發(fā)布時間：2018-07-06 15:38

  本文選題：Wnt信號通路 + PDE5抑制劑　； 參考：《浙江大學(xué)》2014年碩士論文

【摘要】：實驗?zāi)康模篧nt信號通路包括經(jīng)典途徑和非經(jīng)典途徑。研究表明,經(jīng)典Wnt信號不僅在骨發(fā)育與骨形成過程中發(fā)揮重要作用,對于骨量的維持也必不可少。磷酸二酯酶5抑制劑(Phosphodiesterase-5inhibitor, PDE5i)目前在臨床上用于治療勃起功能障礙(Erectile dysfunction, ED)。已有文獻(xiàn)報道磷酸二酯酶5(Phosphodiesterase-5, PDE5)參與調(diào)控非經(jīng)典Wnt信號通路,但是PDE5抑制劑對于Wnt信號所調(diào)控的成骨細(xì)胞分化及骨量的維持是否有影響尚未見報道。本課題通過研究PDE5抑制劑他達(dá)拉非對經(jīng)典Wnt信號的作用,探討PDE5抑制劑對骨量的影響。 實驗方法：HEK293細(xì)胞在L或Wnt3a條件性培養(yǎng)基刺激下,給予不同濃度的他達(dá)拉非(0,1,5,10μM)檢測淋巴增強(qiáng)因子1(Lymphoid enhancer factor-1,Lefl)熒光素酶報告基因的活性。PDE5抑制劑作用使cGMP胞內(nèi)水平上升并進(jìn)一步激活PKG,通過使用不同濃度的PKG抑制劑KT5823(0,0.2,1,5μM)及轉(zhuǎn)染PKG不同亞型(PKG1和PKG2) siRNA,檢測Lefl熒光素酶報告基因的活性。經(jīng)典Wnt信號開放時引起β-catenin在細(xì)胞質(zhì)內(nèi)的穩(wěn)定并進(jìn)入細(xì)胞核。分別用L或Wnt3a條件性培養(yǎng)基刺激HEK293細(xì)胞,并對細(xì)胞核與細(xì)胞質(zhì)蛋白進(jìn)行分離,通過Western Blot檢測他達(dá)拉非(10μM)對于β-catenin蛋白水平及分布的影響。同時通過定量PCR檢測他達(dá)拉非對β-catenin mRNA水平的影響。為了研究他達(dá)拉非對成骨細(xì)胞分化的影響,測定他達(dá)拉非作用下C3H10T1／2細(xì)胞堿性磷酸酶(Alkaline phosphatase, AP)活性的變化,并通過定量PCR檢測成骨細(xì)胞標(biāo)志基因AP、Runt相關(guān)轉(zhuǎn)錄因子2(Runt-related transcription factor2, Runx2)及Osterix (OSX) mRNA水平的變化。最后通過茜素紅染色研究他達(dá)拉非對細(xì)胞形成礦物化結(jié)節(jié)能力的影響。 動物實驗方面,選用C57雌性小鼠,將小鼠隨機(jī)分為四組：對照組(給予生理鹽水)、他達(dá)拉非低(Tadalafil,3mg/kg)、中(Tadalafi1,15mg/kg).高(Tadalafil,45mg/kg)劑量組。小鼠每天灌胃給藥一次,給藥8周。在給藥前、給藥4周及給藥結(jié)束時分別測定小鼠的股骨遠(yuǎn)端骨密度。隨后處死小鼠,取小鼠的一側(cè)股骨做組織切片,進(jìn)行HE染色。分離小鼠另一側(cè)股骨中的骨髓間充質(zhì)干細(xì)胞(Bonemarrow derived mesenchymal stem cells, BMSC)進(jìn)行培養(yǎng)。通過定量PCR檢測成骨細(xì)胞的標(biāo)志基因AP、骨鈣蛋白(Osteocalcin, OC)及Wnt信號靶基因Dickkopf-1(Dkkl)和Lefl的mRNA水平。并通過茜素紅染色檢測BMSC形成礦物化結(jié)節(jié)的能力。 實驗結(jié)果：研究發(fā)現(xiàn),給予他達(dá)拉非顯著降低Wnt3a誘導(dǎo)的Lefl熒光素酶報告基因的活性。給予他達(dá)拉非對β-catenin mRNA水平?jīng)]有影響,但β-catenin的蛋白水平下調(diào),且細(xì)胞核和細(xì)胞質(zhì)的蛋白水平都有所下降。使用PKG抑制劑KT5823增加Wnt3a誘導(dǎo)的Lefl熒光素酶活性,并且敲降PKG2顯著增加Wnt3a誘導(dǎo)的Lefl熒光素酶報告基因活性。在C3H10T1/2細(xì)胞中,給予他達(dá)拉非抑制Wnt3a誘導(dǎo)的成骨標(biāo)志基因AP、Runx2及OSX mRNA水平,細(xì)胞形成礦物化結(jié)節(jié)的能力降低。 動物實驗中,他達(dá)拉非給藥組小鼠股骨遠(yuǎn)端骨密度顯著降低,切片HE染色結(jié)果顯示股骨骨小梁數(shù)量體積明顯減少。與對照組相比,他達(dá)拉非給藥組分離出的BMSC中,AP與OC及Wnt通路靶基因Dkkl與Lefl的表達(dá)下降,形成礦物化結(jié)節(jié)的能力也顯著降低。 實驗結(jié)論：PDE5抑制劑下調(diào)β-catenin蛋白水平,降低Wnt信號通路靶基因Dkkl及Lefl的表達(dá),負(fù)性調(diào)控Wnt/β-catenin信號通路。PDE5抑制劑通過經(jīng)典Wnt信號通路調(diào)控成骨細(xì)胞分化,并具有減少骨量的作用。本研究首次報道使用PDE5抑制劑他達(dá)拉非降低骨量,提示長期使用他達(dá)拉非可能導(dǎo)致骨量丟失,為他達(dá)拉非的臨床應(yīng)用提供了預(yù)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 .
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

【共引文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 周縝;壯筋續(xù)骨湯對骨折大鼠血清GH水平骨痂組織GHR表達(dá)的影響[D];青島大學(xué);2014年

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本文編號：2103267