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目的观察常染色体显性多囊肾病(ADPKD)患者及动物模型中泛素连接酶β-TrCP的表达,初步探讨人囊肿衬里上皮OX161细胞中β-TrCP的作用和调控机制。方法在OX161细胞内转染β-TrCP的siRNA,采用蛋白免疫印迹法检测增殖细胞核抗原(PCNA)的表达,MTT比色法检测吸光度;在OX161细胞中抑制JAK2/STAT3通路或转染STAT3质粒,采用蛋白免疫印迹法检测JAK2、p-JAK2、STAT3、p-STAT3和β-TrCP的表达。收集ADPKD行肾脏切除术患者的肾脏组织和肾旁距癌组织5 cm以上的正常肾脏组织(正常对照组)、雄性Han:SPRD(cy/+)大鼠(ADPKD模型)和野生型Han:SPRD(+/+)大鼠肾组织(对照),均采用蛋白免疫印迹法检测β-TrCP的表达。收集Pkdlflox/flox;tamoxifen-Cre+小鼠(ADPKD模型)和Pkdlflox/flox;tamoxifen-Cre-小鼠(对照)肾组织,采用免疫组织化学法检测β-TrCP的表达和亚定位。结果与UCL93细胞相比,OX161细胞中JAK2/STAT3通路明显活化,β-TrCP表达量增加;β-TrCP敲减后细胞增殖减慢(P<0.05);WP1006抑制JAK2/STAT 3通路后,β-TrCP表达量减低,转染STAT 3质粒后,β-TrCP的表达量增加。与正常对照组、野生型Han:SPRD(+/+)大鼠及Pkdlflox/flox;tamoxifen-Cre-小鼠比较,ADPKD患者肾组织、Han:SPRD(Cy/+)大鼠肾组织及Pkdlflox/flox;tamoxifen-Cre+小鼠肾组织中β-TrCP表达量明显增加。结论在人囊肿衬里上皮OX161细胞株中β-TrCP表达量增多,且受JAK2/STAT3通路调控;在ADPKD患者及其模型鼠肾脏组织中β-TrCP表达量也增加。β-TrCP可能参与促进囊肿的发生和发展。
Objective To investigate the expression of β-TrCP in patients with autosomal dominant polycystic kidney disease (ADPKD) and animal models and to explore the role and regulation of β-TrCP in human cyst-lined epithelial OX161 cells. Methods The siRNA of β-TrCP was transfected into OX161 cells and the expression of proliferating cell nuclear antigen (PCNA) was detected by Western blotting. The absorbance was measured by MTT colorimetric assay. The JAK2 / STAT3 pathway was inhibited or the STAT3 plasmid was transfected into OX161 cells. The expression of JAK2, p-JAK2, STAT3, p-STAT3 and β-TrCP were detected by Western blotting. The normal kidney tissues (normal control group), male Han: SPRD (cy / +) rats (ADPKD model) and wild-type Han: SPRD (+ / +) Rat kidney tissue (control), were detected by Western blot β-TrCP expression. Pkdlflox / flox; tamoxifen-Cre + mice (ADPKD model) and Pkdlflox / flox; tamoxifen-Cre-mice (control) kidney tissue were harvested and the expression and sub-localization of β-TrCP was detected by immunohistochemistry. Results Compared with UCL93 cells, JAK2 / STAT3 pathway was significantly activated in OX161 cells and the expression of β-TrCP was increased. The cell proliferation was decreased after knockdown of β-TrCP (P <0.05). After WP1006 inhibited JAK2 / STAT3 pathway, -TrCP expression decreased, after transfected STAT3 plasmid, the expression of β-TrCP increased. Compared with normal control group, wild-type Han: SPRD (+ / +) rats and Pkdlflox / flox; tamoxifen-Cre-mice, renal tissue of ADPKD patients, flox; tamoxifen-Cre + mouse kidney tissue β-TrCP expression increased significantly. Conclusion The expression of β-TrCP in human cyst-lined epithelial OX161 cell line is increased and regulated by JAK2 / STAT3 pathway. The expression of β-TrCP in ADPKD patients and its model kidneys also increases. β-TrCP may be involved in promoting the occurrence and development of cysts.