纤维蛋白原和纤维蛋白及其降解产物对共培养人脐静脉内皮细胞趋化迁移的影响

目的:研究不同浓度纤维蛋白原(Fg)、纤维蛋白(Fb)及其降解产物(FDPs)对人脐静脉内皮细胞(HUVECs)/平滑肌细胞(SMCs)共培养模型中HUVECs趋化和迁移的影响。方法:以Transwell膜为载体,建立原代培养的HUVECs与兔SMCs共培养体系,应用不同浓度的Fg、Fb和FDPs干预共培养体系,观察HUVECs趋化(通过Transwell膜装置)和迁移(通过细胞刮伤模型)情况。结果:共培养的HUVECs和SMCs在Transwell膜上生长良好,膜两侧的HUVECs和SMCs可以发生细胞连接。Fg≥3.0 mg·mL-1呈浓度依赖性地促使HUVECs的趋化和迁移。Fb≥1.5 mg·mL-1时呈浓度依赖性地促使HUVECs的趋化和迁移数增加(P<0.05)。FDPs≥0.5 mg·mL-1时HUVECs的趋化和迁移数明显增加(P<0.05)。结论:利用Transwell膜建立的HUVECs/SMCs共培养体系能较好地模拟血管壁的结构关系,为进一步开展相关研究奠定了细胞模型的基础。一定浓度的Fg、Fb及其FDPs可以促进HUVECs趋化与迁移过程,在动脉粥样硬化的发生发展中起重要作用。     

纤维蛋白原、纤维蛋白及其降解产物对共培养血管平滑肌细胞表型转换的影响

目的：研究不同浓度纤维蛋白原（Fg）、纤维蛋白（Fb）及其降解产物（FDPs）对共培养血管平滑肌细胞（SMCs）表型转换的影响。方法：建立兔主动脉内皮细胞（ECs）-SMCs共培养体系，分别观察在正常增殖和不同浓度Fg、Fb和FDPs干预后共培养SMCs表型转换标志物——α-SM-actin mRNA的表达。结果：在共培养条件下从第3天开始SMCs呈增殖表型的形态特征，第6天后，呈收缩表型的形态特征；较高浓度（3.0-6.0mg·mL^-1）的Fg、Fb和FDPs均可显著下调α-SM-actin mRNA的表达，抑制SMCs向收缩表型转换。结论：增殖过程中SMCs表型发生有规律的转换；Fg、Fb和FDPs通过影响SMCs的异常表型转换，参与了动脉粥样硬化进程。     

纤维蛋白(原)及其降解产物对共培养人脐静脉内皮细胞的细胞间黏附分子-1表达的影响

目的探讨纤维蛋白原(Fg)、纤维蛋白(Fb)及其降解产物(FDPs)对人脐静脉内皮细胞(HU-VECs)/兔平滑肌细胞(SMCs)共培养模型中细胞间黏附分子-1(ICAM-1)表达的影响。方法建立原代HU-VECS与兔SMC共培养体系,根据干预物及其浓度的不同分为Fg、Fb、FDPs组及相应的0 mg/ml、0.5 mg/ml、3.0 mg/ml和6.0 mg/ml亚组。应用核因子B抑制蛋白(I-B)拮抗剂BAY 11-7082、蛋白激酶C(PKC)拮抗剂Staurosporine分别对0.5 mg/ml、3.0 mg/ml和6.0 mg/ml亚组进行干预。采用反转录(RT)-PCR法检测HU-VECS内ICAM-1 mRNA水平;酶联免疫吸附法(ELISA)检测培养上清液的ICAM-1蛋白含量。结果 Fg 3.0mg/ml和6.0 mg/ml亚组ICAM-1 mRNA水平和蛋白含量显著高于0 mg/ml亚组及相应浓度Fg+BAY 11-7082亚组(均P<0.05);Fg 3.0 mg/ml亚组ICAM-1 mRNA水平和蛋白含量显著高于3.0 mg/ml+Staurosporine亚组(均P<0.05)。Fb 6.0 mg/ml亚组ICAM-1 mRNA水平显著高于0 mg/ml、6.0 mg/ml+BAY11-7082和6.0 mg/ml+Staurosporine亚组(均P<0.05),但Fb 6.0 mg/ml亚组ICAM-1蛋白含量显著高于0 mg/ml亚组(P<0.05)。FDPs 6.0 mg/ml亚组ICAM-1 mRNA水平和蛋白含量显著高于0 mg/ml、相应浓度BAY11-7082和Staurosporine亚组(均P<0.05)。结论中、高浓度Fg、Fb、FDPs能上调血管内皮细胞ICAM-1的表达。     

骨桥蛋白反义核苷酸对大鼠血管平滑肌细胞增殖的影响

背景：骨桥蛋白在血管损伤后新生内膜中的表达明显上调,而且能促进血管平滑肌细胞和外膜细胞的增殖、迁移。 目的：探索骨桥蛋白反义核苷酸对大鼠血管平滑肌细胞增殖的影响。 方法：培养大鼠A10主动脉血管平滑肌细胞,通过MTT比色法测定骨桥蛋白反义核苷酸对平滑肌细胞增殖的影响,流式细胞仪测定骨桥蛋白反义核苷酸对平滑肌细胞周期分布的影响;通过RT-PCR方法检测血管平滑肌细胞转染骨桥蛋白反义核苷酸对增殖细胞核抗原表达的影响。 结果与结论：骨桥蛋白反义核苷酸对血管平滑肌细胞的增殖有明显的抑制作用,随时间延长对细胞增殖抑制率下降。骨桥蛋白反义核苷酸主要作用于G1/S限制点,能阻止血管平滑肌细胞由G0/G1期向S期推进,从而将血管平滑肌细胞阻滞于G0/G1期,抑制血管平滑肌细胞的增殖。血管平滑肌细胞转染骨桥蛋白反义核苷酸后,增殖细胞核抗原的表达水平均较对照组降低(P < 0.05)｡因此,得出骨桥蛋白反义核苷酸通过阻止血管平滑肌细胞由G0/G1期向S期推进,从而抑制血管平滑肌细胞的增殖,进而抑制血管内膜增生。     

增殖细胞核抗原和C-Fos蛋白在人胎胃壁组织中的表达**★

背景：增殖细胞核抗原表达增高可促进细胞DNA合成增加，反映细胞增殖状况。C-Fos与DNA结合可直接调节具有转录活性的转录因子，在胚胎细胞的发育过程中，对细胞的生长、分化及增殖起促进作用。 目的：观察增殖细胞核抗原和C-Fos蛋白在人胎胃组织中的表达。 方法：应用免疫组织化学法和图像分析软件检测第2，3，4三个月龄段，人胎胃组织细胞中PCNA和C-Fos蛋白阳性表达的积分吸光度。 结果与结论：第2~4个月胎龄段，增殖细胞核抗原和C-Fos蛋白在人胎胃壁各层组织细胞均呈阳性表达。随着胎龄的增大，胃壁组织中增殖细胞核抗原蛋白阳性表达先升高再降低，C-Fos蛋白阳性表达则逐渐增高(P < 0.01)。提示增殖细胞核抗原和C-Fos蛋白在人胚胎早期胃组织细胞的生长发育过程中起重要的调节作用。     

纤维蛋白凝胶对大鼠胚胎间充质干细胞增殖与成骨分化的影响：不同质量浓度比较

背景：纤维蛋白是一种天然的可生物降解、组织相容性好的高分子材料，其中血纤维蛋白稳定因子ⅩⅢ已经证明有利于未分化间充质干细胞在高度交联的凝胶支架内迁移，并促进其增殖与分化。 目的：探讨大鼠胚胎间充质干细胞在不同质量浓度纤维蛋白凝胶内的形态学变化，以及生长增殖和成骨分化情况。 方法：无菌条件下取胎鼠四肢，组织块消化法体外分离培养胚胎间充质干细胞。取传至第3代细胞，分别接种于20，10，5 g/L纤维蛋白凝胶内，并设立对照组，接种于无凝胶的正常培养基中。用激光扫描共聚焦显微镜观察细胞在凝胶内的形态学变化，荧光分光光度计测定细胞增殖状态，酶标仪和Von Kossa染色分析细胞碱性磷酸酶活性和钙盐沉积情况。 结果与结论：培养7 d，在纤维蛋白凝胶内的大鼠胚胎间充质干细胞具有较长突起，且连接成网，而对照组细胞呈纺锤形或立方形。与对照组比较，凝胶组细胞增殖活跃，至培养第14天达峰值，以5 g/L凝胶细胞组荧光最强；凝胶组细胞碱性磷酸酶活性从14 d开始增强，21 d达峰值。培养14 d后20 g/L凝胶组在凝胶区出现小矿化结节，随后逐渐融合，而对照组无矿化结节出现。证实纤维蛋白凝胶支架能够支持大鼠胚胎间充质干细胞的存活与生长，且可以促进细胞的增殖和分化。 5 g/L低浓度凝胶有利于细胞形态的发生，20 g/L高浓度凝胶有利于细胞的成骨分化。 关键词：纤维蛋白凝胶；细胞增殖；细胞分化；碱性磷酸酶；细胞培养；胚胎；间充质干细胞 doi:10.3969/j.issn.1673-8225.2010.12.001     

抑肽酶/氨甲环酸改良可注射性纤维蛋白凝胶的体外实验

背景：可注射性纤维蛋白凝胶为软骨缺损组织工程完全再生修复的临床应用带来了新的方向，其降解速度的调控是其中的关键问题之一。 目的：在可注射性纤维蛋白凝胶中引入不同浓度的抑肽酶和氨甲环酸，观察其对纤维蛋白凝胶支架降解速率的影响。 设计、时间及地点：体外细胞学实验，于2008-02/08在广东省构建与检测实验室进行。 材料：以纤维蛋白原、凝血酶及氯化钙制备纤维蛋白凝胶。 方法：取3周龄新西兰幼兔的关节软骨制取软骨细胞，体外单层培养、扩增后植于标准纤维蛋白凝胶支架和改良纤维蛋白凝胶支架（加入抑肽酶7 500，12 500，17 500 MIU/L及氨甲环酸15，20，25 g/L复合液）上，进行体外培养、扩增6周。 主要观察指标：支架降解情况。 结果：支架细胞复合物体外培养3周时，标准组已完全崩解，改良各组体积尚不到原来1/2。培养6周时仍能保持其一定的外形，具有一定的厚度及弹性。不同浓度的抑肽酶和氨甲环酸，在体外环境下均显著地减缓了纤维蛋白胶的降解速度，低于1 2500 MIU/L的抑肽酶和20 g/L氨甲环酸对软骨细胞的繁殖、表型的维持、基质的分泌无明显不良影响，而高于此浓度的抑肽酶和氨甲环酸的加入明显抑制了细胞的增殖、表型的维持和基质的分泌。 结论：通过调节纤维蛋白凝胶中抑肽酶和氨甲环酸的含量，可实现对纤维蛋白凝胶降解速度的调控。     

高纤维蛋白原血症

纤维蛋白原（Fibrinogen,Fg）又称凝血因子I,是由肝细胞合成并分泌的一种糖基化蛋白,其半衰期为3～4d,80％存在于血浆中,正常血浆含量为2～4g／L。目前研究大都将空腹血浆纤维蛋白原水平〉4．0g／L者称为高纤维蛋白原血症（Hypeffibrinogenemia）。在各种病理状态下,血浆纤维蛋白原的异常升高会增加血液黏度、促进血小板聚集、参与调控与炎症细胞、内皮细胞以及其他蛋白质的结合,通过改变血液流变学、损伤血管内皮、介导炎症反应以及参与血栓形成等多种途径参与机体多种疾病的病理过程。     

DNA去甲基化对大鼠骨髓间充质干细胞增殖细胞核抗原的调节

背景：DNA去甲基化是一种重要的表观遗传修饰。 目的：观察DNA去甲基化对骨髓间充质干细胞增殖及增殖细胞核抗原蛋白表达的影响。 方法：全骨髓贴壁培养法分离培养大鼠骨髓间充质干细胞,免疫组化法鉴定骨髓间充质干细胞CD44和CD45蛋白的表达,MTT法检测5-杂氮胞苷对骨髓间充质干细胞增殖的影响,免疫组化法检测5-杂氮胞苷对骨髓间充质干细胞增殖细胞核抗原蛋白表达的影响。 结果与结论：①第3代骨髓间充质干细胞不表达或低表达CD45,高表达CD44。②与未加入5-杂氮胞苷组比较,5-杂氮胞苷干预48 h,6,12,24 μmol/L组显著促进细胞增殖活性(P < 0.05),无浓度依赖性。③与未加入5-杂氮胞苷组比较,5-杂氮胞苷干预48 h,6,12,24 μmol/L 组增殖细胞核抗原蛋白积分吸光度值显著增加 (P < 0.05),组间差异无显著性意义。结果显示在一定浓度范围内,5-杂氮胞苷发挥去甲基化作用,激活增殖细胞核抗原蛋白表达,从而促进骨髓间充质干细胞增殖。     

趋化因子CXCL-8趋化骨髓间充质干细胞迁移的体外效应

背景：早期研究表明趋化因子参与骨髓间充质干细胞的体外趋化,确定骨髓间充质干细胞体内迁移的分子机制,对其介导的细胞治疗中枢神经系统损伤和疾病有重要作用。 目的：观察体外条件下趋化因子CXCL-8对大鼠骨髓间充质干细胞迁移的趋化作用。 设计、时间及地点：细胞学体外观察,于2007-10/2008-06在青岛大学医学院附属医院脑血管病研究所完成。 材料：纯种SPF级成年Wistar大鼠8只,由青岛市实验动物和动物实验中心提供。鼠重组趋化因子CXCL-8为Santa cruz公司产品。 方法：采用全骨髓法分离培养大鼠骨髓间充质干细胞,胰蛋白酶消化传代。取500 μL趋化因子CXCL-8,加入含体积分数为0.5%胎牛血清的DMEM条件培养基,分别将质量浓度调整为5,50,250,500 μg/L加到趋化板下层,对照组单纯添加DMEM条件培养基,以8 μm孔径的聚碳酸酯膜覆盖。调整骨髓间充质干细胞浓度至1.5×109 L-1,取100 μL细胞悬液加到趋化板上层。为验证趋化因子CXCL-8作用的特异性,将经抗CXCR6多克隆抗体孵育12 h后的骨髓间充质干细胞加到趋化板上层,趋化因子CXCL-8加到趋化板下层。 主要观察指标：骨髓间充质干细胞趋化因子受体CXCR1的表达,趋化因子CXCL-8对骨髓间充质干细胞体外趋化迁移作用及其特异性检测。 结果：骨髓间充质干细胞趋化因子受体CXCR1呈阳性表达,位于细胞膜和细胞浆中,RT-PCR琼脂糖凝胶电泳后出现215 bp特异性扩增条带。与对照组比较,加入5~500 μg/L趋化因子CXCL-8后,骨髓间充质干细胞迁移数均明显增加（P < 0.05）。加入抗CXCR1多克隆抗体后,骨髓间充质干细胞迁移数较250 μg/L趋化因子CXCL-8组明显减少（P < 0.05）。 结论：体外条件下趋化因子CXCL-8在5~500 μg/L质量浓度范围可趋化骨髓间充质干细胞迁移,封闭趋化因子受体CXCR1后其趋化迁移作用明显减弱。     

Radioimmunoassay of fibrinogen-fibrin degradation products: Assay for fragment E-related neoantigen -methodological aspects

E fragment and its antigenically-related degradation products of human fibrinogen (Fg) and/or fibrin (Fb) have been measured in both normal and pathological plasmas by a specific radioimmunoassay (RIA) for the neoantigenic expression of E(Eneo) which is engendered after the plasmic cleavage of Fg or Fb. High titer antisera were produced in rabbits against Fg-E (E derived from Fg). The anti-Fg-E sera harvested were absorbed with a cyanogen bromide-activated immunoadsorbent column coupled with intact Fg. The Fg-absorbed antisera were employed to develop a double antibody RIA which detected < 1 ng/ml of Fg-E and exhibited little cross-reactivity of Fg at 1 mg/ml. This Eneo RIA did not discriminate between Fg-E and Fb-E (E derived from Fb) in the competitive inhibition profile, and hence it measured total Eneo antigens. The displacement curves of normal plasmas generally showed a parallel relationship to the standard which permitted the calculation of normal plasma Eneo immunoreactivity (ca. 15 ng/ml). Considerably higher plasma Eneo immunoreactivities have been observed in patients with consumption coagulopathy. Modifications of the Eneo RIA which permit the attainment of this sensitivity are described.     

纤维蛋白对大鼠脑血管内皮细胞血管内皮生长因子表达的影响

目的观察纤维蛋白对大鼠脑血管内皮细胞血管内皮生长因子(VEGF)转录及蛋白水平表达的影响。方法大鼠脑血管内皮细胞分离后培养,加入不同浓度的纤维蛋白,通过Real-time PCR检测VEGF转录水平,应用酶联免疫方法(ELISA)定量检测培养基和细胞裂解液中的VEGF水平。结果纤维蛋白可以特异性诱导大鼠脑血管内皮细胞表达VEGF;加入不同浓度的纤维蛋白(0.03mg/ml、0.1mg/ml、0.3mg/ml和1.0mg/ml),24h后,1.0mg/ml纤维蛋白组的培养基VEGF水平显著增高(P<0.001);1.0mg/ml纤维蛋白与大鼠脑血管内皮细胞分别培养0、2、4、8、24、48h,VEGF浓度在共培养2h已经升高,8h时显著升高,在24h时仍然保持在显著升高表达水平(P<0.005),48h有所下降;Real-time PCR结果提示,大鼠脑血管内皮细胞中VEGF mRNA的上调呈现出剂量和时间依赖性增加。结论纤维蛋白可以上调大鼠血管内皮细胞中的VEGF。     

Evidence for aggregation of fibrin fragments D and E in soluble fibrin complex formation

The capability of fibrin fragments D and E (Fb:D and Fb:E) to participate in soluble fibrin complex (SFC) formation was investigated by gel exclusion chromatography. Non-crosslinked fibrin extensively digested by plasmin and purified Fb:D and Fb:E did not form higher molecular weight aggregates upon incubation with thrombin and upon incubation with fibrinogen in the absence of thrombin. On the other hand, Fb:D-E mixtures, as well as purified Fb:D and purified Fb:E, upon incubation with fibrinogen and thrombin displayed moderate peak shifts toward a higher molecular weight range by agarose gel chromatography, suggesting the formation of complexes in the dimeric-size range. Fb:D and Fb:E differ in this respect from fibrinogen fragments D and E (Fg:D and Fg:E), which fail to form higher molecular weight aggregates under similar experimental circumstances (Smith, G. F. and Bang, N. U., Biochemistry, $\text{11}$:2958–2966, 1972).     

Fibrin gel induces the migration of smooth muscle cells from rabbit aortic explants.

A major step in the pathogenesis of atherosclerosis is the vectorial migration of smooth muscle cells (SMCs) from the arterial media into the intima. Although subcultured SMCs usually show synthetic phenotype, the behaviour of contractile SMCs may be crucial for the subsequent migration of the cells. In the present study, we utilized an in vitro assay system to evaluate the effects of fibrin gels on the migration of SMCs from explants taken from rabbit aorta. After cultured for 5-7 days in a serum-free condition, SMCs appeared from explants covered with fibrin gel. The cells were positive on immunostaining for SMC specific alpha-actin. No migration of SMCs from the control explants without fibrin gel was observed. Then the percentage of explants showing cell migration and the number of migrating cells increased with time. The migration of SMCs into fibrin gels was not dependent on the concentration of fibrinogen used for the preparation of fibrin gel in the range of 1.5-3 mg/ml. Variations of thrombin concentration in the range of 0.25-1.25 U/ml had no significant effect. However, there was less migration of SMCs with higher concentrations of thrombin. Thrombin inhibitors, hirudin and PPACK had no significant effect on the migration of SMCs. An RGD-containing peptide, GRGDS inhibited the migration of SMCs although a control peptide GRGES at the same concentration had no significant effect. A monoclonal antibody to alphavbeta3, LM609, completely inhibited the migration of SMCs from the explants, suggesting that alphavbeta3 integrin is involved in the migration of SMCs into fibrin gels. SMCs which migrated from the explants showed the positive staining with the monoclonal antibodies against SMC myosin heavy chain isoforms, SMemb, SM1 and SM2, suggesting that they are in an intermediate state changing from contractile to synthetic state. In conclusion, the present study showed that fibrin gel induces the migration of SMCs from explants into itself and the process may not need other growth factors or cytokines.     

Fibrinogen-induced increased pial venular permeability in mice

Elevated blood level of Fibrinogen (Fg) is commonly associated with vascular dysfunction. We tested the hypothesis that at pathologically high levels, Fg increases cerebrovascular permeability by activating matrix metalloproteinases (MMPs). Fibrinogen (4 mg/mL blood concentration) or equal volume of phosphate-buffered saline (PBS) was infused into male wild-type (WT; C57BL/6J) or MMP-9 gene knockout (MMP9−/−) mice. Pial venular leakage of fluorescein isothiocyanate-bovine serum albumin to Fg or PBS alone and to topically applied histamine (10⁻⁵ mol/L) were assessed. Intravital fluorescence microscopy and image analysis were used to assess cerebrovascular protein leakage. Pial venular macromolecular leakage increased more after Fg infusion than after infusion of PBS in both (WT and MMP9−/−) mice but was more pronounced in WT compared with MMP9−/− mice. Expression of vascular endothelial cadherin (VE-cadherin) was less and plasmalemmal vesicle-associated protein-1 (PV-1) was greater in Fg-infused than in PBS-infused both mice groups. However, in MMP9−/− mice, VE-cadherin expression was greater and PV-1 expression was less than in WT mice. These data indicate that at higher levels, Fg compromises microvascular integrity through activation of MMP-9 and downregulation of VE-cadherin and upregulation of PV-1. Our results suggest that elevated blood level of Fg could have a significant role in cerebrovascular dysfunction and remodeling.     

Mechanisms involved in the inhibition of neointimal hyperplasia by abciximab in a rat model of balloon angioplasty

Monoclonal antibodies raised against beta(3) integrin are able to inhibit the binding of ligands to certain beta(3) integrins such as alpha(IIb)beta(3) (glycoprotein IIb/IIIa complex) and alpha(v)beta(3) (vitronectin receptor) and as such are inhibitors of platelet aggregation and smooth muscle cell (SMC) migration, both of which are involved in neointimal hyperplasia. The present study was designed to explore the detailed mechanisms of abciximab (Reopro), a monoclonal antibody (mAb) raised against alpha(IIb)beta(3) integrin in neointimal hyperplasia. In this study, carotid arteries of Wistar rats were damaged, and neointimal hyperplasia and lumen occlusion was determined at different time points. Abciximab was administered intravenously by an implanted osmotic pump. Abciximab (0.25 mg/kg/day) time-dependently inhibited both neointimal hyperplasia and lumen occlusion after angioplasty in carotid arteries of rats. Furthermore, the electromicrographs highlighted that SMCs were phenotypically different from the typical contractile, spindle-shaped SMCs normally seen in uninjured vessel walls. Platelet-derived growth factor (PDGF)-BB was strongly produced in thrombus formation and neointimal SMCs after angioplasty, while abciximab significantly reduced PDGF-BB expression in vessel lumens and neointimal SMCs after angioplasty. Balloon angioplasty caused a significant increase of nitrate and cyclic GMP as compared with sham-operated rats. Infusion of abciximab (0.25 mg/kg/day) did not significantly change. Furthermore, the plasma level of thromboxane B(2) (TxB(2)) obviously increased after angioplasty, while abciximab markedly suppressed the elevation of plasma TxB(2) concentration. The results indicate that abciximab effectively prevents neointimal hyperplasia, possibly through the following 2 mechanisms: (1) Abciximab binds to alpha(IIb)beta(3) integrin on platelet membranes resulting in inhibition of platelet adhesion, secretion, and aggregation in injured arteries, followed by inhibition of thromboxane A(2) formation and PDGF-BB release from platelets. (2) Abciximab may also bind to alpha(v)beta(3) integrin on SMCs, thus, subsequently inhibiting cell migration and proliferation.     

PCNA反义脱氧寡核苷酸体外抑制BT325细胞增殖的实验研究

目的 观察增殖细胞核抗原(PCNA)基因反义脱氧寡核苷酸对人多形性胶质母细胞瘤细胞增殖的影响。方法 采用人工合成的PCNA反义和正义脱氧寡核苷酸经阳离子脂质体包裹后转染人多形性胶质母细胞瘤细胞BT325。用MTT比色法检测转染后瘤细胞的生长抑制率;~3H-TdR法检测细胞DNA合成速率;流式细胞仪检测细胞周期分布;RT-PCR检测PCNA mRNA表达;免疫组化方法检测PCNA蛋白表达水平。结果 PCNA反义寡核苷酸可明显抑制BT325细胞的生长;明显减慢其DNA合成速率,阻滞细胞由GO/G1期→S期,PCNA mRNA及其蛋白质表达也同时受到抑制。抑制效应在转染后12h即出现,24h达到高峰,48h后逐渐减弱。抑制作用随反义寡核苷酸浓度的升高而增强,0.8μM PCNA反义寡核苷酸的细胞生长抑制率达84.6％。同样浓度的正义寡核苷酸和脂质体则无明显的细胞生长抑制作用。结论 PCNA反义寡核苷酸在体外能明显抑制人脑多形性胶质母细胞瘤细胞BT325生长、DNA合成、mRNA和PCNA蛋白表达。PCNA基因可望成为胶质瘤基因治疗的新靶点。