siRNA敲减整合素连接激酶影响人阴茎海绵体平滑肌细胞功能的体外研究

目的 探讨整合素连接激酶( ILK)基因对人海绵体平滑肌细胞收缩和舒张功能的影响.方法 人海绵体平滑肌细胞为实验对象,分为非沉默组、非处理对照组和ILK小干扰RNA(siRNA)实验组,采用siRNA干扰技术下调细胞中ILK的表达,伸展实验和基质胶黏附实验分别检测ILK基因敲减前后海绵体平滑肌细胞伸展和黏附能力变化,Transwell小室观察ILK表达对海绵体平滑肌细胞迁移能力的影响,荧光标记的鬼笔环肽染色海绵体平滑肌细胞观察ILK基因敲减前后细胞 骨架的变化.结果 黏附实验中,非沉默siRNA组、非处理对照组和实验组siRNA干扰的平滑肌细胞吸光度(A)值分别为0.184±0.034、0.179±0.028和0.092±0.010;迁移实验中,3组穿过Matrigel胶包被的Transwell小室的平滑肌细胞A值分别为0.184±0.017、0.188 ±0.013和0.106±0.003,实验组与非沉默siRNA组比较差异有统计学意义(P＜0.05);非处理对照组平滑肌细胞应力纤维错综分布于细胞周边与胞质内,而实验组的细胞应力纤维仅分布于细胞边缘.结论 ILK下调降低人海绵体平滑肌细胞的黏附、伸展、迁移能力,并通过影响细胞骨架重构参与调节平滑肌收缩与舒张状态,提示ILK基因有可能成为勃起功能障碍基因治疗的有效靶点.     

携带ROCK2基因siRNA有效片段的慢病毒载体的筛选

目的:筛选能够显著抑制自发性高血压大鼠(SHR)阴茎海绵体平滑肌细胞内ROCK2基因表达的携带ROCK2基因siRNA的慢病毒载体。方法:设计并合成4个靶向ROCK2的siRNA片段,构建并包装成慢病毒载体。随机将5只SHR阴茎海绵体平滑肌细胞分为6组,每组每个样本3×104个细胞,每组5个样本,分别为:A组(未转染对照组)、B组(携带慢病毒转染组)、C~F组(分别携带靶向ROCK2基因siRNA 1~4号靶点的慢病毒转染组),以感染复(MOI)=80转染SHR阴茎海绵体平滑肌细胞,转染后48 h荧光显微镜下观察细胞GFP表达情况,并用RT-PCR检测各组被转染细胞ROCK2 mRNA的表达。结果:荧光显微镜下观察各组细胞转染效率均50%。与A组相比,B组ROCK2 mRNA的表达无明显改变(P﹥0.05);C、D、F组SHR阴茎海绵体平滑肌细胞ROCK2基因mRNA的表达较A组极显著下降(P0.01),抑制效率分别达到(43.91±8.19)%、(47.15±6.64)%、(25.7±6.03)%;E组SHR阴茎海绵体平滑肌细胞ROCK2基因mRNA的表达较A组显著下降(P0.05),抑制效率为(16.81±5.94)%。结论:本研究构建的4种携带ROCK2基因的siRNA慢病毒载体均能够显著抑制SHR阴茎海绵体平滑肌细胞内ROCK2基因的表达,其中有1种慢病毒载体抑制作用最强。     

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.     

小干扰RNA抑制人阴茎海绵体平滑肌细胞间隙连接蛋白connexin43的表达

目的:利用小干扰RNA(siRNA)抑制人阴茎海绵体平滑肌细胞间隙连接蛋白connexin43(Cx43)的表达和检测细胞间间隙连接通讯功能,探讨该技术在阴茎海绵体平滑肌细胞间隙连接和阴茎勃起功能研究中的应用。方法:利用Ambion公司设计软件,构建靶向人Cx43基因的siRNA重组质粒,转染人阴茎海绵体平滑肌细胞48h后,逆转录-聚合酶链反应(RT-PCR)和Western印迹检测Cx43基因和蛋白的相对表达水平、划痕标记荧光染料传输技术检测细胞间间隙通讯功能,并分别与siRNA阴性对照、空白对照组比较。结果:酶切和测序证实siRNA真核表达载体构建成功。siRNA重组质粒转染细胞后的Cx43 mRNA和蛋白相对表达水平分别为(0.45±0.08)%、(0.56±0.06)%,与siRNA阴性对照组(0.72±0.04)%、(0.80±0.08)%和空白对照组(0.74±0.09)%、(0.77±0.11)%相比,差异均有显著性(P(0.05);转染后的细胞间间隙连接通讯功能也显著降低。结论:siRNA能有效抑制人阴茎海绵体平滑肌细胞Cx43的表达和阻断间隙连接介导的间隙连接通讯功能。     

PDE5基因siRNA对人阴茎海绵体平滑肌细胞cGMP的影响

目的:观察PDE5基因小干扰RNA(siRNA)对人阴茎海绵体平滑肌细胞环磷酸鸟苷(cGMP)的影响,为阴茎勃起功能障碍(ED)的基因治疗提供实验依据。方法:使用美国Amb ion公司提供的设计软件设计并合成人PDE5基因的siRNA序列,合成3对PDE5 siRNA和1对阴性对照siRNA,转染人阴茎海绵体平滑肌细胞,同时设定空白转染组为对照组。以酶联免疫法分别检测转染后不同时间(24、48、72、96 h)点海绵体平滑肌细胞内cGMP浓度变化,观察PDE5 siRNA对海绵体平滑肌细胞内cGMP的影响。结果:siRNA1、siRNA2和siRNA3转染后人阴茎海绵体平滑肌细胞cGMP水平显著高于阴性对照siRNA组和空白对照组(P<0.05),在转染后72 h最为显著,siRNA1、siRNA2、siRNA3、阴性对照siRNA和空白对照组cGMP测定值分别为:5.89±0.19、3.52±0.16、2.88±0.08、0.72±0.12、0.60±0.16 pmol/m l,siRNA1组明显高于siRNA2、siRNA3组(P<0.05)。结论:体外化学合成的PDE5 siRNA能有效地增加海绵体平滑肌细胞内cGMP的水平,不同序列siRNA增加cGMP水平的能力不同,为ED的基因治疗提供了新思路。     

靶向人PDE5A3基因的shRNA对人阴茎海绵体平滑肌细胞cGMP的影响

目的:观察腺病毒载体介导特异性短发夹RNA(shRNA)对人阴茎海绵体平滑肌细胞环磷酸鸟苷(cGMP)的影响,为应用RNA干扰(RNAi)技术治疗阴茎勃起功能障碍(ED)提供实验依据。方法:成功构建携带3条针对人PDE5A3基因位点特异性shRNA的重组腺病毒(rAd5-shRNA-PDE5A3),并设立阴性对照病毒组和空白对照组,分别转染人阴茎海绵体平滑肌细胞。以放射免疫法分别检测转染重组腺病毒24、48、72h后细胞内cGMP浓度变化,观察rAd5-shRNA-PDE5A3对海绵体平滑肌细胞内cGMP的影响。结果:实验组rAd5-shRNA-PDE5A3转染人阴茎海绵体平滑肌细胞后胞内cGMP水平显著高于阴性对照组和空白对照组,在转染后72h最为显著。结论:3条特异性针对人PDE5A3mRNA靶位点的shRNA可有效地增加海绵体平滑肌细胞内cGMP的水平,增强对PDE5基因的阻抑效果。     

Expression of functional prostaglandin D (DP) receptors in human corpus cavernosum smooth muscle

Prostaglandin D(2) (PGD(2)) binds to specific G-protein coupled receptors (DP) and induces smooth muscle relaxation by stimulating the synthesis of intracellular cAMP. In this study, we examined the role of PGD(2) and DP receptors in regulating human penile smooth muscle contractility. We determined that human corpus cavernosum tissue and smooth muscle cells in culture expressed functional DP receptor and lipocalin-like prostaglandin D synthase by reverse-transcribed polymerase chain reaction (RT-PCR). Functional PGD synthase activity was confirmed by the synthesis of PGD(2) in human corpus cavernosum smooth muscle cells upon addition of exogenous arachidonic acid. Organ bath preparations of human corpus cavernosum tissue strips, contracted with phenylephrine, relaxed in a dose-dependent fashion to either PGD(2) or the DP selective agonist BW245C. Cultures of human corpus cavernosum smooth muscle cells treated with BW245C showed a two-fold increase in cAMP synthesis. These data are consistent with the expression of functional DP receptors in human corpus cavernosum. This suggests the presence of an intact prostanoid autocrine system that may play a role in regulating penile erectile function.     

Abnormal lipid metabolism down‐regulates adenosine triphosphate synthase β‐subunit protein expression in corpus cavernosum smooth muscle in vitro and in vivo

Metabolic syndrome is closely related to erectile dysfunction (ED), and hyperlipidaemia is considered a major risk factor for ED. Adenosine triphosphate (ATP) synthase is believed to play an important role in metabolic syndrome; it has been hypothesised that ATP synthase contributes to ED development. We have verified this hypothesis using primary cultured human corpus cavernosum smooth muscle (HCCSM) cells treated with excessive free fat acid (FFA) and a high‐fat diet (HFD) mouse model. Our results showed that high fatty factors could cause lipid accumulation in HCCSM cells, which could result in abnormal lipid metabolism, such as high levels of triglycerides, cholesterol and glucose in the HFD mice. There was a remarkable down‐regulation of ATP synthase and p‐Akt after in vivo and in vitro excessive FFA treatments. These results indicated that abnormal lipid metabolism could induce ATP synthase down‐regulation via the Akt phosphorylation pathway and that ATP synthase may be a target of lipotoxicity in corpus cavernosum smooth muscle cells.     

靶向人PDESA3基因的shRNA对人阴茎海绵体平滑肌细胞cGMP的影响

目的：观察腺病毒载体介导特异性短发夹RNA（shRNA）对人阴茎海绵体平滑肌细胞环磷酸鸟苷（cGMP）的影响,为应用RNA干扰（RNAi）技术治疗阴茎勃起功能障碍（ED）提供实验依据。方法：成功构建携带3条针对人PDESA3基因位点特异性shRNA的重组腺病毒（rAd5-shRNA-PDE5A3）,并设立阴性对照病毒组和空白对照组,分别转染人阴茎海绵体平滑肌细胞。以放射免疫法分别检测转染重组腺病毒24、48、72h后细胞内cGMP浓度变化,观察rAd5-shRNA-PDE5A3对海绵体平滑肌细胞内cGMP的影响。结果：实验组rA（15-shRNA-PDE5A3转染人阴茎海绵体平滑肌细胞后胞内cGMP水平显著高于阴性对照组和空白对照组,在转染后72h最为显著。结论：3条特异性针对人PDE5A3mRNA靶位点的shRNA可有效地增加海绵体平滑肌细胞内cGMP的水平,增强对PDE5基因的阻抑效果。     

Effects of sense tankyrase transfection on smooth muscle cells of corpus cavernosum in rat

This study was conducted to construct the eukaryotic expression vectors for sense tankyrase and to investigate the effects of tankyrase transfection on smooth muscle cells of corpus cavernosum in the rat. After the eukaryotic expression vectors for sense tankyrase were constructed and identified smooth muscle cells of rat corpus cavernosum were transfected with the recombinant plasmids of sense tankyrase (pcDNA-TNKS). Levels of DNA and RNA were then evaluated. Measurement of telomerase activity was conducted by TRAP-ELISA assay, the length of telomere by Southern blot and the growth curve by MTT assay. We have found that the eukaryotic expression vectors for sense tankyrase were constructed and the recombinant plasmids of sense tankyrase were transfected into smooth muscle cells of rat corpus cavernosum successfully; no significant differences in telomerase activity were observed between TNKS-transfected cells (SMC-TANKS), zero-load- transfected cells (SMC-Zeo), and non-transfected cells (SMC) (P > 0.05). The length of telomere in SMC-TANKS was longer than that in SMC-Zeo or SMC (P < 0.01), and the OD value of TNKS-transfected cell was significantly higher than that of the non-transfected cells (P < 0.01). These results suggested that the eukaryotic expression vectors for sense tankyrase were constructed successfully, which provides the basis for gene therapy. Transfection of recombinant plasmids of sense tankyrase helps change the telomerase length of smooth muscle cells of corpus cavernosum and extend the cell life span.     

PDE5基因反义寡脱氧核苷酸对人阴茎海绵体平滑肌细胞cNMP的影响

目的 :观察PDE5基因反义寡脱氧核苷酸对人阴茎海绵体平滑肌细胞内cAMP和cGMP的影响 ,为阴茎勃起功能障碍的基因治疗提供理论和实验依据。　方法 :将PDE5基因反义寡脱氧核苷酸 (含第 1外显子部分序列 )与脂质转染试剂DOTAP共同转染人阴茎海绵体平滑肌原代细胞 ,以酶联免疫法分别检测转染后不同时间 (1～ 4 8h)海绵体平滑肌细胞内cAMP和cGMP的浓度变化 ,观察反义寡脱氧核苷酸对平滑肌细胞内cNMP的影响。　结果 :转染后 ,反义实验组平滑肌原代细胞内cGMP水平 (1～ 6h)显著高于对照组 (P <0 .0 1)。　结论 :PDE5基因反义寡脱氧核苷酸可以增加人阴茎海绵体平滑肌细胞cGMP水平 ,本研究有助于了解PDE5基因与cNMP在阴茎勃起中的作用 ,并为阴茎勃起功能障碍的基因治疗提供理论和实验基础。     

短发夹RNA对大鼠阴茎海绵体平滑肌细胞磷酸二酯酶5型基因表达的抑制作用

目的 观察短发夹RNA(shRNA)对大鼠阴茎海绵体平滑肌细胞磷酸二酯酶5型(PDE5)基因表达的抑制作用,探讨运用RNA干扰(RNAi)技术治疗勃起功能障碍(ED)的可行性.方法 构建靶向大鼠PDE5基因的shRNA重组腺病毒rAd-rPDE5-shRNA,将其转染大鼠阴茎海绵体平滑肌细胞48 h后,通过荧光标签进行显微计数确定转染效率,并以逆转录-聚合酶链反应(RT-PCR)及Western blot检测PDE5基因的表达水平.结果 rAd-rPDE5-shRNA构建成功,转染大鼠阴茎海绵体平滑肌细胞效率达95%以上,并使PDE5基因表达在mRNA水平抑制(80.78±2.30)%,在蛋白水平抑制(67.39±3.33)%.结论 以腺病毒为载体表达的shRNA能稳定、有效地抑制大鼠阴茎海绵体平滑肌细胞PDE5基因的表达.     

Human and rabbit cavernosal smooth muscle cells express Rho-kinase

Rho-kinase is an enzyme involved in the Ca2+-sensitizing pathway in smooth muscle cells. Inhibition of this enzyme has been recently demonstrated to elicit penile erection by relaxing cavernosal smooth muscle. We aimed to investigate the presence and activity of Rho-kinase in human cavernosal smooth muscle. Primary culture of smooth muscle cells from human and rabbit penile corpus cavernosum was developed, and cells showed characteristic myocyte morphology and alpha-actin immunoreactivity. The presence of Rho-kinase was demonstrated by indirect immunofluorescence and Western blotting. A specific inhibitor of Rho-kinase, Y-27632, inhibited in a concentration-dependent manner the kinase activity of the protein immunoprecipitated with anti-Rho-kinase antibody. These results demonstrate for the first time expression and activity of Rho-kinase in human penile cavernosal smooth muscle cells and suggest that these cells can provide a cellular model for the study of enzymes involved in Ca2+-sensitizing pathways.     

Misoprostol induces relaxation of human corpus cavernosum smooth muscle: comparison to prostaglandin E1

Prostaglandin E1 (PGE1) relaxes trabecular smooth muscle by interacting with specific G-protein coupled receptors on human corpus cavernosum smooth muscle and increasing intracellular synthesis of cAMP. Misoprostol (Cytotec), is an oral prostaglandin E analogue. The purpose of this study was to compare the functional activity of misoprostol with PGE1 in human corpus cavernosum and cultured human corpus cavernosum smooth muscle cells. Misoprostol, misoprostol free acid or PGE1 induced dose-dependent relaxations in strips of human corpus cavernosum. At concentrations greater than 10(-6) M, tissue recontraction was observed with all three agents. This was abrogated by pretreatment with the thromboxane A2 receptor antagonist SQ29,548. From these observations, we conclude that misoprostol is activated by human corpus cavernosum in situ and relaxes phenylephrine-precontrated tissue strips in vitro. This relaxation response is mediated by the increased cAMP synthesis by these agents.     

Organization and relative content of smooth muscle cells, collagen and elastic fibers in the corpus cavernosum of rat penis

PURPOSE: The corpus cavernosum smooth muscle and extracellular matrix are essential for normal penile erection and are implicated in erectile dysfunction. Although investigations of these issues have used the rat corpus cavernosum, organization of its components is to date not well known. We characterized and quantified the smooth muscle cells and the main extracellular matrix components of the rat corpus cavernosum. MATERIALS AND METHODS: Collagen, elastic fibers and smooth muscle cells were stained on paraffin sections of rat penises using sirius red and Gomori's reticulin, Weigert's resorcin-fuchsin and an anti-smooth muscle cells alpha-actin antibody, respectively. Stained components were then quantified by computer aided morphometry. RESULTS: Smooth muscle cells were restricted to the subendothelial space of corpus cavernosum and had a volumetric density of 9.1%. Collagen was thick, usually in transversely oriented bundles and was the most abundant component of the trabeculae with a volumetric density of 62.7%. Gomori's reticulin disclosed a meshwork of fibrils also in the subendothelial space but did not stain the thicker bundles. Volumetric density of elastic fibers was 4.9%, and at the periphery of the corpus cavernosum the fibers were parallel to the long axis of the penis, while in deeper regions most of them were transversely oriented and at different directions from those of collagen. CONCLUSIONS: Rat corpus cavernosum differs from that of humans by lesser amounts of smooth muscle cells, greater amounts of collagen and the presence of fibrillar collagen and smooth muscle cell subendothelial layers. Therefore, these differences should be considered when using the rat penis for studies on erection.     

腺病毒介导的激活大鼠阴茎海绵体平滑肌细胞中NO／cGMP通路的实验研究

目的：探讨靶向大鼠iNOS基因的shRNA重组腺病毒载体对大鼠阴茎海绵体平滑肌细胞iN0s基因的激活作用,为阴茎勃起功能障碍（ED）的基因治疗提供实验依据。方法：将前期构建的重组腺病毒AdS—iN—OSrshRNA-EGFP（AdU6／shiNOS）和对照病毒AdU6／shControl,分别转染大鼠阴茎海绵体平滑肌细胞,分别在不同病毒MOI（25,50,75）值下72小时后采样检测。采用realtimeRT-PCR半定量检测AdU6／shiNOS对细胞iNOS基因mRNA表达影响;Western—blot法检测海绵体平滑肌细胞iNOS蛋白表达变化。然后培养基中加L—Arg（10mmol／L）,用酶联免疫法检测病毒转染72小时后海绵体平滑肌细胞内cGMP的浓度变化,记录AdU6／shiNOS对平滑肌细胞内cGMP的影响。结果：AdU6／shiNOS转染大鼠阴茎海绵体平滑肌细胞72小时后,和空白对照组、阴性对照组相比iN0s基因在mRNA和蛋白表达水平均显著上调（P〈O．05）,呈剂量依赖性,MOI一75时RNAa效果最好。而且转染72小时后,实验组原代平滑肌细胞内cGMP水平显著高于对照组及空白组（Pd0．05）。结论：利用腺病毒介导的RNAa技术,提高海绵体平滑肌细胞iN0s基因表达获得成功,可以增加阴茎海绵体平滑肌细胞cGMP水平,激活了NO／cGMP通路,这为勃起功能障碍的基因治疗研究开辟了新的方向。     

Cyclic AMP regulates mRNA expression of alpha-1d and alpha-2a adrenergic receptors in cultured human corpus cavernosum smooth muscle cells

While the physiological effects of contractile (e.g. norepinephrine) and relaxatory (e.g. PGE1, forskolin) agents on corpus cavernosum smooth muscle tone have been characterized, the regulation of alpha adrenergic receptor mRNA expression in erectile tissue remains to be investigated. The goal of this study was to investigate the modulation of alpha-1 and alpha-2 adrenergic receptor mRNA expression in cultured human corpus cavernosum smooth muscle cells in response to increased intracellular cAMP induced by prostaglandin E1 and forskolin. Human corpus cavernosum smooth muscle cells were incubated for 24 h with or without PGE1 (5.7 μM), forskolin (10 μM) or an admixture of both. Total RNA was prepared from the cultures. Expression of alpha-1d adrenergic receptor, alpha-2a adrenergic receptor and m2 muscarinic acetylcholine receptor was determined by RNase protection assays. Loading was normalized by RNase protection of the housekeeping gene, cyclophilin A. The relative abundance of mRNAs was quantitated by scanning densitometry. Treatment of human corpus cavernosum smooth muscle cells with PGE1 or forskolin resulted in decreased mRNA expression of alpha-1d and alpha-2a adrenergic receptors and m2 muscarinic acetylcholine receptor when compared to untreated cells. Combinations of PGE1 and forskolin produced a more pronounced decrease in mRNA than either agent alone. PGE1 and forskolin increased intracellular levels of cAMP in human corpus cavernosum smooth muscle cells and combinations of both agents produced a more pronounced increase in cAMP synthesis. These results suggest that cAMP modulates the expression of alpha adrenergic receptors, one of the principal contractile receptor systems in the corpora cavernosa. These observations further support the concept that erectile function is a balance between contractile and relaxatory processes, which in turn regulate structure and function of the corpora cavernosa. International Journal of Impotence Research (2000) 12, Suppl 1, S41-S47     

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。