中药益坎胶囊对动脉性勃起功能障碍大鼠阴茎海绵体平滑肌细胞表型转化的影响

目的:探讨中药益坎胶囊对动脉性勃起功能障碍(ED)大鼠阴茎海绵体平滑肌细胞表型转化的影响,以揭示益坎胶囊治疗ED的机制。方法:选取SD大鼠50只,其中10只设为正常组,另40只通过结扎双髂内动脉造成动脉性ED模型。造模后将其中10只设为模型对照组,其余30只灌胃给予ED治疗中药益坎胶囊,浓度分别为1.88、0.94、0.47 g/kg。给药1个月后,经腹腔注射阿朴吗啡,观察大鼠呵欠次数和阴茎勃起次数;切取大鼠的阴茎组织标本,通过免疫组化法观测海绵体平滑肌细胞收缩型性标志物碱性调宁蛋白(calponin 1)和合成型标志物骨桥蛋白(OPN)的表达,实验组结果与正常组和对照组进行比较。结果:注射阿朴吗啡后,正常组大鼠勃起次数为(4.48±1.25)次,模型对照组为(1.63±0.22)次,两组比较,差异有统计学意义(P0.01);给药后,中药益坎胶囊高、中、低剂量组大鼠勃起次数有明显改善[高剂量:(3.05±1.37)次;中剂量:(2.98±0.16)次;低剂量:(1.75±0.40)次]。免疫组化结果显示,模型对照组大鼠海绵体平滑肌细胞calponin 1表达减少,而OPN表达增多,与正常组相比差异有统计学意义(P0.05);与模型对照组相比,给药后,中药益坎胶囊高、中、低剂量组大鼠海绵体平滑肌细胞calponin 1表达增多,OPN表达减少。结论:中药益坎胶囊能改善动脉性ED大鼠的勃起功能,抑制海绵体平滑肌细胞表型由收缩型向合成型转化。     

阴茎海绵体平滑肌细胞表型转化及其影响因子的研究进展

阴茎海绵体平滑肌细胞是组成阴茎海绵体的主要功能成分,其表型转化是平滑肌细胞增殖和迁移的关键性起始步骤。因此,探讨平滑肌细胞表型转化的机制及其影响因子在阴茎勃起功能障碍的防治过程中具有重要意义。目前通常将平滑肌细胞分为收缩型(分化型)和合成型(未分化型、增殖型或去分化型)两种类型,并发现L转化生长因子(TGF-β)、转录因子E2F1、基本转录元件结合蛋白2(BTEB2)、胰岛素等因素可能影响平滑肌细胞表型转化。本文就近年来阴茎海绵体平滑肌细胞表型转化及其影响因子的研究进展作一简要综述。     

血小板源性生长因子-BB对SD大鼠阴茎海绵体平滑肌细胞表型转化影响的初步研究

目的:研究血小板源性生长因子-BB(PDGF-BB)对SD大鼠阴茎海绵体平滑肌细胞(CCSMC)表型转化的影响。方法:改良组织块法原代培养大鼠CCSMC,并进行细胞免疫荧光鉴定。实验分为空白对照组和不同浓度(5、10、20、40 ng/ml)PDGF-BB组,处理24 h;以及空白对照组和20 ng/ml PDGF-BB作用细胞不同时间(24、48、72 h)组,利用qRT-PCR检测各组细胞α平滑肌肌动蛋白(α-SMA)、平滑肌肌球蛋白重链(SMMHC)、碱性调宁蛋白和骨桥蛋白(OPN)mRNA的表达。结果:原代培养CCSMCα-SMA阳性细胞率达95%以上。与空白对照组比较,不同浓度PDGF-BB作用大鼠CCSMC后α-SMA、SMMHC、碱性调宁蛋白的mRNA表达显著减少(P均0.05);OPN mRNA表达水平显著上调(P0.05)。与空白对照组比较,随20 ng/ml PDGF-BB作用细胞时间的延长,α-SMA、SMMHC、碱性调宁蛋白的mRNA表达显著减少,OPN mRNA表达水平上调,差异均有统计学意义(P均0.05)。结论:PDGF-BB可促使SD大鼠CCSMC表型从收缩型向合成型转化。     

糖尿病性勃起功能障碍大鼠中低睾酮水平对阴茎海绵体平滑肌的影响

目的 观察糖尿病性勃起功能障碍(DMED)大鼠的睾酮水平变化对其阴茎海绵体平滑肌组织和超微结构的影响.方法 将3个月龄雄性Wistar大鼠20只随机等分为两组:正常对照组和DMED组,行放射免疫法测定其血清睾酮浓度和masson染色观察阴茎海绵体平滑肌(CCSM)密度以及通过透射电镜观察海绵体平滑肌细胞的超微结构.结果 DMED组大鼠的血清睾酮水平(17.445 ±2.540) nmol/L较正常组(338.457±63.802) nmol/L明显下降(P＜0.01);同时DMED组大鼠CCSM密度(5.640±0.597)％较正常对照组(19.890±1.957)％亦明显减少(P＜0.01),透射电镜下还观察到DMED组大鼠阴茎CSMC的核浆比增大,高尔基复合体明显肥大以及线粒体水肿.结论 睾酮水平下降可能导致DMED大鼠阴茎海绵体平滑肌发生病理变化.     

酶消化差速贴壁法分离培养老年ED大鼠阴茎海绵体平滑肌细胞的研究

目的 探索培养老年勃起功能障碍(ED)大鼠阴茎海绵体平滑肌细胞(SMCs)生物学特性稳定、细胞纯度高、简单易行的一种新方法. 方法 电生理仪检测24月龄雄性大鼠阴茎海绵体压力和平均动脉压比值(ICP/MAP)来评价阴茎勃起功能,从而选择老年ED大鼠.分别采用组织块法、酶消化法和酶消化差速贴壁法分离培养24月龄ED大鼠SMCs;采用存活率、生长曲线、相差显微镜和HE染色等,比较不同方法培养SMCs的生物学特性;免疫组化、免疫荧光和流式细胞仪通过检测α-SM-actin、Myosin和Desmin的阳性率来鉴定SMCs,并比较不同方法培养SMCs的纯度. 结果 ICP/MAP低于0.45入选老年ED大鼠组.细胞存活率提示酶消化法和组织块法存活率分别为93%和90%,酶消化差速贴壁法显著增加到99%(P＜0.05);生长曲线提示酶消化差速贴壁法细胞数量在第5～7天也显著增加(P＜0.05);酶消化差速贴壁法培养细胞在相差显微镜和HE染色下形态基本一致,呈梭形生长.免疫组化、免疫荧光和流式鉴定是SMCs,组织块法和酶消化法原代培养的SMCs纯度平均分别为45%和82%,酶消化差速贴壁法SMCs纯度显著增加到98%(P＜0.05).α-SM-actin、Myosin和Desmin在SMCs中有大量表达,在11代以内SMCs纯度维持较高水平(95%以上). 结论 酶消化差速贴壁法快速简便,可在一定范围内提高SMCs纯度;用此法培养的SMCs,生物学特性稳定,细胞纯度高,为建立SMCs细胞株奠定基础.     

红景天苷对低氧环境下大鼠阴茎海绵体平滑肌细胞α-肌动蛋白和骨桥蛋白表达的影响

目的:探讨红景天苷对低氧SD大鼠阴茎海绵体平滑肌细胞(CCSMC)α-肌动蛋白、骨桥蛋白(OPN)表达的影响。方法:体外培养SD大鼠CCSMC,免疫组化法鉴定细胞;设正常对照组(21%O2浓度)、低氧组(1%O2浓度)、低氧+红景天苷1 mg/L组、低氧+红景天苷3 mg/L组、低氧+红景天苷5 mg/L组、低氧+前列腺素E1(PGE1)0.4μg/L组,分别培养48 h;RT-PCR法分别测定各组α-肌动蛋白、OPN的相对表达量。结果:体外培养的CCSMC生长良好,抗平滑肌α-肌动蛋白单克隆抗体免疫组化染色阳性;与正常对照组比较,低氧组细胞的α-肌动蛋白表达量下降、OPN表达量升高(P<0.01);与低氧组比较,红景天苷5 mg/L组α-肌动蛋白表达量升高、OPN表达量降低(P<0.01),与PGE1作用相当(P﹥0.05)。结论:低氧可引起SD大鼠CCSMC收缩型标志物α-肌动蛋白表达降低,合成型标志物OPN表达升高,推测低氧可能引起CCSMC由收缩型向合成型转化。红景天苷能抑制低氧引起的CCSMCα-肌动蛋白表达降低、OPN表达升高,浓度为5 mg/L时与PGE1作用几乎相当。     

负压吸引对糖尿病性ED大鼠阴茎海绵体组织一氧化氮合酶表达的影响

目的研究负压吸引对糖尿病性勃起功能障碍（ED）大鼠阴茎组织一氧化氮合酶（NOS）表达水平的影响。方法25只实验鼠中随机选取5只为正常对照组（A组）,其余火鼠用链脲左菌素和阿朴吗啡诱导建立Ⅰ型糖尿病性ED大鼠模型。之后把造模成功的糖尿病性ED大鼠随机分成糖尿病ED吸引组（B组）和糖尿病ED非吸引组（C组）。在B组大鼠负压吸引治疗结束后将A、B、C3组大鼠处死并取阴茎组织进行石蜡包埋。采用免疫组织化学方法检测各组大鼠阴茎组织中三种一氧化氮合酶亚型（nNOS、eNOS、iNOS）的表达情况。结果A组大鼠阴茎组织中nNOS蛋白表达水平高于B组和C组（均P〈0.001）;A组和B组大鼠阴茎组织中eNOS蛋白表达水平高于C组（均P〈0.01）;A组iNOS蛋白表达水平低于B组和C组（P〈0.01,P〈0.001）,同时B组iNOS蛋白表达水平低于C组（P〈0.01）;剩余其他各组间的比较差异无统计学意义（P〉0.05）。结论负压吸引可以通过升高阴茎组织中的eNOS和降低iNOS的表达来改善勃起功能。     

缺氧性ED模型阴茎海绵体微结构改变的研究进展

缺氧是勃起功能障碍(ED)的独立危险因素,其导致ED的机制复杂多样,传统的研究更多关注阴茎海绵体内皮、体内性激素水平的改变,但近年来此方面的研究未有突破性进展。近期研究表明,阴茎海绵体微结构的改变,如收缩型阴茎海绵体平滑肌细胞表型转换、海绵体的纤维化可能是缺氧性ED发生的重要机制。而针对阴茎海绵体微结构改变的一些手段,如基因治疗、干细胞治疗、诱导细胞自噬等有可能为缺氧性ED的治疗带来广阔前景。     

高血压大鼠海绵体平滑肌细胞间连接的变化

目的:比较自发性高血压大鼠(SHR)和正常血压大鼠阴茎海绵体平滑肌细胞间连接改变及与阴茎勃起功能的关系。方法:注射阿朴吗啡(APO)观察14周龄SHR(SHR组,n=5)、W istar-Kyoto大鼠(WKY组,n=5)阴茎勃起情况,用透射电镜观察其阴茎海绵体平滑肌细胞间连接超微结构,RT-PCR测定海绵体平滑肌细胞Connexin 43的mRNA表达,免疫组化观察Connexin 43蛋白表达。结果:SHR组大鼠阴茎勃起次数明显低于WKY组(P<0.05),电镜发现SHR组大鼠阴茎海绵体平滑肌细胞间大量胶原纤维增生,Connexin 43蛋白及其mRNA表达较WKY组显著降低(P<0.05)。结论:高血压影响阴茎勃起功能,阴茎海绵体平滑肌细胞间连接的病理改变可能是高血压性勃起功能障碍的发病机制之一。     

利拉鲁肽对糖尿病大鼠阴茎海绵体eNOS表达的影响

目的:研究胰高血糖素样肽-1(GLP-1)类似物利拉鲁肽对糖尿病(DM)大鼠阴茎海绵体内皮型一氧化氮合酶(e NOS)表达的影响,探讨利拉鲁肽对DM勃起功能障碍(DED)大鼠勃起功能的作用。方法:取6周龄雄性SD大鼠,分为正常对照组(NC,n=10)与实验组(n=20),实验组构建DM大鼠模型,随机将实验组分为DM组(n=8)与GLP-1组(n=8)。干预12周后,检测各组空腹血糖(FPG)、空腹胰岛素(FINS)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、睾酮、白介素-6,计算胰岛素敏感指标Homa-IR与Homa-β,比较各组大鼠勃起功能;Western印迹检测各组大鼠阴茎海绵体组织Akt/p-Akt、e NOS/p-e NOS的表达。结果:DM组大鼠勃起次数(0.90±1.14)及勃起率(37.5%)较GLP-1组(2.90±1.53,25.0%)、NC组(4.20±1.05,100%)均明显减少(P0.05);GLP-1组大鼠勃起率及勃起次数亦少于NC组大鼠(P0.05)。免疫荧光染色提示e NOS主要表达在海绵体血管、血窦内皮细胞的细胞质中,DM组、GLP-1组e NOS蛋白表达水平显著低于正常对照组(P0.05),且GLP-1组明显高于DM组(P0.05)。DM、GLP-1组大鼠阴茎组织e NOS/p-e NOS表达水平较NC组明显下降(P0.01或0.05)。与DM组相比,GLP-1组大鼠阴茎组织p-e NOS表达水平明显升高(P0.05)。3组大鼠阴茎组织Akt比较无显著差异(P0.05)。DM、GLP-1组大鼠阴茎组织p-Akt表达水平较NC组明显下降(P0.01或0.05)。与DM组相比,GLP-1组大鼠阴茎组织p-e NOS表达水平明显升高(P0.05)。结论:GLP-1可能通过调节Akt/e NOS信号通路,保护阴茎海绵体组织内皮细胞功能,改善DED大鼠的勃起功能,提示GLP-1的使用可能是将来治疗和预防DED的重要方法之一。     

Myocardin restores erectile function in diabetic rats: phenotypic modulation of corpus cavernosum smooth muscle cells

This study aimed to investigate whether gene transfer of myocardin to the penis of diabetic rats can modulate corpus cavernosum smooth muscle (CCSM) cells phenotype and restore erectile function. Five normal control rats, and 22 diabetic rats were randomly divided into four groups: rats transfected with adCMV‐myocardin (N = 6), treated with empty vector (N = 6), injected with medium (N = 5), and sham‐operated rats (N = 5). The erectile response was measured 7 days after transfection. The percent of smooth muscle and the expressions of SMα‐actin, smooth muscle myosin heavy chain (SMMHC), calponin were evaluated. The increases in intracorporal pressure(ICP)/mean arterial pressure and total ICP in response to nerve stimulation in the adCMV‐myocardin treated rats were significantly greater than those in the empty vector (P < 0.001 and P < 0.001), medium only (P < 0.001 and P < 0.001), and sham‐operated rats (P < 0.001 and P < 0.001). The suppressed expressions of SMα‐actin, SMMHC and calponin were completely restored, and the amount of smooth muscle in diabetic rats were not restored after treatment. It is concluded that myocardin ameliorated erectile responses in diabetic rats mainly via promoting phenotypic modulation of CCSM cells from a proliferative to a contractile state.     

Human neural crest stem cells transplanted in rat penile corpus cavernosum to repair erectile dysfunction

OBJECTIVE

To investigate the feasibility of applying neural crest stem cells (NCSCs), with multipotent capacity, to repair injury in the penile cavernosum, the HNC10.K10 (K10) immortalized NCSC line was transplanted into the penile cavernosum of adult rats, as one of the causes of erectile dysfunction is damaged penile cavernous smooth muscle cells and sinus endothelial cells.

MATERIALS AND METHODS

The K10 human NCSC line was generated via transfection of primary cultured NCSC with a retroviral vector encoding v‐myc. K10 NCSCs were transplanted into the cavernosum of adult rats. The expression of cell type‐specific markers for endothelial cells (CD31 and von Willebrand factor), and specific markers for smooth muscle cells (smooth muscle cell actin, calponin, and desmin) was determined immunohistochemically in the penile cavernosum of rats 2 weeks after transplantation.

RESULTS

In the rat cavernosum, transplanted K10 NCSCs identified by human nuclear antigen labelling expressed cell type‐specific markers for endothelial cells (CD31 and von Willebrand factor), and specific markers for smooth muscle cells (smooth muscle cell actin, calponin, and desmin) 2 weeks after transplantation. Human NCSCs transplanted into the rat penile corpus cavernosum differentiated into endothelial cells or smooth muscle cells, as shown by their expression of cell type‐specific markers for the cell types.

CONCLUSION

It appears that NCSCs are an ideal cell source for reconstructing endothelial and smooth muscle cells in the corpus cavernosum in cell therapy for patients with erectile dysfunction.     

Effect of tankyrase 1 on autophagy in the corpus cavernosum smooth muscle cells from ageing rats with erectile dysfunction and its potential mechanism

This study compared tankyrase 1 expression and autophagy quantity between erectile dysfunction （ED） and non-ED rats＇ corpus cavernosum smooth muscle cells （CSMCs）. This study aslo explored the effect and possible mechanism of tankyrase 1 on autophagy and cell proliferation in ageing ED rats＇ CSMCs. The intracavernous pres- sure and mean systemic arterial pressure were measured to investigate erectile function so that eight 24-month-old ED and eight 8-month-old male Wistar rats were choosed respectively. The rat CSMCs were isolated and cultured by enzyme digestion, in which tankyrase 1 expression and autophagy quantity were compared. Tankyrase 1 over-expression was induced with plasmid transfection by Lipofectamine^TM. The effect of tankyrase 1 overexpression on proliferation, autophagy and mTOR pathway in 24-month-old ED rats＇ CSMCs was measured by the cell growth curve in MTT assay, cell cycle analysis in flow cytometry （FCM）, key protein expression in Western blot, autophagy quantity in transmission electron microscopy, monodansylcadaverine staining and GFP-LC3 fluorescence. The primary CSMCs were confirmed by immunofluorescence, and the purity was 99.1% in FCM. Compared with that of 8-month-old rats, tankyrase 1 expression and autophagy quantity significantly decreased in 24-month-old ED rats＇ primary CSMCs （P 〈 0.01）. Tankyrase 1 overexpression significantly increased the growth rate （P 〈 0.05） and increased the S phase of cell cycle （P 〈 0.01）. The autophagosome quantity was remarkably increased （P 〈 0.01）, LC3-Ⅰ/Ⅱ and Beclin 1 were upregulated （P 〈 0.01 and P 〈 0.05）, and p-p70S6K （Thr389） was downregulated in 24-month-old ED rat CSMCs （P 〈 0.05）. In conclusion, Tankyrase 1 and autophagy decrease in the CSMCs from aging rats with ED, and tankyrase 1 may have a positive effect on proliferation by enhancing autophagy and regulating the mTOR signalling pathway.     

低氧对SD大鼠阴茎海绵体平滑肌纤维化的影响

目的:探讨低氧对SD大鼠阴茎海绵体平滑肌纤维化的影响。方法:体外培养阴茎海绵体平滑肌细胞,免疫组化鉴定细胞;常规氧浓度(21%O2浓度)分别培养12、24、48、72h作为对照,低氧(1%O2浓度)干预12、24、48、72h,RT-PCR分别测定各组TGF-β1、Ⅰ型胶原、Ⅲ型胶原的相对表达量。结果:体外培养的阴茎海绵体平滑肌细胞生长良好,抗平滑肌α-肌动蛋白单克隆抗体免疫组化染色阳性;RT-PCR结果提示TGF-β1、Ⅰ型胶原、Ⅲ型胶原的相对表达量在48h内与低氧时间成正相关,时间进一步延长不能增加其相对表达量。结论:在低氧环境下,SD大鼠阴茎海绵体平滑肌细胞的TGF-β1、Ⅰ型胶原、Ⅲ型胶原的相对表达量随时间的延长逐渐增加,48h达到最大值。低氧可导致SD大鼠阴茎海绵体平滑肌纤维化。     

粉防己碱(Tet)对兔子阴茎海绵体平滑肌细胞质中自由钙离子浓度的影响(英文)

目的:探讨粉防己碱(tetrandrine,Tet)松弛阴茎海绵体平滑肌的作用机制。方法:体外培养新西兰白兔阴茎海绵体平滑肌细胞,经钙荧光指示剂 Fluo-2/AM 负载后,用荧光离子数字成像系统观察 Tet 对平滑肌细胞内[Ca~(2 )]_i 的影响。结果:Tet(1,10,100μmol/L)对平滑肌细胞内静息[Ca~(2 )]_i无明显影响(P>0.05)。当细胞外钙离子浓度为2.5 mmol/L 时,Tet(1μmol/L,10 μmol/L,100 μmol/L)抑制了高钾和去氧肾上腺素(PE)导致的细胞内[Ca~(2 )]_i 升高(P<0.05),这种抑制作用具有浓度依赖性。在无细胞外钙时,1 μmol/L和10μmol/L Tet 对 PE 引起的细胞内[Ca~(2 )]_i 升高无明显影响(P>0.05);而100 μmol/L Tet 能明显抑制 PE 引起的细胞内[Ca~(2 )]_i 升高(P<0.05)。结论:Tet 通过阻滞电压依赖性钙通道、α_1受体依赖性钙通道和抑制细胞内钙库释放,降低阴茎海绵体平滑肌细胞内[Ca~(2 )]_i 水平,这是 Tet 松弛阴茎海绵体平滑肌的作用机制之一。     

阴茎海绵体平滑肌细胞外钙离子浓度在体检测与神经性ED的相关研究

目的 通过钙离子传感针在体检测阴茎海绵体平滑肌细胞外的钙离子浓度,探讨其与勃起功能障碍(ED)的相关性.方法 40只雄性SD大鼠随机分为损伤组(20只)和对照组(20只),通过剪断双侧海绵体神经制作神经性ED模型,1个月后应用钙离子传感针在体检测大鼠阴茎海绵体平滑肌细胞外的钙离子浓度,比较两组间钙离子浓度的差异.结果 损伤组(ED组)钙离子浓度(电位)为(-0.31±0.35)mV,对照组钙离子浓度(电位)为(0.17±0.34)mV,两组钙离子浓度差异具统计学意义(P<0.05).结论 钙离子传感针可以作为ED在体检测的一种新方法.     

糖尿病性勃起功能障碍相关因素分析

目的 研究糖尿病性勃起功能障碍(ED)的发病情况及其影响ED程度的相关因素。 方法 调查90例男性2型糖尿病病人的性功能状态、年龄、糖尿病病程、测量血压,同时测定其糖化血红蛋白(HbA1C)血脂等指标。结果 糖尿病病人中ED患病率为75.6%(68/90),患病率随年龄的增加而增加,在非ED组与不同程度ED组之间,年龄存在非常显著差异(P<0.01),DM病程、HbA1C存在显著差异(P<0.05)结论 糖尿病病人ED患病率高,糖尿病性ED的程度随年龄、DM病程、HbA1C的增加而加重,长期良好的血糖控制有助于延缓糖尿病性ED的加重。     

Characterization of corpus cavernosum smooth muscle cell phenotype in diabetic rats with erectile dysfunction

Phenotypic modulation from a contractile to a proliferative state within vascular smooth muscle cells has a critical role in the pathogenesis of a variety of cardiovascular diseases. To investigate the characterization of corpus cavernosum smooth muscle cell phenotype in diabetic rats with erectile dysfunction, a group of Sprague-Dawley rats (n=30) were induced by intraperitoneal injection of streptozotocin (60?mg?kg(-1)) and screened by subcutaneous injection of apomorphine (100?μg?kg(-1)) for the measurement and comparison of the penile erections, and then three different groups were defined. Primary corpus cavernosum smooth muscle cells were cultured and passaged. The cavernous tissue segments were subjected to quantitative real-time polymerase chain reaction to determine the expressions of smooth muscle α-actin (SMA), SM myosin heavy chain (SMMHC), smoothelin, calponin and myocardin. Cell contractility in vitro and western blot analysis of SMA and SMMHC in the cavernous tissues and cells were determined. Compared with the control group (n=8) and the diabetes mellitus group (n=5), the expressions of SMA, calponin, SMMHC, smoothelin and myocardin mRNA were decreased in the cavernous tissues in rats of the diabetic erectile dysfunction group (n=15; P=0.001 and 0.02, P=0.014 and 0.012, both P<0.001, P=0.005 and <0.001, P=0.003 and 0.035, respectively). The levels of SMA and SMMHC proteins showed a significant decrease in cavernous tissues and cultured cells in rats of the diabetic erectile dysfunction group. Cells of the diabetic erectile dysfunction group exhibited significantly less contractility compared with those of other groups (P<0.001). Corpus cavernosum SM cell possesses the ability to modulate the phenotype under hyperglycemic conditions, which could have a key role in the pathogenesis of diabetic erectile dysfunction.     

实验性高脂免体外培养阴茎海绵体平滑肌细胞凋亡的研究

目的观察高脂诱导的血管性ED家兔阴茎海绵体平滑肌(Corpus Cavernosum Smooth Muscle,CCSM)细胞形态、结构变化及高脂对CCSM细胞凋亡、增殖的影响。方法雄性新西兰家兔10只,随机分为模型组和对照组,每组各5只。模型组为采用经高脂、高胆固醇饮食结合球囊导管扩张双侧髂内动脉建立、鉴定的血管性ED模型;对照组喂以正常饮食。8周后切取阴茎CCSM组织观察并做体外细胞培养。经处理后分别在光镜、透射电镜下观察,流式细胞仪检测细胞凋亡,MTT比色法观察细胞增殖活性,RT-PCR法研究Caspase-3 mRNA的表达情况,免疫荧光法检测Caspase-3活性。结果与对照组相比,模型组光镜和电镜下CCSM细胞内含较多脂质囊泡,线粒体、内质网等细胞器出现损伤,凋亡小体明显增多。流式细胞仪检测证实模型组CCSM细胞凋亡率较对照组明显增高(1.44±0.20%Vs5.96±1.60%,P<0.01)。MTT比色法显示高脂对CCSM的增殖有明显抑制作用(模型组与对照组A值分别为:0.52±0.08Vs1.42±0.04,P<0.01)。模型组的Caspase-3 mRNA表达和活性值均显著高于对照组(0.74±0.00Vs0.65±0.00,P<0.01;活性值0.03±0.00Vs0.01±0.00,P<0.05)。结论高脂、高胆固醇饮食可严重影响CCSM结构,促进CCSM细胞凋亡并抑制其增殖。CCSM细胞凋亡/增生的失衡可能是高脂导致ED发生发展的重要病理环节。