本文選題：KLF5 + Runx2　； 參考：《河北醫(yī)科大學(xué)》2014年博士論文

【摘要】：血管鈣化是動(dòng)脈粥樣硬化、糖尿病、高血壓、慢性腎臟病的主要并發(fā)癥。血管鈣化導(dǎo)致血管彈性和血管順應(yīng)性降低，使血管疾病突發(fā)事件增加，如心肌梗死，動(dòng)脈粥樣硬化斑塊破裂等。很多因素，如礦物質(zhì)代謝紊亂，慢性炎癥，氧化應(yīng)激等等和血管鈣化的形成有關(guān)。越來(lái)越多的證據(jù)表明，血管鈣化并非簡(jiǎn)單的鈣磷被動(dòng)沉積，而是受到精細(xì)調(diào)控的類似骨的骨化過(guò)程。其中包括血管平滑肌細(xì)胞（vascular smooth cell, VSMC）向成骨樣細(xì)胞轉(zhuǎn)化。體外和活體實(shí)驗(yàn)表明，在高磷刺激下，VSMC中骨相關(guān)蛋白，如堿性磷酸酶（alkaline phosphatase, ALP）、基質(zhì)Gla蛋白（matrix Glaprotein, MGP）、骨橋蛋白（osteopontin, OPN），骨鈣素（osteocalcin, OC）等表達(dá)增加，同時(shí)VSMC分化標(biāo)志基因平滑肌肌動(dòng)蛋白α（smooth muscleα-actin, SM α-actin）和平滑肌22α（smooth muscle22α, SM22α）表達(dá)減少。 最近研究顯示，在動(dòng)脈粥樣硬化患者的血管鈣化組織和小鼠鈣化的VSMC中檢測(cè)到Runt相關(guān)轉(zhuǎn)錄因子2（Runt-related transcription factor2，Runx2，調(diào)節(jié)成骨和軟骨分化的關(guān)鍵轉(zhuǎn)錄因子）的表達(dá)，而正常血管中沒(méi)有檢出。Runx2缺失能夠抑制氧化應(yīng)激誘導(dǎo)的VSMC標(biāo)志基因的下調(diào)，降低成骨樣細(xì)胞的形成。血管緊張素2通過(guò)激活Runx2和NF-кB加劇血管鈣化，表明Runx2在血管鈣化中具有關(guān)鍵作用。 調(diào)控VSMC表型轉(zhuǎn)變的轉(zhuǎn)錄因子也和血管鈣化的發(fā)生發(fā)展有關(guān)。如高磷能夠誘導(dǎo)Krüppel樣因子4（Krüppel-like factor4，KLF4）的表達(dá)，KLF4敲低則可以減輕高磷誘導(dǎo)的VSMC向成骨樣細(xì)胞轉(zhuǎn)化，降低成骨標(biāo)志基因的表達(dá)和鈣沉積。KLF4在腺嘌呤誘導(dǎo)的尿毒癥大鼠的鈣化的主動(dòng)脈中表達(dá)量也增加。我們課題組之前的研究發(fā)現(xiàn)，血管緊張素II（angiotensin II,Ang II）通過(guò)激活Krüppel樣因子5（Krüppel-like factor5，KLF5）（促增殖轉(zhuǎn)錄因子）抑制p21的表達(dá)，促進(jìn)VSMC向合成型細(xì)胞轉(zhuǎn)化。KLF4和KLF5是KLF家族中緊密相關(guān)的轉(zhuǎn)錄因子，分別從正向和反向調(diào)節(jié)細(xì)胞增殖。然而，KLF5在VSMC鈣化中是否發(fā)揮作用還不清楚。此外，KLF5和Runx2介導(dǎo)的VSMC鈣化之間的內(nèi)在關(guān)系也有待探討。本文的目的是闡明KLF5在VSMC鈣化中的作用，明確KLF5和Runx2在調(diào)制VSMC鈣化中的相互關(guān)系。 第一部分高磷促進(jìn)血管平滑肌細(xì)胞的成骨樣分化和KLF5的表達(dá) 目的：探討高磷誘導(dǎo)VSMC鈣化與KLF5表達(dá)的關(guān)系。 方法：以3.8mM無(wú)機(jī)磷處理大鼠VSMC，建立鈣化模型。通過(guò)茜素紅染色發(fā)現(xiàn)鈣化結(jié)節(jié)、ALP活性升高、鈣離子含量測(cè)定以確定VSMC成骨樣轉(zhuǎn)化。以real-time PCR和Western blotting檢測(cè)KLF5、成骨標(biāo)志基因Runx2、VSMC標(biāo)志基因SM α-actin和SM22α表達(dá)。應(yīng)用離體血管環(huán)驗(yàn)證高磷誘導(dǎo)的VSMC鈣化及各基因的表達(dá)變化。 結(jié)果：茜素紅染色顯示，VSMC在3.8mM無(wú)機(jī)磷誘導(dǎo)下，出現(xiàn)明顯的鈣化結(jié)節(jié)，堿性磷酸酶活性增強(qiáng)，鈣離子含量增加，提示大鼠VSMC發(fā)生鈣化。血管環(huán)實(shí)驗(yàn)Von kossa染色顯示，鈣化組出現(xiàn)明顯的黑色鈣沉積。細(xì)胞和離體血管環(huán)中KLF5、Runx2mRNA和蛋白均隨鈣化程度增加表達(dá)上升，SM α-actin和SM22α隨鈣化程度增加表達(dá)下降。 小結(jié)：在高磷誘導(dǎo)下，VSMC向成骨樣細(xì)胞轉(zhuǎn)化，在VSMC向成骨樣細(xì)胞轉(zhuǎn)化和鈣化過(guò)程中伴隨著KLF5和Runx2表達(dá)上調(diào)，二者表達(dá)活性與VSMC鈣化程度呈正相關(guān)。 第二部分KLF5在血管平滑肌細(xì)胞成骨樣分化中的作用及機(jī)制 目的：探尋KLF5在高磷誘導(dǎo)VSMC向成骨樣細(xì)胞分化過(guò)程中的具體機(jī)制。 方法：分別應(yīng)用腺病毒過(guò)表達(dá)KLF5及靶向KLF5的特異性siRNA敲低KLF5，用茜素紅染色和鈣離子含量測(cè)定檢測(cè)VSMC中鈣鹽沉積程度，real-time PCR和Western blotting檢測(cè)KLF5、Runx2、SM α-actin和SM22α表達(dá)。構(gòu)建Runx2啟動(dòng)子指導(dǎo)的熒光素酶報(bào)告基因質(zhì)粒，與KLF5表達(dá)質(zhì)粒共轉(zhuǎn)染血管平滑肌細(xì)胞，以熒光素酶報(bào)告基因檢查KLF5對(duì)Runx2的調(diào)控作用。染色質(zhì)免疫沉淀（chromatin immunoprecipitation, ChIP）分析和oligo pulldown方法分析KLF5在Runx2啟動(dòng)子上的結(jié)合位點(diǎn)。 結(jié)果：鈣離子濃度測(cè)定和茜素紅染色發(fā)現(xiàn)，單純過(guò)表達(dá)KLF5并不會(huì)使VSMC發(fā)生鈣化。然而當(dāng)給予VSMC高磷刺激時(shí)，與轉(zhuǎn)染空病毒組相比，過(guò)表達(dá)KLF5組鈣沉積是轉(zhuǎn)染空病毒組的1.56倍，同時(shí)礦化結(jié)節(jié)的形成顯著增加。高磷刺激后，Runx2表達(dá)量在轉(zhuǎn)染空病毒組和過(guò)表達(dá)KLF5組分別增加1.2和2.3倍。給于高磷刺激后，SM α-actin和SM22α的表達(dá)活性在過(guò)表達(dá)KLF5組較轉(zhuǎn)染空病毒的對(duì)照組分別下降40%和30%。離體動(dòng)脈環(huán)實(shí)驗(yàn)得到相似的結(jié)果。 與轉(zhuǎn)染對(duì)照siRNA組相比，KLF5敲低之后，給予高磷刺激，VSMC中的鈣沉積下降30%，礦化結(jié)節(jié)顯著減少。Runx2表達(dá)活性下降了70%，SM α-actin和SM22α的表達(dá)水平較對(duì)照組明顯增加。 報(bào)告基因分析顯示，不給于高磷刺激時(shí)，KLF5對(duì)Runx2基因啟動(dòng)子的活性無(wú)明顯影響，高磷促進(jìn)KLF5結(jié)合到Runx2啟動(dòng)子區(qū)并誘導(dǎo)其轉(zhuǎn)錄。ChIP分析結(jié)果顯示，在高磷處理的VSMC中， KLF5可結(jié)合到Runx2啟動(dòng)子區(qū)的－185bp—－27bp，這個(gè)區(qū)段包含4個(gè)KLF5結(jié)合位點(diǎn)；oligopulldown分析結(jié)果顯示，高磷促進(jìn)KLF5與Runx2啟動(dòng)子區(qū)近端兩個(gè)KLF5結(jié)合位點(diǎn)結(jié)合的作用大于遠(yuǎn)端的兩個(gè)位點(diǎn)。 小結(jié)：KLF5通過(guò)直接與Runx2啟動(dòng)子結(jié)合激活其轉(zhuǎn)錄，高磷通過(guò)促進(jìn)KLF5與Runx2啟動(dòng)子的結(jié)合，進(jìn)而發(fā)揮其促進(jìn)VSMC向成骨細(xì)胞轉(zhuǎn)化。 第三部分KLF5在慢性腎衰竭血管鈣化中的作用 目的：進(jìn)一步探討KLF5在整體動(dòng)物中調(diào)控慢性腎衰竭血管鈣化中的作用及機(jī)制。 方法：用含大鼠0.75%腺嘌呤的飼料飼喂大鼠，生化分析儀測(cè)定腎衰竭相關(guān)參數(shù)，超聲心動(dòng)圖評(píng)估心臟功能；高分辨率超聲、Von kossa染色和鈣離子測(cè)定檢測(cè)慢性腎衰竭大鼠主動(dòng)脈的鈣化情況；免疫組化方法檢測(cè)KLF5、Runx2和SM α-actin在鈣化動(dòng)脈中的表達(dá)；Real-time PCR和Westernblotting檢測(cè)KLF5和Runx2在主動(dòng)脈中的表達(dá)結(jié)果：腺嘌呤成功誘導(dǎo)大鼠出現(xiàn)腎衰竭，肌酐、尿素氮、血磷和鈣磷乘積增加；同時(shí)合并有心功能衰竭。超聲顯像和Von kossa染色顯示主動(dòng)脈出現(xiàn)明顯的鈣化，，鈣化區(qū)域鈣離子含量增加，并且血磷水平與血管鈣化呈正相關(guān)；鈣化區(qū)域KLF5和Runx2表達(dá)量增加， SM α-actin表達(dá)量降低，與細(xì)胞水平的結(jié)果相一致。 小結(jié)：腺嘌呤飲食誘導(dǎo)的腎衰模型接近尿毒癥血管鈣化的發(fā)病機(jī)理，可作為研究慢性腎衰竭血管鈣化的模型，在主動(dòng)脈鈣化區(qū)域的血管組織中，KLF5和Runx2同時(shí)高表達(dá)，提示在整體動(dòng)物中KLF5表達(dá)上調(diào)與血管鈣化相關(guān)。 結(jié)論： 1.高磷誘導(dǎo)VSMC鈣化和KLF5表達(dá)。 2. KLF5通過(guò)直接與Runx2啟動(dòng)子結(jié)合激活其轉(zhuǎn)錄，高磷通過(guò)促進(jìn)KLF5與Runx2啟動(dòng)子的結(jié)合，進(jìn)而發(fā)揮其促進(jìn)VSMC向成骨樣細(xì)胞轉(zhuǎn)化的作用。 3.慢性腎衰竭大鼠鈣化血管中KLF5表達(dá)增加，VSMC向成骨樣細(xì)胞轉(zhuǎn)化。 4.血管鈣化伴隨著VSMC表型轉(zhuǎn)化，其表型轉(zhuǎn)化與鈣化的耦聯(lián)是通過(guò)KLF5對(duì)Runx2啟動(dòng)子的反式激活而實(shí)現(xiàn)的。
[Abstract]:Vascular calcification is a major complication of atherosclerosis , diabetes , hypertension , and chronic renal disease . The vascular calcification results in decreased vascular elasticity and vascular compliance . There are many factors , such as myocardial infarction , atherosclerotic plaque rupture , etc .


In recent studies , the expression of Runt - related transcription factor 2 ( Runt - related transcription factor2 , Runx2 , the key transcription factor regulating the differentiation of bone and cartilage ) was detected in vascular calcification and calcification in atherosclerotic patients .


KLF4 and KLF5 are closely related transcription factors in KLF4 . KLF4 and KLF5 are closely related transcription factors in KLF4 .


The first part of high phosphorus promotes the osteoblast - like differentiation of vascular smooth muscle cells and the expression of KLF5


Objective : To investigate the relationship between high phosphorus - induced calcification and KLF5 expression .


Methods : The rats were treated with 3.8 mM inorganic phosphorus to establish a calcification model . The calcification nodules , ALP activity and calcium ion content were determined by alizarin red staining . The expression of KLF5 , KLF5 and SM22偽 were detected by real - time PCR and Western blotting .


Results : Alizarin red staining showed that , under the induction of 3.8 mM inorganic phosphorus , there appeared obvious calcification nodules , increased alkaline phosphatase activity and increased calcium ion content , which suggested that the calcium ion content increased and the calcium ion content increased . The expression of KLF5 , Runx2 mRNA and protein in calcification group increased with the degree of calcification . The expression of SM 偽 - actin and SM22偽 decreased with the degree of calcification .


Conclusion : The expression of KLF5 and Runx2 is up - regulated in the process of transforming and calcification into bone - like cells . Both of them are positively correlated with the degree of calcification .


The Role and Mechanism of the Second Part KLF5 in Osteoblast - like Differentiation of Vascular Smooth Muscle Cells


Objective : To explore the specific mechanism of KLF5 induced by KLF5 in the differentiation of bone - like cells .


Methods : The expression of KLF5 , Runx2 , SM 偽 - actin and SM22偽 were detected by using the specific siRNA targeting KLF5 and KLF5 respectively . The luciferase reporter plasmid was constructed by PCR and Western blotting . The luciferase reporter plasmid was co - transfected with KLF5 expression plasmid , and the regulatory action of KLF5 on Runx2 was examined by luciferase reporter gene . The binding sites of KLF5 on the Runx2 promoter were analyzed by chromatin immunoprecipitation ( ChIP ) analysis and oligo - assay .


Results : The calcium ion concentration and alizarin red staining showed that the simple overexpression of KLF5 did not lead to calcification . However , after high phosphorus stimulation , the expression of SM 偽 - actin and SM22偽 increased by 1.2 and 2.3 times , respectively . After high phosphorus stimulation , the expression of SM 偽 - actin and SM22偽 decreased by 40 % and 30 % respectively after high P stimulation .


Compared with control siRNA group transfected with KLF5 , after KLF5 was low , calcium deposition decreased by 30 % and mineralized nodules decreased significantly after KLF5 was knocked down . The expression of Runx2 decreased by 70 % , and the expression level of SM 偽 - actin and SM22偽 increased significantly compared with control group .


The results showed that KLF5 could bind to the promoter region of Runx2 and induce its transcription . The results of ChIP analysis showed that KLF5 could bind to - 185bp - -27bp in the promoter region of Runx2 , and the segment contained 4 KLF5 binding sites .
The results showed that the combination of KLF5 and KLF5 binding sites at the proximal end of the promoter region of Runx2 promoter region was greater than that at the distal end of KLF5 promoter region .


Summary : KLF5 activates transcription of KLF5 directly in combination with the Runx2 promoter to promote the binding of KLF5 to the Runx2 promoter , which in turn plays its role in promoting the transformation of the VV5 to the osteoblast .


Role of the third part KLF5 in vascular calcification of chronic renal failure


Objective : To investigate the role and mechanism of KLF5 in the regulation of vascular calcification of chronic renal failure .


Methods : Rats were fed with 0.75 % adenine in rats . The parameters of renal failure were measured by biochemistry analyzer , and the function of heart was evaluated by echocardiography .
High - resolution ultrasound , Von kossa staining and calcium ion assay were used to detect the calcification of aorta in rats with chronic renal failure .
Immunohistochemical method was used to detect the expression of KLF5 , Runx2 and SM 偽 - actin in calcified arteries .
Real - time PCR and Western blotting were used to detect the expression of KLF5 and Runx2 in aorta .
At the same time , heart failure was combined . Ultrasound imaging and Von kossa staining showed significant calcification in the aorta , the calcium ion content in calcified area increased , and the level of blood phosphorus was positively correlated with calcification of the vessel ;
The expression of KLF5 and Runx2 in calcified areas increased , and the expression of SM 偽 - actin decreased , which was consistent with the result of cell level .


Summary : The renal failure model induced by adenine diet is close to the pathogenesis of vascular calcification in uremia , and can be used as a model to study the vascular calcification of chronic renal failure . In the vascular tissue of the aortic calcification area , KLF5 and Runx2 are expressed simultaneously , suggesting that the up - regulation of KLF5 expression in the whole animal is related to calcification .


Conclusion :


1 . High phosphorus - induced calcification and KLF5 expression .


2 . KLF5 can activate the transcription of KLF5 and Runx2 promoter by directly binding to the promoter of Runx2 promoter .


3 . The expression of KLF5 in calcified vessels was increased in rats with chronic renal failure .


4 . The phenotypic transformation of vascular calcification is associated with the phenotypic transformation , and the coupling of phenotypic transformation and calcification is achieved by transactivation of the Runx2 promoter via KLF5 .

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692.5

【共引文獻(xiàn)】

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6 甘q

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