体外培养人网膜前脂肪细胞分泌瘦素、脂联素的研究
发布时间：2018-07-20 12:21
【摘要】： 目的:通过体外培养人大网膜前脂肪细胞,并诱导分化为成熟脂肪细胞,研究这一过程中瘦素、脂联素的分泌规律。 方法: 1、选择择期开腹手术病人,要求无全身代谢及内分泌疾病,无服用干预糖及脂肪代谢的药物史。年龄范围(40±8)岁,体重指数(BMI)小于25kg/m~2。 2、采用胶原酶消化的方法分离培养人大网膜前脂肪细胞,通过对细胞形态学观察、MTT比色法作细胞生长曲线、油红O脂肪染色抽提法对细胞鉴定,观察原代培养人前脂肪细胞增殖和分化过程。 3、分别在前脂肪细胞增殖过程、及21天的分化过程中收集细胞上清液。用酶联免疫吸附法同批检测所收集细胞上清液中瘦素和脂联素蛋白含量。 4、统计学处理:分析结果用“均数±标准差”表示,P＜0.05有统计学意义。 结果: 1、成功地培养了原代人网膜前脂肪细胞。前脂肪细胞形态与成纤维细胞相似,增殖期的4～10天为对数增长期;人工诱导分化后,第4天左右可见脂质小滴,随分化进程脂滴逐渐增多,21天左右大部分分化为成熟脂肪细胞。 2、前脂肪细胞增殖期,在所收集的前脂肪细胞培养液中可检测到低水平的瘦素分泌,但始终未测到脂联素蛋白分泌。 3、前脂肪细胞分化期,瘦素分泌量随诱导分化进程而持续增多,第17天达高峰,之后保持高水平分泌状态至诱导分化完成。脂联素则在分化诱导到第7天才检测到低水平的分泌,此时倒置显微镜下已可观察到有内含脂滴的脂肪细胞出现;第17天分泌量达最高峰;第21天分泌量可见明显下降(P＜0.05),而此时胞内脂质含量未降。 结论: 1、成功建立了原代人网膜前脂肪细胞体外培养模型,为进一步研究中心性肥胖及胰岛素抵抗相关疾病提供了细胞学平台。 2、在原代培养的人网膜脂肪细胞上,瘦素和脂联素呈现不同的分泌模式。其中,脂联素可以作为鉴定前脂肪细胞分化成熟的特异性标志。 3、不同阶段、不同状态的脂肪细胞,其分泌瘦素、脂联素的功能有明显差异。为研究肥胖患者体内低脂联素和高瘦素的分泌机制提供了实验室证据:即肥胖患者血中低脂联素和高瘦素的特点可能与前脂肪细胞大小有关,而与其数量关系不是十分密切。 4、脂联素可能存在自分泌的反馈抑制作用,且其分泌下调机制可能与分化有关,而与脂质积聚关系不是十分密切,为其调节机制提供离体的实验室证据。
[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 卤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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329
本文编号：2133497
[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 卤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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329
【引证文献】
相关硕士学位论文 前1条
1 杨坤;性激素对人网膜前脂肪细胞分泌瘦素和脂联素的影响[D];山西医科大学;2008年
，本文编号：2133497
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