脂肪因子Omentin-1、Chemerin、Vaspin体外表达与GDM胰岛素抵抗研究
发布时间：2018-07-24 09:56
【摘要】：研究背景妊娠期糖尿病(gestational diabetes mellitus,GDM)是在妊娠期首次发生或发现的糖尿病,严重威胁孕妇及其子代健康。至今为止GDM的病因尚不清楚,众多研究一致认为是多种因素共同作用的结果。胰岛素抵抗(insulin resistance,IR)及由于胰岛素抵抗带来的糖脂代谢紊乱是目前公认的GDM发病的病理生理基础,而胰岛素信号通路的异常是IR发生的主要原因。近年来,来源于脂肪组织的脂肪因子(Adipokines)与GDM的关系倍受关注,已成为新的研究热点。课题组前期研究发现,在GDM孕妇血清、体内网膜组织以及胎盘组织中,Omentin-1明显降低,而Chemerin、Vaspin则明显升高。研究结果表明脂肪因子Omentin-1、Chemerin、Vaspin与GDM的胰岛素抵抗可能存在关联。但前期研究局限于血清和人体内组织学的研究,只是揭示了三者与GDM之间存在的这种表象,同时,带来了新的问题,即Omentin-1、Chemerin、Vaspin与GDM的胰岛素抵抗是否存在因果关系问题,如果存在,那么是脂肪因子的变化导致了 GDM胰岛素抵抗的发生?还是GDM胰岛素抵抗的发生导致了脂肪因子的变化?脂肪因子与GDM胰岛素抵抗的机制,着重在于脂肪因子与胰岛素受体后信号分子的表达及磷酸化水平之间的关联。为进一步探讨脂肪因子Omentin-1、Chemerin、Vaspin与GDM胰岛素抵抗是否存在必然联系及具体机制,本研究在前期研究基础上,建立GDM大网膜前脂肪细胞原代培养及增殖分化模型,以体外培养GDM脂肪细胞为载体,检测脂肪因子Omentin-1、Chemerin、Vaspin在不同程度过表达状态下,脂肪细胞中胰岛素受体下游信号分子(胰岛素受体底物-1/2(insulin receptor substrate,IRS-1/2)、磷脂酰肌醇-3 激酶(phosphatidyl inositol-3 kinase,PI3K))mRNA及蛋白表达,IRS-1/2酪氨酸磷酸化水平的变化,并最终以脂肪细胞葡萄糖摄取率的变化为验证,从胰岛素经典信号传导通路(IRS-1/-2、PI3K)探讨脂肪因子Omentin-1、Chemerin、Vaspin与GDM胰岛素抵抗的内在联系,为研究GDM发病机理和治疗策略提供线索。实验分为两部分第一部分GDM大网膜前脂肪细胞原代培养及增殖分化模型的建立[目的]建立妊娠期糖尿病(GDM)患者大网膜前脂肪细胞原代培养及向成熟脂肪细胞增殖分化的模型,为GDM患者脂肪因子体外脂肪细胞表达等后续研究奠定基础。[方法]使用改良的细胞培养法,以GDM剖宫产患者大网膜纯脂肪组织为原材料,作前脂肪细胞的原代培养及传代,绘制传代前脂肪细胞的生长曲线,并对传代前脂肪细胞做诱导分化,对已诱导分化细胞进行油红O染色、甘油磷酸脱氢酶(GPDH)动态变化测定、油红O染色提取法测定脂肪细胞内脂肪含量及RT-PCR检测脂联素mRNA表达,鉴定是否已诱导为成熟脂肪细胞,并进行细胞冻存与复苏实验。[结果]1、培养6 h即有部分细胞贴壁,13 h贴壁率70%,24 h后基本全部贴壁。2、培养出的前脂肪细胞基本均为梭形细胞,增殖旺盛,第4天细胞开始快速增殖,从生长曲线可见倍增时间大约为48 h左右。3、第7天原代前脂肪细胞逐渐由梭形变为椭圆形或圆形,胞内开始出现脂肪颗粒,而传代细胞仍然保持梭形和无脂滴。4.、第9天原代前脂肪细胞已开始出现大量脂肪颗粒,而传代细胞至第9天后仍继续保持梭形,细胞基本单层融合,排列紧密,油红O染色显示仍未着色。5、传代后前脂肪细胞前4天细胞形态及生长状况与原代前脂肪细胞极为相似,基本为梭形,大小较为均匀。第5-6天后细胞进入快速增殖,排列平行紧密,通常第7天即可完成单层融合。6、前脂肪细胞在一定浓度胰岛素(10μg/ml)和地塞米松(1umol/L)的诱导作用下,GPDH开始上升并迅速增加,9天左右到达高峰并维持在高水平,形态上表现为3天后部分细胞开始出现单个散在脂滴,随着分化时间延长,脂滴逐渐增多,胞质中出现围绕细胞核的小脂滴,11天左右到达高峰,与油红O染色定量结果相吻合。7、RT-PCR检测脂联素mRNA表达,由电泳结果可见分化后的细胞有脂联素mRNA表达,前脂肪细胞无脂联素mRNA表达。[结论]1、GDM大网膜脂肪组织中具有前脂肪细胞,以课题组改良法培养出的人源前脂肪细胞成分均一,增殖旺盛,倍增时间为48h左右。可进行连续传代和大量扩增,一般在5代以内细胞增殖迅速,再往后则增殖和分化率开始降低。2、原代前脂肪细胞在培养增殖过程中,会自然出现向成熟脂肪细胞的分化,而传代后前脂肪细胞则失去了分化的能力,可能与原代细胞仍然会携带某些体内诱导因子有关;3、前脂肪细胞可被诱导而分化,一定量的胰岛素(10μg/ml)和地塞米松(lumol/L)在前脂肪细胞的分化中起重要作用,是其启动和促进因子,诱导后分化率高,可达80%左右;4、诱导后细胞内脂肪含量的增加比GPDH的出现要晚6天左右,酶的出现在先,脂肪出现在后;5、传代前脂肪细胞可进行冻存和复苏,经适当诱导后可定向分化为成熟脂肪细胞,脂肪细胞模型的建立,为后续脂肪细胞体外实验研究做好了前期准备。第二部分Omentin-1、Chemerin、Vaspin体外过表达对胰岛素信号传导通路的影响[目的]以体外培养GDM前脂肪细胞为载体,探讨脂肪因子Omentin-1、Chemerin、Vaspin在过表达状态下,脂肪细胞胰岛素受体下游信号分子胰岛素受体底物-1/2(IRS-1/2)、磷脂酰肌醇-3激酶(PI3K)mRNA及蛋白表达,IRS-1/2酪氨酸磷酸化水平的变化,以及细胞葡萄糖摄取率变化,从胰岛素IRS-1/2、PI3K(P85α)信号传导通路探讨脂肪因子Omentin-1、Chemerin、Vaspin与GDM胰岛素抵抗的内在联系。[方法]复苏、传代及诱导分化GDM前脂肪细胞,构建人源Omentin-1、Chemerin、Vaspin过表达质粒,利用大肠杆菌表达系统进行质粒转化、扩大培养和提取;以3个过表达梯度(1.0μμg 2.5μg、5.0μg)转染GDM人源第3代传代脂肪细胞(每个浓度转染6孔,3系共18个数据),以无转染组为对照;Q-PCR检测细胞中Omentin-1、Chemerin、Vaspin、胰岛素受体底物-1/2(IRS-1/2)、磷脂酰肌醇-3激酶(PI3K(P85α))mRNA 表达,Western Blot 检测细胞中 Omentin-1、Chemerin、Vaspin、IRS-1、IRS-2、PI3K(P85α)蛋白表达及 IRS-1/2 酪氨酸磷酸化水平,[3H]-2-脱氧-D-葡萄糖摄取测定法检测不同浓度转染组细胞葡萄糖摄取率的变化。统计分析时各系以0.0μg组均值为标准,其余3组以相应比值的x±s进行标化和量化,SPSS 20.0软件统计处理。[结果]1、Omentin-1、Chemerin、Vaspin mRNA及蛋白表达随转染浓度梯度升高而表达量增加(P均0.05),所构建过表达载体有效;2、随Omentin-1表达增加,转染人源Omentin-1脂肪细胞中IRS-1、PI3K(P85α)mRNA及蛋白表达明显增加(P均0.05),IRS-2 mRNA及蛋白表达未发生明显变化(P均0.05),IRS-1磷酸化程度明显升高(P=0.031),IRS-2磷酸化程度未发生明显变化(P=0.685),葡萄糖摄取率上升(P=0.024);3、随Chemerin表达增加,转染人源Chemerin脂肪细胞中IRS-1/2、PI3K(P85α)mRNA及蛋白表达未出现明显变化(P均0.05),但存在IRS-1磷酸化程度明显增加(P=0.041),IRS-2磷酸化程度变化不明显(P=0.585),脂肪细胞葡萄糖摄取率略有增加,但无统计学差别(P=0.064);4、随Vaspin表达增加,转染人源Vaspin脂肪细胞中IRS-1、IRS-2、PI3K(P85α)mRNA及蛋白表达均未发生明显变化(P均0.05),IRS-1、IRS-2酪氨酸磷酸化程度均未出现明显变化(P均0.05),葡萄糖摄取率变化不明显(P=0.656)。[结论]1、Omentin-1与GDM胰岛素抵抗存在紧密的联系,Omentin-1是机体的保护因子,对肥胖、糖尿病等具有拮抗作用,Omentin-1的表达下降是机体发生胰岛素抵抗的重要原因之一,GDM胰岛素抵抗发生,是Omentin-1表达下降的果。其增敏机制是Omentin-1的表达可能通过某种途径激活了 IRS-1/PI3K(P85 α)信号通路,导致了 IRS-1表达增加和磷酸化活化,促进了 PI3K的活化和表达,从而提高脂肪细胞葡萄糖的摄取,发挥其胰岛素增敏作用,而与IRS-2没有明显关联。2、Chemerin在GDM血清及网膜脂肪组织中的高表达,可能与GDM的胰岛素抵抗存在一定的关联,但更多的可能,是对GDM肥胖、慢性炎症状态及胰岛素抵抗的一种补偿机制,更倾向于是GDM胰岛素抵抗的结果。其一定程度促进IRS-1酪氨酸磷酸化的机制,以及是否通过了其他途径一定程度上增加了机体对胰岛素敏感,尚不明确,还有待于进一步研究。3、Vaspin 与 IRS-1、IRS-2、PI3K(P85α)并不存在明显关联,Vaspin 的升高不会导致GDM的胰岛素抵抗,Vaspin在GDM血清及网膜脂肪组织中的高表达,不是胰岛素抵抗的因,而是GDM机体存在的脂肪蓄积、高血糖及高脂血症状态与Vaspin的一种交互影响的结果。临床上可以此作为一种衡量孕妇脂代谢紊乱程度和是否存在慢性炎症状态的“代谢紊乱标志”,但与GDM胰岛素抵抗的发生没有直接的必然的联系。
[Abstract]:Background gestational diabetes mellitus (GDM) is the first occurrence or discovery of diabetes in pregnancy, which is a serious threat to the health of pregnant women and their offspring. So far, the cause of GDM is not clear. Many studies have agreed to be a result of the combination of various factors. Insulin resistance (insulin resistance, IR) and the cause of the pancreas. The disorder of glycolipid metabolism caused by Isle resistance is the pathophysiological basis of GDM, and the abnormal insulin signaling pathway is the main cause of IR. In recent years, the relationship between adipose tissue derived from adipose tissue (Adipokines) and GDM has attracted much attention and has become a new research hotspot. In the women's serum, in the omentum tissue and in the placenta, the Omentin-1 was significantly reduced, while the Chemerin and Vaspin were significantly increased. The results showed that the insulin resistance of Omentin-1, Chemerin, Vaspin and GDM may be associated. However, the previous study was limited to the study of serum and human histology, but only revealed the relationship between the three and GDM. The existence of this representation brings new questions: whether there is a causal relationship between the insulin resistance of Omentin-1, Chemerin, Vaspin and GDM, if there is a change in the fat factor that leads to the occurrence of GDM insulin resistance, or is the occurrence of GDM insulin resistance leading to a change in the fat factor, the fat factor and the GDM The mechanism of insulin resistance lies in the association between the expression of adipose factors and the expression of post insulin receptor signaling molecules and the level of phosphorylation. In order to further investigate whether there is an inevitable relationship and specific mechanism of adipose factor Omentin-1, Chemerin, Vaspin and GDM insulin resistance, this research is based on the preliminary Study on the establishment of GDM great omentum fat. The primary culture and proliferation and differentiation model of the adipose cells were used to culture GDM adipocytes in vitro, and to detect the adipose factor Omentin-1, Chemerin, and Vaspin at different levels of overexpression. The downstream signal molecules of insulin receptor (-1/2 (insulin receptor substrate, IRS-1/2) and phosphatidyl inositol -3 kinase (PHO) in adipocytes Sphatidyl inositol-3 kinase, PI3K) mRNA and protein expression, the changes in the level of IRS-1/2 tyrosine phosphorylation, and finally verified by the changes in the glucose uptake rate of adipocytes. The internal relationship between the fat factor Omentin-1, Chemerin, Vaspin and GDM insulin resistance is discussed from the insulin classical signal transduction pathway (IRS-1/-2, PI3K). The pathogenesis and treatment strategy provide clues. The experiment is divided into two parts: the first part of the two part of the primary adipocyte preadipocyte culture and proliferation and differentiation model establishment [Objective] to establish the primary culture of preadipocyte preadipocytes and the proliferation and differentiation of mature adipocytes in patients with gestational diabetes mellitus (GDM), for the in vitro adipose factor of GDM patients. A modified cell culture method was used to use the modified cell culture method. The primary adipose tissue of the GDM caesarean section was used as the raw material, the primary culture and generation of preadipocytes, the growth curve of preprepassable adipocytes were plotted, and the pre passages were induced and differentiated, and the differentiated cells were induced into the differentiated cells. Oil red O staining, glycerol phosphate dehydrogenase (GPDH) dynamic change determination, oil red O staining method to determine fat content in adipocytes and RT-PCR detection of adiponectin mRNA expression, identification has been induced into mature adipocytes, and cell cryopreservation and resuscitation experiment. [results]1, 6 h, a part of cell adherence, 13 H adherence rate 70%, 24 h After almost all the adherent.2, the cultured preadipocytes were basically spindle cells, proliferating and proliferating, and the cells began to proliferate quickly on the fourth day. The multiplication time was about 48 h.3 from the growth curve, and the primary adipocytes were gradually oval or round, and the fat particles began to appear in the cell, and the passages were still preserved in the seventh days. With shuttle and fat free.4., a large number of fat particles have begun to appear in the ninth day pregeneration adipocytes, while the cells of the passages continue to remain spindle shaped after ninth days. The cells are basically monolayer and closely arranged. The oil red O staining shows that the.5 is still not stained, and the fine cell morphology and growth status of the preadipocyte before the generation and the pre generation adipocytes are very different from those of the original preadipocytes. Similar, basically spindle shape, the size is more uniform. After 5-6 days, the cells enter the rapid proliferation, the arrangement is parallel and close, usually seventh days can complete the single layer fusion.6, the preadipocytes in a certain concentration of insulin (10 mu g/ml) and dexamethasone (1umol/L) induced, GPDH began to rise and rapidly increase, 9 days to reach the peak and maintain high Level, after 3 days, the cells began to appear single scattered in the lipid droplets. As the time of differentiation extended, the lipid droplets increased gradually, and the small fat droplets around the nucleus appeared in the cytoplasm, reaching the peak at about 11 days, and anastomosing.7 with the quantitative results of the oil red O staining. The expression of lipoplex mRNA was detected by RT-PCR, and the differentiated cells were visible from the electrophoresis results. The expression of adiponectin mRNA, the preadipocytes without adiponectin mRNA expression. [conclusion]1, GDM omentum adipose tissue has preadipocytes in the adipose tissue, and the preadipocytes of human preadipocytes are homogenized, proliferate, and the multiplication time is about 48h. After that, the proliferation and differentiation rate began to decrease.2, and the preadipocytes would naturally appear to mature adipocytes during the process of culture and proliferation, while preadipocytes lost their ability to differentiate, which may be related to the primary cells, and 3, preadipocytes can be induced and differentiated. A certain amount of insulin (10 g/ml) and dexamethasone (lumol/L) play an important role in the differentiation of preadipocytes. It is the promoter and promoting factor, and the induced differentiation rate is high, up to about 80%. 4, the increase of intracellular fat content after induction is 6 days left right later than that of GPDH, the enzyme appears first, fat appears in the post, and 5, fat fine before passage. The cell can be frozen and resuscitation, and can be differentiated into mature adipocytes after proper induction. The establishment of adipocyte model is ready for the subsequent study of adipocytes in vitro. Second the effect of overexpression of Omentin-1, Chemerin and Vaspin on insulin signaling pathway in vitro [Objective] to cultivate preadipose fat before GDM in vitro Cells were used to explore the expression of insulin receptor substrate -1/2 (IRS-1/2), phosphatidylinositol -3 kinase (PI3K) mRNA and protein expression, the changes in the level of IRS-1/2 tyrosine phosphorylation, and the changes in the glucose uptake rate of cells from the islets of the adipocyte Omentin-1, Chemerin and Vaspin. The intrinsic relationship between adipose factor Omentin-1, Chemerin, Vaspin and GDM insulin resistance was investigated by IRS-1/2, PI3K (P85 alpha) signal transduction pathway. [Methods] resuscitation, generation and induction of differentiation of pre GDM preadipocytes, constructing human Omentin-1, Chemerin, Vaspin overexpressed plasmids, using Escherichia coli expression system for plasmid transformation, and expanding culture and extraction; GDM human third generation adipocytes were transfected with 3 overexpression gradients (1 mu g, 2.5 mu g, 5 g), which were transfected with 6 pores and 3 lines with 18 data, and Q-PCR was used to detect Omentin-1, Chemerin, Vaspin, the insulin receptor substrate -1/2 (IRS-1/2), and phosphatidylinositol -3 kinase (PI3K (PI3K)). The expression of Omentin-1, Chemerin, Vaspin, IRS-1, IRS-2, PI3K (P85 alpha) protein and the level of IRS-1/2 tyrosine phosphorylation were detected. The changes of glucose uptake in the cells of different concentrations were detected by the [3H]-2- deoxy -D- glucose uptake assay. The statistical analysis was based on the mean of the 0 mu g group, and the other 3 groups were fed with the corresponding ratio X. SPSS 20 software statistical processing. [results]1, Omentin-1, Chemerin, Vaspin mRNA and protein expression increased with the increase of transfection concentration gradient (P 0.05), and the expression vector was constructed effectively; 2, with the increase of Omentin-1 expression, the expression of IRS-1, PI3K (P85 alpha) and protein expression increased obviously. Addition (P 0.05), IRS-2 mRNA and protein expression did not change significantly (P 0.05), IRS-1 phosphorylation level increased significantly (P=0.031), IRS-2 phosphorylation level did not change significantly (P=0.685), glucose uptake rate increased (P=0.024); 3, as Chemerin expression increased, transfected into human Chemerin fat cells, IRS-1/2, protein expression and protein expression There was no obvious change (P 0.05), but there was a significant increase in the degree of phosphorylation of IRS-1 (P=0.041), the degree of IRS-2 phosphorylation was not significantly changed (P=0.585), and the glucose uptake rate of adipocytes was slightly increased, but there was no statistical difference (P=0.064); 4, with the increase of Vaspin expression, the transfection of IRS-1, IRS-2, PI3K (P85 alpha) and protein tables in human Vaspin adipocytes There was no obvious change (P 0.05), IRS-1, IRS-2 tyrosine phosphorylation level did not change significantly (P 0.05), glucose uptake rate changes (P=0.656). [conclusion]1, Omentin-1 and GDM insulin resistance is closely linked, Omentin-1 is the body's protective factor, obesity, diabetes and other antagonistic effects, Omentin-1. The decrease of expression is one of the important causes of insulin resistance in the body. GDM insulin resistance is the result of the decline of Omentin-1 expression. Its sensitization mechanism is that the expression of Omentin-1 may activate the IRS-1/PI3K (P85 alpha) signaling pathway through some way, which leads to the activation of IRS-1 expression and phosphorylation, and promotes the activation and table of PI3K. As a result, it improves the uptake of glucose in adipocyte and plays its insulin sensitizing effect, but has no obvious association with IRS-2, and the high expression of Chemerin in GDM serum and omentum adipose tissue may be associated with the insulin resistance of GDM, but more likely, it is a kind of GDM obesity, chronic inflammatory state and insulin resistance. The compensation mechanism is more likely to be the result of GDM insulin resistance. To a certain extent, the mechanism of the tyrosine phosphorylation of IRS-1, and whether the body is sensitid to insulin to some extent through other ways, is not clear, and further study of.3, Vaspin and IRS-1, IRS-2, and PI3K (P85 alpha) have no obvious association, Vaspi The increase of N does not lead to insulin resistance in GDM. The high expression of Vaspin in GDM serum and omentum adipose tissue is not the cause of insulin resistance, but is the result of the accumulation of fat in GDM body, the interaction of hyperglycemia and hyperlipidemia with Vaspin. This can be used as a measure of the degree of lipid metabolism disorder in pregnant women. And whether there is a "metabolic disorder marker" in chronic inflammatory state, but it is not directly related to the occurrence of GDM insulin resistance.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R714.256
[Abstract]:Background gestational diabetes mellitus (GDM) is the first occurrence or discovery of diabetes in pregnancy, which is a serious threat to the health of pregnant women and their offspring. So far, the cause of GDM is not clear. Many studies have agreed to be a result of the combination of various factors. Insulin resistance (insulin resistance, IR) and the cause of the pancreas. The disorder of glycolipid metabolism caused by Isle resistance is the pathophysiological basis of GDM, and the abnormal insulin signaling pathway is the main cause of IR. In recent years, the relationship between adipose tissue derived from adipose tissue (Adipokines) and GDM has attracted much attention and has become a new research hotspot. In the women's serum, in the omentum tissue and in the placenta, the Omentin-1 was significantly reduced, while the Chemerin and Vaspin were significantly increased. The results showed that the insulin resistance of Omentin-1, Chemerin, Vaspin and GDM may be associated. However, the previous study was limited to the study of serum and human histology, but only revealed the relationship between the three and GDM. The existence of this representation brings new questions: whether there is a causal relationship between the insulin resistance of Omentin-1, Chemerin, Vaspin and GDM, if there is a change in the fat factor that leads to the occurrence of GDM insulin resistance, or is the occurrence of GDM insulin resistance leading to a change in the fat factor, the fat factor and the GDM The mechanism of insulin resistance lies in the association between the expression of adipose factors and the expression of post insulin receptor signaling molecules and the level of phosphorylation. In order to further investigate whether there is an inevitable relationship and specific mechanism of adipose factor Omentin-1, Chemerin, Vaspin and GDM insulin resistance, this research is based on the preliminary Study on the establishment of GDM great omentum fat. The primary culture and proliferation and differentiation model of the adipose cells were used to culture GDM adipocytes in vitro, and to detect the adipose factor Omentin-1, Chemerin, and Vaspin at different levels of overexpression. The downstream signal molecules of insulin receptor (-1/2 (insulin receptor substrate, IRS-1/2) and phosphatidyl inositol -3 kinase (PHO) in adipocytes Sphatidyl inositol-3 kinase, PI3K) mRNA and protein expression, the changes in the level of IRS-1/2 tyrosine phosphorylation, and finally verified by the changes in the glucose uptake rate of adipocytes. The internal relationship between the fat factor Omentin-1, Chemerin, Vaspin and GDM insulin resistance is discussed from the insulin classical signal transduction pathway (IRS-1/-2, PI3K). The pathogenesis and treatment strategy provide clues. The experiment is divided into two parts: the first part of the two part of the primary adipocyte preadipocyte culture and proliferation and differentiation model establishment [Objective] to establish the primary culture of preadipocyte preadipocytes and the proliferation and differentiation of mature adipocytes in patients with gestational diabetes mellitus (GDM), for the in vitro adipose factor of GDM patients. A modified cell culture method was used to use the modified cell culture method. The primary adipose tissue of the GDM caesarean section was used as the raw material, the primary culture and generation of preadipocytes, the growth curve of preprepassable adipocytes were plotted, and the pre passages were induced and differentiated, and the differentiated cells were induced into the differentiated cells. Oil red O staining, glycerol phosphate dehydrogenase (GPDH) dynamic change determination, oil red O staining method to determine fat content in adipocytes and RT-PCR detection of adiponectin mRNA expression, identification has been induced into mature adipocytes, and cell cryopreservation and resuscitation experiment. [results]1, 6 h, a part of cell adherence, 13 H adherence rate 70%, 24 h After almost all the adherent.2, the cultured preadipocytes were basically spindle cells, proliferating and proliferating, and the cells began to proliferate quickly on the fourth day. The multiplication time was about 48 h.3 from the growth curve, and the primary adipocytes were gradually oval or round, and the fat particles began to appear in the cell, and the passages were still preserved in the seventh days. With shuttle and fat free.4., a large number of fat particles have begun to appear in the ninth day pregeneration adipocytes, while the cells of the passages continue to remain spindle shaped after ninth days. The cells are basically monolayer and closely arranged. The oil red O staining shows that the.5 is still not stained, and the fine cell morphology and growth status of the preadipocyte before the generation and the pre generation adipocytes are very different from those of the original preadipocytes. Similar, basically spindle shape, the size is more uniform. After 5-6 days, the cells enter the rapid proliferation, the arrangement is parallel and close, usually seventh days can complete the single layer fusion.6, the preadipocytes in a certain concentration of insulin (10 mu g/ml) and dexamethasone (1umol/L) induced, GPDH began to rise and rapidly increase, 9 days to reach the peak and maintain high Level, after 3 days, the cells began to appear single scattered in the lipid droplets. As the time of differentiation extended, the lipid droplets increased gradually, and the small fat droplets around the nucleus appeared in the cytoplasm, reaching the peak at about 11 days, and anastomosing.7 with the quantitative results of the oil red O staining. The expression of lipoplex mRNA was detected by RT-PCR, and the differentiated cells were visible from the electrophoresis results. The expression of adiponectin mRNA, the preadipocytes without adiponectin mRNA expression. [conclusion]1, GDM omentum adipose tissue has preadipocytes in the adipose tissue, and the preadipocytes of human preadipocytes are homogenized, proliferate, and the multiplication time is about 48h. After that, the proliferation and differentiation rate began to decrease.2, and the preadipocytes would naturally appear to mature adipocytes during the process of culture and proliferation, while preadipocytes lost their ability to differentiate, which may be related to the primary cells, and 3, preadipocytes can be induced and differentiated. A certain amount of insulin (10 g/ml) and dexamethasone (lumol/L) play an important role in the differentiation of preadipocytes. It is the promoter and promoting factor, and the induced differentiation rate is high, up to about 80%. 4, the increase of intracellular fat content after induction is 6 days left right later than that of GPDH, the enzyme appears first, fat appears in the post, and 5, fat fine before passage. The cell can be frozen and resuscitation, and can be differentiated into mature adipocytes after proper induction. The establishment of adipocyte model is ready for the subsequent study of adipocytes in vitro. Second the effect of overexpression of Omentin-1, Chemerin and Vaspin on insulin signaling pathway in vitro [Objective] to cultivate preadipose fat before GDM in vitro Cells were used to explore the expression of insulin receptor substrate -1/2 (IRS-1/2), phosphatidylinositol -3 kinase (PI3K) mRNA and protein expression, the changes in the level of IRS-1/2 tyrosine phosphorylation, and the changes in the glucose uptake rate of cells from the islets of the adipocyte Omentin-1, Chemerin and Vaspin. The intrinsic relationship between adipose factor Omentin-1, Chemerin, Vaspin and GDM insulin resistance was investigated by IRS-1/2, PI3K (P85 alpha) signal transduction pathway. [Methods] resuscitation, generation and induction of differentiation of pre GDM preadipocytes, constructing human Omentin-1, Chemerin, Vaspin overexpressed plasmids, using Escherichia coli expression system for plasmid transformation, and expanding culture and extraction; GDM human third generation adipocytes were transfected with 3 overexpression gradients (1 mu g, 2.5 mu g, 5 g), which were transfected with 6 pores and 3 lines with 18 data, and Q-PCR was used to detect Omentin-1, Chemerin, Vaspin, the insulin receptor substrate -1/2 (IRS-1/2), and phosphatidylinositol -3 kinase (PI3K (PI3K)). The expression of Omentin-1, Chemerin, Vaspin, IRS-1, IRS-2, PI3K (P85 alpha) protein and the level of IRS-1/2 tyrosine phosphorylation were detected. The changes of glucose uptake in the cells of different concentrations were detected by the [3H]-2- deoxy -D- glucose uptake assay. The statistical analysis was based on the mean of the 0 mu g group, and the other 3 groups were fed with the corresponding ratio X. SPSS 20 software statistical processing. [results]1, Omentin-1, Chemerin, Vaspin mRNA and protein expression increased with the increase of transfection concentration gradient (P 0.05), and the expression vector was constructed effectively; 2, with the increase of Omentin-1 expression, the expression of IRS-1, PI3K (P85 alpha) and protein expression increased obviously. Addition (P 0.05), IRS-2 mRNA and protein expression did not change significantly (P 0.05), IRS-1 phosphorylation level increased significantly (P=0.031), IRS-2 phosphorylation level did not change significantly (P=0.685), glucose uptake rate increased (P=0.024); 3, as Chemerin expression increased, transfected into human Chemerin fat cells, IRS-1/2, protein expression and protein expression There was no obvious change (P 0.05), but there was a significant increase in the degree of phosphorylation of IRS-1 (P=0.041), the degree of IRS-2 phosphorylation was not significantly changed (P=0.585), and the glucose uptake rate of adipocytes was slightly increased, but there was no statistical difference (P=0.064); 4, with the increase of Vaspin expression, the transfection of IRS-1, IRS-2, PI3K (P85 alpha) and protein tables in human Vaspin adipocytes There was no obvious change (P 0.05), IRS-1, IRS-2 tyrosine phosphorylation level did not change significantly (P 0.05), glucose uptake rate changes (P=0.656). [conclusion]1, Omentin-1 and GDM insulin resistance is closely linked, Omentin-1 is the body's protective factor, obesity, diabetes and other antagonistic effects, Omentin-1. The decrease of expression is one of the important causes of insulin resistance in the body. GDM insulin resistance is the result of the decline of Omentin-1 expression. Its sensitization mechanism is that the expression of Omentin-1 may activate the IRS-1/PI3K (P85 alpha) signaling pathway through some way, which leads to the activation of IRS-1 expression and phosphorylation, and promotes the activation and table of PI3K. As a result, it improves the uptake of glucose in adipocyte and plays its insulin sensitizing effect, but has no obvious association with IRS-2, and the high expression of Chemerin in GDM serum and omentum adipose tissue may be associated with the insulin resistance of GDM, but more likely, it is a kind of GDM obesity, chronic inflammatory state and insulin resistance. The compensation mechanism is more likely to be the result of GDM insulin resistance. To a certain extent, the mechanism of the tyrosine phosphorylation of IRS-1, and whether the body is sensitid to insulin to some extent through other ways, is not clear, and further study of.3, Vaspin and IRS-1, IRS-2, and PI3K (P85 alpha) have no obvious association, Vaspi The increase of N does not lead to insulin resistance in GDM. The high expression of Vaspin in GDM serum and omentum adipose tissue is not the cause of insulin resistance, but is the result of the accumulation of fat in GDM body, the interaction of hyperglycemia and hyperlipidemia with Vaspin. This can be used as a measure of the degree of lipid metabolism disorder in pregnant women. And whether there is a "metabolic disorder marker" in chronic inflammatory state, but it is not directly related to the occurrence of GDM insulin resistance.
【学位授予单位】：昆明医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R714.256
【参考文献】
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2 潘宝龙;马润玫;;血清脂肪因子omentin-1、chemerin的水平与妊娠期糖尿病的相关性[J];南方医科大学学报;2016年09期
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