抑制糖酵解酶ENO1活性联合IL-28B下调调节性T细胞对宫颈癌的治疗作用研究

Aerobic glycolysis is one of important metabolic properties of cervical cancer cells. Meanwhile, immunosuppression induced by local regulatory T cells (Treg) infiltration and the increased peripheral Treg cells contribute to the development of cervical cancer. However, there are little effective agents to inhibit the aerobic glycolysis of tumor, and we do not know if there is any cooperative effect of inhibition of glycolysis and down-regulation of Tregs. This study is to investigate the cooperative therapeutic effect of inhibiting tumor cell metabolism and down-regulation of Tregs. In our previous works, enolase1 (ENO1) was found expressed highly in cervical cancer, and its expression decreased with effective TP (taxol and cisplatin) chemo-therapy. Silencing of ENO1 gene by SiRNA inhibited the proliferation and metastasis of cervical cancer cells, and enhanced the sensitivity to taxol and cisplatin-treatment. In addition, we investigated the immunotherapeutic effect of IL-28B against cervical cancer in U14 cervical cancer cells-bearing mice and found that Ad-mIL-28B treatment significantly decreased the number of regulatory T cells. Subsequently, there is a significant decrease in the size and the numbers of heteromorphic tumor cells. The tumor metastasis in lung and liver of the Ad-mIL-28B group also decreased. It suggests that IL-28B can inhibit the growth and metastasis of cervical cancer in U14 tumor-bearing mice by down-regulating the regulatory T cells. In this study, we are to investigate the methods to inhibit ENO1, and to investigate their therapeutic effect to cervical cancer and the cooperative effect together with IL-28B. First, ENO1 monoclonal antibodies and intracellular antibodies will be constructed, and the effect of them and SiRNA to inhibit ENO1 activity and tumor development are going to be evaluated in vitro and in vivo. Second, we will study the therapeutic effect of IL-28B for cervical cancer in detail. Third, the cooperative effect of ENO1 antibodies and IL-28B is to be analyzed. This study will provide useful information on the treatment of cervical cancer.

晚期或复发宫颈癌的传统治疗效果不佳，亟需研究新型治疗方法。研究表明有氧糖酵解和免疫抑制分别是宫颈癌形成的重要代谢特性和外部条件，后者主要由调节性T细胞（Treg）升高引起。然而，目前针对糖酵解和Treg的治疗措施还很少，并鲜有对它们联合治疗作用的报道。本课题拟从干预肿瘤代谢和调节肿瘤免疫内外两个方面，研究对宫颈癌的综合治疗措施。我们前期研究发现宫颈癌高表达糖酵解限速酶烯醇化酶1（ENO1）；将ENO1基因沉默能明显降低宫颈癌细胞成瘤性和侵袭转移能力，增强化疗的敏感性。我们亦发现IL-28B可有效下调宫颈癌荷瘤小鼠Treg细胞，并显著缩小瘤体积。本课题拟进一步制备ENO1单克隆抗体和胞内抗体，比较它们和RNA干扰对ENO1活性的抑制效果，以及对宫颈癌的治疗作用；进而将抑制ENO1活性的制剂和IL-28B联合，研究抑制糖酵解和下调Treg的协同治疗作用。本课题可为宫颈癌治疗提供新的思路和依据。

本课题拟制备ENO1单克隆抗体（ENO1mAb），抑制肿瘤糖酵解；同时应用IL-28B下调调节性T细胞（Treg），增强抗肿瘤免疫，研究二者的协同抗肿瘤作用。我们按计划书完成了主要工作，研究了IL-28B与糖酵解酶抑制剂的协同抗肿瘤作用，制备了具有较高抗肿瘤活性的ENO1mAb，进而设计纳米颗粒及病毒载体，使ENO1mAb进入宫颈癌细胞发挥作用。主要研究内容及结果如下：（1）IL-28B与糖酵解酶抑制剂的协同抗肿瘤作用。我们构建了小鼠U14宫颈癌皮下移植瘤模型，发现应用腺病毒介导的IL-28B下调Treg，具有抗肿瘤作用；筛选获得具有较强的抗肿瘤作用的糖酵解酶抑制剂三溴丙酮酸（3-BrPA），发现IL-28B联合3-BrPA具有协同抑制小鼠移植瘤的作用。（2）ENO1mAb的制备及抗肿瘤作用研究。应用杆状病毒表达载体，表达纯化出ENO1蛋白，通过杂交瘤技术制备了5个ENO1mAb-H1-H5。通过检测抗体对肿瘤侵袭迁移的抑制作用，筛选出具有较好抗肿瘤活性的ENO1mAb-H1和ENO1mAb-H5。进而制备PLGA及介孔二氧化硅（MSN）纳米颗粒，经叶酸及透明质酸修饰，靶向介导ENO1mAb进入肿瘤细胞。初步研究显示ENO1mAb-H1和ENO1mAb-H5可以抑制宫颈癌Hela细胞糖酵解、抑制肿瘤细胞增殖、降低肿瘤成瘤性。（3）制备重组ENO1mAb可变区基因的腺相关病毒（rAAV-H1-H5）。从杂交瘤细胞的mRNA上扩增编码ENO1mAb重链和轻链可变区基因，制备rAAV-H1-H5，在宫颈癌细胞内表达ENO1mAb可变区。初步研究表明ENO1mAb-H1-H5具有一定的抗肿瘤作用。（4）IL-28B蛋白制备研究。将IL-28B基因构建在表达载体上，得到Ppic9k-his-IL-28B，转化入毕赤酵母表达载体，虽经反复优化表达条件，但蛋白表达量过低，未能纯化获得足够蛋白进行后续研究。（5）干扰丙酮酸激酶（PKM2）对宫颈癌的治疗作用研究。我们应用RNA干扰技术沉默PKM2基因，对宫颈癌也具有一定的治疗作用。