卷曲乳杆菌S层蛋白SlpA抑制宫颈癌细胞增殖的作用与分子机制研究

Cervical cancer is one of the leading causes of cancer-related death in women. Lactobacillus crispatus can inhibit cervical cancer cell proliferation and affect the risk of HPV infection and carcinogenesis, although the precise mechanism remains to be elucidated. In our preliminary research, one probiotic L. crispatus strain has been isolated from the cervicovaginal secretions of healthy women of childbearing age. SlpA, the major component of L. crispatus S-layer, has been shown to suppress SiHa cell proliferation and mediate cell adhesion. .In this study, the SlpA protein will be expressed and purified to verify its role in cell adhesion by indirect immunofluorescence. Its influence factors and involved mechanism will be revealed by flow cytometry combined with site-directed mutagenesis. Besides, MTT, soft agar colony formation and EdU assays will be performed to confirm the inhibitory effect on cervical cancer cell proliferation. The inhibitory manner will be evaluated via detecting cell apoptosis, autophagy and cell cycle. At last, the inhibitory mechanism will be analyzed by RNA-Seq and verified by siRNA-mediated gene depletion..The results of this study will help to elucidate the inhibitory effect and mechanism of SlpA on cervical cancer cell proliferation, and reveal the potential protective role of L. crispatus in the development of cervical cancer. Our results may also help to provide basic information about new strategy for the prevention and treatment of cervical cancer, and has both theoretical significance and application prospect.

宫颈癌是严重威胁女性生命健康的恶性肿瘤。卷曲乳杆菌可抑制宫颈癌细胞增殖，影响HPV感染及宫颈癌患病风险，具体机制尚不明确。本项目组前期从健康育龄女性的宫颈-阴道分泌物中，分离筛选到一株卷曲乳杆菌益生菌株，发现其S-层蛋白SlpA可抑制宫颈癌细胞增殖并介导细胞黏附。现拟通过表达、纯化SlpA，使用间接免疫荧光和流式细胞术研究其对宫颈上皮细胞的黏附作用及影响因素，结合定点突变揭示黏附机制；采用MTT、软琼脂克隆形成和EdU实验证实其抑制宫颈癌细胞增殖的作用，通过检测细胞凋亡、自噬及细胞周期的变化探究其作用方式；运用RNA-Seq探寻相关的调控通路并进行验证。本项目的研究，不仅能够明确SlpA抑制宫颈癌细胞增殖的作用与分子机理，也有助于揭示卷曲乳杆菌在宫颈癌发生发展过程中潜在的保护机制，为宫颈癌的预防和治疗提供新的思路，为新药研究和临床应用提供理论基础，具有重要的理论意义和应用前景。

卷曲乳杆菌是女性下生殖道重要的益生菌，可降低高危型HPV感染及宫颈癌患病风险，但具体机制尚不明确。本项目分离筛选得到两株卷曲乳杆菌益生菌株Lc31和Lc83（CGMCC No. 21348和21349），黏附实验发现卷曲乳杆菌通过S 层蛋白 SlpA 结合角蛋白K10黏附宿主上皮细胞实现定植，具体互作区域为SlpA N-His片段与KRT10 541-661aa片段；细胞功能实验发现卷曲乳杆菌代谢产物等自身成分能抑制宫颈癌细胞的增殖和迁移，促进宫颈癌细胞凋亡和自噬；转录组测序发现卷曲乳杆菌刺激后宫颈癌细胞铁死亡通路被激活，关键指标MDA、GSH和ROS显著提高，提示铁死亡可能是介导卷曲乳杆菌抑制宫颈癌细胞的关键途径；本研究还发现卷曲乳杆菌黏附上皮细胞和抑制宫颈癌细胞的作用具有菌株和细胞特异性，提示在今后筛选预防治疗宫颈癌的益生菌时需要考虑到菌株水平的差异。本项目的研究结果不仅揭示了卷曲乳杆菌黏附上皮细胞、抑制宫颈癌细胞的作用和机制，也为宫颈癌的预防治疗及相关新药研究和临床应用提供了基础，具有重要的理论意义和应用前景。