PDE5抑制剂通过经典Wnt信号降低骨量的研究

发布时间：2018-07-06 15:38

本文选题：Wnt信号通路 + PDE5抑制剂　；参考：《浙江大学》2014年硕士论文

【摘要】：实验目的：Wnt信号通路包括经典途径和非经典途径。研究表明,经典Wnt信号不仅在骨发育与骨形成过程中发挥重要作用,对于骨量的维持也必不可少。磷酸二酯酶5抑制剂(Phosphodiesterase-5inhibitor, PDE5i)目前在临床上用于治疗勃起功能障碍(Erectile dysfunction, ED)。已有文献报道磷酸二酯酶5(Phosphodiesterase-5, PDE5)参与调控非经典Wnt信号通路,但是PDE5抑制剂对于Wnt信号所调控的成骨细胞分化及骨量的维持是否有影响尚未见报道。本课题通过研究PDE5抑制剂他达拉非对经典Wnt信号的作用,探讨PDE5抑制剂对骨量的影响。实验方法：HEK293细胞在L或Wnt3a条件性培养基刺激下,给予不同浓度的他达拉非(0,1,5,10μM)检测淋巴增强因子1(Lymphoid enhancer factor-1,Lefl)荧光素酶报告基因的活性。PDE5抑制剂作用使cGMP胞内水平上升并进一步激活PKG,通过使用不同浓度的PKG抑制剂KT5823(0,0.2,1,5μM)及转染PKG不同亚型(PKG1和PKG2) siRNA,检测Lefl荧光素酶报告基因的活性。经典Wnt信号开放时引起β-catenin在细胞质内的稳定并进入细胞核。分别用L或Wnt3a条件性培养基刺激HEK293细胞,并对细胞核与细胞质蛋白进行分离,通过Western Blot检测他达拉非(10μM)对于β-catenin蛋白水平及分布的影响。同时通过定量PCR检测他达拉非对β-catenin mRNA水平的影响。为了研究他达拉非对成骨细胞分化的影响,测定他达拉非作用下C3H10T1／2细胞碱性磷酸酶(Alkaline phosphatase, AP)活性的变化,并通过定量PCR检测成骨细胞标志基因AP、Runt相关转录因子2(Runt-related transcription factor2, Runx2)及Osterix (OSX) mRNA水平的变化。最后通过茜素红染色研究他达拉非对细胞形成矿物化结节能力的影响。动物实验方面,选用C57雌性小鼠,将小鼠随机分为四组：对照组(给予生理盐水)、他达拉非低(Tadalafil,3mg/kg)、中(Tadalafi1,15mg/kg).高(Tadalafil,45mg/kg)剂量组。小鼠每天灌胃给药一次,给药8周。在给药前、给药4周及给药结束时分别测定小鼠的股骨远端骨密度。随后处死小鼠,取小鼠的一侧股骨做组织切片,进行HE染色。分离小鼠另一侧股骨中的骨髓间充质干细胞(Bonemarrow derived mesenchymal stem cells, BMSC)进行培养。通过定量PCR检测成骨细胞的标志基因AP、骨钙蛋白(Osteocalcin, OC)及Wnt信号靶基因Dickkopf-1(Dkkl)和Lefl的mRNA水平。并通过茜素红染色检测BMSC形成矿物化结节的能力。实验结果：研究发现,给予他达拉非显著降低Wnt3a诱导的Lefl荧光素酶报告基因的活性。给予他达拉非对β-catenin mRNA水平没有影响,但β-catenin的蛋白水平下调,且细胞核和细胞质的蛋白水平都有所下降。使用PKG抑制剂KT5823增加Wnt3a诱导的Lefl荧光素酶活性,并且敲降PKG2显著增加Wnt3a诱导的Lefl荧光素酶报告基因活性。在C3H10T1/2细胞中,给予他达拉非抑制Wnt3a诱导的成骨标志基因AP、Runx2及OSX mRNA水平,细胞形成矿物化结节的能力降低。动物实验中,他达拉非给药组小鼠股骨远端骨密度显著降低,切片HE染色结果显示股骨骨小梁数量体积明显减少。与对照组相比,他达拉非给药组分离出的BMSC中,AP与OC及Wnt通路靶基因Dkkl与Lefl的表达下降,形成矿物化结节的能力也显著降低。实验结论：PDE5抑制剂下调β-catenin蛋白水平,降低Wnt信号通路靶基因Dkkl及Lefl的表达,负性调控Wnt/β-catenin信号通路。PDE5抑制剂通过经典Wnt信号通路调控成骨细胞分化,并具有减少骨量的作用。本研究首次报道使用PDE5抑制剂他达拉非降低骨量,提示长期使用他达拉非可能导致骨量丢失,为他达拉非的临床应用提供了预警。
[Abstract]:It is shown that the canonical Wnt signaling plays an important role in bone formation and bone formation and is essential for the maintenance of bone mass . The phosphodiesterase 5 inhibitor is currently used clinically in the treatment of erectile dysfunction ( ED ) . It has been reported that phosphodiesterase 5 is involved in the regulation of non - classical Wnt signaling pathways , but it has not been reported as to whether the differentiation of osteoblast and the maintenance of bone mass regulated by the Wnt signaling have not been reported .

In order to study the effect of tadalafil ( 10 渭M ) on the level and distribution of 尾 - catenin protein , the effect of tadalafil ( 10 渭M ) on the level and distribution of 尾 - catenin was detected by means of quantitative PCR . The effects of tadalafil ( 10 渭M ) on the level and distribution of 尾 - catenin were determined by quantitative PCR .

In animal experiment , C57 female mice were randomly divided into four groups : control group ( normal saline ) , tadalafil ( 3 mg / kg ) , medium ( Tadalafi1 , 15 mg / kg ) , and high ( Tadashi , 45 mg / kg ) dose group .

The results showed that the level of 尾 - catenin was not significantly reduced by the administration of tadalafil significantly , but the protein level of 尾 - catenin was downregulated , but the protein level of 尾 - catenin was downregulated . The activity of Wnt3a - induced luciferase reporter gene was increased significantly .

Compared with the control group , the expression of Dkkl and the Wnt pathway target genes Dkkl and Lefl decreased in the BMSC isolated from the tadalafil administration group , and the ability to form the mineralized nodules was also significantly reduced .

Conclusion : The downregulation of 尾 - catenin protein level and decreasing the expression of Wnt signaling pathway target gene Dkkl and Lefl , and negatively regulate Wnt / 尾 - catenin signaling pathway .
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

【共引文献】