿病造模成功的共有18只,再隨機(jī)將18只分為對(duì)照組(n=9)和蘇木組(n=9),所有存活大鼠均毛色、活動(dòng)正常,造模組體重減輕。血糖較造模前增加(P0.05),造模組大鼠體重低于正常組,造模組中蘇木組體重低于對(duì)照組(P0.05)。HE染色觀察大鼠主動(dòng)脈形態(tài)結(jié)構(gòu),蘇木組和對(duì)照組,較正常組血管平滑肌均有損傷,但蘇木組較對(duì)照組,內(nèi)膜雖有破壞,卻已有改善。透射電鏡觀察糖尿病大鼠血管內(nèi)皮細(xì)胞中的自噬結(jié)構(gòu),蘇木組較正常組,胞漿內(nèi)自噬體數(shù)目增多、體積變大,線粒體呈嵴紊亂或空泡化。對(duì)照組糖尿病大鼠血管中的自噬體數(shù)量較正常組減少。細(xì)胞實(shí)驗(yàn):MTT實(shí)驗(yàn)選定用于實(shí)驗(yàn)的巴西蘇木素濃度為15 ug/ml;Beclin1、LC3-Ⅱ的蛋白表達(dá),高糖處理組較正常組低,巴西蘇木素組較高糖處理組高(P0.05)。p62的蛋白表達(dá),高糖處理組較正常組高,巴西蘇木素組低于正常組和高糖處理組(P0.05)。結(jié)論中藥蘇木及其主要有效成分巴西蘇木素通過調(diào)控自噬而對(duì)高糖誘導(dǎo)血管內(nèi)皮細(xì)胞發(fā)揮保護(hù)作用,為治療糖尿病大血管病變提供實(shí)驗(yàn)依據(jù)。
[Abstract]:Objective To investigate the protective effect and mechanism of Caesalpinia Sapan L and its main active component Brazilin on vascular endothelial cell injury induced by high glucose, and to provide experimental basis for its treatment of diabetic macroangiopathy. (n=20), the normal group was fed normally, and the model group was fed with high fat and high sugar diet for 4 weeks. The diabetic rats were randomly divided into control group and hematoxylin group. The hematoxylin group was fed with hematoxylin granules dissolved in 2 ml distilled water at 810 mg/kg body weight per day. The blood glucose and body weight were measured after 4 weeks. The aorta of diabetic rats were stained with HE. The autophagy structure of vascular endothelial cells in diabetic rats was observed by transmission electron microscopy (tem). Effects. Cell experiment: Cultured human umbilical vein endothelial cells (HUVEC), determined the concentration of Brazilian hematoxylin by MTT method; added glucose solution with a concentration of 33.3 mmol/l, established high glucose-induced HUVEC injury in vitro model, given concentration of 15 ug/ml Brazilian hematoxylin, Western-blot detection of autophagy-related genes. Results Animal experiment: 18 diabetes mellitus rats were successfully established, and then randomly divided into control group (n=9) and hematoxylin group (n=9). All the surviving rats were hair color, normal activity, weight loss of model group. Blood glucose increased (P 0.05), body weight of model group was lower than that of normal group. HE staining was used to observe the morphology and structure of aorta in diabetic rats (P 0.05). The vascular smooth muscle of hematoxylin group and control group were damaged, but the intima of hematoxylin group was damaged, but it was improved. The autophagy structure of vascular endothelial cells in diabetic rats was observed by transmission electron microscope. The number of autophages in the blood vessels of diabetic rats in the control group was lower than that of the normal group. Cell experiment: MTT assay selected Brazilian hematoxylin concentration for the experiment was 15 ug/ml; Beclin1, LC3-II protein expression, high glucose treatment group was lower than the normal group, Brazilian hematoxylin group was higher. The expression of p62 protein was higher in the sugar treatment group than in the normal group, and lower in the Brazilian hematoxylin group than in the normal group and the high glucose treatment group (P 0.05). Conclusion The Chinese herbal hematoxylin and its main effective component Brazilian hematoxylin can protect the vascular endothelial cells induced by high glucose by regulating autophagy, and can be used to treat diabetic macroangiopathy. For experimental basis.
【學(xué)位授予單位】：錦州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.2

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