常染色体显性多囊肾病中泛素连接酶β-TrCP的表达及其作用和调控机制

Expression,Function and Regulation of Ubiquitin Ligaseβ-TrCP in Autosomal Dominant Polycystic Kidney Disease

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 observe the expression of ubiquitin ligaseβ-TrCP in animal model and patients with autosomal dominant polycystic kidney disease（ADPKD）,and to preliminarily inves-tigate the function and regulatory mechanism ofβ-TrCP in human OX161 cells.Methods OX161 cells were transfected with β-TrCP siRNA.The proliferation of OX161 cells was measured by＆nbsp;MTT assay,and the expression of PCNA was detected by Western blot.Furthermore,OX161 cells were treated with JAK2/STAT3 inhibitor WP1006 or transfected with STAT3 expression plas-mids.The expression of JAK2,p-JAK2,STAT3,p-STAT3 andβ-TrCP was examined by Western blot.Moreover,kidney tissues and normal tissues 5 cm from cancer tissues of ADPKD patients undergoing nephrectomy and kidney tissues of male Han：SPRD（cy/＋）rats and Pkd1flox/flox：tamoxifen-Cre＋ mice and wild type Han：SPRD（＋/＋）rats and Pkd1flox/flox：tamoxifen-Cre-mice were collected to determine the expression and localization of β-TrCP.Results Compared with UCL93 cells,JAK2/STAT3 pathway was activated andβ-TrCP expression was increased in OX161 cells.The proliferation of OX161 cells was suppressed after knockdown of β-TrCP.The expression of β-TrCP decreased after treatment with WP1006,but increased after transfection with STAT3 plasmids.Compared with normal kidney tissues from ADPKD patients,kidney tis-sues from wild type Han：SPRD（＋/＋）rats and kidney tissues from Pkd1flox/flox：tamoxifen-Cre-mice,the expression of β-TrCP respectively increased in kidney tissues from ADPKD pa-tients,Han：SPRD（cy/＋）rats and Pkd1flox/flox：tamoxifen-Cre＋ mice.Conclusion The ex-pression ofβ-TrCP increases in human OX161 cells,as well as in kidney tissues from ADPKD pa-tients and animal models,suggesting thatβ-TrCP is involved in the occurrence and development of ADPKD.In addition,β-TrCP expression is regulated by JAK2/STAT3 pathway.