纳米细菌在泌尿系结石形成过程中的作用及其机制的研究

Etiology of urinary stones are complex.It is not clear now. In recent years, research on nanobacteria, for the elucidation of the pathogenesis of urinary stones, provide a new entry point. In the research,the kidney stones models of male wistar rats use nanobacteria were established. Kidney stone fomation,tubular injury were observed dynamically in different time,and the lateral pelvis urine was collected to culture and detect nanobacteria.Then removal of both kidneys to observed renal tubular epithelial cell morphology, ultrastructural changes from laser scanning confocal,check CaSR and Claudin-14 by west-blptting .Finally HK-2 cells randomly were divided into four groups: NB group, tetracycline interference group, negative control and positive control group,to study the renal tubular epithelial cell injury and effection of crystal remain by nanobacteria,check CaSR and Claudin-14 by west-blptting.Through the research, the understanding of the etiology of urinary stones can be deepened.It can benefit to looking for new clues and basis for further study of the pathogenesis of urinary stones.

泌尿系结石病因复杂，目前尚不明了。近年来，本课题组及其他学者对纳米细菌的研究，为泌尿系结石发病机制的阐明提供了新的切入点。本研究通过建立纳米细菌Wistar雄性大鼠肾结石模型，不同时段行Micro-CT检查，动态观察肾结石形成过程，同时，各时段收集肾盂尿，研究纳米细菌感染情况；然后处死大鼠，冰冻切片观察结石结晶数量、大小及分布，制作石蜡切片，用免疫电镜动态观察肾小管上皮细胞损伤情况，蛋白水平检测CaSR和Claudin-14蛋白表达；最后通过体外细胞培养，分别对纳米细菌组、四环素干扰组、阴性对照组、阳性对照组的HK-2细胞损伤、COM粘附及HK-2细胞形态进行观察，Western blotting检测CaSR和Claudin-14表达，研究纳米细菌对CaSR-Claudin-14调控机制、肾小管上皮细胞损伤及晶体滞留的影响。通过上述研究，探讨纳米细菌在泌尿系结石形成过程中的作用及其机制。

纳米细菌（nanobacteria, NB）是迄今为止发现的，唯一可在人体内产生羟基磷灰石的一种生物，NB所特有的生物矿化功能与多种钙化性疾病息息相关。本课题从个体、组织、细胞三个层次证实，NB系泌尿系结石这一典型性钙化性疾病的成因之一，在泌尿系结石形成发展过程中起到一定作用。.首先通过收集肾结石病人清洁中段晨尿，经分离、提取得到NB，经细菌培养制备纳米细菌悬液，测浓度纯，大鼠尾静脉注射，构建Wistar雄性大鼠肾结石模型；采集各组大鼠血、尿进行生化检测，利用Micro-CT动态观测各组大鼠成石情况，并对结石进行评分与成分分析，实验数据显示，NB组成石率与对照组差异有统计学意义，从而肯定了NB的成石效能；对NB组成石大鼠肾脏组织，运用Western-Blotting法、QRT-PCR法等试验方法，检测其肾脏组织内CaSR、Claudin-14的表达改变，与阳性对照组比较差异有统计学意义，揭示了NB通过激活CaSR-Claudin-14调控机制，进一步影响大鼠体内钙离子代谢；运用激光共聚焦等检测手段对肾小管上皮HK-2细胞NB感染情况进行观察，可见NB通过诱导脂质过氧化反应对HK-2细胞造成损伤，损伤程度随作用时间增加而增加，HK-2细胞损伤后对一水草酸钙（COM）晶体的粘附性增加，晶体黏附量与NB作用时间成正比。综上所述，本课题从三个层次证实，纳米细菌为肾结石成因之一，为肾结石病因学的研究提供了理论依据与数据支持。项目资助发表核心论文14篇，培养硕士研究生3名，其中3人均已获硕士学位，项目投入经费47万元，支出34.2129万元，各项支出基本与预算相符，结余12.7871万元，剩余经费计划用于本项目研究后续支出。