【作者】 杨俊霞；

【导师】 宋方洲；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2007， 博士

【摘要】 肥胖症是由于体脂过度堆积而产生的慢性疾病,是心脑血管疾病、糖尿病、高血压、高脂血症以及癌症等多种疾病的高危因素,现已成为全球性的健康问题。多年来研究发现肥胖的发生与下丘脑对摄食的神经内分泌调控密切相关,由下丘脑分泌的促食肽黑色素浓集激素（MCH）及其受体（MCHR1和MCHR2）的发现为肥胖发生机制及防治的研究提供了新的靶点。通过构建转基因和基因敲除动物模型已对MCH及MCHR1与肥胖及能量代谢的关系进行了深入细致的研究,但有关MCHR2的功能及其与肥胖的关系至今仍不清楚。因此,本课题应用DNA重组技术构建人MCHR2真核表达载体,并稳定转染哺乳动物细胞,分析其表达、定位及生物学活性,进而以3T3-L1前脂肪细胞为研究对象,探讨MCHR2是否具备调控能量平衡的生理功能,为深入研究MCHR2与肥胖的关系奠定基础。研究内容包括以下三个部分:1. MCHR2真核表达载体的构建与鉴定。方法:以人胎脑cDNA文库为模板PCR扩增获取MCHR2基因全长cDNA编码区序列,定向插入到真核表达载体pcDNA3.1（+）中,并转化大肠杆菌,经抗生素平板筛选获得阳性克隆,再分别进行酶切、PCR及DNA测序鉴定。结果:获得了长为1023bp的人MCHR2 cDNA全长的目的片段,并插入到pcDNA3.1（+）载体的CMV启动子下游构建了表达MCHR2的重组质粒,酶切和PCR鉴定证实重组质粒带有目的基因片段,DNA测序确认插入片段的序列准确无误。结论:表达人MCHR2蛋白的真核表达载体pcDNA3.1-MCHR2构建成功。2. MCHR2在CHO细胞中的稳定表达及其生物学活性的分析。方法:脂质体转染法将pcDNA3.1-MCHR2真核表达载体导入CHO细胞,经G418筛选抗性细胞克隆,建立稳定转染细胞系,RT-PCR、Western blot检测MCHR2转录水平和蛋白水平的表达,免疫荧光检测MCHR2在细胞中的定位,放射性配体结合实验检测受体的密度和亲和力,通过测定细胞内cAMP、Ca²⁺浓度的改变分析MCHR2的信号转导通路。结果:经脂质体转染和G418抗性筛选6⁸周,建立了稳定转染pcDNA3.1-MCHR2质粒的CHO细胞系;在该细胞系中MCHR2基因能被有效地转录和翻译,并准确定位于细胞膜上,与125I-MCH的结合具有特异、高亲和及可饱和的配体-受体结合特性,其最大结合容量Bmax值为309.97±1.14fmol/mg protein,平衡解离常数Kd值为0.170±0.0006 nmol/L;MCH作用于MCHR2后不影响细胞内cAMP生成,可促使细胞内钙库释放Ca²⁺进而使胞内Ca²⁺浓度出现明显而短暂的升高,Ca²⁺振荡过程在MCH刺激后30⁵0s内完成,其对Ca²⁺释放的作用呈剂量依赖关系,半数有效浓度EC50为2.32±0.01nmol/L,提示MCHR2的信号转导通路是与Gq蛋白偶联。结论:建立了稳定转染MCHR2的CHO细胞系,从MCHR2的表达、定位、受体活性及信号转导通路等方面证实了真核表达载体pcDNA3.1-MCHR2稳定转染哺乳动物细胞后能有效表达具有生物学活性的MCHR2蛋白,为后续体外研究MCHR2基因的功能提供了可靠的实验依据。3. MCHR2对脂肪细胞生物学特性的影响。方法:通过脂质体转染和G418筛选建立稳定转染pcDNA3.1-MCHR2质粒的3T3-L1前脂肪细胞系,命名为3T3-L1-MCHR2细胞,作为对照的稳定转染空质粒的细胞命名为3T3-L1-mock细胞;RT-PCR、Western blot、免疫荧光检测、放射性配体结合实验及细胞内Ca²⁺检测鉴定MCHR2的表达及活性;MTT法检测细胞增殖,FCM检测细胞周期分布;激素鸡尾酒法（MIX+DEX+insulin）诱导前脂肪细胞分化,油红O染色观察脂肪细胞分化的形态学改变及量化分化程度,RT-PCR检测脂肪细胞分化成熟的标记基因;检测成熟脂肪细胞中TG的生成、FFA及Leptin的分泌。结果:建立了稳定转染细胞系——3T3-L1-MCHR2,并通过了MCHR2表达和活性的鉴定;对前脂肪细胞增殖和分化特性作比较分析,可见MCH对3T3-L1-MCHR2和3T3-L1-mock细胞的增殖无影响,对两株细胞的分化则有一定的促进作用,两者相比,3T3-L1-MCHR2细胞的分化速度更快、程度更高,具体表现为油红O比色在分化第9天开始出现脂质聚集增多（3T3-L1-mock细胞为第12天）,分化成熟标记基因PPARγ2、C/EBPα和aP2从分化第3天起上调,Leptin自分化第6天起上调,而3T3-L1-mock细胞的PPARγ2和Leptin于分化第6天、C/EBPα和aP2于分化第9天才上调,且增幅较弱;对成熟脂肪细胞的脂质代谢特性的分析显示,3T3-L1-MCHR2和3T3-L1-mock细胞在MCH作用下FFA的含量无明显差异,而TG的含量明显升高,升高率分别为22.61%和12.17%;ELISA法检测成熟脂肪细胞Leptin的分泌,3T3-L1-MCHR2细胞在MCH作用6h及24h后Leptin分泌明显增多,增幅分别为28.17%和18.25%,而3T3-L1-mock细胞则在24h后才增多,增幅为14.57%。结论:MCHR2可介导MCH对前脂肪细胞分化、脂肪合成代谢及Leptin分泌的正调控作用,对前脂肪细胞增殖和脂肪分解代谢则无影响,初步证实MCHR2的生理功能参与了能量平衡的调控。更多还原

【Abstract】 Obesity, a chronic disease characterized by excessive accumulation of body fat, has become a global health problem and is a significant risk factor for developing many serious diseases, including cardio- and cerebro- vascular diseases, diabetes, hypertension, dyslipidemias and some cancers. In recent years, it has been well established that obesity is associated with the neuroendocrine control of the hypothalamus. The identification of the hypothalamic orexigenic neuropeptide melanin-concentrating hormone （MCH） and its two receptors （MCHR1 and MCHR2） provide new targets for treatment of obesity. Major advances have been made in identifying the relevance of MCH and MCHR1 to obesity and energy homeostasis by transgenic animal and gene knockout animal. However, the function of MCHR2 and its relevance to obesity is still indistinct. In this study, we first construct the eukaryotic expression vector of human MCHR2 by DNA recombination technique. Then, we stably transfect mammalian cell line with the vector and analyse the expression, localization and biologic activity of MCHR2. Finally, we investigate the physiological function of MCHR2 in 3T3-L1 preadipocyte to evaluate whether it is involved in mediating the effects of MCH on energy balance and its relevance to obesity. This study includes three parts:1. Construction and identification of eukaryotic expression vector of human MCHR2. Methods: The full-length MCHR2 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and was oriently inserted into eukaryotic expression vector pcDNA3.1（+）. The positive clone was obtained after transformation and antibiotic screening and was identified by restriction endonuclease digestion, PCR, and DNA sequencing. Results: The 1023bp MCHR2 cDNA fragment was obtained and cloned into the downstream of CMV promoter of pcDNA3.1（+）. The recombinant plasmid was identified by restriction endonuclease digestion and PCR. DNA sequencing confirmed that the sequence of the inserted element was correct. Conclusion: The eukaryotic expression vector pcDNA3.1-MCHR2 was successfully constructed.2. Stably expression and biological activity of MCHR2 in CHO cells. Methods: CHO cells were transfected with the recombinant plasmids using lipofectamine. Stable cell line was established by G418 selection. Expression of MCHR2 in transcriptional level and protein level was detected by RT-PCR and western blot. Localization of MCHR2 in CHO cells was assayed by immunofluorence technique. Radioligand binding assays were performed to survey receptor density and affinity of MCHR2. Signal transduction pathways mediated by MCHR2 were analyzed by measurement of intracellular cAMP and calcium. Results: CHO cell line stably transfected with pcDNA3.1-MCHR2 plasmids was generated after liposome transfection and G418 selection. MCHR2 gene was efficiently transcribed and translated, and its protein exactly localized on the cytomembrane. MCHR2 could specific bind with ¹²⁵I-MCH with high-affinity and saturability （Bmax=309.97±1.14fmol/mg protein, Kd=0.170±0.0006nmol/L）. MCH stimulation had no effect on the production of cAMP. MCH induced a clear and transient increase of intracellular calcium, and this procedure was completed within 30⁵0s. The effect of MCH on intracellular calcium showed dose-dependent response with an effective half-maximal concentration （EC50） of 2.32±0.01nmol/L. It is suggested that MCHR2 is only coupled to Gq protein. Conclusion: Stably transfected CHO cell line was established successfully. It is confirmed that MCHR2 expressed in mammalian cells is a functional receptor for the MCH by the analysis of its expression, localization, receptor characteristics, and signal pathways. This present study provides a reliable experimental foundation for further studies on the function of MCHR2 in vitro.3. Influence of MCHR2 on the biological characteristics of adipocyte. Methods: 3T3-L1 cell line stably transfected with pcDNA3.1-MCHR2 plasminds was generated by transfection and G418 selection and named as 3T3-L1-MCHR2 while the control cell line transfected with pcDNA3.1（+） was named as 3T3-L1-mock. The expression and biological activity of MCHR2 were identified by RT-PCR, western blot, immunofluorence, radioligand binding and intracellular calcium assay. Cell proliferation was determined by MTT assay. Cell cycle was detected by flow cytometry. Cell differentiation was induced by hormone cocktail （MIX+DEX+insulin） and examined by Oil red O staining. The marker genes of adipocyte differentiation were examined by RT-PCR. The production of triglyceride and secretions of FFA and leptin were measured. Results: Stably transfected cell line named 3T3-L1-MCHR2 was established and passed the identification of expression and biological activity of MCHR2. MCH had no effect on the proliferation of 3T3-L1-MCHR2 and 3T3-L1-mock cells. Both the two cell lines were induced to differentiate to mature adipocytes by MCH. The speed and degree of differentiation in 3T3-L1-MCHR2 cells were higher than those in 3T3-L1-mock cells. Following was the detailed display of differentiation in 3T3-L1-MCHR2 cells: accumulation of lipid increased at day 9 （in 3T3-L1-mock it increased at day 12）; marker genes of mature adipocyte including PPARγ2、C/EBPαand aP2 increased at day 3, leptin increased at day 6, while in 3T3-L1-mock cells, up-regulation of PPARγ2 and leptin appeared at day 6, up-regulation of C/EBPαand aP2 appeared at day 9, and the increasing levels were lower. There was no difference of the FFA secretion between MCH-treated group and control group. The triglyceride content in 3T3-L1-MCHR2 and 3T3-L1-mock mature adipocytes obviously increased by MCH, and the rising rate were 22.61% and 12.17%, compared with corresponding untreated cells. MCH stimulated leptin releases from 3T3-L1-MCHR2 mature adipocytes at 6h and 24h, the rising rate were 28.17% and 18.25% compared with the untreated cells. In contrast, secretion of leptin from 3T3-L1-mock mature adipocytes only increased by 14.57% at 24h. Conclusion: MCHR2 mediates the positive control of MCH on preadipocyte differentiation, triglyceride synthesis and leptin secretion without the influence on preadipocyte proliferation and lipolysis. The present study tentatively confirms that the physiological function of MCHR2 involvs in the regulation of energy balance.更多还原