肝癌细胞stefin B表达在肿瘤微环境中的作用及机制研究

Numerous studies have shown that high expression of stefin B in hepatocellular carcinoma (HCC) showed significant positive correlation with tumorigenesis, growth, recurrence and metastasis. The data suggested that there may have some other mechanisms of stefin B on tumor progression. However, little is known about the precise mechanism of stefin B function in HCC. In previous study, we examined the expression of stefin B and its association with MMP1 in stroma of HCC. We speculated that stefin B might have positive influence on the progression of HCC through involving fibroblasts function in tumor microenviroment. To confirm the hypothesis, the expression of stefin B will be measured in tumor and adjacent normal tissue of HCC, emphasizing on differential expression between stromal cells and tumor cells. Clinical correlation between expression of stefin B and pathological parameter will be evaluated. The effect of stefin B on normal normal fibroblasts (NFs) activation will be observed. Stefin B cDNA will be transfected into MHCC97H cells to establish the cell line showing stable stefin B expression (MHCC97H/stefin B). Three-dimensional (3D) co-cultrue model of MHCC97H/stefin B cells and NFs or carcinoma-associated fibroblasts (CAFs) will be constructed. The effects by stefin B on the behavior of NFs(CAFs), activity of MMPs-TIMP and secretion of cytokine will be approached the mechanisms. Model of co-cultrue of MHCC97H/stefin B cells and (NFs) CAFs in mixing gelatin including Fibxinogen, Thrombin and Aprotinin to investigate the morphous and chemotaxis of two kinds of cells. Spontaneous metastasis assay in nude mice by MHCC97H/stefin B cells and (NFs) CAFs will be investigated the effects of cross talk between two kinds of cells on cancer invasion and metastasis and underline the mechanisms. Our findings will provide the first evidence that the expression of stefin B in hepatoma carcinoma cells may play important parts in tumorigenesis, growth, recurrence and metastasis involvement function of stromal fibroblasts in tumor microenviroment. It will also implicate a causal mechanism defining how tumor stromal fibroblasts support neoplastic progression by manipulating the expression of stefin B in hepatoma carcinoma cells, and may contribute to anticancer research in HCC.

临床研究发现肝细胞癌(HCC)中stefin B高表达与肿瘤进展正相关，但确切机制尚未阐明。本课题组发现stefin B调控HCC间质MMP1表达。推测stefin B可能通过影响肿瘤微环境间质成纤维细胞的功能发挥对HCC的正向调控作用。为证实假设，本课题检测stefin B在临床HCC癌与间质中的差异表达，分析其与病理学特征的关系；观察stefin B在成纤维细胞(NFs)活化中的作用；通过共培养体系探讨肝癌细胞stefin B表达对NFs 和癌相关成纤维细胞(CAFs)功能的影响及其机制；观察过表达stefin B肝癌细胞与NFs和CAFs分别进行混合胶胶内共培养的细胞形态与趋化性；探讨过表达stefin B肝癌细胞与CAFs在裸鼠荷瘤体内交互串话及机制。研究结果将为探讨stefin B影响肿瘤进展机制和HCC复发与转移机制奠定基础，为从微环境途径寻找HCC防治新靶点提供新思路。

目的:肝细胞癌 (HCC) 中stefin B (CSTB) 的表达可能通过肿瘤微环境影响肿瘤的进展，但其作用与机制仍不明确。本课题通过临床、细胞及动物三个水平来探讨肝癌细胞sefin B表达对微环境间质成纤维细胞的作用及其机制。方法与结果：(1) 检测临床HCC癌与间质中stefin B表达，分析其与病理学特征的关系，结果表明stefin B在癌及间质中呈现高表达(P<0.05)，且与肿瘤大小、转移及Edmondson分级存在密切正相关 (均 P<0.001)，stefin B高表达是HCC转移的预测因子之一 (OR=17.324, P<0.001)；(2) 构建stefin B过表达慢病毒载体，获得LV-CSTB-MHCC97H肝癌细胞株及对照细胞株 (LV-NULL-MHCC97H)，分别与LX2共培养，探讨肝癌细胞stefin B表达对LX2功能的影响，结果表明LV-CSTB-MHCC97H条件培养液(CM) (25%、50%、75%) 及Transwell共培养 (12h、24h、48h) 均能促进LX2活化、α-SMA表达、迁移与增殖能力 (P<0.05)，MMP2、MMP9、PDGF、TGF-β (仅Transwell共培养) 的mRNA与蛋白表达增强 (P<0.05)，PDGF、HGF、TNFα分泌增强 (P<0.05)，MMP9活性增强 (P<0.05)，其中50%CM及48h的Transwell共培养组变化最明显；(3)分别用MHCC97H (1×107/只), LV-NULL-MHCC97H+LX2 (1×107+1×107/只，下同) 和LV-CSTB-MHCC97H+LX2细胞建立荷瘤模型，42天后观察原位瘤病理学变化，检测相关蛋白表达。结果表明LV-CSTB-MHCC97H+LX2组原位瘤增殖、LX2活化增强(P<0.05)及肺转移率增强，MMP2、MMP9、PDGF表达增强(P<0.05)。结论与意义：stefin B在癌及间质中高表达与肿瘤大小、转移及Edmondson分级紧密相关，且是转移的有效预测因子；肝癌细胞stefin B表达能够通过肿瘤微环境对HCC发挥正向调控作用，且可能与促进MMP2、MMP9酶活性相关。研究结果能为探讨stefin B影响HCC进展的机制提供理论依据，为从肿瘤微环境途径探寻HCC防治新思路与新靶点。