量子点荧光标记用于PCOS及PCOS伴2型糖尿病大鼠卵巢热休克蛋白的研究

发布时间：2018-04-21 06:13

  本文选题：多囊卵巢综合征 + 2型糖尿病　； 参考：《武汉大学》2014年博士论文

【摘要】：目的：多囊卵巢综合征(polycystic ovary syndrome, PCOS)是一种以内分泌紊乱为主,多种代谢异常的异质性临床综合征,发病率为4%～8%。该病发病机制至今尚不清楚。PCOS患者代谢异常不容忽视,应作为该疾病长期预防和治疗的重要内容,PCOS发展为2型糖尿病(2-DM)的机制及其病理生理特点目前尚未研究清楚。热休克蛋白(heat shock protein,HSP)是所有原核细胞和真核细胞在生理、病理及环境因素(高温、缺氧或病毒感染、应激)下均可产生的一组高度保守的蛋白质分子家族,具有多种生物学功能,已有多个研究发现HSP与2-DM的发生发展具有密切关系。然而在PCOS伴2-DM中HSP的表达情况目前尚未有研究报道,因此研究PCOS及PCOS伴2-DM大鼠中HSP的表达情况,可以进一步明确HSP在PCOS及其远期并发症中发病机制的可能作用机理。量子点是一种新型的纳米荧光探针,其在生物医学的多个领域均有广泛的应用前景。本文利用量子点荧光标记技术对PCOS及PCOS伴2-DM大鼠卵巢内HSP的检测,并与传统的免疫组化法进行比较,探讨HSP在PCOS及PCOS伴2-DM发病中的可能机制,并对量子点荧光标记技术进行初步的探讨,为进一步拓展其应用领域提供依据。 方法：将23日龄雌性SD大鼠分为PCOS组,PCOS伴2-DM组及对照组,PCOS组及PCOS伴2-DM组以硫酸普拉睾酮钠90μg(/g·d)皮下注射20d,同时给予高脂高糖饲料喂养；PCOS伴2-DM组则在硫酸普拉睾酮钠停药后第2天给予链脲佐菌素(STZ)45mg/kg腹腔注射1次,PCOS组及对照组注射等量柠檬酸盐缓冲液。注射后72h处死大鼠,观察卵巢外观、光镜(HE染色)改变,测定血清性激素、胰岛素及血糖水平,并对各组大鼠卵巢组织中HSP70和HSP90的表达进行QDs-SA605荧光标记和免疫组化染色,观察两种蛋白的表达情况,并比较两种染色方法对上述两种蛋白检测的异同。 结果：1、硫酸普拉睾酮钠诱导SD幼年雌性大鼠PCOS动物模型卵巢病理改变及血内分泌改变与PCOS病人相似,卵巢呈多囊样改变,并存在着明显的高胰岛素血症。硫酸普拉睾酮钠联合低剂量STZ诱导的PCOS伴2-DM的动物模型表现出与PCOS伴2-DM患者相似的内分泌特点。 2、大鼠卵巢组织中有HSP70及HSP90的表达,颗粒细胞中表达较多,主要为胞质表达,HSP70的表达趋势为正常组PCOS组PCOS伴2-DM组,HSP90的表达趋势为正常组≈PCOS伴2-DM组PCOS组。 3、QDs-SA605标记可以检测HSP70及HSP90的表达,其表达部位和表达强度趋势与免疫组化相似,且由于颜色对比较强,更易于观察。持续观察30天,荧光强度虽略有衰减,但荧光基本未泯灭,标本仍清晰可见。 结论：硫酸普拉睾酮钠联合低剂量STZ可诱导PCOS伴2-DM的动物模型,但该模型的稳定性及持久性还有待进一步研究。PCOS及PCOS伴2-DM卵巢中均存在HSP70及HSP90的表达。QDs标记可以用于HSP的检测,其荧光强度在相当长的时间内可保持稳定,能满足生物医学实际应用的要求。
[Abstract]:Objective: polycystic ovary syndrome (PCOS) is a heterogeneous clinical syndrome characterized by endocrine disorders and metabolic abnormalities. The pathogenesis of PCOS is still unclear. The metabolic abnormality of PCOS patients can not be ignored. The mechanism and pathophysiological characteristics of PCOS developing into type 2 diabetes mellitus 2-DM2 should be considered as an important part of long-term prevention and treatment of PCOS. Heat shock shock protein (HSPs) is a highly conserved family of proteins that can be produced by all prokaryotes and eukaryotic cells under physiological, pathological and environmental factors (hyperthermia, hypoxia or viral infection, stress). Several studies have found that HSP is closely related to the occurrence and development of 2-DM. However, the expression of HSP in PCOS with 2-DM has not been reported. Therefore, studying the expression of HSP in PCOS and PCOS with 2-DM can further clarify the possible mechanism of HSP in the pathogenesis of PCOS and its long-term complications. Quantum dots (QDs) are a new type of nanoscale fluorescent probes, which are widely used in many fields of biomedicine. In this paper, the detection of HSP in ovary of PCOS and PCOS with 2-DM rats was studied by using quantum dot fluorescence labeling technique. Compared with the traditional immunohistochemical method, the possible mechanism of HSP in the pathogenesis of PCOS and PCOS with 2-DM was discussed. The fluorescence labeling technique of quantum dots is discussed preliminarily, which provides the basis for further expanding its application field. Methods: Twenty-three day-old female SD rats were divided into PCOS group (PCOS with 2-DM group) and control group (PCOS group and PCOS with 2-DM group) by subcutaneous injection of 90 渭 g of testosterone sulfate (90 渭 g / g 路g / d) for 20 days. At the same time, they were given high-fat and high-sugar diet and fed with 2-DM group. On the second day after withdrawal, 45 mg / kg streptozotocin (STZ) was injected intraperitoneally into PCOS group and control group with the same amount of citrate buffer. The rats were killed 72 hours after injection. The changes of ovary appearance and HE staining were observed. Serum sex hormone, insulin and blood glucose were measured. The expression of HSP70 and HSP90 in ovarian tissues of each group was labeled with QDs-SA605 fluorescence and immunohistochemical staining. The expression of the two proteins was observed and the differences and similarities between the two staining methods were compared. Results the ovarian pathological changes and the changes of blood endocrine were similar to those of PCOS patients, and there were obvious hyperinsulinemia in the ovaries of male SD young female rats induced by Pretestosterone sulfate sodium. The animal model of PCOS with 2-DM induced by low dose STZ combined with pretestosterone sulfate showed similar endocrine characteristics to that of PCOS with 2-DM. 2. The expression of HSP70 and HSP90 was found in the ovary of rats, and the expression of HSP70 in the granulosa cells was more than that in the granulosa cells. The tendency of the expression of HSP70 in the cytoplasm was that in the normal PCOS group, PCOS accompanied with 2-DM group was the normal group 鈮，

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