Maspin抑制膀胱癌转移和提高化疗疗效的作用功能及其机制研究

The main reason for the difficult treatment of advanced bladder cancer is the tumor metastasis and resistance to chemotherapy. Solving these two problems will bring a great benefit in the treatment of bladder cancer. The applicant's pre-national natural science foundation of china(30700832) showed that the expression of Maspin had the significant difference in the lymph node metastasis, the primary cancer tumor and the normal bladder tissue. Also,other researches showed that Maspin could control the cancer cell invasion and metastasis and induce the apoptosis. So we proposed that it could inhibit the lymph node metastasis and improve the efficacy of chemotherapy for the bladder cancer by enhancing the expression of Maspin. This subject intends to start with the cellular and animal models to explore if increasing Maspin expression could reduce the ability of invasion and metastasis of bladder cancer and improve the efficacy of chemotherapy.Then,we use the technology of tandem affinity purification(TAP) and targeted proteomics to isolate and identificate the interacting protein of Maspin, and to explore the network characteristics of these Protein interaction. In the last part,we use the research of clinical retrospective study to explore the value of Mapin in the diagnosis and prognosis of bladder cancer. We hope to find the mechanism of Maspin in bladder cancer progression and find a new target to suppress the invasion and metastasis of bladder cancer and improve the efficacy of chemotherapy.

晚期膀胱癌因肿瘤远处转移、化疗耐药，往往治疗比较棘手。如果同时解决这两个问题，则膀胱癌的治愈率明显提高。申请人前国家自然科学基金课题（30700832）研究显示Maspin在膀胱癌淋巴结转移灶、原发灶及正常膀胱组织中的表达显著差异；而目前研究也提示Maspin可调控癌细胞浸润转移和诱导凋亡。那么，我们设想：上调Maspin表达是否可以同时抑制膀胱癌淋巴道转移与提高化疗疗效呢？本课题拟先从分子细胞水平、动物模型层面，探讨Maspin的表达上调是否可达到同时降低膀胱癌的浸润转移能力和提高化疗疗效的目的；再以串联亲和纯化耦联质谱技术为主的靶向蛋白质组学等技术分离、鉴定Maspin的相互作用蛋白，探索这些蛋白相互作用时动态分子网络特征；最后临床样本回顾性研究，分析Maspin在膀胱癌诊断与预后中的价值。结果有望揭示Maspin在膀胱癌进展中的作用机制，为膀胱癌转移抑制、化疗增敏提供分子靶标。

Maspin蛋白是丝氨酸抑制蛋白超级家族的重要成员之一。在多种肿瘤中发挥抑癌基因的作用。本项目通过建立Maspin过表达细胞系，采用了MTT和克隆形成实验检测Maspin对膀胱癌细胞T24、5637增值能力和对顺铂的化疗敏感性。通过流式细胞仪检测Maspin是否是通过细胞凋亡机制发挥抑制膀胱癌细胞增殖的作用。通过western blots检测AKT, phospho(p)-AKT, PI3K, mTOR, Caspase3 和 Bcl-2的蛋白表达量，研究Maspin增加膀胱癌细胞对顺铂的化疗敏感性的可能机制。在临床样本方面，我们采用免疫组织化学染色的方法检测72例膀胱癌组织和12例正常膀胱黏膜组织的Maspin表达；分析72例膀胱癌患者术前盆腔CT扫描图片以及术后患者淋巴结标本的病理资料，计算诊断标准的敏感性、特异性、阳性预测值、阴性预测值，比较分析Maspin检测、螺旋CT在术前诊断膀胱癌是否有淋巴结转移方面的诊断效果。以上结果显示Maspin在诊断膀胱癌患者淋巴结转移中是一个有价值的肿瘤生物标志物，并且是研究膀胱癌化疗耐药性的一个重要潜在靶点。