【作者】 左中；

【导师】 雷寒；

【作者基本信息】 重庆医科大学 ， 内科学， 2011， 博士

【摘要】 微小RNA（microRNA,miRNA）作为一种内源性的非编码的RNA，通过对靶基因mRNA的降解或翻译抑制来调节基因表达。miRNA150在免疫调节作用已被知晓，而其潜在的心血管系统调节作用还不清楚。本研究内容包括：1、系统分析miRNA150敲除(knockout,KO)小鼠的心血管系统稳态（Homeostasis），是否具有心肌细胞肥大，肾脏损害等表现；2、从整体水平，分子水平到细胞水平对内皮功能损害，氧化应激等指标分析，探索出现上述表现的可能机制，并予依普利酮（Eplereone,EPL）进行短期干预；3、通过衰老相关肾脏损害的机制分析，分析导致肾脏损害可能的病理生理机制，为进一步探索miRNA150在调节血压以及对肾脏损害的机制提供线索。全文分为三部分。第一部分miRNA150敲除小鼠的心血管系统稳态[目的]系统研究miRNA150-KO小鼠的心血管系统表现。[方法]miRNA150-KO小鼠（KO组，n=8）与野生型小鼠比较（WT组，n=8），尾袖法（tail-cuff method）监测血压变化，16周后观察心脏、肾脏、肾上腺的形态学改变及重量指数，HE染色观察心肌组织结构变化和心肌细胞横切面积（cardiomyctye cross-sectional area,CSA）；三色法（Masson-Trichrome staining）观察肾脏组织结构改变；免疫荧光（immunofluorescence）肾上腺皮质改变；流式细胞仪监测T淋巴细胞、B淋巴细胞以及B1细胞表达。[结果]（1）miRNA150-KO小鼠在11周龄始出现收缩压升高，并持续存在（；2）肾上腺重量指数升高（p<0.05）,伴有肾上腺皮质增生；（3）KO脾脏B1细胞含量是WT组的2.6倍；（4）较WT组，KO组CSA显著增加；（5） KO组肾小球和肾小管间质胶原沉积明显。[结论] miRNA150-KO小鼠表现出高血压的特点，脾脏B1细胞扩增，心肌细胞肥大，肾上腺增大及肾皮质增厚和肾脏结构性损害。第二部分miRNA150在血压调节中作用机制的初步研究第一节miRNA150-KO小鼠CYP11B2表达和内皮功能变化研究[目的]通过研究醛固酮合成酶(aldosterone synthase) CYP11B2和内皮功能的变化来初步探究miRNA150-KO小鼠高血压形成的机制。[方法]miRNA150-KO小鼠（KO组，n=8）与野生型WT小鼠，在20周龄时采血浆测醛固酮水平，免疫组化和免疫印迹法检测CYP11B2的表达；选取主动脉行内皮舒张功能检测；DHE staining检测主动脉，肾脏，心脏原位超氧化物的表达；苏木精化学发光法检测主动脉的NADPH氧化酶的活性。[结果]（1）KO组血浆醛固酮水平较WT组高（P＜0.05）;（2）CYP11B2水平，IHC和Western-blot检测KO组较WT组均高（P＜0.05，P＜0.01）；（3）与WT组比较，KO组主动脉内皮舒张功能受损（P＜0.05）；（4）KO组超氧化物生成较WT组为高（P＜0.05），其NADPH氧化酶活性也明显增强（P＜0.01）。[结论]醛固酮合成酶表达增加和主动脉内皮舒张功能受损可能是miRNA150-KO小鼠血压升高的机制。第二节依普利酮短期干预实验[目的]观察依普利酮（Eplereone，EPL）对miRNA150-KO小鼠血压水平、主动脉内皮功能及氧化应激产物的影响，以进一步探讨miRNA150-KO小鼠血压发生的可能机制。[方法] miRNA150-KO小鼠（n=12）随机分为DMSO组（KO-DMSO）和EPL组（KO-EPL），（n=6）；WT小鼠分成DMSO组（WT-DMSO）和EPL组（WT-EPL）（n=6）。EPL组干预为50mg/Kg.day腹腔注射，连续干预5天。（1）观察小鼠体重、尿量变化；（2）并观察组织器官重量指数变化及血浆醛固酮水平变化；(3) DHE staining法观察肾脏、主动脉超氧化物变化；（4）NADPH氧化酶活性变化情况；（5）免疫组化8-OHDG的表达；（6）EPL组对肾脏结构的保护作用。[结果]（1）EPL组（KO-EPL）血压水平下降，较KO-DMSO为显（P＜0.01）;（2）EPL干预使KO组尿量增加（P<0.05），而血浆醛固酮水平没有显著性变化;（3） EPL改善KO小鼠内皮功能（P＜0.05），并下调NADPH氧化酶活性（P＜0.05）；（4）EPL减少肾脏和主动脉的超氧化物产生（P＜0.05，P＜0.01）；（5）EPL促使8-OHDG表达降低。[结论]依普利酮短期干预研究能降低KO小鼠血压水平，抑制NADPH氧化酶活性，改善主动脉内皮功能，逆转肾脏损害。第三部分衰老相关的肾脏损害因素分析[目的]检测在老年大鼠肾脏中klotho，内皮素（ET）受体和超氧化物的变化，以及是否这些变化在认知功能障碍的大鼠中是否加剧。[方法]根据认知功能测试，将20只大鼠（雄性，27个月）随机分为两组，损伤的老年大鼠组（n=9）和未损伤的老年大鼠组（n=11）。12个月龄大鼠（n=10）作为年轻未损伤组。采集血清ELISA测定血清肌酐和ET-1的浓度。采用PAS染色和三色染色评价肾组织学改变。DHE染色测定肾脏原位超氧化物。采用免疫组化和Western印迹检测Klotho蛋白，内皮素受体ET，Nox2，IL-6，和MnSOD在肾皮质和髓质的表达。[结果]认知功能损伤的老年大鼠组血清肌酐显着增加（p<0.05），提示肾功能受损。老年大鼠出现肾小球硬化和小管间质性纤维化。认知功能损伤的老年大鼠组较认知功能未损伤的老年大鼠上述这些病理变化显著的加剧（p<0.01）。老年大鼠肾皮质和髓质klotho蛋白表达显著下降，IL-6，Nox2，ET-a受体和超氧化物超的表达增加，然而老年大鼠肾脏的线粒体超氧化物歧化酶（MnSOD）和ETb受体的表达明显降低。认知功能损伤的老年大鼠组较认知功能未损伤的老年大鼠组上述这些变化更为明显。[结论]认知功能损伤的老年大鼠年龄相关的肾脏损害加剧。在年龄相关的肾脏损害中，Klotho，ETB，和MnSOD表达下调，但ETA,和IL-6，Nox2，超氧化物表达上调。这些变化在认知功能障碍的大鼠更为明显。更多还原

【Abstract】 As an endogenous uncoded RNA, miRNA regulates gene expressionthrough degradation or translation inhibition. The role of miRNA150inimmune regulation has been known, and its potential role in regulating thecardiovascular system is unclear. This research includes the followingitems:1. To analyze the homeostasis of the cardiovascular system ofmiRNA150knockout mouse, to see if there is myocardial cell hypertrophyor renal impairment;2. To analyze, from a whole level to a molecular level,impairment of endothelium as well as oxidative stress, so as to explore thepossible mechanism for the above manifestations, and to conductshort-term intervention with Eplereone;3. To analyze the possiblepathological and physiological mechanisms that might cause renal damagethrough analyzing the mechanism of age-related renal impairment, so as toprovide clues for further exploration of the regulation of miRNA150inblood pressure and its impairment to renal function. This paper can bedivided into three parts. Part Ⅰ. Homeostasis of the Cardiovascular System of miRNA150-knockout MiceObjective: To research systematically the manifestations of thecardiovascular system of miRNA150knockout mice. Methods: miRNA150knockout mice （KO Group，n=8）and wild-type mice （WT Group，n=8）were compared. Blood pressure was monitored with tail-cuff method; themorphological changes and weight indexes of hearts, kidneys and adrenalglands were observed after16w. The changes of myocardial tissue structureand cardiomycyte cross-sectional area (CSA) were observed with HEstaining. The changes of renal tissue structure were observed withMasson-trichrome staining. The changes of adrenal cortex were observedwith immunofluorescence. The expressions of T cell, B cell and B1cellwere monitored by flow cytometry. Results:(1)The systolic pressure ofmiRNA150-KO mice increased from11w and continued;(2)the mass indexof adrenal glands increased (P<0.05), accompanied with adrenal cortexproliferation;(3)The B1cell content in KO spleen is2.6times of that inWT Group;(4) Compared with WT Group, CSA in KO Group increasedsignificantly;(5) Glomerular and tubular interstitial collagen depositionwas obvious in KO Group. Conclusion: miRNA150-KO mice presentedwith the manifestations of hypertension, which showed proliferation ofspleen B1cells, cardiocytes hypertrophy, adrenal cortex thickening andrenal structural damage. Part Ⅱ Research of the Mechanism of miRNA150in RegulatingBlood PressureSection One CYP11B2Expression and Endothelial FunctionChanges of miRNA150-KO MiceObjective: To preliminarily investigate into the mechanism ofhypertension forming in miRNA150-KO mice through analyzing thechanges of aldosterone synthase CYP11B2and endothelial function.Methods: In20w, plasma of miRNA150knockout mice （KO Group，n=8）and wild-type mice （WT Group，n=8）was taken to determine the plasmaaldosterone level; the CYP11B2expression was detected byimmuno-histochemistry and Western blot; endothelium function in aorticwas detected; the expression of superoxide in situ in aorta, kidney and heartwas detected with DHE staining; the activity of NADPH oxidase in aortawas detected with lucigenin chemiluminescence method. Results:(1) KOGroup had a higher plasma aldosterone level than WT Group (P<0.05);(2)KO Group had a higher level of CYP11B2in both IHC and Western-blotdetection（P＜0.05，P＜0.01）;(3) Compared with WT Group, theendothelial diastolic function was impaired （P＜0.05);(4) KO Group hada higher level of superoxide production（P＜0.05）, and the activity ofNADPH oxidase was also increased obviously（P＜0.01）. Conclusion: The increase of CYP11B2expression and the endothelial dysfunction in aortamight be the mechanism for the high blood pressure of miRNA150-KOmice. Section Two Eplereone Short-term Intervention Experimenton miRNA150-KO MiceObjective: To observe the effect of Eplereone on the blood pressure,aorta endothelial function and the production of oxidative stress, so as tofurther discuss the possible mechanism for the blood pressure ofmiRNA150-KO mice. Methods: miRNA150-KO mice were randomlydivided into DMSO group（KO-DMSO, n=6）and EPL group（KO-EPL,n=6）; WT mice were divided into DMSO group (WT-DMSO，n=6) and theEPL group (WT-EPL, n=6）. The intervention for EPL group wasintraperitoneal injection50mg/Kg per day for5days.(1) The body massand urine volume were observed;(2) The changes of tissue and organ massindexes and plasma aldosterone were observed;(3) Superoxide productionin kidney and aorta was observed by DHE staining;(4) The activity ofNADPH oxidase was measured;(5) The expression of8-OHDG wasmeasured by IHC;(6) The protective function of EPL group to the kidneywas observed. Results:(1)Compared with KO-DMSO group, KO-EPLgroup showed a decreased blood pressure（P＜0.01）;(2) EPL intervention increased the urine volume of KO group.There was no any change ofplasma aldosterone level;(3) EPL improved the endothelial function of KOmice（P＜0.05）, and down-regulated the activity of NADPH oxidase（P＜0.05）;(4) EPL reduced the production of superoxide in kidney and aorta（P＜0.05，P＜0.01）；(5) EPL reguced the expression of8-OHDG protein.Conclusion: The short-term intervention with Eplereone can decrease theblood pressure of KO mice, inhibit the activity of NADPH oxidase,improve the endothelial function in aorta and reverse renal damage. Part Ⅲ Research of Related Factors Leading to Aging-relatedKidney DamageObjective: Aging is associated with a decline in renal function.Klotho is a recently-discovered anti-aging gene. The purpose of this studywas to determine changes in klotho, endothelin (ET) receptors, andsuperoxide production in kidneys of aged rats. Methods: Twenty aged rats(male,27months) were divided into an Old Impaired group (n=9) and anOld Intact group (n=11) according to a cognitive function test. A group of12month-old rats (n=10) was used as a Young Intact group. Blood wascollected for measuring serum creatinine and ET-1concentration. Renalhistology was evaluated using PAS staining and trichrome staining. The in situ superoxide production was measured in kidneys usingdihydroethidium staining. Klotho protein, ET receptors, Nox2,interleukin-6(IL-6), and MnSOD expression in renal cortex and medullawere assessed by immunohistochemistry and western-blotting. Results:Plasma creatinine was increased significantly in the Old Impaired group,suggesting impaired renal function. Aged rats showed glomerulosclerosisand tubulointerstitialfibrosis. These pathological changes were markedlyaggravated in the old cognitively impaired than in the old cognitively intactanimals. Notably, aged rats demonstrated a significant decrease in klothoprotein expression in renal cortex and medulla. Protein expression of IL-6,Nox2, ETA receptors and superoxide production were increased whereasMnSOD and ETb receptors expression were decreased in kidneys of theaged rats. Interestingly, these changes were more pronounced in the oldimpaired than in the old intact rats. Conclusions: The aging-relatedkidney damage paralleled with the cognitive function impairment. Theaging-related kidney damage was associated with downregulation of klotho,ETB, and MnSOD expression but upregulation of ETA, IL-6, and Nox2expression and superoxide production. These findings raise the necessityto further assess the roles of these factors and their relationship inaging-related kidney damage.更多还原