PSA糖链在血小板介导小细胞肺癌血行转移中的作用及分子机制初步研究

Hematogenous metastasis is one of the main reasons for the poor prognosis of small cell lung carcer( SCLC ). This study is based on literatures and the applicant's early study finds: platelets can mediate and then promote the hematogenous metastasis of lung cancer, high expression of polysialic acid (PSA) and polysialic acid transfer enzymes are in the presence in highly metastasis SCLC cells , and inhibition of PSA glycochain in surface of SCLC cells can obviously inhibit platelet and SCLC cell adhesion, so we hypothesize that the high expression of PSA glycochain in surface of SCLC cells may has an important role in the platelets mediated SCLC hematogenous metastasis, PSA enhance platelets interaction with SCLC cells and promote lung cancer cell 's invasion and metastasis ability, probably is one of the important mechanisms of platelets mediated SCLC hematogenous metastasis. This study intends to employ the RNA interference, cell transfection methods,and nude mice tumor metastasis model ,et al ,to establish a stable and high expression of PSA and a sustained inhibition of the expression of PSA SCLC cell lines, to study from two aspects of positive and negative the effect of PSA glycochain on metastasis ability of the SCLC cells and the nude mice metastasis tumors, and their probably signal pathway, in order to lays a preliminary foundation for further understanding the hematogenous metastasis mechanism of lung cancer and exploring new effective control targets .

血行转移是小细胞肺癌（SCLC）预后不佳的主要原因之一。本研究基于文献和申请者的前期发现：血小板能介导并促进肺癌的血行转移，高转移SCLC中存在多聚唾液酸 (PSA)及其转移酶的高表达，而抑制细胞表面PSA糖链后可明显抑制血小板与SCLC细胞的粘附，据此推测SCLC细胞表面PSA糖链高表达，应在血小板介导的SCLC血行转移中有重要作用，PSA增强血小板与SCLC细胞的粘附并继而促进肺癌细胞获得更强的侵袭和转移能力，很可能是血小板介导SCLC血行转移的重要机制之一。本项目拟通过RNA干扰、细胞转染、裸鼠转移瘤模型等方法，建立稳定高表达PSA和持续抑制其表达的SCLC细胞株，从正反两方面研究PSA糖链对上述细胞和裸鼠肿瘤转移能力的影响，并进一步研究血小板通过PSA促进SCLC血行转移可能的分子机制和信号通路，为深入明确肺癌血行转移的机理继而探索新的有效防治靶点奠定基础。

本项目调整后为一年期项目，计划研究任务和目标为：1、检测所筛选的小细胞肺癌细胞株（SCLC）多聚唾液酸（PSA）及神经细胞粘附分子（NCAM）的表达；2、检测SCLC细胞株两种唾液酸转移酶ST8SiaⅡ和ST8SiaⅣ的表达；3、在上述基础上，筛选一种唾液酸转移酶，建立稳定高表达PSA的SCLC肺癌细胞株和建立稳定下调表达PSA的SCLC肺癌细胞株；4、明确NCAM表面PSA糖链增强或抑制表达后，对于SCLC与血小板粘附作用的影响；5、进一步试验各组细胞粘附和转移侵袭能力的改变，及以信号通路为主线初步探索其潜在的机制。目前完成的情况包括：1、已培养小细胞肺癌细胞株NCI-H446，完成细胞PSA-NCAM检测；2、已完成H446细胞唾液酸转移酶ST8SiaⅡ和ST8SiaⅣ的表达检测，提示ST8SiaⅡ有良好的表达，并筛选用于进一步试验；3、采用慢病毒转染和ShRNA, 通过ST8SiaⅡ转移酶的作用，已构建高表达PSA的SCLC细胞株和下调表达PSA的SCLC细胞株，培养传代备用；4、已提取纯化富血小板悬液，检测各组细胞与血小板的粘附作用；5、已进行细胞粘附和侵袭检测试，并准备信号通路干预试验，完善进一步的试验研究。