【作者】 刘松；

【导师】 姚丽君；

【作者基本信息】 华中科技大学 ， 内科学（肾脏病学）， 2016， 硕士

【摘要】 目的:探究SGK3以及Nedd4-2对OK细胞中NPT2a分布及活性的影响,初步探讨血清糖皮质激素诱导激酶3（SGK3）、Nedd4-2在PTH介导的OK细胞中NPT2a的分布及活性变化中的作用。方法:选用肾脏近曲小管的细胞培养模型（OK细胞）作为研究对象,分别用SGK3灭活型质粒SGK3 K191M、Nedd4-2野生型质粒瞬时转染OK细胞,提取OK细胞膜成分,用免疫印迹法检测膜上NPT2a表达;提取OK细胞总蛋白,用免疫印迹法检测SGK3、Nedd4-2及总蛋白中NPT2a的表达,用放射免疫技术检测OK细胞对³²Pi的摄取,评价NPT2a活性。并用浓度100n M的重组人甲状旁腺素（1-34）即PTH（1-34）刺激OK细胞0、30min、2h,通过测定OK细胞对放射性³²Pi的摄取检测磷的转运,评价NPT2a活性;提取OK细胞膜成分用免疫印迹法检测膜上NPT2a表达,提取OK细胞总蛋白用免疫印迹法检测NPT2a的表达变化及SGK3、Nedd4-2磷酸化水平。结果:1)SGK3灭活型质粒SGK3 K191M、Nedd4-2野生型质粒瞬时转染OK细胞后,OK细胞膜上NPT2a表达减少,细胞总蛋白中NPT2a表达未见明显变化,OK细胞对³²Pi的摄取减少;2)PTH刺激后,NPT2a在OK细胞膜上的表达减少,细胞总蛋白中NPT2a表达未见明显变化,OK细胞对³²Pi的摄取减少,SGK3、Nedd4-2磷酸化水平降低。结论:SGK3以及Nedd4-2参与调节OK细胞中NPT2a的分布及活性;SGK3、Nedd4-2可能参与调节PTH介导的OK细胞中NPT2a的分布及活性变化。更多还原

【Abstract】 Purpose: To investigate whether SGK3 and Nedd4-2 modulate the distribution and activity of NPT2 a in OK cells, and to preliminary study the potential roles of SGK3 and Nedd4-2 in PTH induced NPT2 a relocation and activity in OK cells.Method: Constitutively inactived SGK3 K191 M and wild-type Nedd4-2 WT plasmids were transfected into OK cells respectively, the transfection efficiency and expression level of NPT2 a in the membrane and the whole cell lysate were evaluated by western blot analysis,and the ³²Pi uptake were measured by radioimmunoassay to determine NPT2 a activity. Furthermore, OK cells were treated with 100 n M PTH for 30 min or 2h, NPT2 a activity was determined by ³²Pi- radiolabeled phosphate uptake into OK cells. The membranous expression of NPT2 a, the phosphorylation level of SGK3 and Nedd4-2 of the whole cell lysate were detected by Western blot analysis.Results: 1) After being transfected with SGK3 K191 M and Nedd4-2 WT plasmids respectively, the membranous expression of NPT2 a and the ³²Pi uptake was decreased significantly compared with control cells, while the expression level of NPT2 a in the whole cell lysate was no difference between the transfected group and the control group. 2) PTH significantly decreased NPT2 a membrane expression after 30 min and 2h treatment, which was coupled with decreased ³²Pi uptake. However, the expression level of NPT2 a in the whole cell lysate didn’t show significantly change. Acute PTH stimulation decreased the phosphorylation level of SGK3 and Nedd4-2 in the total protein.Conclusion: It is concluded that SGK3 and Nedd4-2 modulate the distribution and activity of NPT2 a, SGK3 and Nedd4-2 may play a role in PTH induced NPT2 a relocation and activity in OK cells.更多还原