成人及家兔阴茎海绵体平滑肌细胞培养和鉴定

目的 :探索阴茎海绵体平滑肌细胞 (CCSM)体外培养及鉴定方法。　方法 :采用勃起功能正常的成年男子及新西兰家兔的新鲜阴茎标本 ,经处理后采用原代细胞培养法 ,对阴茎海绵体组织块进行培养 ,获得CCSM后用光镜、透射电镜和免疫组化等多种方法从细胞形态学及CCSM生长特性等不同侧面对培养的人及新西兰家兔的CCSM进行鉴定 ,并测定CCSM在体外培养时的细胞增殖情况。　结果 :经 2 4h潜伏期 ,体外培养的CCSM进入指数增长期 ,维持约 96h后进入平台期 ;从接种组织块至长成单层细胞约需 15～ 2 0d ,传代后约 4～ 7d后长成单层 ;传代后的细胞增殖旺盛 ,成束状生长 ,并呈现层叠排列及“峰 谷”现象 ;CCSM在体外可稳定传 7～ 12代 ,传代期间其形态及增殖活性变化不明显 ;各种检测鉴定均证实所获细胞为CCSM。　结论 :CCSM原代细胞培养法简便、稳定、可靠 ,对研究阴茎的勃起机制及勃起功能障碍的发生、发展、调控及其治疗药物筛选有重要理论及现实意义。     

The effects of isolated lipoproteins and triglyceride, combined oxidized low density lipoprotein (LDL) plus triglyceride, and combined oxidized LDL plus high density lipoprotein on the contractile and relaxation response of rabbit cavernous smooth muscle

The aim of this study was to investigate the effects of isolated lipoproteins and triglyceride (TG), and the effects of combined oxidized low density lipoprotein (LDL) plus TG and the combined oxidized LDL plus high density lipoprotein (HDL) on the contractility and relaxation response of rabbit cavernous smooth muscle. Cavernous muscle strips from New Zealand White rabbits were studied in organ chambers for isometric tension measurement. The strips were exposed to HDL, LDL, oxidized LDL, TG, combined oxidized LDL plus TG and combined oxidized LDL plus HDL for 30 min. Both HDL and LDL did not affect contraction and relaxation responses of the cavernous muscles. The oxidized LDL did not affect norepinephrine (NE)-induced contractility of the strips, but significantly ( p < 0.05) decreased the relaxation response to endothelium-dependent agonist, acetylcholine (Ach). Non-specific NO synthase inhibitor (L-NAME) completely inhibited the relaxation response to Ach, and L-arginine partially improved the diminished relaxation. TG did not significantly change the relaxation responses to Ach, but decreased the contractility of cavernous muscle to NE. Neither the combined oxidized LDL plus TG nor oxidized LDL plus HDL had significant synergistic or detoxication effects on the contractility and relaxation responses. In conclusion, oxidized LDL may have acute toxic effects on the endothelium-dependent, NO-mediated relaxation, but not on the contractility, of rabbit cavernous smooth muscle. TG may decrease contractility of the cavernous muscle. There may be neither synergistic nor detoxication effects on the contractility and relaxation response when TG or HDL is added to the oxidized LDL.     

A novel mouse model of autologous venous graft intimal hyperplasia

BACKGROUND: To investigate the molecular mechanism of autologous venous graft intimal hyperplasia, a mouse model is needed. Currently only vein to carotid artery mouse models are available and are hampered by a high thrombosis rate. We hypothesized that operating on the aorta would lead to intimal hyperplasia with decreased risk of thrombosis. MATERIALS AND METHODS: In C57BL/6J mice, the left external jugular vein was grafted into the infrarenal abdominal aorta by end-to-end anastomosis with 11-0 Ethilon. Grafts harvested at 1, 2, 4, 8, and 16 weeks postoperatively were subjected to histological and immunohistochemical analysis. RESULTS: Thirty-one of 35 mice survived; 2 mice were sacrificed secondary to thrombosis. The percentage lumen narrowing (+/-SE) was 7.8 +/- 0.3, 16.4 +/- 0.9, 19.2 +/- 0.9, 22.3 +/- 0.8, and 23.9 +/- 1.6% at 1, 2, 4, 8 and 16 weeks, respectively. Nuclear density decreased with each successive time point. The percentage of alpha-smooth-muscle actin-positive cells within the neointima peaked at 16 weeks (53%), and the percentage of cells positive for proliferating cell nuclear antigen peaked at 2 weeks (39%). CONCLUSIONS: We thus report on a novel mouse model of intimal hyperplasia in autologous venous grafts with a low thrombosis rate. Further studies using this model, coupled with genetic and bone marrow transplantation mouse models, should lead to significant enhancement in understanding of the mechanism of intimal hyperplasia.     

Vascular endothelial growth factor stimulates embryonic urinary bladder development in organ culture

OBJECTIVES: To determine whether vascular endothelial growth factor A (VEGF) and its receptors are expressed during bladder development in mice when capillaries are forming, and whether exogenous VEGF might enhance the growth of endothelia and other types of bladder cells, using an embryonic organ-culture model. MATERIALS AND METHODS: Whole bladders from wild-type mice, at embryonic day (E) 14, were grown in serum-free organ culture in an air/5% CO2 atmosphere; some cultures were supplemented with VEGF and/or with VEGF receptor 1/Fc chimera (VEGFR1/Fc), which blocks VEGF bioactivity. Organs were harvested after 6 days and the expression of VEGF and related molecules assessed using immunohistochemistry. RESULTS: VEGF, VEGFR1 and VEGFR2 positive cells were immunodetected in E14 and E18 bladders. Exogenous VEGF increased whole-organ growth, as assessed by explant areas, total cell numbers, DNA and protein content; proliferation was enhanced, and apoptosis decreased, in urothelium and surrounding tissues. VEGF also increased the proportions of cells expressing endothelial (CD31) and smooth muscle (alpha smooth muscle actin) markers. VEGFR1/Fc blocked the growth-enhancing effects of exogenous VEGF. CONCLUSIONS: In organ culture, exogenous VEGF not only stimulated embryonic bladder endothelial cells but also strikingly enhanced the growth of the whole organ. Whether the effects of VEGF on diverse bladder cell populations are direct or indirect requires further investigation. The finding that VEGF protein is present in embryonic bladders in vivo raises the possibility that it has similar actions during normal development. The results also illuminate the pathobiology of certain bladder diseases in which VEGF levels have been shown to be increased.     

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     

Effects of cyclophilin A on cell proliferation and gene expressions in human vascular smooth muscle cells and endothelial cells

BACKGROUND: Cyclophilin A (CypA) is a cytosolic protein which involves many biological functions including immune modulation, cell growth, tumorigenesis, and vascular disease. The objective of this study was to determine the effect of CypA on cell proliferation and several gene expressions in human endothelial cells and vascular smooth muscle cells. METHODS: Human coronary artery endothelial cells (HCAEC), human lung microvascular endothelial cells (HMVEC-L), and human aorta smooth muscle cells (HAoSMC) were used in this study. Cells were treated with 10 nM CypA for 24 h. The cell proliferation was determined by [3H]thymidine incorporation. The mRNA levels of 13 genes including CD147 (receptor for CypA), PDGF-BB, endothelin-1 (ET-1), vascular endothelial growth factor receptor-1 (VEGFR-1), VEGFR-2, VEGFR-3, neuropilin-1 (NRP-1), NRP-2, eNOS, iNOS, nNOS, ICAM-1, and PECAM-1 were semiquantitatively determined by real time RT-PCR as standardized with a house keeping gene beta-actin. RESULTS: CypA significantly increased cell proliferation of HAoSMC and HMVEC-L by 31% and 45%, respectively, as compared to controls, but had no effect on HCAEC. Blocking CD147 did not affect the mitogenic action of CypA. In addition, CypA also significantly increased the mRNA expression of CD147 by 43% and VEGFR-2 by 65% in HAoSMCs (P < 0.05, t test). HAoSMCs expressed much higher CD147 and neuropilin-1 (NRP-1) mRNA than HMVECs-L and HCAECs (P < 0.017, ANOVA). Furthermore, CypA increased ET-1 mRNA by 22% and VEGFR-1 mRNA by 23% in HMVECs-L, but had limited effects on HCAECs. HMVECs-L had much higher expressions of PDGF-BB, ET-1, VEGFR-2, VEGFR-1, VEGFR-3, and NRP-2 than HAoSMCs and HCAECs (P < 0.017, ANOVA). By contrast, HCAECs had much higher ICAM-1 mRNA levels than HMVECs-L and HAoSMCs (P < 0.017, ANOVA). CONCLUSIONS: These data demonstrate that CypA has a mitogenic effect on HAoSMCs and HMVECs-L, but not HCAECs. CD147 may not mediate the action of CypA. In addition, CypA substantially alters the mRNA levels of several key genes in human vascular cells, indicating potential multifunctional roles of CypA in vascular system. Furthermore, this study provides several new aspects of gene expressions in vascular cells.     

VEIN TO ARTERY GRAFTS: A MORPHOLOGICAL AND HISTOCHEMICAL STUDY OF THE HISTOGENESIS OF INTIMAL HYPERPLASIA

Failure of vein to artery grafts has been associated with intimal thickening (hyperplasia) and atherosclerosis. Current theories of intimal development, derived from arterial studies, show that smooth muscle cells migrate from the media to the intima after endothelial damage, where they proliferate and produce intimal hyperplasia. However, little is known of the histogenesis of these lesions in vein grafts. Experimental ilio-lumbar vein to iliac artery autografts were placed in 52 rats and analysed by light microscopy and histochemistry from 2 to 140 days after surgery. On day 2 the grafts and adjacent artery were severely damaged. Regeneration of damaged arterial tissue occurred by day 5, and thickening was already evident in the arterial intima. The intimal cells had histochemical characteristics of smooth muscle. By day 15, this hyperplastic intima was continuous across the anastomosis from the artery into the graft. After day 28 a wedge of densely packed cells was present in the vein graft intima for approximately 2 mm into the graft. By day 140, all the grafts were fully re-endothelialized. Intimal hyperplasia was present in all grafts and varied in thickness from 3 to 20 cells. Histochemical staining of these cells showed them to be of smooth muscle origin.     

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.     

The effect of six different statins on the proliferation, migration, and invasion of human smooth muscle cells

Intimal hyperplasia (IH) can occur after any vascular injury and results from smooth muscle cells (SMC) proliferation, migration, and invasion into the subintimal space. The purpose of this study was to investigate the effect of six different statins on the proliferation, migration, and invasion of human venous SMC. The statins were all used at their Cmax concentrations. SMCs were used to construct growth curves in the presence of 10% fetal calf serum or 10% fetal calf serum supplemented with the six statins. Migration and invasion experiments were performed using modified Boyden chambers. The invasion experiments were performed using Matrigel coated plates. We found that all of the statins significantly inhibited SMC proliferation compared to the platelet-derived growth factor control (ranging from fluvastatin 33% of control to pravastatin 72% of control, P = 0.03). SMC migration through uncoated polycarbonate membranes in presence of the six statins was significantly reduced (ranging from lovastatin 43% to pravastatin 57% of control, P = 0.006). All six statins also significantly reduced SMC invasion (ranging from fluvastatin 65% to simvastatin 87% of control, P = 0.002). We conclude that the inhibitory effect of statins on SMC proliferation, migration, and invasion is a class, rather than drug specific effect.     

Arrangement pattern of new-lining endothelial cells

New-lining endothelial cells in the neointimae of two types of synthetic vascular prostheses were examined by light- and electron microscopy. Examination was carried out at one to 846 days post implantation of the prostheses in the thoracic aortae of 57 canines. The surfaces of both types of prostheses were covered by a continuous layer of endothelial cells. The long axes of the cells were found to be parallel to the blood stream. This observation indicates that the arrangement pattern of endothelial cells in the neointimae is largely dependent on the direction of the blood stream rather than on the direction of the tension exerted on the vascular prostheses.     

肝硬化患者脾静脉和胃冠状静脉壁类粥样硬化样改变

研究肝硬化门静脉高压症患者脾静脉和胃冠状静脉壁类粥样硬化样改变,并探讨其发生机理及临床意义。方法５０例肝硬化门静脉高压症患者,在行脾切除贲门周围血管离断术时取一段脾静脉和胃冠状静脉供研究,１０例十二指肠球部溃疡患者和１０例外伤性脾破裂患者为对照组,行光镜和电镜观察。     

Methylmethacrylate monomer produces direct relaxation of vascular smooth muscle in vitro

Methylmethacrylate bone cement is associated with severe hypotensive reactions during surgery and anesthesia. The purpose of this in vitro study was to determine if methylmethacrylate monomer could produce hypotension by acting directly on vascular smooth muscle.
Segments of human saphenous vein or rabbit thoracic aorta were cut into rings. The rings were mounted in isolated tissue chambers in order to measure isometric tension development.
Methylmethacrylate monomer (methylmethacrylic acid ester) produced direct relaxation of venous or aortic rings preconstricted with either potassium ion or noradrenaline. The relaxation was concentration-dependent, occurring at concentrations from 10^-1 to 10^-1 M. The relaxation of rabbit aortic rings {preconstricted with noradrenaline) was unaffected by pre-treatment with atropine, propranolol, cimetidine, indomethacin, or methylene blue. Endothelial stripping with Triton X-100, sufficient to completely abolish acetylcholine-induced relaxation, also had little effect on methylmethacrylate-induced relaxation. Methylmethacrylate produced direct relaxation of rabbit aortic rings constricted with either potassium or noradrenaline in calcium-deficient media, and inhibited subsequent calcium-induced constriction.
These results suggest that methylmethacrylate monomer may interfere with intracellular and extracellular calcium mobilization and excitation/contraction coupling in vascular smooth muscle. The direct relaxation of venous and arterial smooth muscle produced by methylmethacrylate monomer may contribute in part to the hypotension that can occur when acrylic bone cement is employed during orthopedic procedures.     

缺氧诱导因子-1α与同源盒基因共转染在移植静脉重塑态的表达

目的 探讨缺氧诱导因子(HIF-1α)和同源盒基因(gax)共转染在移植静脉组织中的表达状态.方法 Wistar大鼠16只随机分为实验组和对照组,每组8只,均行自体静脉移植术,实验组移植静脉行缺氧诱导因子(HIF-1α)和同源盒基因(gax)共转染,对照组则未行基因转染,于术后14 d取出移植的静脉,分别采用逆转录-聚...     

Vascular smooth muscle cells derived from atherosclerotic human arteries exhibit greater adhesion, migration, and proliferation than venous cells

BACKGROUND: Phenotypic variation of vascular smooth muscle cells (VSMC) may result in altered biological behavior and responses. Within the vessel wall, arterial VSMC have a greater propensity to form atherosclerotic lesions as compared to venous VSMC. In this study the rates of proliferation, adhesion, and migration were compared between VSMC of atherosclerotic arterial and venous origin. MATERIALS AND METHODS: Human VSMC cultures were isolated from 18 infragenicular arteries at the time of below knee amputation and from 20 saphenous veins during lower extremity revascularization surgery. Cell cultures were isolated from the media of each specimen and maintained in distinct cell lines for all assays. Cells from passages 2 and 3 were assayed for their proliferative capacity using total DNA fluorescence photometry and for adhesion and migration using a modified Boyden chamber. RESULTS: Patient age and the incidence of atherosclerotic risk factors did not vary significantly between the arterial and the venous patient groups. VSMC of atherosclerotic arterial origin demonstrated greater proliferation (arterial, 162 +/- 59 absorption units, vs. venous, 106 +/- 56 absorption units, P < 0.001), adhesion (arterial, 74.1 +/- 22.6 cells/microscopic field, vs. venous, 41.3 +/- 12.8 cells/microscopic field, P < 0.001) and migration (arterial, 427 +/- 185 cells/microscopic field, vs venous, 119 +/- 101 cells/microscopic field, P < 0.001) than VSMC of venous origin. CONCLUSION: Human atherosclerotic arterial VSMC exhibit significantly increased rates of proliferation, adhesion, and migration as compared to human venous VSMC. These observations of VSMC in culture are consistent with the clinical predilection for the hyperplasic responses that result in the development of atherosclerosis in the arterial wall. Possible intrinsic differences in VSMC phenotype should be considered in designing methods to limit atherosclerosis.     

2-脱氧-D-葡萄糖对人脐静脉内皮细胞的作用

目的观察2-脱氧-D-葡萄糖对人脐静脉内皮细胞增殖、迁移、管腔形成及细胞凋亡的影响,试图寻找治疗血管瘤的新药。方法用四甲基偶氮唑盐比色法、倒置显微镜下形态学观察、体外划痕实验的方法,观察2-脱氧-D-葡萄糖对人脐静脉内皮细增殖、迁移的作用;用成管法,检测2-DG对血管形成的影响;用流式细胞仪检测2-DG对血管内皮的凋亡影响、结果2-DG作用于人脐静脉内皮细胞后,对其增殖,迁移和成管能力有明显抑制作用,大大地增加细胞的凋亡率,且有显著剂量和时间依赖性．结论2-DG对HUVEC增殖、迁移和成管有显著的抑制作用,且能促使细胞凋亡。     

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。     

淫羊藿苷对兔阴蒂海绵体平滑肌细胞NO及NOS活性的影响

目的 探讨淫羊藿苷对体外培养的兔阴蒂海绵体平滑肌细胞一氧化氮(NO)产生及一氧化氮合酶(NOS)活性的作用效果。 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,利用免疫细胞化学染色法通过检测α-actin进行细胞鉴定;利用硝酸还原酶法及NOS试剂盒测定不同浓度淫羊藿苷对阴蒂海绵体平滑肌细胞NO生成及NOS活性的影响。 结果 培养的兔阴蒂海绵体平滑肌细胞呈现典型的平滑肌细胞形态特征;淫羊藿苷浓度依赖性增强家兔阴蒂海绵体平滑肌细胞NOS活性并增加NO生成(P<0.01),且被NOS抑制剂L-硝基精氨酸(LNNA)所抑制(P<0.01)。结论 淫羊藿苷可能通过增强NOS活性而提高阴蒂海绵体平滑肌细胞NO生成,增强性刺激下阴蒂海绵体平滑肌的松弛作用而增强阴蒂胀大勃起功能。     

胰腺内皮细胞共培养对C3A细胞功能变化的影响

目的 探讨与胰腺内皮细胞共培养时C3A细胞功能变化.方法 选择C3A细胞与胰腺内皮细胞的密度比例为10:1分别进行直接和间接共培养7 d,同时设立空白对照组,观察各实验组细胞生长和形态特征,分别用杜邦全自动生化分析仪、放射免疫分析法和ELISA法检测培养液中AST、ALT漏出量,白蛋白含量和安定代谢量.结果 C3A细胞与胰腺内皮细胞直接共培养第5、7天能显著降低ALT、AST的漏出量,第7天提高白蛋白合成量最高达12.977μg/ml和提高安定代谢能力;C3A细胞与胰腺内皮细胞间接共培养在第5、7天也能显著提高白蛋白合成量到7.380μg/ml、10.773μg/ml.结论 与胰腺内皮细胞直接与间接共培养均能明显改善C3A细胞的功能.