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1.
目的:探讨子痫前期血清对细胞滋养细胞-平滑肌细胞共培养体系平滑肌细胞凋亡的影响。方法:采用组织块贴壁法培养人早孕期细胞滋养细胞(CTB)和人脐动脉平滑肌细胞(HUASMC),传代后建立滋养细胞与人脐动脉平滑肌细胞共培养模型并分别加入正常和子痫前期患者静脉血清培养24h,并设立相对应的单培养组;Transwell小室检测CTB的侵袭能力;细胞计数试剂盒(CCK-8)测定HUASMC活力;Annexin V-FITC检测HUASMC凋亡率;RT-PCR检测滋养细胞FasL mRNA与HUASMC的Fas mRNA的表达;W estern blot检测HUASMC的Fas蛋白的表达。结果:子痫前期患者血清能显著降低共培养体系中CTB的侵袭能力,上调HUASMC活力,降低HUASMC早期凋亡率、FasmRNA和Fas蛋白的表达。结论:子痫前期血清可能通过降调细胞滋养细胞-平滑肌细胞共培养体系滋养细胞的侵袭能力,降调共培养体系HUASMC Fas的表达,从而抑制HUASMC凋亡。  相似文献   

2.
目的:探讨子痫前期血清对滋养-内皮细胞共培养体系滋养细胞侵袭力改变及对内皮细胞凋亡的影响.方法:选择正常足月妊娠剖宫产孕妇10例(正常对照组),子痫前期患者20例(子痫前期组).采用组织块贴壁法培养人孕早期细胞滋养细胞(CTB),胰酶消化法培养正常妊娠人脐静脉内皮细胞(HUVEC),传代后建立滋养细胞与脐静脉内皮细胞共培养模型,并分别加入正常对照组和子痫前期组患者静脉血清培养24小时;Transwell小室检测细胞滋养细胞的侵袭能力;流式细胞学检测人脐静脉内皮细胞凋亡率;逆转录-聚合酶链反应(RT-PCR)检测细胞滋养细胞MMP-2、MMP-9mRNA及人脐静脉内皮细胞自杀相关因子(Fas)mRNA的表达.结果:子痫前期患者血清共培养体系的细胞滋养细胞的侵袭能力低于正常对照组(P<0.01);人脐静脉内皮细胞凋亡率明显低于正常对照组(P<0.05);MMP-2、MMP-9和FasmRNA的表达明显下降(P<0.05).结论:子痫前期血清可能通过降调细胞滋养细胞MMP-2、MMP-9表达而影响滋养-内皮细胞共培养体系滋养细胞的侵袭能力,并且其滋养-内皮细胞共培养体的内皮细胞Fas的表达异常可能与内皮细胞凋亡下降有关.提示滋养细胞MMP-2、MMP-9、Fas/Fasl的表达异常可能参与了子痫前期子宫螺旋动脉重铸障碍的病理过程.  相似文献   

3.
目的:探讨子痫前期(PE)血清激活血管平滑肌细胞蛋白激酶C-α(PKC-α)及前胶原Ⅰ表达是否由磷脂酶C-γ1(PLC-γ1)信息分子介导。方法:原代培养脐静脉内皮细胞(HUVEC)与脐动脉平滑肌细胞(HUASMC),体外建立HUVEC与HUASMC共培养体系,用PE血清处理共培养HUASMC细胞。MTT法检测HUASMC细胞活力;Annexin VFITC-流式细胞仪检测HUASMC凋亡;Western blot法检测PLC-γ1(磷酸化)、PKC-α(包膜/胞浆比值)活性以及前胶原Ⅰ表达。siRNA沉默PLC-γ1表达后,观察PE血清对共培养HUVEC细胞PKC-α活性及前胶原Ⅰ表达的影响。结果:PE血清可促进共培养HUASMC细胞PLC-γ1磷酸化、PKC-α膜转位及前胶原Ⅰ的表达(P0.05);siRNA沉默HUASMC细胞PLC-γ1蛋白表达可逆转PE血清引起的PKC-α膜转位及前胶原Ⅰ的表达(P0.05)。结论:PE血清通过PLC-γ1-PKC-α信息途径促进HUASMC前胶原Ⅰ的表达,可能在胎盘血管病理过程中起着重要的作用。  相似文献   

4.
目的:探讨子痫前期子痫患者脐血清诱导脐动脉平滑肌细胞(HUASMC)前胶原I、Ⅲ的表达、NF-κB活性及维生素E对其的影响。方法:采用组织块培养法培养正常妊娠HUASMC,传代后待细胞长满至70% ~80%后,加或不加维生素E作用30min后分别加入子痫前期子痫患者脐静脉血清,培养2h,Westernblotting测定细胞胞浆I -κB、胞核NF κB含量;培养48h,MTT法测定细胞增殖活力,流式细胞学测定细胞周期,RT -PCR测定平滑肌细胞前胶原I、Ⅲ的表达。结果:子痫前期子痫患者脐静脉血清培养的HUASMC胞浆I- κB含量明显低于对照组,胞核NF- κB含量、细胞增殖活力、前胶原ImR NA的表达、S及G2 /M期细胞百分比明显高于正常妊娠组(P<0 .01),G0 +G1 期细胞百分比明显低于妊娠组(P<0. 01)。维生素E处理可以抑制子痫前期子痫患者血清引起的NF -κB核转位、细胞增殖活力、前胶原I的表达(P<0 .01),G0 +G1 期细胞百分比明显增加(P<0 .01)。结论:子痫前期子痫患者脐静脉血清HUASMC增殖、NF- κB核转位及I型胶原表达,VitE可抑制子痫前期子痫患者脐静脉血清诱导的HUASMCNF -κB核转位、细胞增生及I型胶原表达。  相似文献   

5.
目的:探讨子痫前期子痫患者脐血清诱导脐动脉平滑肌细胞(HUASMC)前胶原I、Ⅲ的表达、NF-κB活性及维生素E对其的影响。方法:采用组织块培养法培养正常妊娠HUASMC,传代后待细胞长满至70% ~80%后,加或不加维生素E作用30min后分别加入子痫前期子痫患者脐静脉血清,培养2h,Westernblotting测定细胞胞浆I -κB、胞核NF κB含量;培养48h,MTT法测定细胞增殖活力,流式细胞学测定细胞周期,RT -PCR测定平滑肌细胞前胶原I、Ⅲ的表达。结果:子痫前期子痫患者脐静脉血清培养的HUASMC胞浆I- κB含量明显低于对照组,胞核NF- κB含量、细胞增殖活力、前胶原ImR NA的表达、S及G2 /M期细胞百分比明显高于正常妊娠组(P<0 .01),G0 +G1 期细胞百分比明显低于妊娠组(P<0. 01)。维生素E处理可以抑制子痫前期子痫患者血清引起的NF -κB核转位、细胞增殖活力、前胶原I的表达(P<0 .01),G0 +G1 期细胞百分比明显增加(P<0 .01)。结论:子痫前期子痫患者脐静脉血清HUASMC增殖、NF- κB核转位及I型胶原表达,VitE可抑制子痫前期子痫患者脐静脉血清诱导的HUASMCNF -κB核转位、细胞增生及I型胶原表达。  相似文献   

6.
目的:探讨绒毛外滋养细胞基质金属蛋白酶-9(MMP-9)的表达对血管内皮细胞凋亡的影响。方法:MMP-9的siRNA转染绒毛外滋养细胞株(TEV-1),利用实时定量逆转录-聚合酶链反应(real time RT-PCR)和ELISA法检测转染前后细胞中MMP-9、FasL基因及蛋白表达的变化;通过trans well共培养技术,研究滋养细胞对内皮细胞凋亡的影响。结果:(1)MMP-9 siRNA转染后24h,滋养细胞MMP-9 mRNA的表达较对照组明显降低(P0.05),而FasL mRNA表达与对照组无明显差异(P0.05);转染后48h,细胞培养液中MMP-9蛋白及FasL蛋白表达均较对照组显著降低(P0.05);(2)与未转染者比较,转染MMP-9 siRNA的滋养细胞与内皮细胞共培养48h后,内皮细胞凋亡率显著降低,差异有显著性(P0.05)。结论:绒毛外滋养细胞表达MMP-9的水平影响其诱导内皮细胞凋亡的作用,此作用可能与MMP-9调节FasL蛋白的分泌表达有关。  相似文献   

7.
侯蕾  陈必良  赵尹霄  柏露 《生殖与避孕》2007,27(10):653-657
目的:探讨甲氧滴滴涕(MXC)对绒毛外滋养细胞侵袭能力的影响。方法:用胰蛋白酶消化后再经流式细胞仪分离得到人绒毛外滋养细胞进行原代培养及鉴定。细胞分为MXC(1μmol/L,3μmol/L、5μmol/L、7μmol/L)处理组及空白对照组,培养12h、24h和48h。用侵袭小室检测每组各时间点的侵袭能力,Western blot检测各组各时间点MMP-2、MMP-9、TIMP-2、TIMP-1表达,RT-PCR法检测MMP-9 mRNA、MMP-2 mRNA表达。结果:MXC处理组滋养细胞侵袭能力呈时间剂量依赖型下降,MMP-9 mRNA、MMP-2 mRNA表达下降,MMP-9、MMP-2表达下降,TIMP-2、TIMP-1表达增高。MMP-2及其mRNA下降程度与MXC有时间剂量效应。结论:MXC可通过改变基质金属蛋白酶及其抑制物的比例降低绒毛外滋养细胞侵袭能力。  相似文献   

8.
目的:探讨G-蛋白偶联受体30(GPR30)在妊娠期胎盘中的表达,以及其对人胎盘滋养细胞侵袭力的影响。方法:免疫组化法检测早孕期绒毛、正常产妇胎盘和重度子痫前期患者胎盘组织中GPR30表达。用17-β-雌二醇(17β-E2)、GPR30激动剂G1和阻滞剂G15预处理体外培养绒毛组织和人绒毛外滋养细胞株HTR8/SVneo。显微镜下观察体外绒毛组织滋养细胞生长侵袭范围,Transwell侵袭实验检测HTR8/SVneo细胞侵袭能力。免疫荧光法检测绒毛组织和HTR8/SVneo细胞中GPR30蛋白表达。Western blot法检测HTR8/SVneo细胞中GPR30和MMP-9蛋白表达。结果:GPR30在早期绒毛组织和正常末期胎盘滋养细胞上都有表达,且早孕期的表达水平高于正常末期胎盘,但在重度子痫前期胎盘上GPR30蛋白表达明显减少。E2及GPR30激动剂G1可增加滋养细胞的侵袭能力,阻滞剂G15则可下调其侵袭性;E2、G1可诱导滋养细胞GPR30蛋白表达上调,而G15则下调其表达。GPR30蛋白水平与侵袭相关蛋白MMP-9表达水平有相关性。结论:GPR30可能参与人类滋养细胞侵袭力的调节,对滋养细胞的侵袭力有正性促进作用。  相似文献   

9.
目的探讨低氧对子痫前期滋养细胞中可溶性血管内皮生长因子受体-1(sFlt-1)和缺氧抑制因子-1α(HIF-1α)的表达及滋养细胞凋亡和侵袭能力的影响。方法取正常妊娠及子痫前期孕妇终止妊娠后的胎盘绒毛组织,分离培养滋养细胞。分为:对照组(正常孕妇):21%O2浓度培养;子痫前期组:分别行21%O2浓度(21%O2组)和1%O2浓度(1%O2组)培养。采用细胞免疫荧光检测滋养细胞密度,实时定量-多聚酶链反应(real tima-PCR)和蛋白印记(Western Blot)法检测滋养细胞中sFlt-1和HIF-1αmRNA及蛋白的表达,TUNNEL法检测滋养细胞调亡,Transwell测定滋养细胞的侵袭能力。结果子痫前期21%O2组、1%O2组与对照组sFlt-1mRNA的表达分别为(2.87±1.94)ng/ml、(3.51±1.25)ng/ml和(1.93±0.77)ng/ml,HIF-1αmRNA的表达分别为(2.79±0.64)ng/ml、(1.77±0.23)ng/ml和(3.63±0.38),三组比较,差异均有统计学意义(P均〈0.01)。21%O2组、1%O2组与对照组sFlt-1蛋白分别为(4.99±1.77)μg/ml、(7.02±1.23)μg/ml和(3.83±0.63)μg/ml,HIF-1α蛋白分别为(3.83±0.63)μg/ml、(3.06±0.25)μg/ml和(7.73±1.02)μg/ml,三组比较,差异均有统计学意义(P均〈0.01)。21%O2组、1%O2组与对照组72h滋养细胞凋亡分别为(15.81±3.93)个、(20.18±3.47)个和(6.64±1.37)个,侵袭数目分别为(10.05±1.92)个、(18.65±4.17)个和(2.22±0.43)个,三组比较,差异均有统计学意义(P均〈0.01)。结论子痫前期和正常妊娠孕妇滋养细胞均对低氧敏感,低氧对滋养细胞sFlt-1和HIF-1α的表达有影响,且低氧条件下促使滋养细胞凋亡及侵袭能力强化。  相似文献   

10.
Fas/FasL是凋亡信号传导的一条途径.正常妊娠滋养细胞表达FasL诱导母体活化淋巴细胞凋亡是胎儿免疫耐受的重要机制.Fas/FasL在妊娠滋养细胞疾病中表达的研究刚起步.葡萄胎滋养细胞表达FasL引起蜕膜Fas+淋巴细胞凋亡促进免疫耐受和滋养细胞的存活.恶性滋养细胞Fas介导的凋亡敏感性降低及与FasL同时表达反击淋巴细胞可传授免疫赦免.Fas/FasL可能代表一个机制,通过这个机制恶性滋养细胞抵抗凋亡,逃避免疫监视和发生转移.  相似文献   

11.
利用体外培养系统,通过3H-TdR掺入和MTT法观察了甾体激素和血管活性物质内皮素(ET)与一氧化氮(NO)在培养的人子宫动脉平滑肌细胞(HUASMC)增殖中的作用。结果:雌激素对HUASMC的DNA合成和细胞增殖无影响。孕激素和ET-1可促进HUASMC DNA合成和细胞增殖,其作用呈剂量依赖性。孕激素可明显增强HUASMC对ET-1的增殖反应,NO供体硝普钠(SNP)抑制 HUASMC DNA合成和细胞增殖,其作用强度与浓度呈正相关。雌激素可增强SNP对HUASMC的抑制作用,孕激素无此作用。结果提示 ET-1和 NO在子宫血流调节和内膜血管重建中具有重要作用并受卵巢激素调节。  相似文献   

12.
IntroductionAging process has been related to erectile dysfunction (ED) possibly due to morphological changes in corpus cavernosum among many other causes.AimTo evaluate smooth muscle and collagen content in human corpus cavernosum and to correlate it to age.MethodsCadaveric human cavernosal tissue was collected during the period of 1 year. Morphological analysis of a whole corpus cavernosum was performed in tissue sections stained with Masson's trichromic method to differentiate smooth muscle (red) from collagen (blue) content.Main Outcome MeasuresAnalysis was performed with specialized micrographs image analysis software. Pearson's correlation test was used to establish correlation between corpus cavernosum morphology (smooth muscle and collagen content) and age.ResultsA total sample of 89 tissues from different male cadavers were analyzed. The average age of the sample was 49.2 ± 19.1 years, with a range between 14 and 90 years. There was a statistically significant inverse correlation between age and the percentage of smooth muscle content (P = 0.012), direct correlation between age and percentage of collagen content (P = 0.019), and inverse correlation between age and the ratio of smooth muscle : collagen content (P = 0.007).ConclusionsAge-related morphological changes in terms of smooth muscle and collagen content are observed in human corpus cavernosum as a possible contributing factor to the development of ED. Ferrer JE, Velez JD, and Herrera AM. Age-related morphological changes in smooth muscle and collagen content in human corpus cavernosum.  相似文献   

13.
缝隙连接是细胞间的直接通讯方式。缝隙连接蛋白43(CX43)是构成细胞缝隙连接的主要蛋白,在子宫平滑肌呈特异性表达,可调控子宫平滑肌细胞内信号通路,影响子宫收缩的协调性和节律性。就缝隙连接与CX43的关系、CX43在子宫平滑肌细胞内的表达及通过对其磷酸化位点的磷酸化作用调控子宫平滑肌细胞内信号通路等综述。探讨CX43如何调控子宫收缩协调性、同步性和极性,为早产和宫缩乏力性产后出血的预防和治疗提供理论支持。  相似文献   

14.
BackgroundStore-operated calcium entry and its key players, stromal interaction molecule (STIM) and Orai calcium channels, have been proposed as emergent therapeutic targets in cardiovascular pathophysiology. We hypothesize alteration of STIM/Orai signaling in erectile dysfunction (ED).AimTo evaluate the contribution of STIM/Orai to human penile tissue contraction and to analyze the influence of ED on STIM/Orai signaling at functional and expression levels in human penile vascular tissues.MethodsHuman penile resistance arteries (HPRA) and human corpus cavernosum (HCC) were dissected from cavernosal specimens from 30 organ donors without history of ED (No ED) and from 48 patients with ED undergoing penile prosthesis insertion and functionally evaluated in wire myographs and organ chambers, respectively. Expression of STIM-1, Orai1, and Orai3 in HCC was localized and quantified by immunofluorescence.Main Outcome MeasuresThe main outcome measures are functional responses in HCC and HPRA and STIM/Orai channel protein expression in human cavernosal tissue.ResultsInhibition of Orai channels with YM-58483 (20 μM) significantly reduced norepinephrine–induced contractions in both HCC and HPRA from either No ED or ED subjects, but the effects were more marked in ED (−20.1 ± 5.9% vs −45.5 ± 13.2% and −15.9 ± 4.0% vs −31.4 ± 6.9% reduction in Emax to norepinephrine in HCC and HPRA, respectively). Thromboxane-induced contractions were reduced and neurogenic contractile and relaxant responses modulated by Orai inhibition in penile tissues from patients with ED. In fact, addition of YM-58483 concentration dependently relaxed precontracted HPRA and HCC. These relaxations were significantly more pronounced in tissues from patients with ED (EC50 7.5 vs 1.3 μM and 10.5 vs 1.3 μM, for HCC and HPRA, respectively). All HCC specimens displayed expression of STIM-1, Orai1, and Orai3. Significantly increased expression of Orai1 and Orai3 but not STIM-1 was observed in patients with ED.Clinical TranslationInhibition of enhanced Orai activity in human penile vascular tissue could facilitate erectile responses, alleviating ED.Strengths and LimitationsEnhanced STIM/Orai activity contribution to penile smooth muscle tone in ED is demonstrated at functional and structural levels in human tissues from a representative sample of patients with ED and in comparison with healthy tissue. We cannot differentiate the specific contribution of risk factors associated with ED to hyperactivity of the Orai system.ConclusionsOrai channels significantly contribute to human penile smooth muscle contraction. Orai contribution to penile smooth muscle tone is functionally enhanced in ED accompanied by increased expression of Orai channels in cavernosal tissue. Orai inhibition could be a potential therapeutic strategy to reduce penile smooth muscle contraction in ED.Sevilleja-Ortiz A, El Assar M, García-Rojo E, et al. Enhanced Contribution of Orai Channels to Contractility of Human Penile Smooth Muscle in Erectile Dysfunction. J Sex Med 2020;17:881–891.  相似文献   

15.
The fetus depends upon adequate uteroplacental blood flow for normal growth and development. Inaccessibility of the fetus has made assessment of uteroplacental blood flow difficult. Doppler ultrasound of umbilical artery blood flow provides a noninvasive method for assessing pregnancies at risk for poor outcomes. This article describes blood flow studies using the Doppler ultrasound technique and provides related nursing implications.  相似文献   

16.
缝隙连接是细胞间的直接通讯方式。缝隙连接蛋白43(CX43)是构成细胞缝隙连接的主要蛋白,在子宫平滑肌呈特异性表达,可调控子宫平滑肌细胞内信号通路,影响子宫收缩的协调性和节律性。就缝隙连接与CX43的关系、CX43在子宫平滑肌细胞内的表达及通过对其磷酸化位点的磷酸化作用调控子宫平滑肌细胞内信号通路等综述。探讨CX43如何调控子宫收缩协调性、同步性和极性,为早产和宫缩乏力性产后出血的预防和治疗提供理论支持。  相似文献   

17.
目的:了解单脐动脉胎儿的转归,围生结局及处理方式。方法:回顾我院近4年170例单脐动脉胎儿的临床资料(经超声、MRI、羊水或脐血染色体检查、引产胎儿尸体解剖结果等),随访出生后婴儿情况并进行临床评估及分析。结果:单脐动脉合并其他结构畸形的发生率为49.3%,合并一种畸形的发生率为15.9%,合并多种畸形发生率为33.3%;单脐动脉胎儿合并染色体异常发生率为18.4%;行MRI检查37例,与超声结果相符29例,8例与超声结果有差异,与超声符合率为78.4%。结论:超声检查易早期诊断单脐动脉,单脐动脉具有胎儿畸形的高风险性。单脐动脉合并其他结构畸形者建议行胎儿染色体检查。超声、染色体检查诊断未发现合并畸形的胎儿出生后仍有异常可能。单脐动脉不是剖宫产指征。  相似文献   

18.
Placement of umbilical artery catheters was retrospectively reviewed in 181 newborns to evaluate random placement of catheter tip in the "high" position between T7 and T9 in the thoracic aorta of 127 infants and in the "low" position below 4 in the abdominal aorta of 54 infants. Group differences in gestational age, asphyxia, hypotension, respiratory disease, duration of catheterization, or infusate type were not significant. Cyanosis or blanching in the low extremities occurred in 67% of the "low" group and 21% of the "high" group ( P < .001). Hematuria occurred in 39% of the "low" group and 21% of the "high" group ( P < .05). High placement appears to have fewer complications. Prompt intervention by neonatal nurse practitioners can help reverse complications that occur during umbilical artery catheterizations.  相似文献   

19.
目的:探讨产前超声筛查胎儿单脐动脉声像图的特征及其临床意义。方法:选择2007年12月—2009年7月于天津市中心妇产科医院进行产前超声检查的38 215例患者,发现单脐动脉366例,其中经分娩或引产证实的40例住院患者获得全部胎儿单脐动脉的临床和超声资料。结果:胎儿单脐动脉检出率约0.96%(366/38 215),40例超声均诊断为胎儿单脐动脉,超声显示在胎儿盆腔膀胱及脐轮斜切面仅见膀胱一侧脐动脉走行。其中,超声发现合并胎儿其他系统发育异常5例而引产,因宫内死胎引产1例;继续妊娠并生产的34例中,有12例在妊娠期间和产程中出现异常,7例胎儿发现异常。结论:彩色多普勒超声检查能准确诊断胎儿单脐动脉,超声检查发现胎儿单脐动脉时需多角度动态观察以明确是否合并其他器官发育畸形,对于继续妊娠者需密切注意胎儿发育情况和产程。  相似文献   

20.
IntroductionRelaxation of cavernous smooth muscle cells (SMCs) is a key component in the control of the erectile mechanism. SMCs can switch their phenotype from a contractile differentiated state to a proliferative and dedifferentiated state in response to a change of local environmental stimuli. Proliferation and contraction are both regulated by the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are degraded by phosphodiesterases (PDEs). The most abundant PDE present in corpora cavernosa is the electrolytic cGMP‐specific phosphodiesterase type 5 (PDE5).AimWe investigated the cellular localization of PDE5 in in vitro cultured corpora cavernosa cells and the effect of mitogenic stimulation on PDE5 expression.MethodsBiochemical ad molecular techniques on cultured SMCs from human and rat penis.Main Outcome MeasuresWe studied the ability of the quiescent SMC phenotype vs. the proliferating phenotype in modulation of PDE5 expression.ResultsWe demonstrated that PDE5 is localized in the cytoplasm, in the perinuclear area, and in discrete cytoplasmic foci. As previously demonstrated in human myometrial cells, the cytoplasmic foci may correspond to centrosomes. In corpora cavernosa, PDE5 protein levels are strongly regulated by the mitotic activity of the SMCs, as they were increased in quiescent cultures. In contrast, treatment with platelet‐derived grow factor (PDGF), one of the most powerful mitogenic factors for SMCs, reduces the expression of PDE5 after 24 hours of treatment.ConclusionWe found that PDGF treatment downregulates PDE5 expression in proliferating SMCs, suggesting that PDE5 may represent one of the markers of the contractile phenotype of the SMCs of corpora cavernosa. Carosa E, Castri A, Forcella C, Sebastiani G, Di Sante S, Gravina GL, Ronchi P, Cesarini V, Dolci S, Di Stasi S, Lenzi A, and Jannini EA. Platelet‐derived growth factor regulation of type‐5 phosphodiesterase in human and rat penile smooth muscle cells. J Sex Med 2014;11:1675–1684.  相似文献   

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