兔骨关节炎软骨细胞中bcl-2、bax mRNA及蛋白表达的意义

目的探讨bcl-2、bax基因在兔骨关节炎软骨细胞凋亡中的意义。方法新西兰白兔12只,随机分为模型组,按照石膏外固定兔后膝关节造模,以及空白对照组,6周后处死动物取得膝关节软骨。采用免疫组织化学和原位杂交检测bcl-2、bax mRNA及蛋白表达。结果模型组软骨细胞bcl-2、bax mRNA表达明显多于对照组(P＜0．05)。免疫组织化学模型组软骨细胞bcl-2、bax阳性表达灰度值明显降低,与对照组比较差异有统计学意义(P＜0．05)；阳性细胞比例明显提高,与对照组比较差异有统计学意义(P＜0．01)。模型组bcl-2/bax阳性细胞率比值比对照组低,差异有统计学意义(P＜0．05)。结论bcl-2和bax共同参与了兔骨关节炎模型软骨细胞凋亡的调节,结果导致软骨细胞凋亡加快。     

bcl-2蛋白在H460和H446中的表达及其应激变化

培养正常肺细胞系MRC-5和两株肺肿瘤细胞系H460、H446,应用Western blot法检测bcl-2蛋白的表达;采用120 mj/cm2剂量紫外线(UV)照射,继续培养并对bcl-2蛋白进行免疫组化观察.Western blot结果示,在肺肿瘤细胞系H460和H446中的bcl-2蛋白表达明显高于正常细胞系MRC-5;免疫组化检测示,UV照射后培养10.5 h,bcl-2的表达在3种细胞系中均显著高于未照射组.认为bcl-2蛋白过表达与肺肿瘤的发生有关,但肺肿瘤细胞中bcl-2蛋白的过表达未影响其对UV照射引起的应激反应,从而使肿瘤细胞具有了在应激条件下较正常细胞更强的生存能力.     

自发性高血压大鼠心肌凋亡及bcl-2、bax蛋白的表达

1材料与方法 8周龄自发性高血压大鼠(SHR)30只,雌雄各半.随机分为两组,分别为缬沙坦组和无药组各15只.8周龄雄性Wistar大鼠15只作为正常血压对照组.缬沙坦组给予血管紧张素Ⅱ1型受体拮抗剂,缬沙坦20mg·kg-1·d-1,溶解于饮用水中每天1次胃管灌入.无药组及正常血压对照组每天饮用水10 ml/kg灌胃.每2周测尾动脉血压.至16周龄所有大鼠乙醚麻醉后断头处死,迅速取左心室组织(包括室间隔)称重.计算心脏指数=心脏重量/体重(mg/g).采用末端脱氧核糖核酸转移酶介导的dUTP缺口末端标记(TUNEL)技术原位检测凋亡细胞.采用免疫组织化学定位研究心肌组织bcl-2及bax蛋白表达情况.同时应用Western印迹定量研究心肌bcl-2及bax蛋白表达.研究数据以-x±s表示,P＜0.05为差异有显著性意义.     

曲美他嗪与雷米普利对慢性心力衰竭大鼠心肌细胞凋亡及bax、bcl-2蛋白表达的影响

目的观察曲美他嗪与雷米普利单用及联合用药对慢性心力衰竭(CHF)大鼠心肌细胞凋亡程度及凋亡相关蛋白bax、bcl-2蛋白表达的影响。方法雄性Wistar大鼠50只,随机分为模型组、曲美他嗪组、雷米普利组、联合用药组,用腹主动脉缩窄法复制CHF模型,另取假手术组作为对照,每组10只大鼠。观察和比较各组大鼠左心室重量指数(LVMI)及心功能,原位缺口末端标记法(TUNEL)检测心肌细胞凋亡指数,SP免疫组化法检测各组大鼠bax、bcl-2蛋白的表达情况,用RT-PCR法测定各组大鼠bax mRNA及bcl-2 mRNA的表达水平。结果与模型组比较,药物干预组大鼠心功能明显改善,凋亡指数明显降低,bcl-2阳性表达及其mRNA水平显著升高,bax阳性表达及其mRNA水平显著降低,其中以联合用药组变化最显著。结论曲美他嗪与雷米普利单用及联合应用均可有效改善心功能、抑制细胞凋亡、延缓CHF,联合用药在抗心肌细胞凋亡方面的疗效优于单独用药。     

bcl-2、p53、C-myc蛋白产物在胃癌表达的研究

许多研究证明，肿瘤的发生．发展与癌基固和抑癌基因的异常有关．且与肿瘤细胞凋亡被抑制有关，本探讨bcl-2、p53、C-myc、蛋白产物在胃癌中的表达。     

氟对软骨细胞凋亡因子bcl-2和Bax蛋白表达的影响

目的 观察氟对体外培养软骨细胞中凋亡相关因子bcl-2和Bax蛋白表达的影响,探讨氟在致软骨细胞凋亡中的作用机制.方法 采用软骨细胞体外培养方法,原代培养乳鼠关节软骨细胞,传第3代后按染氟剂量不同分为0(对照)、5、20、40mg/L组,培养10 d后,透射电镜下观察软骨细胞的超微结构改变,采用Western印迹法检测软骨细胞bcl-2和Bax蛋白表达.结果 透射电镜下,对照组和5 mg/L组软骨细胞呈球形,粗面内质网发达,线粒体膜性结构完整;20、40 mg/L组软骨细胞内可见脂滴增多,胞质内出现大量空泡类物质,细胞内膜结构不清,部分细胞出现核固缩.20、40 mg/L组软骨细胞bcl-2蛋白表达(0.626±0.042、0.531±0.039)较对照组(0.876±0.035)明显降低(P均＜0.01);而Bax蛋白表达(0.966±0.047、1.289±0.156)较对照组(0.642±0.050)明显增加(P均＜0.01).5 mg/L组软骨细胞bcl-2、Bax蛋白表达(0.885±0.065、0.657±0.045)与对照组比较,差异无统计学意义(P均＞0.05).此外,40 mg/L组与20 mg/L组比较,bcl-2、Bax蛋白表达差异有统计学意义(P均＜0.01).结论 染氟20、40 mg/L可对软骨细胞超微结构造成损伤,其通过减少抑凋亡因子bcl-2的表达和增加促凋亡因子Bax的表达,从而产生促进软骨细胞凋亡的作用.

Abstract：

Objective To study the effect of fluoride on the expression of bcl-2 and Bax in chondrocyte in vitro, and investigate the mechanism of action of chondrocyte apoptosis induced by fluoride. Methods Articular chondrocytes of neonate rat were cultured in vitro and treated with 0(control),5,20,40 mg/L of fluoride,respectively, for 10 days. Then observed the u]trastructure of chondrocytes under eletronicmicroscope, and tested the expression of bcl-2 and Bax in chondrocyte in different groups by Western blotting. Results Abundant rough endoplasmic reticulums (RERs) and complete structure of mitochondria membranes were presented in globular chondrocytes in the control group and 5 mg/L group; but more lipid droplets and vacuoles were seen in the cytoplasm, and the structure of intracellular membranes became incomplete, and some shrieked chromatin and pyknosis were seen in the chondrocytes of the 20,40 mg/L groups. The expression of bcl-2 markedly decreased in 20 mg/L group(0.626 ± 0.042) and 40 mg/L group(0.531± 0.039) compared to the control group(0.876 ± 0.035,all P ＜ 0.01 ). And the expression of Bax significantly increased in 20 mg/L group(0.966 ± 0.047) and 40 mg/Lgroup ( 1 .289 ± 0.156) compared to the control group(0.642 ± 0.050, all P ＜ 0.01). But there was no statistical significant difference of the expression of bcl-2 or Bax between 5 mg/L group(0.885 ± 0.065,0.657 ± 0.045) and control group (all P ＞ 0.05 ). However there were statistical differences of expressions of bcl-2 and Bax between 20 and 40 mg/L groups(all P ＜ 0.01 ). Conclusions Twenty and 40 mg/L fluoride can cause damage to the ultrastructure of chondrocyte, and fluoride possibly promotes chondrocyte apoptosis by reducing the expression of antiapoptotic factor bcl-2 and increasing the expression of Bax.     

甲状腺癌组织中BAG-1、bcl-2蛋白表达及临床意义

目的 探讨BAG-1、bcl-2在甲状腺癌发生、发展中的作用。方法采用免疫组化法检测48例甲状腺癌、11例甲状腺腺瘤组织中BAG-1、bcl-2蛋白的表达情况，并进行相关性分析。结果BAG-1、bcl-2蛋白在甲状腺癌中的表达率（66．7％、75．0％）均高于甲状腺腺瘤（18．2％、27．3％）：甲状腺癌中两基因的表达呈正相关，BAG-1表达水平在甲状腺癌组织学分型、临床分期及淋巴结转移均有相关性。结论BAG-1特异性表达于甲状腺癌组织中，其可能与bcl-2协同参与甲状腺癌的发生、发展；BAG-1表达状态可作为评估甲状腺癌生物学行为和预后的重要参考指标。     

缬沙坦对自发性高血压大鼠心肌凋亡及bcl-2、bax蛋白的表达的影响

目的　探讨自发性高血压大鼠 (SHR) ,左心室肥厚过程中 ,心肌组织凋亡情况及凋亡调节蛋白bcl 2、bax表达的变化 ,AT1受体拮抗剂缬沙坦对心肌细胞凋亡的影响。方法　实验动物为 8周龄分为缬沙坦治疗组和非治疗组(SHR无药组 ) ,以Wistar鼠作为正常血压对照组 ,观察期限为 8周。采用TdT介导的dUTP缺口末端标记技术(TUNEL)判定心肌细胞凋亡 ,并行免疫组化、Western印迹等方法检测实验动物心肌组织凋亡调节蛋白bcl- 2、bax的表达情况。结果　SHR心肌组织中存在细胞凋亡现象 ,并有bax高表达。缬沙坦治疗 8周后 ,SHR心肌组织bax蛋白表达显著降低至接近Wistar组。Bcl 2蛋白在SHR治疗组及Wistar组表达较SHR非治疗组有增高趋势 (P >0 0 5 )。前两组bax/bcl 2比值较后者显著降低 (P <0 0 5 )。结论　心肌细胞凋亡是代偿性心肌肥厚可能机制之一。高血压病早期缬沙坦在降压同时可有效抑制心肌细胞凋亡 ,具有积极的抗心室重建作用。     

匹罗卡品对兔眼睫状体细胞bcl-2、bax蛋白表达的影响

匹罗卡品是眼科常用药。我们发现长期使用匹罗卡品治疗的青光眼患者较发病后立即手术者其虹膜基质萎缩与瞳孔括约肌瘫痪均较明显。     

多原发癌组织中bcl-2表达状况的研究

国内对多原发癌的研究相对较少,尚未见有对多原发癌分子病理学研究的报道[1～3].以往的研究表明,bcl-2基因的过表达在许多单发肿瘤的发生发展中起很重要作用[4～6],本实验应用免疫组织化学方法对多原发癌组织中的bcl-2蛋白表达进行检测.     

FK506治疗大鼠早期膜性肾病的实验研究

组相比出现大量蛋白尿,血清白蛋白水平降低.免疫组化检测结果示M组TGF-β1和NF-κB的表达均高于N组,而FK506组的水平低于M组.Western印迹法显示M组及FK506组TGF-β1高于N组,FK506组的水平低于M组. 结论 FK506能降低膜性肾病大鼠尿蛋白,提高血清白蛋白,可抑制肾组织TGF-β1与NF-κB的表达,用其防治大鼠早期膜性肾病有较好的疗效.     

环孢素A及其衍生物FK506体内抗长爪沙鼠日本血吸虫病的研究

目的本文报告了曼氏血吸虫有明显杀虫效果的环孢素A及其衍生物FK506对长爪沙鼠日本血吸虫病保护作用的初步研究。结果显示,环孢素A对长爪沙鼠日本血吸虫病有明显的保护作用,减虫率为41.62%;减卵率为72.91%;经环孢素A作用后,日本血吸虫虫体表面出现肿胀、水泡、破损、感觉乳突变形或消失等改变;而FK506则几乎没有作用,减虫率仅4.99%;减卵率仅6.39%;经FK506处理后,与对照组相比,虫体活力和皮层无明显变化。环孢素A与FK506两者间的杀虫区别值得进一步探讨。     

他克莫司在移植肾功能延迟恢复患者中的临床应用

目的:探讨他克莫司(FK506)在移植肾功能延迟恢复(DGF)患者中的临床应用价值与合理用药方案.方法:17例DGF患者临床结合移植肾病理确立诊断.肾移植术后早期均接受三联(FK506 MMF Pred)免疫抑制药物治疗至少3个月.不用任何生物制剂诱导治疗,观察临床疗效及副作用.结果:17例患者无一例死亡或摘除移植肾.15例在术后第2～3天开始血液透析(HD)/连续性血液净化(CBP)治疗,2例在术后第5天开始HD/CBP.HD/CBP治疗2～15次后,10例在术后7天内停止,7例在术后7天后仍需CBP治疗,最长1例在术后第18天停止透析.FK506治疗后8～17天患者尿量开始明显增多,SCr开始明显下降.17例患者诊断DGF时SCr水平在489～1028μmol/L,14例在治疗后8～17天降至＜200μmol/L,另3例中2例SCr分别在术后第24天,28天降至＜200μmol/L.副作用主要是腹泻(3例),血糖升高(1例)及手颤,肢体麻木(4例),但未出现CMV等严重感染病例.结论:FK506 MMF Pred三联免疫抑制治疗方案治疗肾移植DGF安全有效,可作为肾移植术后DGF患者的过渡治疗方法之一.     

他克莫司对转化生长因子-β诱导的肾小管上皮细胞转分化的抑制作用及对核因子-κB活性的影响

目的:探讨他克莫司(FK506)对转化生长因子-β(TGF-β)诱导的肾小管上皮细胞转分化的作用,及其与核因子-κB(NF-κB)活性变化的关系.方法:以人类肾脏近曲小管上皮细胞株(Human kidney cell,HKC)为研究对象,分为以下4组:①阴性对照组;②TGF-β(8 ng/ml)组:③FK506(0.1、1、10、50 ng/ml)组:④TGF-β(8 ng/ml) FK506(0.1、1、10、50 ng/ml)组.应用形态学、间接免疫荧光法,免疫组化技术和酶联免疫吸附法观察FK506对TGF-β诱导的HKC细胞转分化的作用、细胞培养上清液纤连蛋白、Ⅰ型胶原的变化及FK506对HKC细胞NF-κB活性的影响.结果:FK506(1,10,50 ng/ml) TGF-β组比TGF-β组诱导的HKC细胞E-钙粘蛋白和细胞角蛋白表达有所增强,波形蛋白和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达比TGF-β组减弱(P＜0.05),同时HKC细胞NF-κB的活性比TGF-β组减弱(P＜0.05),培养上清液纤连蛋白及Ⅰ型胶原的浓度比TGF-β组降低(P＜0.05);FK506(1、10、50 ng/ml)使基础状态下HKC细胞NF-κB的活性明显下降(P＜0.05)和上清液中纤连蛋白及Ⅰ型胶原的水平明显降低(P＜0.05).结论:①FK506剂量依赖性地抑制TGF-β诱导的肾小管上皮细胞转分化和纤连蛋白及Ⅰ型胶原的形成及基础状态下和TGF-β诱导的HKC细胞NF-κB的表达.②FK506抑制TGF-β诱导的HKC细胞转分化很可能与NF-κB的表达下调有关,需要进一步研究.     

Enhanced liver regeneration in rats treated with 15-deoxyspergualin alone and in combination with FK 506

The effects of 15-deoxyspergualin (DSG) alone and in combination with FK 506 (FK) on liver regeneration after 2/3 hepatectomy was studied. The administration of 5 mg/kg DSG increased the liver weight as a percentage of the body weight (RLW) in the 5 days following the hepatectomy. This enhanced regeneration was not affected if interleukin(IL)-2 was also given. The combination of 1 mg/kg DSG and 0.05 mg/kg FK induced the highest values for RLW. The amounts of food and water intake increased in the DSG and FK combined group. Blood chemistry indicated that the combined administration of DSG and FK could reduce side-effects more effectively than separate administration. These observation led us to conclude that DSG has a stimulating potency in liver regeneration by means of direct supression of T cell activation, and there is a synergistic effect on regeneration by DSG and FK.     

Effect of FK506 on the activation state of hepatic macrophages in Propionibacterium acnes-treated rats

Activated hepatic macrophages can provoke massive liver necrosis following endotoxin stimulation through microcirculatory disturbances due to sinusoidal fibrin deposition in rats pretreated with heat-killed Propionibacterium acnes. In these rats, FK506 (tachlorinus) administered 24 h before and at the time of endotoxin injection, significantly attenuated liver injury compared with the rats given no FK506. The effect of FK506 on hepatic macrophage activation and its action sites were studied in Propionibacterium acnes-treated rats. When rats received Propionibacterium acnes intravenously, hepatic-mRNA expression of interferon-γ-inducing factor and interleukin-2 and splenic-mRNA expression of interferon-γ were significantly increased compared with normal rats. Hepatic-mRNA expression of CD14, a receptor for lipopolysaccharide and its binding protein complex, was also increased preceding the expressions of the three cytokines in the liver and spleen. FK506 administration attenuated hepatic-mRNA expression of interleukin-2 and both superoxide anions as well as tumour necrosis factor-α production by hepatic macrophages, but did not change CD14-mRNA expression in Propionibacterium acnes-treated rats. It is suggested that a cytokine network through interferon-γ-inducing factor, interferon-γ and interleukin-2 may operate during activation of hepatic macrophages in rats treated with heat-killed Propionibacterium acnes, while CD14 expression on the cells may increase independently of this network. FK506 seems to attenuate such activation by suppressing hepatic interleukin-2 expression, without affecting CD14 expression on the cells.     

Enhancing and suppressive effects of immunosuppressants cyclosporin A,FK506, and KM2210 on the colony formation of murine bone marrow cells

Immunosuppressants cyclosporin A (CsA), FK506, and KM2210 modulated colony formations of murine hematopoietic progenitor cells. In a 4-h treatment with CsA, 10 g/ml increased the formation of colony-forming units of mixed lineages (CFU-Mix) but decreased the formation of highly proliferative potential colony-forming units (CFU-HPP); 1 g/ml of CsA increased the formations of CFU-HPP, CFU-Mix, and colony-forming units of granulocytes/macrophages (CFU-GM); 0.1 g of CsA increased the formation of CFU-Mix and burst-forming units of erythroid lineage (BFU-E). Lower doses of CsA appeared to induce an increase in various colony formations. FK506 increased CFU-HPP and CFU-Mix formations at lower doses. Another immunosuppressant, KM2210, increased CFU-HPP and CFU-GM formations but decreased CFU-Mix and BFU-E formations. In a 24-h treatment, 10 g/ml and 1 g/ml of CsA inhibited all the colony formations, but 0.1 g/ml of CsA increased CFU-Mix, CFU-GM, and BFU-E formations. Similarly, 100 ng/ml and 10 ng/ml of FK506 decreased all the colony formations but 1 ng/ml of FK506 increased CFU-HPP and CFU-GM formations. KM2210 inhibited all the colony formations. These findings showed that lower doses of CsA and FK506 appeared to increase the colony formations, although higher doses of these drugs decreased the colony formations, similar to the findings in a 4-h treatment. On the other hand, KM2210 showed opposing effects on colony formation with 4-h and 24-h treatments.     

Effects of FK506, an immunosuppressive agent,on genesis of water-immersion stress-induced gastric lesions in rats

We examined the effects of FK506, an immunosuppressive agent, on the genesis of water immersion stress-induced gastric lesions in rats. Using high-performance liquid chromatography, four kinds of prostaglandins, ie, 6-keto-prostaglandin F₁, prostaglandin F₂, prostaglandin E₂, and prostaglandin D₂, were detected, and no leukotrienes were detected in gastric mucosa in rats without stress. After 6 hr of stress, gastric lesions developed with decreases in all prostaglandin contents, and the emergence of peptide leukotrienes was observed. Intramuscular administration of FK506 (0.1, 0.25, 0.5, 1.0, and 2.0 mg/kg) reduced lesion index dose-dependently. Administration of FK506 at doses over 0.25 mg/kg decreased all prostaglandin contents, but did not affect the increase in leukotriene contents. Pretreatment with famotidine or omeprazole reduced lesion index, and the protective effects were equivalent to those of 1.0 mg/kg of FK506, although FK506 did not affect gastric secretion during water-immersion stress. Water-immersion stress did not change the activities of xanthine oxidase in either stomach or serum. Polyoxyethylenemodified superoxide dismutase did not prevent gastric lesions. Water-immersion stress significantly increased myeloperoxidase activity in gastric mucosa, and FK506 reduced the increase in myeloperoxidase activity induced by stress. From our results, other factors besides gastric acid secretion and tissue eicosanoid contents, such as chemoattractant factor, might also be involved in the genesis of water-immersion stress-induced gastric lesions in rats.     

Post-Ischemic Alterations in [3H]FK506 Binding in the Gerbil and Rat Brains

We investigated post-ischemic changes in FK506 binding protein (FKBP) in the brain after transient global ischemia in gerbils or transient focal ischemia in rats. [³H]FK506 was used to label FKBP as a immunophilin. In transient global ischemia, [³H]FK506 binding showed a transient reduction in the frontal cortex only 1 h after recirculation. In the striatum, the dorsolateral part exhibited a significant increase in [³H]FK506 binding 5, 24 and 48 h after ischemia. However, the ventromedial part showed a transient elevation in [³H]FK506 binding 24 h after ischemia. Thereafter, the ventromedial part showed no conspicuous change in [³H]FK506 binding up to 7 days after ischemia. The dorsolateral part also showed no significant change in [³H]FK506 binding 7 days after ischemia. In the hippocampus and thalamus, [³H]FK506 binding was unchanged in the stratum radiatum of the hippocampal CA₁ sector, hippocampal CA₃ sector, dentate gyrus and thalamus up to 7 days after ischemia. However, the stratum oriens of the hippocampal CA₁ sector showed a significant reduction in [³H]FK506 binding 48 h and 7 days after ischemia. A histological study showed that transient cerebral ischemia caused a severe damage in the striatum and hippocampal CA₁ sector. In a model of transient focal ischemia, a marked increase in [³H]FK506 binding was also found in the striatum and cerebral cortex where severe infarctions were observed. These results demonstrate that post-ischemic change in [³H]FK506 binding between the striatum and hippocampus may be produced by different mechanisms. Furthermore, our findings suggest that immunophilins may play some role in the pathogenesis of ischemic diseases.