血清-蛋白质组学逆向识别肝癌微血管侵犯特异性生物标记物及功能鉴定

Microvascular invasion (MVI) is one of the major predictors of recurrence and metastasis following curative resection or liver transplantation for hepatocellular carcinoma (HCC). It was only identified on the postoperative specimens and occurred in more than 50% of HCC patients. Since it can not be identified preoperatively,appropriate choice of treatment strategies can be influenced, and leading to the waste of liver transplantation donors. Therefore, it is urgent to require solution preoperatively. Our preliminary result supports the existence of HCC antigens associated with MVI in light of specific antibodies detected in patients' serum. Thus, we will use the reversed sero-proteomics approach followed by in vivo micro vascular invasion assays to study the following aims including: 1) Screen the MVI associated HCC antigens by sero-proteomics; 2) Validate MVI associated antigens with sera from HCC patients with and without MVI and identify optimal serologic antibody responses as biomarkers for preoperative prediction of MVI; 3) Examine the expression and distribution of these putative HCC antigens in HCC tissues and the correlation of their expression patterns with the occurrence of MVI; 4) Investigate their functions in MVI by using shRNA interference and chicken embryo micro vascular invasion assays. The ultimate goals of this project are to identify serologic biomarkers for preoperative diagnosis of MVI and develop new vaccine and targeted therapies for HCC with MVI by targeting MVI associated antigens.

微血管侵犯（Microvascular Invasion, MVI）是肝癌复发转移的关键因素，术后检测其发生率超过50%。术前不能有效识别肝癌伴有MVI将影响治疗手段的选择，并造成肝移植供体的浪费。因此，术前识别亟待解决。我们前期研究已发现肝癌细胞存在血管侵犯相关抗原及血清中存在相应抗体。为此，我们首次采用逆向血清-蛋白质组学技术结合在体微血管侵袭实验对如下内容进行研究：（1）研究肝癌细胞在有无MVI时生物学特性变化，筛选可能与肝癌MVI相关抗原；（2）用有无合并MVI的肝癌患者血清认证MVI相关抗原，并鉴别血清中同MVI相关特异性最强的生物标记物；（3）检测临床肝癌组织标本中MVI相关抗原表达；（4）用shRNA基因沉默技术及在体鸡胚绒毛尿囊膜模型评估其在MVI过程中的功能。该项目将为术前诊断肝癌MVI确定血清生物标记物提供可能，并为将来针对肝癌MVI的特异性疫苗研究提供实验基础。

微血管侵犯（Microvascular Invasion, MVI）是肝癌复发转移的关键因素，术后检测其发生率超过50%。术前不能有效识别肝癌伴MVI将影响治疗手段的选择，并造成肝移植供体的浪费。我们前期研究发现，肝癌细胞存在与血管侵犯相关的抗原及血清中存在相应抗体。在本项目中，我们采用逆向血清-蛋白质组学技术，鉴别血清中同MVI相关的特异性最强的生物标记物，并用MVI阴性和阳性的肝癌患者血清来认证MVI抗原，检测肝癌组织标本中MVI抗原的表达水平，在体外实验中用MVI阴性和阳性肝癌病人的血清干预肝癌细胞后观察其侵袭能力的变化。我们已收集包含血清和肿瘤组织的肝癌标本200余份，并通过术后病理回顾区分为MVI阳性或阴性组；筛选出了5个与MVI阳性相关的肝癌抗原，其中alpha-enolase表达最强，21个与MVI阴性相关的肝癌抗原，其中heat shock protein 90-alpha，heat shock 70 kDa protein 4和annexin A2表达最强。肝癌细胞体外侵袭实验显示，抗α-enolase抗体对肝癌细胞的侵袭性具有显著的抑制作用。本项目的科学意义在于为术前诊断MVI确定血清生物标记物提供可能，并为将来针对MVI的特异性疫苗研究提供实验基础。