基于Wnt/β-catenin信号通路调控Foxn1研究先天肾虚影响胸腺发育的分子机制

According to traditional Chinese medicine (TCM) theory, kidney stores essence, and then refines sublimation into power. Kidney deficiency causes lack of kidney essence. Congenital deficiency of kidney essence causes neonatal developmental disorders, deformities or dysplasia. Wnt/beta -catenin signaling pathway plays an important role in regulating the proliferation, differentiation and apoptosis of thymus cells. Foxn1 is the key factor for the migration, differentiation and maturation of thymic epithelial cells (TECs) during thymic development. Based on previous study, we found many pathological changes showing in the thymus of congenital kidney deficiency rats, such as cortical thinning, medullary enlargement, and a large number of epithelial cells exhibited karyopyknosis. The research team speculated that Wnt/β-catenin signaling pathway promoted Foxn1 expression, which primarily causes dysplasia of thymic epithelial cell. We take the Foxn1 protein expression as the breakthrough point, and establish kidney deficiency of pregnant animal by using the method of which compound horroring stresses stimulate pregnant rats. The potential mechanism regards thymic development of congenital kidney deficiency rats and regulation of kidney were discussed by the dynamic observation of embryonic regarding the protein expression of Wnt, β-catenin, Foxn1 with using the Wnt inhibitor and tonifying kidney essence. Meanwhile, the changes of markers on the surface of thymic epithelial cells and the development of T cells was observed, and the underlying mechanism of thymic development and kidney regulation of thymic development in the congenital kidney rats were clarified. Thus, the result might enrich the theory of TCM about the kidney dominates growth and development.

肾藏精，精化气，肾虚则精亏，气不足。先天之精无以充养而亏虚，可致日后生长发育不良、障碍甚至畸形。在胸腺的发育期，Wnt/β-catenin信号通路在调控胸腺细胞增殖、分化、凋亡过程中发挥重要作用，其下游的Foxn1基因是影响胸腺上皮细胞迁移、分化和成熟的关键因子。基于前期研究发现先天肾虚仔鼠的胸腺出现皮质变薄，髓质区扩大，胸腺上皮细胞出现大量核固缩等病理变化，课题组推测Wnt/β-catenin信号通路调节Foxn1是胸腺发育异常的关键环节。本研究拟以Foxn1为切入点，复制先天肾虚模型，并给予Wnt抑制剂和补肾填精方左归丸干预，采用动态观察胚胎期仔鼠Wnt4、β-catenin、Foxn1等蛋白的表达，结合胸腺上皮细胞表面标志蛋白的变化特点和T细胞的发育程度，进一步探索先天肾虚仔鼠胸腺发育异常及肾调控胸腺发育的可能机制，充实中医肾主生长发育的理论。

《灵枢·天年》云：“以母为基,以父为楯。”明代医家万全在《幼科发挥》中提到：“父母强者，生子亦强，父母弱者，生子亦弱。”提示母代的强弱会影响到子代的强弱。课题组前期研究发现先天肾虚仔鼠的胸腺出现皮质变薄，髓质区扩大，胸腺上皮细胞出现大量核固缩等病理变化，故本项目以胸腺发育为切入点，围绕假说开展实验研究。.选用SFP级SD大鼠雌雄配对，每日以恐吓为主的复合应激法刺激孕鼠，以孕鼠和仔鼠的一般状态、孕鼠胎产数、孕鼠尿17羟皮质类固醇的含量确定孕鼠的肾虚模型造模成功。以仔鼠出现畸形、体重小，身长短、皮肤皱缩、颜色暗红、活动度较差等一般表现，结合甲状腺激素FT3、FT4水平确定了仔鼠肾虚造模成功。.为了研究肾虚仔鼠胸腺的发育情况，课题组选用HE和电镜观察胸腺组织的形态和超微结构；选用流式细胞学技术检测仔鼠胸腺和脾脏内T细胞的分型；运用RT-PCR和Western Blot技术检测Wnt4、β-catenin、Foxn1基因和蛋白在仔鼠胸腺组织内的表达。从胸腺组织中培养SD仔鼠胸腺上皮细胞，通过广谱细胞角蛋白免疫荧光染色进行细胞类型的鉴定；CCK-8法测定左归丸含药血清和Wnt4抑制剂对细胞增值率的影响，Western Blot检测不同ICG-001浓度的抑制效果，两者结合判断左归丸含药血清和ICG-001的最佳实验浓度。将分离培养的TECs分为：对照组、补肾组、Wnt抑制剂对照组、Wnt抑制补肾组。处理24小时后，运用RT-PCR和Western Blot技术检测Wnt4、β-catenin、Foxn1基因和蛋白的表达，运用Elisa技术检测胸腺素β4与胸腺素α1的含量。.本研究成功复制了先天肾虚模型，通过动物实验和胸腺上皮细胞实验，明确了胸腺内Wnt/β-catenin信号通路表达异常，是先天肾虚影响T细胞分化发育和迁徙的机制。补肾填精法能够通过激活胸腺内Wnt/β-catenin信号通路，增加Foxn1的表达，促进TECs分泌胸腺肽，调控T细胞分化发育和迁徙，改善先天肾虚免疫紊乱状态。为临床免疫系统发育异常从肾论治提供科学依据，对于提高整个国民素质有着重要意义。