miR-92a在糖尿病勃起功能障碍大鼠中的作用及机制初探

发布时间：2018-04-16 05:10

本文选题：阿扑吗啡 + 链脲佐菌素　；参考：《华中科技大学》2014年博士论文

【摘要】：第一部分糖尿病印大鼠模型的建立 [目的]建立PDE5i治疗效果不佳的糖尿病ED大鼠模型 [方法]采用腹腔注射链脲佐菌素诱导糖尿病大鼠,10周后,对存活的糖尿病大鼠进行阿扑吗啡(APO)筛选实验。以大鼠阴茎体增长、露出作为勃起功能良好的标志,即APO(+)；反之,为APO(-)。随后,对APO(-)和APO(+)糖尿病大鼠进行海绵体压力测定以及PDE5i治疗效果的评价。最后,对APO(-)糖尿病大鼠阴茎组织中NO/cGMP通路、RhoA/Rho激酶通路及细胞凋亡进行检测。 [结果]与正常对照大鼠相比,APO(-)糖尿大鼠和APO(+)糖尿病大鼠勃起功能均明显降低,但APO(-)糖尿大鼠勃起功能更差。当给予APO(+)糖尿病大鼠PDE5i(艾力达2.08mg/kg,海绵体测压半小时前灌胃给予,相当60kg成人20mg的等效剂量)治疗后,其勃起功能明显改善,基本上可以达到正常水平,而APO(-)糖尿病大鼠对PDE5i治疗的反应性差,尽管海绵体内压有所提高,但仍远低于正常水平。机制研究发现APO(-)糖尿病大鼠阴茎组织中P-eNOS/eNOS明显下降,ROCK2表达水平和P-MYPT1/MYPT1明显上升,且凋亡细胞显著增多。 [结论]阿扑吗啡实验可成功筛选出对PDE5i治疗效果不佳的糖尿病ED大鼠。第二部分糖尿病ED大鼠阴茎组织内miRNAs表达谱分析及验证 [目的]筛选出可能与糖尿病大鼠勃起功能相关的重要]miRNAs [方法]提取糖尿病ED大鼠(APO(-)糖尿病大鼠)阴茎组织RNA,采用Agilent Rat miRNA V18.0芯片,对已知的677个miRNAs进行检测。采用Real-time PCR,对差异显著的miRNAs进行验证,同时检测其下游靶基因的mRNA表达水平。 [结果]在APO(-)糖尿病大鼠阴茎组织中,3个miRNAs变化明显(下调1/3以上或上调至少3倍,P0.05vs正常对照组,且探针平均信号值大于4)。其中,miR-133b表达明显下调,miR-92a和miR-29b表达明显上调。采用Real-time PCR方法,发现miR-133b表达下降,miR-92a和miR-29b表达上升,与miRNA芯片结果一致,同时发现niR-92a在阴茎海绵体、主动脉、大脑、肾脏、心脏和肝脏中均有表达。与其他组织和器官相比,miR-92a在阴茎组织中表达丰度相对较低。进一步对niR-92a的下游靶基因进行了检测,结果发现eNOS和DKK3的mRNA表达水平在APO(-)糖尿病大鼠阴茎组织中明显下降,而CD49e、klf2和klf4的mRNA表达水平无明显变化。 [结论]miR-92a在糖尿病ED大鼠阴茎组织中高表达可能与eNOS和DKK3的表达水平下调相关。第三部分高糖环境下主动脉内皮细胞中miR-92a及其靶基因的表达变化研究 [目的]在体外水平研究高糖对内皮细胞中miR-92a及其靶基因的表达变化 [方法]采用贴壁法培养原代大鼠主动脉内皮细胞,在高糖环境下培养后,提取主动脉内皮细胞RNA,对miR-92a、eNOS和DKK3的表达水平进行检测。同时,提取内皮细胞蛋白,对eNOS和DKK3的蛋白表达水平进行检测。在高糖培养下,给予miR-92a拮抗剂antagomir-miR-92a后,提取主动脉内皮细胞RNA和蛋白,检测eNOS和DKK3的表达水平。 [结果]在高糖环境下培养48小时和72小时后,miR-92a的表达水平在主动脉内皮细胞中均明显上调,且呈递增趋势；eNOS和DKK3的mRNA表达水平均明显下调,且呈递减趋势。同时,eNOS和DKK3的蛋白表达水平在高糖刺激72小时的主动脉内皮细胞中明显降低。当使用antagomir-miR-92a预处理主动脉内皮细胞后,在高糖环境下培养72小时后,与对照组(antagomir-miR-NC)相比,主动脉内皮细胞中eNOS和DKK3蛋白表达水平明显升高。 [结论]主动脉内皮细胞中miR-92a在高糖环境下表达上调,进而抑制eNOS和DKK3表达。第四部分 miR-92a-inhibition对糖尿病ED大鼠勃起功能的作用及机制初探 [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机制的探索 [方法]以慢病毒为载体,构建表达与miR-92a序列互补的miR-92a-inhibition (Lv-miR-92a-inhibition)。用贴壁法培养原代大鼠主动脉内皮细胞,主动脉细胞转染Lv-miR-92a-inhibition后,检测eNOS和DKK3的表达水平。链脲佐菌素诱导糖尿病大鼠后,采用阿扑吗啡实验筛选出APO(-)糖尿病大鼠。向APO(-)糖尿病大鼠阴茎海绵体内局部注射Lv-miR-92a-inhibition,治疗2周后,采用电刺激海绵体神经测定大鼠阴茎海绵体内压,评价勃起功能。留取大鼠阴茎组织后,对eNOS和DKK3的蛋白表达水平进行检测。 [结果]成功构建了慢病毒介导的miR-92a-inhibition,当其转染主动脉内皮细胞96小时后,主动脉内皮细胞中eNOS和DKK3的mRNA和蛋白表达水平明显上调。向APO(-)糖尿病大鼠注射Lv-miR-92a-inhibition2周后,其勃起功能明显改善。初步的机制研究发现Lv-miR-NC-inhibition治疗后,APO(-)糖尿病大鼠阴茎组织中eNOS蛋白表达水平明显上调,接近于正常水平,同时DKK3蛋白表达水平也明显上调,但低于正常水平。 [结论]miR-92a-inhibition可能通过上调eNOS和DKK3的表达水平,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 .

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.1;R698

【参考文献】