本文選題：多囊卵巢綜合征 + 2型糖尿病��； 參考：《武漢大學(xué)》2014年博士論文

【摘要】：目的：多囊卵巢綜合征(polycystic ovary syndrome, PCOS)是一種以內(nèi)分泌紊亂為主,多種代謝異常的異質(zhì)性臨床綜合征,發(fā)病率為4%～8%。該病發(fā)病機制至今尚不清楚。PCOS患者代謝異常不容忽視,應(yīng)作為該疾病長期預(yù)防和治療的重要內(nèi)容,PCOS發(fā)展為2型糖尿病(2-DM)的機制及其病理生理特點目前尚未研究清楚。熱休克蛋白(heat shock protein,HSP)是所有原核細(xì)胞和真核細(xì)胞在生理、病理及環(huán)境因素(高溫、缺氧或病毒感染、應(yīng)激)下均可產(chǎn)生的一組高度保守的蛋白質(zhì)分子家族,具有多種生物學(xué)功能,已有多個研究發(fā)現(xiàn)HSP與2-DM的發(fā)生發(fā)展具有密切關(guān)系。然而在PCOS伴2-DM中HSP的表達情況目前尚未有研究報道,因此研究PCOS及PCOS伴2-DM大鼠中HSP的表達情況,可以進一步明確HSP在PCOS及其遠(yuǎn)期并發(fā)癥中發(fā)病機制的可能作用機理。量子點是一種新型的納米熒光探針,其在生物醫(yī)學(xué)的多個領(lǐng)域均有廣泛的應(yīng)用前景。本文利用量子點熒光標(biāo)記技術(shù)對PCOS及PCOS伴2-DM大鼠卵巢內(nèi)HSP的檢測,并與傳統(tǒng)的免疫組化法進行比較,探討HSP在PCOS及PCOS伴2-DM發(fā)病中的可能機制,并對量子點熒光標(biāo)記技術(shù)進行初步的探討,為進一步拓展其應(yīng)用領(lǐng)域提供依據(jù)。 方法：將23日齡雌性SD大鼠分為PCOS組,PCOS伴2-DM組及對照組,PCOS組及PCOS伴2-DM組以硫酸普拉睪酮鈉90μg(/g·d)皮下注射20d,同時給予高脂高糖飼料喂養(yǎng)；PCOS伴2-DM組則在硫酸普拉睪酮鈉停藥后第2天給予鏈脲佐菌素(STZ)45mg/kg腹腔注射1次,PCOS組及對照組注射等量檸檬酸鹽緩沖液。注射后72h處死大鼠,觀察卵巢外觀、光鏡(HE染色)改變,測定血清性激素、胰島素及血糖水平,并對各組大鼠卵巢組織中HSP70和HSP90的表達進行QDs-SA605熒光標(biāo)記和免疫組化染色,觀察兩種蛋白的表達情況,并比較兩種染色方法對上述兩種蛋白檢測的異同。 結(jié)果：1、硫酸普拉睪酮鈉誘導(dǎo)SD幼年雌性大鼠PCOS動物模型卵巢病理改變及血內(nèi)分泌改變與PCOS病人相似,卵巢呈多囊樣改變,并存在著明顯的高胰島素血癥。硫酸普拉睪酮鈉聯(lián)合低劑量STZ誘導(dǎo)的PCOS伴2-DM的動物模型表現(xiàn)出與PCOS伴2-DM患者相似的內(nèi)分泌特點。 2、大鼠卵巢組織中有HSP70及HSP90的表達,顆粒細(xì)胞中表達較多,主要為胞質(zhì)表達,HSP70的表達趨勢為正常組PCOS組PCOS伴2-DM組,HSP90的表達趨勢為正常組≈PCOS伴2-DM組PCOS組。 3、QDs-SA605標(biāo)記可以檢測HSP70及HSP90的表達,其表達部位和表達強度趨勢與免疫組化相似,且由于顏色對比較強,更易于觀察。持續(xù)觀察30天,熒光強度雖略有衰減,但熒光基本未泯滅,標(biāo)本仍清晰可見。 結(jié)論：硫酸普拉睪酮鈉聯(lián)合低劑量STZ可誘導(dǎo)PCOS伴2-DM的動物模型,但該模型的穩(wěn)定性及持久性還有待進一步研究。PCOS及PCOS伴2-DM卵巢中均存在HSP70及HSP90的表達。QDs標(biāo)記可以用于HSP的檢測,其熒光強度在相當(dāng)長的時間內(nèi)可保持穩(wěn)定,能滿足生物醫(yī)學(xué)實際應(yīng)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，

本文編號：1781252