肾上腺素能受体家族在肿瘤恶病质脂肪分解过程中的调控机制研究

Extensive loss of body fat is a hallmark of cancer cachexia, and the underlying mechanisms are poorly understood. Lately, it has been demonstrated that mice with the deficiency of any of the lipases, adipose triglyceride lipase (ATGL) or hormone sensitive lipase (HSL), are resistant to tumor-associated depletion of both adipose tissue and skeletal muscle. This reveals the pivotal role of lipolysis in the pathogenesis of cancer cachexia, suggesting that drugs targeting adipose lipases may prevent or reverse cachexia. In our previous study, we found that patients with cancer cachexia have increased expression of β1-adrenergic receptor (β1-AR) in adipose tissue at both mRNA and protein level, which may stimulate lipid mobilization by upregulating HSL expression. This indicates the involvement of β1-AR in the enhanced lipolysis of cancer cachexia. On this basis, our current study aims to find out the adrenergic receptor subtype regulating lipolysis in cancer cachexia by analyzing the association of adrenergic receptors (β1-AR, β2-AR, β3-AR, α2C-AR) expression with catecholamines serum level, ATGL and HSL gene expression and activity, lipolytic rate, and cachectic phenotypes in clinical specimens. In addition, we will establish the adipocyte model of cachectic fat loss from the mouse 3T3-L1 preadipocytic cell line using MDI criteria, as well as two mouse models of cancer cachexia, heterotopic model of Lewis lung cancer and orthotopic B16 mouse melanoma. Both in vitro and in vivo functional studies will be carried out to confirm the involvement of target receptor in lipolysis of cancer cachexia. Furthermore, the relationship between the target receptor and the lipases (ATGL and HSL), and the underlying mechanisms will be studied through the detection of downstream pathways (AC/cAMP/PKA/ATGL/HSL). Finally, the target receptor will be evaluated in mouse models of cancer cachexia as a potential therapeutic target for cachexia. This project may provide new insights into the pathogenesis of cancer cachexia and lead to novel therapeutic targets to ameliorate this clinical syndrome.

脂肪消耗是肿瘤恶病质的主要特征之一，目前对其发病机制所知甚少。最新发现敲除脂肪分解酶ATGL或HSL可防止小鼠发生肿瘤恶病质，提示干预脂肪分解有利于改善肿瘤恶病质。我们前期研究发现，肿瘤恶病质病人脂肪组织中肾上腺素能受体β1亚型表达增加，可能通过激活下游的HSL促进脂肪分解。本课题拟通过临床标本检测，分析肾上腺素能受体（β1、β2、β3、α2C亚型）表达与儿茶酚胺水平、ATGL和HSL表达及活性、脂肪分解率、恶病质表型的关系，找到调控恶病质脂肪分解的受体亚型；利用脂肪分解体外研究模型（用MDI方案诱导3T3-L1细胞分化）及Lewis肺癌/B16黑素瘤小鼠恶病质模型验证其调控作用；检测下游信号通路：AC/cAMP/PKA/ATGL/HSL，阐明其与ATGL和HSL的关系及相关机制；用动物模型评价其作为恶病质治疗靶点的可行性。本课题有望加深对肿瘤恶病质发病机制的认识，为临床治疗提供理论依据。

脂肪消耗是肿瘤恶病质最显著的改变之一，与恶性肿瘤的高致残率和致死率直接相关，目前对其分子机制所知甚少。恶病质脂肪消耗反应了甘油三酯合成与分解失衡，一般认为脂肪分解增加在其中起主要作用。激素敏感性酯酶（HSL）激活是引起恶病质脂肪分解增强的关键环节。我们前期研究发现，肿瘤恶病质病人脂肪组织中肾上腺素受体β1亚型（ADRB1）表达增加，并与HSL表达呈正相关，推测ADRB1表达上调通过激活HSL导致脂肪消耗。.本研究首先构建了基于3T3-L1前脂肪细胞株的人类ADRB1基因稳定过表达的细胞模型。将ADRB1 cDNA克隆片段和慢病毒载体连接；将重组载体与慢病毒包装质粒共转染293T细胞，收集上清液感染3T3-L1前脂肪细胞，利用GFP流式分选法筛选稳转细胞株。Western blot检测证明，ADRB1过表达组的ADRB1蛋白水平显著高于mock组。通过MDI方案诱导脂肪分化，使之模拟肿瘤恶病质病人脂肪细胞的特征性改变，为后续研究奠定了模型基础。.在此基础上，我们通过体外实验研究了ADRB1在肿瘤恶病质脂肪消耗中的调控作用。我们设立了正常对照组、mock组、ADRB1过表达组、抑制剂干预组。将ADRB1基因稳定转染进3T3-L1前脂肪细胞，以MDI方案诱导分化，利用Western blot检测ADRB1和HSL蛋白水平。油红O染色证实，正常对照组和mock组能够分化，ADRB1过表达组无法分化，且可被ADRB1抑制剂逆转。ADRB1过表达组中HSL蛋白表达增强，且可被ADRB1抑制剂逆转。该结果证明，ADRB1表达增加通过激活HSL的表达刺激脂肪分解，参与肿瘤恶病质脂肪消耗过程。本研究加深了对肿瘤恶病质发生机制的认识。