阴蒂海绵体中磷酸二酯酶5表达及淫羊藿甙对cGMP浓度的影响

目的　探讨阴蒂海绵体中磷酸二酯酶 5表达及淫羊藿甙对cGMP浓度的影响。　方法　通过反转录聚合酶链反应技术 (RT PCR)检测兔阴蒂海绵体中PDE5mRNA的表达 ;12 5I放射免疫法测定不同浓度淫羊藿甙对阴蒂海绵体内PDE5酶底物cGMP浓度的影响 ,西地那非作为阳性对照 ,分别计算EC50 。　结果　阴蒂海绵体组织中有PDE5的表达 ,淫羊藿甙可明显提高阴蒂海绵体内的cGMP浓度 ,具有显著的浓度依赖性。　结论　淫羊藿甙对阴蒂海绵体平滑肌细胞内cGMP水平的影响可能是通过PDE5发挥作用 ,与NO cGMP信号通路有关。     

淫羊藿苷对破骨细胞活性的影响

目的:观察淫羊藿苷对破骨细胞骨吸收及凋亡的影响,探讨淫羊藿苷的抗骨质疏松作用机制。方法:体外分离、培养兔破骨细胞,与玻片及骨磨片共同培养,用10-7、10-6、5×10-6、10-5mol/L浓度的淫羊藿苷刺激破骨细胞,倒置相差显微镜下观察活体细胞、HE染色、TRAP染色及骨吸收陷窝甲苯胺蓝染色,鉴定破骨细胞,并进行骨吸收陷窝计数和面积测量,吖啶橙染色观察凋亡破骨细胞所占的比例。结果:与空白对照组比较,10-6、5×10-6、10-5mol/L浓度的淫羊藿苷组破骨细胞凋亡率均明显增高,骨吸收陷窝数目、面积明显减少,随浓度增加抑制作用增强,差异有显著性意义(P<0.05)。结论:淫羊藿苷可诱导破骨细胞凋亡,抑制骨吸收,并随浓度增加抑制作用增强。     

雌兔阴蒂海绵体平滑肌细胞的体外培养及生物学特性

目的 :探讨雌兔阴蒂海绵体平滑肌细胞的体外培养方法及其生物学特性。　方法 :采用酶消化法对阴蒂海绵体平滑肌细胞进行体外培养 ,倒置显微镜下观察细胞形态 ,计数细胞贴壁率及生长情况 ,用免疫组织化学方法鉴别培养的平滑肌细胞。　结果 :体外培养的细胞证实为兔阴蒂海绵体平滑肌细胞 ,梭形 ,呈长轴平行排列 ,贴壁快 ,生长迅速而平稳。　结论 :体外培养的阴蒂海绵体平滑肌细胞可在合适的条件下生长和传代 ,并能够保持稳定的生物学特性 ,可为阴蒂生理等方面的研究提供细胞材料     

淫羊藿次苷Ⅰ与其原型药物淫羊藿苷对rBMSCs成骨性分化的影响比较

目的观察淫羊藿次苷Ⅰ(IcarisideⅠ)与其原型药物淫羊藿苷(Icariin,ICA)对大鼠骨髓间充质干细胞(rat bone marrow stromal cells,r BMSCs)成骨分化过程中碱性磷酸酶(ALP)、骨钙素(BGP)蛋白表达量及ALP、BGP、Osterix和Runx-2 mRNA表达的影响。方法贴壁筛选法体外培养r BMSCs,以1×10-5mol/L的ICA和IcarisideⅠ对r BMSCs的成骨性分化进行药物干预。ELISA法检测ICA组、IcarisideⅠ组和空白对照组及转化液组之间ALP活性、BGP分泌量;RT Real-Time PCR法检测ALP、BGP、Osterix和Runx-2 mRNA基因表达。结果与空白对照组相比,IcarisideⅠ与其原型药物ICA均可显著增强r BMSCs的ALP活性,促进BGP的分泌,提高ALP、BGP、Osterix和Runx-2的mRNA水平(P0.01)。IcarisideⅠ与ICA组比较,其ALP、BGP蛋白表达量及基因表达明显高于ICA组(P0.01),Osterix和Runx-2的基因表达两组无显著性差异。结论 IcarisideⅠ也能促进r BMSCs成骨性分化,并且其活性高于原型药物ICA,也是淫羊藿发挥抗骨质疏松活性的主要成分。(2)提示补肾中药淫羊藿其补肾壮骨作用与促进ALP、BGP蛋白分泌量,上调ALP、BGP、Osterix和Runx-2的mRNA水平有关。     

兔阴蒂海绵体平滑肌细胞的体外培养方法及生物学特性

目的：探索新西兰白兔阴蒂海绵体平滑肌细胞的体外培养方法及生物学特性。方法：取兔阴蒂海绵体，采用酶消化法进行平滑肌细胞体外培养；在倒置显微镜下观察培养细胞的生长状况、形态特征及贴壁过程；用计数法测定细胞贴壁率；用MTT法描绘原代培养细胞的生长曲线；利用逆转录聚合酶链反应(RT—PCR)方法检测平滑肌细胞特征性标记物α-actin表达。结果：培养的兔阴蒂海绵体平滑肌细胞具有典型的平滑肌细胞形态特征，为梭形，呈长轴平行排列，具有明显的方向性；体外贴壁快，生长迅速，体外培养的阴蒂海绵体平滑肌细胞在合适的传代条件和比例下能够生存并保持其稳定的生物学特性。结论：体外培养的兔阴蒂海绵体平滑肌细胞模型可用于进一步研究阴蒂组织细胞学、分子生物学机制以及受体介导的平滑肌收缩信号转导机制及药理学作用等。     

淫羊藿提取物淫羊藿苷在细胞水平防治骨质疏松症的研究概况

随着我国人口老龄化加剧,骨质疏松症的患病率亦显著上升。近年来对传统补肾中药淫羊藿防治骨质疏松症的研究较多,研究发现淫羊藿单体淫羊藿苷具有较强的抗骨质疏松症活性,能够通过调节骨代谢来有效发挥抗骨质疏松的作用。笔者从淫羊藿苷对骨髓间充质干细胞、成骨细胞及破骨细胞的调节作用及相关信号通路来综述淫羊藿苷防治骨质疏松症的研究进展,旨在为淫羊藿苷的实验研究及临床应用提供思路及借鉴。     

淫羊藿苷对雌性大鼠阴道血流影响的研究

目的 研究淫羊藿苷对雌性大鼠阴道充血功能的作用.方法 15只健康成年雌性Wistar大鼠(200～250g)分为三组,5只/组,A组为生理盐水灌胃组,B组以淫羊藿苷2mg/kg灌胃给药,C组以西地那非2mg/kg灌胃给药.均间隔10min行盆神经阴道支电刺激,观察随时间变化的阴道血流变化,进行组间比较.结果 A组不同时间电刺激后阴道血流未见明显变化(P＞0.05).B组于60min起电刺激后阴道血流明显升高(P＜0.05),至90min阴道血流峰值达到最高点,然后开始缓慢下降,至120min回落至起始水平.C组(西地那非)40min起电刺激后阴道血流明显升高(P＜0.05),至70min阴道血流峰值达到最高点,然后开始缓慢下降,至110min回落至开始水平.比较最高点阴道血流峰值,淫羊藿苷组高于西地那非组(P＜0.05).结论 淫羊藿苷能够增强雌性性唤起功能大鼠模型阴道血流,增强其性唤起过程中阴道充血功能.     

淫羊藿苷对肝癌细胞株SMMC-7721增殖与凋亡的影响

目的:探讨淫羊藿苷对肝癌细胞株SMMC-7721的抑增殖和促凋亡作用及其机制。方法:以不同浓度(0,6.25,12.5,25,50,100,200,400,800μg/mL)的淫羊藿苷作用于SMMC-7721细胞株不同时间(24,48,72 h)后,用MTT法检测药物对SMMC-7721细胞增殖状态;以不同浓度淫羊藿苷作用SMMC-7721细胞48 h后,用流式细胞仪检测细胞周期及凋亡情况,用Western blot检测细胞PCNA,Bcl-2,Bax蛋白的表达。结果:与对照组(0μg/mL)比较,各浓度的淫羊藿苷均明显抑制SMMC-7721细胞的增殖,并呈明显的时间与浓度依赖性(均P<0.05);淫羊藿苷呈浓度依赖性诱导SMMC-7721细胞发生G0/G1期阻滞和凋亡,下调SMMC-7721细胞PCNA蛋白和Bcl-2蛋白,上调Bax蛋白的表达(均P<0.05)。结论:淫羊藿苷可抑制SMMC-7721细胞增殖并诱导其凋亡。     

淫羊藿不同提取物对去势大鼠PINP、NTx影响的实验研究

目的通过PINP、NTx指标的变化研究淫羊藿抗骨质疏松的作用机制。方法采用去卵巢方法诱发大鼠骨质疏松模型,淫羊藿不同提取物进行灌胃,检测PINP、NTx等相关指标的变化,评价淫羊藿抗骨质疏松的疗效。结果与空白对照组相比,模型组E2、PINP含量明显降低(P0.001),NTx水平升高(P0.001);与模型组比较,淫羊藿不同提取物各剂量组均可提高去势大鼠E2水平(P0.05),提高PINP水平(P0.05),降低NTx水平(P0.05);淫羊藿水煎液各组PINP和NTx含量高于其他各组,差异具有统计学意义。结论淫羊藿具有雌激素样作用,淫羊藿不同提取物均可改善去势大鼠骨质疏松症。     

氯化锂联合淫羊藿苷对成骨细胞增殖分化的影响

目的研究氯化锂(LiCl)与淫羊藿(ICA)苷联合应用对成骨细胞增殖、分化的影响,为LiCl与ICA联合应用提高成骨细胞矿化能力提供研究基础。方法采用酶消化法和茜素红染色法鉴定并获得大量细胞为成骨细胞,随机分为4组即空白组、ICA组(80 nmol/L), LiCl(5 nmol/L),ICA(80 nmol/L)+LiCl(5 nmol/L)组,诱导21 d后采用茜素红法及碱性磷酸酶活性测定各组成骨细胞矿化能力及活性。采用CCK-8和Western blot法测定各组对体外成骨细胞增殖活性及成骨细胞中p-GSK3β,β-catenin水平。结果倒置显微镜下观察细胞多为单核、多边形及梭形,局部有细胞密集的细胞团,茜素红染色将钙化结节染成橘红色; LiCl及ICA单独作用于成骨细胞时,都可以明显促进成骨细胞的增殖,增加ALP活性及矿化能力,同时明显促进p-GSK3β、β-catenin蛋白的表达;但是LiCl与ICA联合应用能够明显促进成骨细胞的增殖,并且在分子水平能够明显抑制p-GSK3β、β-catenin的表达,且差异具有统计学意义(P0.05)。结论 LiCl与ICA联合应用可以通过wnt信号通路促进成骨细胞的增殖、分化。     

体外分离培养兔阴茎海绵体平滑肌细胞的纯度分析

目的比较和评价不同条件下体外培养兔阴茎海绵体平滑肌细胞的纯度。方法应用胶原酶消化法和组织贴块法分离培养幼兔海绵体平滑肌细胞。差速贴壁法纯化海绵体平滑肌细胞。采用α-平滑肌肌动蛋白(α-SM-actin)和肌球蛋白(Myosin)免疫荧光鉴定海绵体平滑肌细胞,波形蛋白(Vimentin)衬染检测成纤维细胞。流式细胞仪检测海绵体细胞表达α-SM-actin的阳性率,比较不同培养条件下海绵体平滑肌细胞的纯度。结果体外培养的海绵体来源细胞在荧光显微镜下对α-SM-actin和Myosin仅有少量表达,随传代次数的增加,细胞表达α-SM-actin和Myosin的阳性率进一步降低。流式细胞仪检测酶消化法和贴块法原代培养的海绵体平滑肌细胞的纯度分别为16.91%和11.13%。经差速贴壁纯化后第1代海绵体平滑肌细胞的纯度分别提高到26.88%和21.98%。Vimentin免疫荧光检测到大量阳性表达。结论目前常规方法体外分离、培养的海绵体平滑肌细胞纯度较低,大量海绵体间质来源的成纤维细胞混杂生长,差速贴壁法可在一定范围内提高海绵体平滑肌细胞的纯度。     

Effect of the Bowman-Birk inhibitor (a soy protein) on in vitro bladder neck/urethral and penile corporal smooth muscle activity

AIMS: The Bowman-Birk inhibitor (BBI), is a serine protease inhibitor derived from soy beans, which is presently being evaluated in clinical trials for its ability to serve as a cancer preventive or anti-inflammatory agent. The form of BBI currently in clinical trials is known as Bowman-Birk inhibitor concentrate (BBIC). There have been anecdotal reports from patients of improved voiding and sexual functions in the ongoing BBIC trials. The objective of this study was to quantify the effect of BBI and BBIC on urethral and corporal smooth muscle activity. METHODS: In vitro muscle strip studies of New Zealand White rabbit urethra/bladder neck and penile corpora in the presence or absence of BBI or BBIC incubation (5 mg/mL) were performed. RESULTS: In dose-response curves to alpha stimulation, BBI mediated a shift to the right (decreased receptor sensitivity in bladder/urethra as well as corpora with no change in the maximal response). Bladder base/ urethra contraction by field stimulation was significantly inhibited by BBI at higher frequencies (1-32 Hz) (12.2 + 0.8 g vs. 6.3 + 0.75 g, P < 0.05). BBI inhibited field stimulated relaxation of corporal muscle at lower frequencies. Muscarinic contraction of the bladder neck/urethra in alpha prestimulated tissue was significantly inhibited by BBI (5.3 + 0.2 g vs. 2.7 + 0.1 g, P < 0.05). BBI has an inhibitory effect on alpha adrenergic dose-response curves in bladder neck/urethral and corpora smooth muscle. BBI also significantly inhibited neurohumoral cholinergic release and in vitro muscarinic contraction of the urethra. The effects on corpora relaxation were less pronounced. CONCLUSIONS: The data suggest that the phytochemical BBI may promote physiologic effects of urethral relaxation and improved voiding by unique mechanisms and deserves further study as a pharmacologic agent for lower urinary tract symptoms.     

Construction of cavernosum smooth muscle using umbilical artery smooth muscle cells seeded on acellular corporal collagen matrices

The objective of this study was to investigate the feasibility of tissue engineering of corpus cavernosal smooth muscle. Acellular corporal collagen matrices (ACCMs) were obtained from the penis of adult rabbits by a cell removal procedure. ACCMs were implanted into the back muscles of allogenic rabbits to investigate the resulting immunological reaction. Human umbilical artery smooth muscle cells (HUASMCs) were isolated from human umbilical arteries through explant techniques and expanded in vitro . Subsequently, third and fifth passage HUASMCs were seeded to ACCMs at a concentration of 30 × 10⁶ cells/mL. Then, seeded ACCMs were implanted subcutaneously in athymic mice. The implants were retrieved at 10, 20 and 40 days after implantation. Histochemistry, immunohistochemistry and scanning electron microscopy were performed to analyse the morphological characteristics of the engineered tissues. Additionally, organ bath studies were performed to address the contractility of the engineered tissues. The decellularization process successfully extracted all cellular components while preserving the original collagen fibers. The immunological reaction to ACCMs consisted of only a transient nonspecific inflammatory response. Light and scanning electron microscopy demonstrated that HUASMCs extended onto the three-dimensional ACCMs scaffolds in vitro . Histological analyses of the explants from all time points demonstrated a progressive regeneration of smooth muscle, with structures very similar to native corpus cavernosum smooth muscle. The maximum contraction force induced by phenylephrine and electrical stimulation were 3.64 ± 0.18 g/100 mg and 2.50 ± 0.21 g/100 mg, respectively. Our study demonstrates that HUASMCs can be seeded on three-dimensional ACCM scaffolds and will develop tissues similar to that of the native corpus cavernosum smooth muscle.     

兔血管平滑肌细胞和聚羟基丁酯(PHB)的细胞相容性实验研究

目的：探索血管平滑肌细胞和新型可降解材料聚羟基丁酯（PHB）的细胞相容性，为组织工程血管的构建寻找理想的支架材料。方法：将组织块法体上培养与兔血管平滑肌细胞种植在PHB膜片和PHB三维微孔支架上，在相差显微镜下观察细胞的粘附和生长情况。用MTT法测定细胞粘附率和细胞增殖指数，复合培养7天后进行扫描电镜观察并用流式细胞仪（FCM）的测定细胞周期，DNA指数。结果：兔血管平滑肌细胞在PHB膜片上粘附率为77％，细胞增殖符合细胞的生长曲线，在PHB三维微孔支架上生长情况良好，并被证实为二倍体细胞。结论：兔血管平滑肌细胞和聚羟基丁酯（PHB）的细胞相容性较好，但细胞与材料间的粘附有待进一步改善。     

依托咪酯对兔离体气管平滑肌收缩力的影响

目的：探讨依托咪酯对气管平滑肌收缩力的直接影响。方法：采用电刺激兔离体气管平滑肌并测量其张力。结果：接近于临床有效血浆浓度的依托咪酯即可显著抑制气管平滑肌的收缩,并呈剂量依赖性。结论：对有气管高反应的患者可安全地选用依托咪酯作为静脉全麻药,并有可能起到一定的治疗作用。     

兔阴茎海绵体平滑肌细胞的快速分离及鉴定

目的探讨兔阴茎海绵体平滑肌细胞快速分离方法,为应用膜片钳技术研究阴茎勃起机制提供实验材料.方法采用木瓜蛋白酶和胶原酶两步酶解消化法,快速分离出新西兰大白兔阴茎海绵体平滑肌细胞并应用免疫组化鉴定.结果分离的细胞成活率较高,贴壁呈长梭形,胞膜光滑完整,胞浆均匀,可用于膜片钳记录.免疫组化鉴定为兔阴茎海绵体平滑肌细胞.结论酶解消化快速分离兔阴茎海綿体平滑肌细胞为全细胞膜片钳技术研究阴茎勃起功能障碍的电生理机制提供了较好的实验材料.     

Comparison of rabbit corporal smooth muscle cell-culture Methods in vitro

Objective: To apply series or convenient and effective methods to produce large amount of pure corporal smooth muscle cells in vitro in terms of experimental requirement. Methods: The explant cell culture and digestion cell culture methods were compared. Results: The cell yield and purity were significantly higher in digestion cell culture compared with explant cell culture; cell growth velocity was more rapid in digestion cell culture also; but the cell culture success rates were higher in explant cell culture compared with digestion cell culture and the explant cell culture method was much easier to be mastered. Conclusions: Each method had individual merit and shortcoming. The best one of the two methods could be chosen according to experimental requirement.The SMC cultured in vitro are proved to be used to evaluate and investigate the effect of some medicine on penile erection.     

Human prostatic smooth muscle cells in culture: Estradiol enhances expression of smooth muscle cell-specific markers

Smooth muscle cells (SMCs) constitute a major cellular component of prostatic stroma. SMC tension plays an important role in urethral obstruction secondary to benign prostatic hyperplasia (BPH). We have developed an in vitro procedure for the propagation of human prostatic SMCs. Tissue specimens from patients undergoing radical prostatectomy or cystectomy were enzymatically disaggregated and cultured in MCDB-131 medium supplemented with horse serum, insulin, conditioned medium from the tumor cell line CRL-5813, and steroid hormones. The medium was assembled on the basis of the effects these supplements have on the growth of SMC cultures and on the expression of the two markers desmin and smooth muscle myosin. Addition of 0.1 μM of estradiol to the growth medium dramatically increased expression of these SMC-specific markers. Dihydrotestosterone (DHT) and hydrocortisone had a similar, albeit less pronounced effect. At three to five passages, about two thirds of the cells were immunohistologically positive for smooth muscle myosin or desmin. Almost all cells were positive for the myofibroblast marker smooth muscle α-actin throughout 10 passages and more. In SMC cultures, cells staining for smooth muscle myosin and desmin were found to seek direct contact to myofibroblasts. They grew in aggregates on a layer of myofibroblasts which adhered to the surface of the culture vessel. As revealed by transmission electron microscopy the cultured cells exhibited morphological features of myofibroblasts. Characteristics of smooth muscle cells, such as prominent bundles of microfilaments associated with dense bodies, basal laminae investing the cells, and numerous caveolae at the cell surfaces were regularly observed in cultures of low passages. After several passages, these features were markedly decreased and organelles of the biosynthetic system became more prominent. In summary, we present an in vitro model of prostatic SMCs and demonstrate that steroid hormones have characteristic effects on these cells. SMC cultures are expected to facilitate investigation of the functions and properties of human prostatic SMCs. Prostate 30:117–129, 1997. © 1997 Wiley-Liss, Inc.     

海绵体脱细胞基质和平滑肌细胞相容性的研究

目的探讨海绵体脱细胞基质（ACCM）与人脐动脉平滑肌细胞（HUASMCs）的相容性。方法以1％的Triton-X100与0．1％NH3H2O制备ACCM。异体肌肉内埋植实验评价ACCM的生物相容性。分离培养HUASMCs，噻唑蓝（MTT）法测定ACCM浸提液对HUASMCs增殖的影响。将3—5代的HUASMCs接种ACCM，共培养3、5、10d后观察HUASMCs与ACCM复合情况。结果ACCM无细胞残留，异体肌肉内埋植实验证实ACCM生物相容性良好。浸提液实验显示体外培养第4天和第6天，浸提液组A值明显高于对照组（P〈0．05）。HUASMCs能渗人ACCM内部，并分化形成平滑肌柬。结论ACCM与HUASMCs相容性良好，两者复合有望构建组织工程海绵体平滑肌。