肝硬化门脉高压症脾亢脾促进肝癌发生发展的分子机制研究

Background and objectives:.The relationship between spleen from hypersplenism due to portal hypertension(PH) and development and progression of hepatocellular carcinoma (HCC) receives increasingly attention. Our previous study demonstrated that there were 26 differential cytokines between PH hypersplenic and normal splenic tissue, some of which were cytokines associated with development and progression of HCC such as TNF-β, IGFBP-2, TGF-β and VEGF. Furthermore,our study also found that serum from patients with hypersplenism due to PH remarkably induced growth and morphological change of MHCC97H cells. These results initially indicated that the spleen from hypersplenism due to PH might promote hepatocarcinogenesis, however, the underlying mechanism remains unclear. Based on above findings, objectives of the present study were to explore promotion of spleen from hypersplenism due to PH on development and progress in HCC and its molecular mechanism..Methods: .1. Serum from patients with hypersplenism due to PH will be collected. Then we evaluate effects of the serum on growth, proliferation, epithelial-mesenchymal transition (EMT), invasion and metastasis in human HCC cell lines. 2. We seek to establish a model by orthotopic implantation of human HCC in nude mice and further validate the results of in vitro experiment. 3. We will further analyze the differential expression of cytokine in serum from patients with hypersplenism due to PH and the control groups by cytokine array and bioinformatics. Next, human HCC cells are cultured and stimulated with different concentrations of the selective cytokines for the indicated time. And growth, proliferation, EMT, invasion and metastasis of the cells are assayed.Then the blocker of cytokine will be used in the present experiment. Moreover, the cells will be transiently transfected with cytokine small-interfering RNA (siRNA) or negative control siRNA (NC siRNA) using DharmaFECT 4 transfection reagents according to the manufacturer's instructions, and the above methods are used the sequent experiments. .Prospective conclusions:.The present study will reveal that serum from patients with hypersplenism due to PH has the ability to promote growth, proliferation, EMT, invasion and metastasis of HCC via investigation of animal, cell culture and molecular biology experiments. More importantly, our study would further clarify that the spleen from hypersplenism due to PH might promote development and progression of HCC and its molecular mechanisms. These findings could provide several new clues to elucidate the relationship between the spleen and the progression of HCC, and are also expected to open up a new avenue for clinical prevention and treatment of HCC.

门脉高压症(PH)脾亢脾与肝癌发生发展的关系日益受到重视。本课题组既往研究发现（国家自然科学基金资助,No.30471692）：PH脾亢脾组织中与肝癌发生发展相关的一些细胞因子表达上调。此外，PH脾亢患者血清明显促进肝癌细胞生长及形态改变，提示PH脾亢脾可促进肝癌发生，但具体机制尚不清楚。为此本项目拟通过：1.以PH脾亢患者脾切除前后血清为干预因素，观察肝癌细胞生长、增殖、上皮间质转化(EMT)及侵袭转移等指标的变化；2.建立人肝癌裸鼠原位种植瘤模型进一步验证上述体外实验结果；3.细胞因子芯片及生物信息学分析筛选出脾切除前后与肝癌发生发展高度相关的细胞因子2-3种，检测其在肝癌细胞生长、增殖、EMT及侵袭转移中的作用，继而利用阻断及RNA干扰等方法，进一步探讨PH脾亢脾促进肝癌发生发展的分子机制，为阐明脾脏与肝癌的关系提供直接的实验依据，亦有望为临床上肝癌的防治开辟一条新途径。

研究背景：.脾脏在肝癌的发生发展中起着重要作用，但目前尚缺乏脾脏影响肝癌发生发展的直接实验依据，确切的作用及机制也有待深入探讨。因此，本项目拟观察肝硬化门脉高压症脾亢脾对人肝癌细胞生长增殖的影响，进而明晰相关分子机制，旨在为阐明脾脏与肝癌进展的关系提供一些新线索，开辟一条从脾脏入手防治肝癌的新途径。.研究内容：.1. 收集肝硬化门脉高压症脾功能亢进行脾切除的患者术前、术后1w、术后2w外周血清。培养不同转移潜能的人肝癌细胞株，上述各组血清作用于不同时间点(24、48、72 h)后，观察各组细胞的形态变化， MTT法分析细胞生长情况；平板克隆实验观察并计算细胞克隆形成率；Brdu ELISA法测定细胞增殖; ELISA法测定IGF-II及HGF的含量；细胞免疫荧光法观PCNA及C-myc表达，分析细胞增殖情况；Real-time PCR及Western blot法检测癌基因（C-myc、N-ras）mRNA及蛋白表达；.2. 采用细胞因子芯片检测肝硬化门脉高压症脾亢患者脾切除前后外周血清细胞因子差异表达，结合生物信息学分析筛选出与肝癌生长增殖相关的细胞因子1-2种；.3. 收集肝硬化门脉高压症脾功能亢进行脾切除的患者术前、术后1w、术后2w外周血清, ELISA法检测IGF-II、IGF-1R及HGF含量，进一步验证芯片结果。.4. 采用IGF-1R阻断剂鬼臼苦素( PPP)及IGF-II siRNA干扰等方法，深入探讨肝硬化门脉高压症脾亢脾促进肝癌发生发展的分子机制。.研究结果：.1. 肝硬化门脉高压症脾亢患者外周血清可诱导人肝癌细胞(HepG2、SMMC-7721、MHCC97-H)生长增殖； .2. 肝硬化门脉高压症脾亢患者术前与术后两周外周血清中的细胞因子表达有差异，其中与肝癌发生发展相关的细胞因子如IGF-II等表达有所下调；.3. 肝硬化门脉高压症脾亢患者术前外周血清中IGF-II、IGF-1R、HGF的水平升高，而术后2 w外周血清中IGF-II、IGF-1R、HGF的水平降低；.4. 肝硬化门脉高压症脾亢患者术前外周血清可能部分通过IGF-1R促人肝癌细胞生长增殖。.科学意义：.揭示肝硬化门脉高压症脾亢脾可能通过IGF-II/IGF-1R信号通路促进肝癌进展，有望为临床上肝癌的防治开辟从脾脏入手的新方法和新思路。