本文選題：阿撲嗎啡 + 鏈脲佐菌素��； 參考：《華中科技大學(xué)》2014年博士論文

【摘要】：第一部分 糖尿病印大鼠模型的建立 [目的]建立PDE5i治療效果不佳的糖尿病ED大鼠模型 [方法]采用腹腔注射鏈脲佐菌素誘導(dǎo)糖尿病大鼠,10周后,對存活的糖尿病大鼠進(jìn)行阿撲嗎啡(APO)篩選實驗。以大鼠陰莖體增長、露出作為勃起功能良好的標(biāo)志,即APO(+)；反之,為APO(-)。隨后,對APO(-)和APO(+)糖尿病大鼠進(jìn)行海綿體壓力測定以及PDE5i治療效果的評價。最后,對APO(-)糖尿病大鼠陰莖組織中NO/cGMP通路、RhoA/Rho激酶通路及細(xì)胞凋亡進(jìn)行檢測。 [結(jié)果]與正常對照大鼠相比,APO(-)糖尿大鼠和APO(+)糖尿病大鼠勃起功能均明顯降低,但APO(-)糖尿大鼠勃起功能更差。當(dāng)給予APO(+)糖尿病大鼠PDE5i(艾力達(dá)2.08mg/kg,海綿體測壓半小時前灌胃給予,相當(dāng)60kg成人20mg的等效劑量)治療后,其勃起功能明顯改善,基本上可以達(dá)到正常水平,而APO(-)糖尿病大鼠對PDE5i治療的反應(yīng)性差,盡管海綿體內(nèi)壓有所提高,但仍遠(yuǎn)低于正常水平。機(jī)制研究發(fā)現(xiàn)APO(-)糖尿病大鼠陰莖組織中P-eNOS/eNOS明顯下降,ROCK2表達(dá)水平和P-MYPT1/MYPT1明顯上升,且凋亡細(xì)胞顯著增多。 [結(jié)論]阿撲嗎啡實驗可成功篩選出對PDE5i治療效果不佳的糖尿病ED大鼠。 第二部分 糖尿病ED大鼠陰莖組織內(nèi)miRNAs表達(dá)譜分析及驗證 [目的]篩選出可能與糖尿病大鼠勃起功能相關(guān)的重要]miRNAs [方法]提取糖尿病ED大鼠(APO(-)糖尿病大鼠)陰莖組織RNA,采用Agilent Rat miRNA V18.0芯片,對已知的677個miRNAs進(jìn)行檢測。采用Real-time PCR,對差異顯著的miRNAs進(jìn)行驗證,同時檢測其下游靶基因的mRNA表達(dá)水平。 [結(jié)果]在APO(-)糖尿病大鼠陰莖組織中,3個miRNAs變化明顯(下調(diào)1/3以上或上調(diào)至少3倍,P0.05vs正常對照組,且探針平均信號值大于4)。其中,miR-133b表達(dá)明顯下調(diào),miR-92a和miR-29b表達(dá)明顯上調(diào)。采用Real-time PCR方法,發(fā)現(xiàn)miR-133b表達(dá)下降,miR-92a和miR-29b表達(dá)上升,與miRNA芯片結(jié)果一致,同時發(fā)現(xiàn)niR-92a在陰莖海綿體、主動脈、大腦、腎臟、心臟和肝臟中均有表達(dá)。與其他組織和器官相比,miR-92a在陰莖組織中表達(dá)豐度相對較低。進(jìn)一步對niR-92a的下游靶基因進(jìn)行了檢測,結(jié)果發(fā)現(xiàn)eNOS和DKK3的mRNA表達(dá)水平在APO(-)糖尿病大鼠陰莖組織中明顯下降,而CD49e、klf2和klf4的mRNA表達(dá)水平無明顯變化。 [結(jié)論]miR-92a在糖尿病ED大鼠陰莖組織中高表達(dá)可能與eNOS和DKK3的表達(dá)水平下調(diào)相關(guān)。 第三部分 高糖環(huán)境下主動脈內(nèi)皮細(xì)胞中miR-92a及其靶基因的表達(dá)變化研究 [目的]在體外水平研究高糖對內(nèi)皮細(xì)胞中miR-92a及其靶基因的表達(dá)變化 [方法]采用貼壁法培養(yǎng)原代大鼠主動脈內(nèi)皮細(xì)胞,在高糖環(huán)境下培養(yǎng)后,提取主動脈內(nèi)皮細(xì)胞RNA,對miR-92a、eNOS和DKK3的表達(dá)水平進(jìn)行檢測。同時,提取內(nèi)皮細(xì)胞蛋白,對eNOS和DKK3的蛋白表達(dá)水平進(jìn)行檢測。在高糖培養(yǎng)下,給予miR-92a拮抗劑antagomir-miR-92a后,提取主動脈內(nèi)皮細(xì)胞RNA和蛋白,檢測eNOS和DKK3的表達(dá)水平。 [結(jié)果]在高糖環(huán)境下培養(yǎng)48小時和72小時后,miR-92a的表達(dá)水平在主動脈內(nèi)皮細(xì)胞中均明顯上調(diào),且呈遞增趨勢；eNOS和DKK3的mRNA表達(dá)水平均明顯下調(diào),且呈遞減趨勢。同時,eNOS和DKK3的蛋白表達(dá)水平在高糖刺激72小時的主動脈內(nèi)皮細(xì)胞中明顯降低。當(dāng)使用antagomir-miR-92a預(yù)處理主動脈內(nèi)皮細(xì)胞后,在高糖環(huán)境下培養(yǎng)72小時后,與對照組(antagomir-miR-NC)相比,主動脈內(nèi)皮細(xì)胞中eNOS和DKK3蛋白表達(dá)水平明顯升高。 [結(jié)論]主動脈內(nèi)皮細(xì)胞中miR-92a在高糖環(huán)境下表達(dá)上調(diào),進(jìn)而抑制eNOS和DKK3表達(dá)。 第四部分 miR-92a-inhibition對糖尿病ED大鼠勃起功能的作用及機(jī)制初探 [目的]拮抗miR-92a是否可以改善糖尿病ED大鼠勃起功能及初步機(jī)制的探索 [方法]以慢病毒為載體,構(gòu)建表達(dá)與miR-92a序列互補(bǔ)的miR-92a-inhibition (Lv-miR-92a-inhibition)。用貼壁法培養(yǎng)原代大鼠主動脈內(nèi)皮細(xì)胞,主動脈細(xì)胞轉(zhuǎn)染Lv-miR-92a-inhibition后,檢測eNOS和DKK3的表達(dá)水平。鏈脲佐菌素誘導(dǎo)糖尿病大鼠后,采用阿撲嗎啡實驗篩選出APO(-)糖尿病大鼠。向APO(-)糖尿病大鼠陰莖海綿體內(nèi)局部注射Lv-miR-92a-inhibition,治療2周后,采用電刺激海綿體神經(jīng)測定大鼠陰莖海綿體內(nèi)壓,評價勃起功能。留取大鼠陰莖組織后,對eNOS和DKK3的蛋白表達(dá)水平進(jìn)行檢測。 [結(jié)果]成功構(gòu)建了慢病毒介導(dǎo)的miR-92a-inhibition,當(dāng)其轉(zhuǎn)染主動脈內(nèi)皮細(xì)胞96小時后,主動脈內(nèi)皮細(xì)胞中eNOS和DKK3的mRNA和蛋白表達(dá)水平明顯上調(diào)。向APO(-)糖尿病大鼠注射Lv-miR-92a-inhibition2周后,其勃起功能明顯改善。初步的機(jī)制研究發(fā)現(xiàn)Lv-miR-NC-inhibition治療后,APO(-)糖尿病大鼠陰莖組織中eNOS蛋白表達(dá)水平明顯上調(diào),接近于正常水平,同時DKK3蛋白表達(dá)水平也明顯上調(diào),但低于正常水平。 [結(jié)論]miR-92a-inhibition可能通過上調(diào)eNOS和DKK3的表達(dá)水平,改善糖尿病ED大鼠勃起功能。
[Abstract]:the first portion

Establishment of Diabetic Rat Model

Objective : To establish a model for the treatment of diabetic ED rats with poor therapeutic effect on PDEs

In this study , apomorphine ( APO ) screening experiment was carried out on the surviving diabetic rats after 10 weeks of induction of diabetic rats by intraperitoneal injection of streptozocin , and the sign of erectile dysfunction was exposed to the growth of the penis body of rats , that is APO ( + ) ;
In the end , the NO / cGMP pathway in penile tissue of APO ( - ) diabetic rats , RhoA / rho kinase pathway and apoptosis were detected .

Results Compared with normal control rats , APO ( - ) diabetic rats and APO ( + ) diabetic rats had significantly lower erectile function , but APO ( - ) diabetic rats had a better erectile function .

Conclusion The experimental results suggest that apomorphine can successfully screen the diabetic ED rats with poor therapeutic effect on PDEs .

the second part

Analysis and validation of expression of miRNA in penile tissue of diabetic ED rats

Objective : To screen out the important role related to erectile dysfunction in diabetic rats

Methods : The RNA of penile tissue of diabetic ED rats ( APO ( - ) diabetic rats ) was extracted from diabetic ED rats ( APO ( - ) diabetic rats ) .

The results showed that in APO ( - ) diabetic rat penile tissue , the changes of 3 were obviously ( down - regulated 1 / 3 or up - regulated at least 3 - fold , P0.05 vs control group , and the average signal value of probe was greater than 4 ) . Compared with other tissues and organs , the expression levels of miR - 92a and miR - 29b were significantly lower than those of other tissues and organs , and the mRNA expression levels of eNOS and DKK3 were significantly decreased in the penile tissue of APO ( - ) diabetic rats , while the levels of mRNA expression of CD49e , klf2 and klf4 were not significantly changed .

Conclusion The high expression of miR - 92a in penile tissue of diabetic ED rats may be correlated with downregulation of eNOS and DKK3 .

PART III

Expression of miR - 92a and its target gene in aortic endothelial cells under high glucose environment

Objective : To study the expression of miR - 92a and its target genes in endothelial cells by high glucose level in vitro

The expression level of eNOS and DKK3 was detected . At the same time , the expression level of eNOS and DKK3 was detected by extracting endothelial cell protein , and then the expression level of eNOS and DKK3 was detected .

RESULTS : The expression level of miR - 92a was up - regulated in aortic endothelial cells after 48 - hour and 72 - hour incubation in high - sugar environment , and the expression level of miR - 92a increased gradually .
At the same time , the levels of eNOS and DKK3 mRNA were down - regulated and decreased gradually . At the same time , the protein expression level of eNOS and DKK3 was significantly decreased in 72 - hour aortic endothelial cells stimulated by high glucose . After 72 - hour incubation in high - sugar environment , the expression level of eNOS and DKK3 in aortic endothelial cells was significantly increased compared with control group ( . omir - miR - NC ) .

Conclusion The expression of miR - 92a in aortic endothelial cells is up - regulated in high - sugar environment , which can inhibit the expression of eNOS and DKK3 .

PART IV

Effect and mechanism of miR - 92a - inhibition on erectile function in diabetic ED rats

Objective To study whether miR - 92a can improve erectile function and preliminary mechanism of diabetic ED rats

The expression level of eNOS and DKK3 was determined by using slow virus as the vector . The expression level of eNOS and DKK3 was determined by the adherent method . After 2 weeks of treatment with apomorphine , the expression level of eNOS and DKK3 was detected . After 2 weeks of treatment , the rat penile cavernous nerves were injected into APO ( - ) diabetic rats to evaluate the erectile function . After the rat penile tissue was retained , the protein expression level of eNOS and DKK3 was detected .

The expression level of eNOS and DKK3 in the penile tissue of APO ( - ) diabetic rats was significantly higher than that of normal level , but the level of expression of DKK3 protein was also significantly increased , but the level of DKK3 protein was lower than normal level .

Conclusion miR - 92a - inhibition may improve the erectile function of diabetic ED rats by up - regulating the expression level of eNOS and DKK3 .

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R587.1;R698

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 韋安陽;楊勇;何書華;羅新貴;張濤;劉洋;;miR-145在糖尿病性勃起功能障礙大鼠發(fā)病機(jī)制中的作用[J];南方醫(yī)科大學(xué)學(xué)報;2011年06期

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本文編號：1757431