【摘要】： 目的:通過體外培養(yǎng)人大網(wǎng)膜前脂肪細(xì)胞,并誘導(dǎo)分化為成熟脂肪細(xì)胞,研究這一過程中瘦素、脂聯(lián)素的分泌規(guī)律。 方法: 1、選擇擇期開腹手術(shù)病人,要求無全身代謝及內(nèi)分泌疾病,無服用干預(yù)糖及脂肪代謝的藥物史。年齡范圍(40±8)歲,體重指數(shù)(BMI)小于25kg/m~2。 2、采用膠原酶消化的方法分離培養(yǎng)人大網(wǎng)膜前脂肪細(xì)胞,通過對細(xì)胞形態(tài)學(xué)觀察、MTT比色法作細(xì)胞生長曲線、油紅O脂肪染色抽提法對細(xì)胞鑒定,觀察原代培養(yǎng)人前脂肪細(xì)胞增殖和分化過程。 3、分別在前脂肪細(xì)胞增殖過程、及21天的分化過程中收集細(xì)胞上清液。用酶聯(lián)免疫吸附法同批檢測所收集細(xì)胞上清液中瘦素和脂聯(lián)素蛋白含量。 4、統(tǒng)計(jì)學(xué)處理:分析結(jié)果用“均數(shù)±標(biāo)準(zhǔn)差”表示,P＜0.05有統(tǒng)計(jì)學(xué)意義。 結(jié)果: 1、成功地培養(yǎng)了原代人網(wǎng)膜前脂肪細(xì)胞。前脂肪細(xì)胞形態(tài)與成纖維細(xì)胞相似,增殖期的4～10天為對數(shù)增長期;人工誘導(dǎo)分化后,第4天左右可見脂質(zhì)小滴,隨分化進(jìn)程脂滴逐漸增多,21天左右大部分分化為成熟脂肪細(xì)胞。 2、前脂肪細(xì)胞增殖期,在所收集的前脂肪細(xì)胞培養(yǎng)液中可檢測到低水平的瘦素分泌,但始終未測到脂聯(lián)素蛋白分泌。 3、前脂肪細(xì)胞分化期,瘦素分泌量隨誘導(dǎo)分化進(jìn)程而持續(xù)增多,第17天達(dá)高峰,之后保持高水平分泌狀態(tài)至誘導(dǎo)分化完成。脂聯(lián)素則在分化誘導(dǎo)到第7天才檢測到低水平的分泌,此時(shí)倒置顯微鏡下已可觀察到有內(nèi)含脂滴的脂肪細(xì)胞出現(xiàn);第17天分泌量達(dá)最高峰;第21天分泌量可見明顯下降(P＜0.05),而此時(shí)胞內(nèi)脂質(zhì)含量未降。 結(jié)論: 1、成功建立了原代人網(wǎng)膜前脂肪細(xì)胞體外培養(yǎng)模型,為進(jìn)一步研究中心性肥胖及胰島素抵抗相關(guān)疾病提供了細(xì)胞學(xué)平臺。 2、在原代培養(yǎng)的人網(wǎng)膜脂肪細(xì)胞上,瘦素和脂聯(lián)素呈現(xiàn)不同的分泌模式。其中,脂聯(lián)素可以作為鑒定前脂肪細(xì)胞分化成熟的特異性標(biāo)志。 3、不同階段、不同狀態(tài)的脂肪細(xì)胞,其分泌瘦素、脂聯(lián)素的功能有明顯差異。為研究肥胖患者體內(nèi)低脂聯(lián)素和高瘦素的分泌機(jī)制提供了實(shí)驗(yàn)室證據(jù):即肥胖患者血中低脂聯(lián)素和高瘦素的特點(diǎn)可能與前脂肪細(xì)胞大小有關(guān),而與其數(shù)量關(guān)系不是十分密切。 4、脂聯(lián)素可能存在自分泌的反饋抑制作用,且其分泌下調(diào)機(jī)制可能與分化有關(guān),而與脂質(zhì)積聚關(guān)系不是十分密切,為其調(diào)節(jié)機(jī)制提供離體的實(shí)驗(yàn)室證據(jù)。
[Abstract]:Aim: to study the secretion of leptin and adiponectin by cultured human omentum preadipocytes and differentiated into mature adipocytes in vitro. Methods: 1. Patients undergoing elective laparotomy should have no systemic metabolic and endocrine diseases, and no history of taking drugs to interfere with the metabolism of sugar and fat. The age range was (40 鹵8) years, and the body mass index (BMI) was less than 25 kg / m ~ (-2). The preomentum adipocytes were isolated and cultured by collagenase digestion. The cell growth curve was determined by MTT colorimetric method. The proliferation and differentiation of human preadipocytes in primary culture were observed by oil red O fat staining. 3. The supernatants were collected during the process of preadipocyte proliferation and 21 days of differentiation. The contents of leptin and adiponectin in supernatant were detected by enzyme-linked immunosorbent assay (Elisa). Results: 1. Primary human preomentum adipocytes were successfully cultured. The morphology of preadipocytes was similar to that of fibroblasts, and 4 days after proliferation was logarithmic growth period, and lipid droplets were observed on the 4th day after induced differentiation. With the increase of lipid droplets during the course of differentiation, most of the adipocytes differentiated into mature adipocytes about 21 days. 2. During the proliferation of preadipocytes, low levels of leptin secretion could be detected in the preadipocyte culture medium collected. However, adiponectin secretion was not detected all the time. During preadipocyte differentiation, leptin secretion continued to increase with the process of differentiation, reached its peak on the 17th day, and then maintained a high level of secretion until differentiation was completed. Adiponectin detected low levels of adipocytes on the 7th day after differentiation induction, and adipocytes with lipid droplets were observed under inverted microscope, and the secretion reached the highest level on the 17th day. On the 21st day, the secretion decreased significantly (P < 0.05), but the intracellular lipid content did not decrease at this time. Conclusion: 1. The primary culture model of human omentum preadipocytes in vitro was successfully established. 2. Leptin and adiponectin present different secretory patterns in primary cultured human omental adipocytes. Adiponectin can be used as a specific marker of adipocyte differentiation and maturation. 3The function of leptin and adiponectin in adipocytes at different stages and different states were significantly different. It provides laboratory evidence to study the secretory mechanism of hypoadiponectin and hyperleptin in obese patients: the characteristics of hypoadiponectin and hyperleptin in obese patients may be related to the size of preadipocytes. 4. Adiponectin may have an autocrine feedback inhibitory effect, and its down-regulation may be related to differentiation, but not to lipid accumulation. To provide in vitro laboratory evidence for its regulatory mechanism.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 楊坤;性激素對人網(wǎng)膜前脂肪細(xì)胞分泌瘦素和脂聯(lián)素的影響[D];山西醫(yī)科大學(xué);2008年

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本文編號：2133497