P-Erk1/2和P-Akt1在高血压大鼠阴茎海绵体中的表达
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摘要
目的：了解磷酸化Erk1/2 (P-Erk1/2)和磷酸化Akt1(P-Akt1)在自发性高血压大鼠(SHR)和正常血压大鼠阴茎海绵体中的表达及与勃起功能的关系。方法：1.实验动物：健康成年雄性SPF级自发性高血压大鼠(SHR)与对照组WKY各8只,14周龄,体重250-300g。2.阴茎勃起功能测定：麻醉前大鼠称重,以戊巴比妥钠对实验大鼠进行麻醉(30mg/Kg,腹腔内注射),暴露颈总动脉,用充满肝素盐水的30G针头成功穿刺,连接压力转换器,计算机连续监测记录平均动脉压(MAP)。取大鼠下腹正中切口,在10倍显微镜下仔细游离前列腺两侧后方,显露盆腔星状神经节,沿前列腺表面与直肠之间将denonvillies筋膜剪开,于前列腺背侧找到海绵体神经(corpus nerve,CN),用温生理盐水棉片覆盖,保护,备电刺激时使用。暴露阴茎海绵体后,以充满肝素盐水的24G针头插入右侧海绵体,连接另一个压力转换器,持续监测大鼠阴茎海绵体内压((intracavernosal pressure, ICP)。计算机记录一段ICP基础压后,将不锈钢电极置于海绵体神经,施加不同的电刺激(刺激电压分别为3V、5V,频率12Hz,波幅5ms,持续30s-60s),连续记录电刺激后ICP/MAP的变化。3.取材及制备：阴茎勃起功能测定完毕后,即在靠近阴茎海绵体根部处截取阴茎海绵体,以PBs液(0.01 moL／L,pH 7.4)冲洗净残存血液后去掉尿道及阴茎龟头部分,阴茎海绵体标本分为两部分,立即放入EP管,一部分中性甲醛固定后用于免疫组化分析,一部分于-70℃超低温冰箱保存用于RT-PCR实验。4.免疫组化测定P-Erk1/2和P-Akt1蛋白在大鼠阴茎海绵体中的表达：SP二步法,阴茎海绵体标本经石蜡包埋后,按免疫组化常规处理石蜡切片,PBS洗10min；0.3%H2O2-甲醇处理、30min；高压修复5min；10%正常羊血清室温封闭15min；滴加一抗于标本上,4℃过夜；PBS洗3次,每次10 min；滴加二抗,室温孵育30min; DAB显色。5. RT-PCR检测P-Erk1/2mRNA和P-Akt1 mRNA在大鼠阴茎海绵体中的表达：依据小量组织／细胞总RNA抽提试剂盒说明书及分子克隆实验指南(第二版)进行操作,在无菌工作台内提取阴茎海绵体组织总RNA,保存于-80℃超低温冰箱中,用Nanodrop按设备使用说明测量总RNA浓度,依常规RT-PCR步骤完成RT-PCR,电泳后用LAS-300成像仪扫描图像,对P-Erk1/2mRNA和P-Akt1mRNA进行半定量分析。6.实验数据用(?)±s表示,采用SPSS14.0软件行t检验分析,P<0.05认为有统计学意义。结果：3V和5V电刺激海绵体神经后SHR组ICP/MAP比值(0.26±0.06、0.28±0.04)均较WKY组(0.46±0.12、0.76±0.13)显著降低(P<0.05),P-ERK1、P-ERK2的mRNA和P-ERK1/2蛋白的相对表达量(积分光密度值IOD)在SHR组(0.81±0.05、0.91±0.06、47.22±10.05)较WKY组(0.42±0.04、0.68±0.14、7.49±1.45)显著升高(P<0.05)；P-Akt1的mRNA和P-Akt1蛋白的相对表达量在SHR组(0.90±0.05、11.17±2.21)与WKY组(0.92±0.06、10.91±1.86)无显著差异(P>0.05)。
结论：高血压性勃起功能障碍的发生与阴茎海绵体P-Erk1/2的过度表达有关,而与P-Akt1的表达水平无明显相关。
Object:To understand the expression of phospho-Erkl/2 and phospho-Aktl in corpus cavernosum between Spontaneous Hypertensive Rats(SHR) and normatensive rats. Methods:1.Experim-ental animal:Healthy adult male SHR and control group WKY each 8,14 weeks, weight 250～300g.2.Erectile functional examination:before anesthesia, rats weighing, to anesthetize the rats by pentobarbital sodium(30mg/Kg,intraperitoneal injection). Then, to expose the arteria carotis communis,the 30G pin which be filled with hepatinate puncture the arteria carotis communis, and connect the pressure translator, computer monitoring and recording MAP continuously. The rat abdominal midline incision, carefully under the microscope at 10 times free prostate on both sides of the rear to expose the pelvic ganglion, along the surface of prostate and rectum will denonvillies fascia between the cut, find the cavernous nerves in the dorsal prostate (corpus nerve, CN), cotton pad covered with warm saline, protection, maintenance of electrical stimulation used.After the penis exposed to the 24G needle filled with heparin saline into the right corpus cavernosum, connected to another pressure converters, continuous monitoring of intracavernosal internal pressure ((intracavernosal pressure, ICP).The basis of computer records a pressure ICP, will stainless steel electrodes placed in the cavernous nerve, applying different electrical stimulation (stimulation voltage was 3V,5V, frequency 12Hz, amplitude 5ms, sustained 30s～60s), continuous record of electrical stimulation ICP/MAP changes.3. draw the materials and preparation:After determination of erectile function, namely, near the corpus cavernosum penis root interception Department to PBs solution (0.01 moL/L, pH 7.4) washed clean of blood remaining after the urethra and penis glans removed parts of penis specimens divided into two parts, and immediately placed in EP tube, part of the neutral formalin-fixed for immunohistochemical analysis, part of the refrigerator at-70℃Ultra-low temperature preservation for RT-PCR experiments.4. Immunohistochemical determination of P-Erk1/2 and P-Aktl protein expression in rat corpus cavernosum:SP two-step method, penis specimens were embedded in paraffin after conventional treatment by immunohistochemical paraffin section, PBS wash 10min; 0.3% H2O2-methanol treatment 30min; pressure restoration 5min; 10% normal goat serum at room temperature closed 15min; drops plus an anti-in specimens,4℃overnight; PBS wash three times, each time 10 min; dropping the second antibody and incubated at room temperature 30min; DAB color.5. RT-PCR detect the expression of P-Erkl/2mRNA and P-Aktl mRNA in rat corpus cavernosum:Based on a small amount of tissue/cell total RNA extraction kit and molecular cloning manual Guide (Second Edition) to operate within the table in the corpus cavernosum tissue extract total RNA, stored at -80℃Ultra-low temperature refrigerator by equipment for use with the Nanodrop measurement of total RNA concentration, according to conventional RT-PCR steps to complete the RT-PCR, electrophoresis apparatus with a LAS-300 imaging scans, semi-quantitative analysis P-Akt1mRNA on P-Erk1/2mRNA. 6.Statistical Analysis:all datas were expressed as mean±standard error, statistical analysis was accomplished with SPSS 14.0 software, the two groups using t test, P<0.05 for the difference was significant. Results: Compared with the controls, ICP/MAP was lower significantly in the SHR group (P＜0.05), The expression of mRNA of P-ERK1, P-ERK2 and protein of P-ERK1/2 in the SHR group (0.81±0.05, 0.91±0.06 and 47.22±10.05, respectively) was significantly increased than that of in the WKY group (0.42±0.04,0.68±0.14 and 7.49±1.45, respectively) (P＜0.05). The level of mRNA of P-Akt and protein of P-Akt in the SHR group (0.90±0.05 and 11.17±2.21) was not significantly with that of in the WKY group (0.92±0.06 and 10.91±1.86). (P＞0.05).
Conclusion:The occurrence of erectile dysfunction in hypertension is in relation to the overexpressing of P-Erkl/2 in the cavernous tissues, but is not obvious correlation with the expressing of P-Aktl.
结论：高血压性勃起功能障碍的发生与阴茎海绵体P-Erk1/2的过度表达有关,而与P-Akt1的表达水平无明显相关。
Object:To understand the expression of phospho-Erkl/2 and phospho-Aktl in corpus cavernosum between Spontaneous Hypertensive Rats(SHR) and normatensive rats. Methods:1.Experim-ental animal:Healthy adult male SHR and control group WKY each 8,14 weeks, weight 250～300g.2.Erectile functional examination:before anesthesia, rats weighing, to anesthetize the rats by pentobarbital sodium(30mg/Kg,intraperitoneal injection). Then, to expose the arteria carotis communis,the 30G pin which be filled with hepatinate puncture the arteria carotis communis, and connect the pressure translator, computer monitoring and recording MAP continuously. The rat abdominal midline incision, carefully under the microscope at 10 times free prostate on both sides of the rear to expose the pelvic ganglion, along the surface of prostate and rectum will denonvillies fascia between the cut, find the cavernous nerves in the dorsal prostate (corpus nerve, CN), cotton pad covered with warm saline, protection, maintenance of electrical stimulation used.After the penis exposed to the 24G needle filled with heparin saline into the right corpus cavernosum, connected to another pressure converters, continuous monitoring of intracavernosal internal pressure ((intracavernosal pressure, ICP).The basis of computer records a pressure ICP, will stainless steel electrodes placed in the cavernous nerve, applying different electrical stimulation (stimulation voltage was 3V,5V, frequency 12Hz, amplitude 5ms, sustained 30s～60s), continuous record of electrical stimulation ICP/MAP changes.3. draw the materials and preparation:After determination of erectile function, namely, near the corpus cavernosum penis root interception Department to PBs solution (0.01 moL/L, pH 7.4) washed clean of blood remaining after the urethra and penis glans removed parts of penis specimens divided into two parts, and immediately placed in EP tube, part of the neutral formalin-fixed for immunohistochemical analysis, part of the refrigerator at-70℃Ultra-low temperature preservation for RT-PCR experiments.4. Immunohistochemical determination of P-Erk1/2 and P-Aktl protein expression in rat corpus cavernosum:SP two-step method, penis specimens were embedded in paraffin after conventional treatment by immunohistochemical paraffin section, PBS wash 10min; 0.3% H2O2-methanol treatment 30min; pressure restoration 5min; 10% normal goat serum at room temperature closed 15min; drops plus an anti-in specimens,4℃overnight; PBS wash three times, each time 10 min; dropping the second antibody and incubated at room temperature 30min; DAB color.5. RT-PCR detect the expression of P-Erkl/2mRNA and P-Aktl mRNA in rat corpus cavernosum:Based on a small amount of tissue/cell total RNA extraction kit and molecular cloning manual Guide (Second Edition) to operate within the table in the corpus cavernosum tissue extract total RNA, stored at -80℃Ultra-low temperature refrigerator by equipment for use with the Nanodrop measurement of total RNA concentration, according to conventional RT-PCR steps to complete the RT-PCR, electrophoresis apparatus with a LAS-300 imaging scans, semi-quantitative analysis P-Akt1mRNA on P-Erk1/2mRNA. 6.Statistical Analysis:all datas were expressed as mean±standard error, statistical analysis was accomplished with SPSS 14.0 software, the two groups using t test, P<0.05 for the difference was significant. Results: Compared with the controls, ICP/MAP was lower significantly in the SHR group (P＜0.05), The expression of mRNA of P-ERK1, P-ERK2 and protein of P-ERK1/2 in the SHR group (0.81±0.05, 0.91±0.06 and 47.22±10.05, respectively) was significantly increased than that of in the WKY group (0.42±0.04,0.68±0.14 and 7.49±1.45, respectively) (P＜0.05). The level of mRNA of P-Akt and protein of P-Akt in the SHR group (0.90±0.05 and 11.17±2.21) was not significantly with that of in the WKY group (0.92±0.06 and 10.91±1.86). (P＞0.05).
Conclusion:The occurrence of erectile dysfunction in hypertension is in relation to the overexpressing of P-Erkl/2 in the cavernous tissues, but is not obvious correlation with the expressing of P-Aktl.
引文
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[4]Jensen J, Axel L, Stimpel H. et al The prevalence and etiology of impotence in 101 male hypertens ve outpatients[J]. Am J Hypertens, 1999,12(3):271-275
[5]Dimmeler S, Fleming I, Fisslthaler B et al. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation[J]. Nature,1999,399:601-605.
[6]Hurt KJ, Musicki B, Palese MA,et al. Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection[J]. Proc Nail Acad Sci USA,2002,99(6):4061-4066.
[7]O'Donnell AB, Araujo AB, McKinlay JB. The health of normally aging men:The Massachusetts Male Aging Study (1987-2004)[J]. Exp Gerontol.2004 Jul;39(7):975-984.
[8]姜睿,刘军祥,殷民,等.原发性高血压与勃起功能的关系[J].泸州医学院学报,2007,30(1)：5-7.
[9]Jiang R, Chen JH, Jin J et al. Ultrastructural comparison of penile cavernous tissue between hypertensive and normotensive rats[J]. Int J Impot Res,2005,17(5):417-423.
[10]朱平宇,姜睿,邓青富,等.Rho激酶及血红素氧合酶在自发性高血压大鼠阴茎海绵体中的表达[J].中华男科学杂志,2008,14(3)：215-219.
[11]丁玉强,王亚奇,秦秉志,等.一氧化氮合酶在大鼠主盆神经及阴茎勃起组织中的分布[J].解剖学报,1994,25(3)：236-239。
[12]胡礼泉,张银高,镇万华,等.一氧化氮等神经递质与阴茎勃起关系的实验观察[J].中华外科杂志,1996,34(6)：377-379.
[13]Musicki B, Burnett AL. eNOS function and dysfunction in the penis[J]. Exp Biol Med,2006,231(2):154-165.
[14]Bivalacqua TJ, Musicki B, Usta MF, et al. Endothelial nitric oxide synthase gene therapy for erectile dysfunction [J]. Curr Pharm Des, 2005, 11(31):4059-4067.
[15]Staal S P. Molecular clonging of the Akt oncogene and its human homologues Akt1 and Akt2:amplification of Akt1 in a primary human gastric adenocarcinoma[J]. Proc Natl Acad Sci USA,1987,84: 5034-5037.
[16]Jones P F, Jakubowicz T, Pitossi F J, et al. Molecular cloning and identification of a serine/threonine protein kinase of the second- messenger subfamily[J], Proc Natl Acad Sci USA,1991,88:4171-4175.
[17]Fontana J, Fulton D, Chen Y, et al. Domain mapping studies reveal that the M domain of hsp90 severs as a molecular scaffold to regulate Akt-dependen phosphorylation of endothelial nitric oxide synthase and NO release. Circ Res.2002.90,866-873.
[18]Bernier S G, Haldar S, Michel T, et al. Bradykinin-regulated interactions of the mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase[J]. J Biol Chem,2000,275:30707-30715
[19]Rikitake Y, Hirata K, Kawashima S, et al. Involvement of endothelial nitric oxide in sphingosine-1-phosphate-induced angiogenes.Arterioscler Thromb Vasc Biol,2002,22(1):108-114.
[20]Babaei S,, Teiehert-Kuliszewska K, Zhang Q, et al. Angiogenic actions of angiopoietin-1 require endothelium-derived nitric oxide.. Am J Pathol,2003,162:1927-1936.
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