同步化法处理软骨细胞后对病毒转染效率影响的研究

目的探讨同步化法对反转录病毒转染软骨细胞效率的影响。方法将构建并经过包装后的反转录病毒载体转染到经低温处理后的同步化兔膝关节软骨细胞,运用免疫荧光显微镜观察荧光显色,并用硝酸还原法检测细胞培养液中一氧化氮(NO)的含量,并与非同步化转染组对照,酶联免疫吸附测定(ELISA)法检测细胞培养液中人白细胞介素-1受体(hIL-1Ra)的表达量,与非同步化的细胞上清液中hIL-1Ra的含量做空白对照。结果酶切鉴定重组基因正确;免疫荧光显微镜观察可见绿色荧光显色;同步化组上清液中NO含量为(145±3)μmol/L,非同步化组上清液中NO含量为(89±3)μmol/L;同步化组上清液中的hIL-1Ra的含量为(65±4)ng/L,明显高于非同步化组上清液中的hIL-1Ra的含量(41±3)ng/L(P<0.05)。结论软骨细胞经同步化后能显著提高反转录病毒载体的转染效率。     

逆转录病毒载体介导iNOS基因转染人CD3AK细胞的初步研究

目的　构建免疫细胞性一氧化氮供体CD3AK/iNOS细胞。方法　用脂质体介导重组逆转录病毒质粒 pLNC iNOS转染入包装细胞PA317中 ,经G4 18筛选出抗性克隆。用NIH3T3细胞测定病毒上清滴度。用病毒上清感染人CD3AK细胞。测定CD3AK/iNOS细胞培养上清中iNOS活性及NO含量。结果　脂质体转染后G4 18筛选出 38个抗性克隆 ,能稳定合成并分泌重组逆转录病毒颗粒。NIH3T3细胞测定病毒滴度为 1 2× 10 5cfu/ml。CD3AK/iNOS细胞合成并分泌的NO含量及iNOS活性比CD3AK细胞明显增高。结论　成功构建CD3AK/iNOS细胞 ,并能分泌高浓度NO。     

真核表达载体介导白细胞介素24在HepG2细胞的表达及其体外抗肿瘤效应研究

目的  研究真核表达载体pcDNA3.1(+) 介导白细胞介素（interleukin, IL）24在肝癌细胞系HepG2细胞中表达的可行性,以及IL-24体外对HepG2细胞的抗瘤效应和可能机制。方法 采用脂质体转染法将重组质粒pcDNA3.1(+)-IL-24 及空质粒分组转染HepG2细胞,在倒置显微镜下观察细胞形态变化。用噻唑蓝比色方法测量细胞增殖指数,流式细胞仪研究细胞早期凋亡率及其细胞周期分布。结果  IL-24 mRNA成功表达于HepG2细胞中。重组质粒转染组可见明显的凋亡细胞形态,48 h增殖抑制率（F=27.058,P<0.01）、72 h增殖抑制率( F=63.481,P<0.01)和早期细胞凋亡率( F=326.220,P<0.01)与对照组的差异均有统计学意义。细胞分布呈明显的G2/M周期阻滞。结论 真核表达载体pcDNA3.1(+)可以有效地介导IL-24在HepG2细胞的表达。IL-24对HepG2细胞有明显的增殖阻滞和凋亡诱导效应,G2/M细胞周期阻滞或许是IL-24抗瘤效应的机制。       

突变型人白细胞介素-2基因在大肠杆菌中的表达

目的：建立突变型人白细胞介素-2（rhIL-2）基因大肠杆菌表达系统，为rhlL-2的生产及临床应用奠定基础。方法：自人胎盘组织中提取总RNA，逆转录PCR（RT-PCR）扩增突变型人IL-2基因cDNA，通过对下游引物的设计将编码125位半胱氨酸（c125）的密码子TGT更改为编码丝氨酸的密码子TCT。构建表达型重组质粒pBV-IL-2，温度诱导表达．表达产物行SDS一聚丙烯酰氨凝胶电泳（SDS-PAGE）及WesternBlot鉴定。结果：DNA序列分析证实，重组质粒pBV-IL-2含有突变型人IL-2编码序列及正确的开放读码框架。SDS-PAGE显示，诱导表达碎菌后的沉淀中有分子质量大小约为16．5ku的外源蛋白产生。经Western印迹（WesternBlot）证明为rhlL-2。结论：成功构建了突变型人白细胞介素-2大肠杆菌表达系统。表达产物主要以包涵体的形式存在于碎菌后的沉淀中。     

白细胞介素-1β对大鼠软骨细胞MMP-13 表达的影响及 miR-27b 的调控作用

目的观察白细胞介素（IL）-1β对大鼠软骨细胞基质金属蛋白酶（MMP）-13 表达的影响及miR-27b 的调控作用。方法雄性Wistar 大鼠7 只提取软骨细胞。Western blot 检测IL-1β刺激软骨细胞0 h、24 h、48 h 各时间点 MMP-13 表达变化;miRNAs 微阵列分析48 h 内软骨细胞差异表达的miRNAs;Real-time PCR 定量分析筛选出下调最为明显的miRNAs;荧光素酶报告基因实验验证miR-27b 与MMP-13 的靶定调控关系。结果IL-1β刺激软骨细胞后,MMP-13 蛋白在0 h、24 h、48 h 各时间点表达逐渐增加（P < 0.05）;miRNAs 微阵列分析发现48 h 内软骨细胞有36 个miRNAs 出现表达变化,变化最为明显的有6 个,分别为miR-27b、miR-31、miR-26a、miR-26b、miR-23、 miR-204;Real-time PCR 显示miR-27b 下调最为明显;miR-27b 拟似物和荧光素酶表达质粒共转染软骨细胞后,荧光素酶活性受到明显抑制（P < 0.05）。结论IL-1β刺激软骨细胞后,出现miR-27b 的表达下调和MMP-13 蛋白的表达上调,miR-27b 与MMP-13 存在靶定调控关系。     

同步化法处理细胞后对病毒转染效率影响的研究

目的 探讨同步化法对反转录病毒转染软骨细胞效率的影响.方法 将构建并经过包装后的反转录病毒载体转染到经低温处理后的同步化兔膝关节软骨细胞,运用免疫荧光显微镜观察荧光显色,并用硝酸还原法检测细胞培养液中一氧化氮(NO)的含量,并与非同步化转染组对照,酶联免疫吸附测定(ELISA)法检测细胞培养液中人白细胞介素-1受体(hIL-1Ra)的表达量,与非同步化的细胞上清液中hIL-1Ra的含量做空白对照.结果 酶切鉴定重组基因正确;免疫荧光显微镜观察可见绿色荧光显色;同步化组上清液中NO含量为(145±3)μmol/L,非同步化组上清液中NO含量为(89±3)μmol/L;同步化组上清液中的hIL-1Ra的含量为(65±4)ng/L,明显高于非同步化组上清液中的hIL-1Ra的含量(41±3)ng/L(P＜0.05).结论 软骨细胞经同步化后能显著提高反转录病毒载体的转染效率.     

异维A酸治疗尖锐湿疣对白细胞介素-2、可溶性白细胞介素-2受体影响

目的 :观察异维A酸治疗尖锐湿疣对血清白细胞介素 2 (IL 2 ) ,可溶性白细胞介素 2受体(sIL 2R)的变化 ,以探讨异维A酸治疗尖锐湿疣的作用机制。方法 :3 2例尖锐湿疣病人 ,初发 1 3例 ,复发 1 9例 ,给异维A酸胶丸 1 0mg,po,tid或bid ,随访 3mo ,治疗前及治疗后mo 4查血清sIL 2R ,IL 2。结果 :痊愈 2 4例 (75 % ) ,显效 3例 ,有效 2例 ,无效 3例 (9% ) ;血清sIL 2R初发病人治疗前为(2 0 1±s 1 2 1 ) pmol·L-1,治疗后mo 4为 (1 93±1 3 2 ) pmol·L-1,P <0 .0 5 ;血清sIL 2R复发病人治疗前为 (693± 1 2 1 )pmol·L-1,治疗后mo 4为 (5 1 9± 1 0 3 ) pmol·L-1,P <0 .0 1 ;血清IL 2初发病人治疗前 (7.0± 1 .2 ) μg·L-1,治疗mo 4为 (7.2± 1 .2 )μg·L-1,P <0 .0 1 ;血清IL 2复发病人治疗前 (6±3 ) μg·L-1,治疗后mo 4为 (8.5± 2 .7) μg·L-1,P<0 .0 1。结论 :异维A酸通过调节免疫及其他多种机制达到治疗尖锐湿疣的作用     

白藜芦醇对兔实验性骨关节炎白细胞介素-1β表达的影响

目的:研究白藜芦醇干预兔实验性骨关节炎模型动物后关节软骨、滑膜细胞白细胞介素-1β合成的变化,探讨其治疗骨关节炎的效果和作用机制。方法:36只新西兰大白兔,随机抽取6只,作为正常对照组(A组),其余30只参考文献方法,制备兔OA模型,术后4周,经X线和病理改变证实造模成功后,将造模动物随机分为:模型组(B组)、阳性药物对照组(C组)(双醋瑞因1 mg·kg-1·d-1)、受试药物高剂量组(D1组)(白藜芦醇120 mg·kg-1·d-1)、中剂量组(D2组)(白藜芦醇60 mg·kg-1·d-1)、低剂量组(D3组)(白藜芦醇30 mg·kg-1·d-1),每组6只。并按设定剂量每日灌胃给药,连续给药6周后,处死动物,切取股骨内髁软骨标本,脱水石蜡包埋后切片,用免疫组化法检测软骨细胞中IL-1β水平。结果:受试药物组(D1组、D2组、D3组)软骨细胞中IL-1β阳性细胞分别为(21.8±3.8)%,(26.1±13.3)%,(30.4±4.9)%,明显低于模型组(B组)(49.1±5.5)%和阳性药物对照组(C组)(36.0±4.0)%,(P<0.01)。结论:高、中剂量的白藜芦醇能抑制模型组动物软骨细胞合成IL-1β,其能力优于阳性对照药物。     

周期性张应变对重组人白细胞介素-1β诱导的一氧化氮表达作用的实验研究

目的在P1代1月龄雄性新西兰大白兔膝关节软骨细胞水平,观察周期性张应变(CTS)对重组白细胞介素(IL)-1β诱导的一氧化氮(NO)表达的拮抗作用,从而了解力学信号在软骨修复中的活动机制。方法将体外培养的兔膝关节P1代软骨细胞随机分为5组,每组有18个样本,分为空白对照组、IL-1β作用组及IL-1β和CTS共同作用组,CTS分别作用8、16、24h,24h后收取培养细胞上清液测定NO含量,比较各组变化。结果正常兔膝关节P1代软骨细胞表达一定量的NO,IL-1β作用组NO含量明显升高,差异具有统计学意义(P<0.05);IL-1β和CTS共同作用组NO含量明显减低,同IL-1β作用组相比,各组间差异具有统计学意义(P<0.05),CTS作用8h最佳阻止了NO的表达。结论CTS对rhIL-1β诱导的NO表达具有强大的拮抗作用,并且CTS产生的力学信号转化成强大的生物化学信号后能够持续性地以特定方式阻滞分解介质的产生。     

携带反义MDR1基因的逆转录病毒载体的构建和表达

目的　建立逆转录病毒介导的反义多药耐药MDR1基因转移体系 ,探讨反义RNA封闭白血病耐药细胞中MDR1基因表达的能力。方法　逆转录 聚合酶链反应 (RT PCR)法从白血病耐药细胞株HL 6 0 /VCRmRNA中扩增出含有启始密码子ATG的 5 2 4bp的MDR1基因cDNA片段 ,反向插入逆转录病毒载体 pLXSN ,通过脂质体转和乒乓感染单、双噬型包装细胞GP +E86和GP +envAM 12 ,以双嗜型病毒上清感染白血病耐药细胞K5 6 2 /MDR ,半定量RT PCR和流式细胞术(FCM)分析MDR1基因转录和P 糖蛋白 (P gp)的表达。 结果　酶切、PCR和DNA测序证实反义MDR1基因重组逆转录病毒载体 pLASSN构建正确 ;双嗜型病毒生产细胞的滴度为 1 3× 10 5CFU/ml,巢式PCR和补救分析均未检测到辅助病毒存在 ;PCR和RT PCR分析证实病毒生产细胞和反义修饰的耐药细胞K5 6 2MDR/LASSN中均有反义MDR1基因整合和MDR1基因反义RNA的转录 ;FCM分析K5 6 2MDR/LASSN细胞中P gp表达强度和表达率比对照细胞均有明显下降。 结论　逆转录病毒介导的反义基因转移系统安全有效 ,可用于肿瘤耐药逆转的研究     

蚓激酶重组逆转录病毒载体pLN-BCP-LK~R的构建及其在山羊乳腺组织中的表达

目的在山羊奶中表达蚓激酶。方法将含有蚓激酶基因的表达盒插入到逆转录病毒载体pLNCL中获得了蚓激酶重组逆转录病毒载体,转染泌乳期奶山羊乳腺小叶并获得暂时表达。利用脂质体转染PA317包装细胞,G418进行筛选得到阳性细胞克隆,克隆扩大培养后病毒上清感染妊娠期奶山羊乳腺小叶组织,山羊产后采奶纤维蛋白平板溶圈法检测蚓激酶活性。结果产后奶样经纤维蛋白平板溶圈法检测羊奶具有明显纤溶活性。结论蚓激酶基因已经整合到奶山羊乳腺组织并能实现相对稳定的表达。     

Cyclin D_1基因蛋白在乳腺癌中的表达

目的 探讨Cyclin D_1基因在乳腺癌中的表达和意义.方法 应用ABC免疫组化法检测14例癌旁组织及49例乳腺癌中Cyclin D_1蛋白的表达.结果 Cyclin D_1在乳腺癌中有较高表达,过表达率49%(24/49);在14例癌旁乳腺组织中无过表达;乳腺癌的临床病理特征无明显相关性.结论 Cyclin D_1基因过表达是乳腺癌发生发展过程中的重要事件     

Im人成熟型TGF-β1重组表达载体的构建

目的构建人类成熟型TGF-β1（hTGF-β1）基因的表达载体,探索TGF-β1在大肠埃希菌中的表达,获得重组表达载体pET32a-hTGFβ1。方法Trizol法抽提人新鲜胎盘组织的总RNA,RT-PCR法扩增人成熟型TGF-β1基因,PCR产物纯化后经BglⅡ和HindⅢ双酶切后与相同酶切的pET32a（＋）载体连接,构建重组载体pET32a-hTGF-β1。结果成功构建了hTGF-β1重组表达载体pET32a-hTGFβ。结论hTGF-β1基因的原核表达载体的成功构建,为下一步表达、纯化该基因的蛋白产物以及hTGF-β1多克隆抗体的制备提供了依据。     

Expression of heme oxygenase-1 and regulation by cytokines in human osteoarthritic chondrocytes

Heme oxygenase-1 (HO-1) is implicated in the protection against tissue injury. We investigated the expression of this protein in cartilage sections and chondrocytes obtained from osteoarthritic patients. HO-1 was immunodetected in preparations from cartilage and also in chondrocytes cultured in the absence of stimulation. We found that HO-1 can be modulated by cytokines since the pro-inflammatory cytokines interleukin (IL)-1β, IL-17 and tumour necrosis factor-α (TNF-α) down-regulated this protein, whereas the anti-inflammatory cytokine IL-10 exerted the opposite effect. Our results suggest a role for HO-1 as part of protective mechanisms against tissue injury in human cartilage.     

无肝状态下丙泊酚代谢相关UGT1A6在大鼠肝外器官的基因表达

目的:研究无肝期前、后与丙泊酚代谢相关的代谢酶UGT1A6在大鼠小肠、肾、肺、脑器官组织中基因表达的变化,以期初步阐释丙泊酚无肝期肝外代谢特点形成的原因。方法:15只大鼠单次注射丙泊酚后随机分为3组(n=5),A组为对照组,B组阻断肝门30min,C组阻断肝门60min,截取各组大鼠小肠、肾、肺、脑器官组织,采用逆转录聚合酶链式反应(RT-PCR)技术,检测各组UGT1A6mRNA的表达。结果:大鼠各受检器官组织中UGT1A6的基因表达水平B、C组明显高于A组(P<0.01);B组与C组相比,没有显著性差异(P>0.05)。结论:大鼠受检组织中UGT1A6基因表达在无肝期比无肝期前增多,系可能促进丙泊酚无肝期肝外代谢增强的原因。     

ERO1L蛋白在胃癌组织中表达水平变化及临床意义

目的 探讨了内质网氧化物蛋白(ERO1L)在胃癌组织中的表达变化以及与患者预后的关系.方法 选取2009年6月—2011年6月接受胃癌根治术的128例患者的胃癌组织作为研究对象,并选择相同患者的癌旁无病变组织作为对照.采用实施荧光定量PCR、Western blot和免疫组织化学分析肿瘤组织和癌旁组织ERO1L mRNA和蛋白质表达水平.并分析ERO1L蛋白的表达水平与胃癌患者预后的关系.结果 与癌旁组织比较,胃癌组织中ERO1L mRNA和蛋白质水平明显增加(P<0.05).ERO1L蛋白主要表达于细胞质中,经评定其中102例ERO1L表达呈阳性,26例表达呈阴性.ERO1L阳性组患者术后1、3和5年的累积生存率明显低于阴性组(P<0.05).结论 ERO1L在胃癌组织中呈高表达状态,并与患者预后密切相关.     

Balb／C小鼠金属硫蛋白—1基因在大肠杆菌中的高效表达

将Balb/C小鼠金属硫蛋白－1基因克隆于质粒表达载体pET-21a上,克隆采用限制性内切酶BamH1与EcoR1酶切pET21a及金属硫蛋白－1基因PCR产物,连接成pET21a-MT重组质粒,并将其转化进入大肠杆菌表达受体菌BL21（DE3）中,经异丙基－β-硫代半乳糖苷（IPTG）诱导,SDS－聚丙烯酰胺凝胶电泳（PAGE）证实产生高效表达金属硫蛋白－1基因的金属硫蛋白融合蛋白。表达蛋白在胞内主要以不溶性包涵体存在。     

Modulation of IL-1-induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities

1. Nitric oxide (NO) is produced in diseased joints and may be a key mediator of IL-1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU)] and classical [N^ω-monomethyl-L-arginine (L-NMMA), N^ω-nitro-L-arginine methyl ester (L-NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin-1β (rhIL-1β) on rat cartilage anabolism. Three different culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps.
2. Chondrocyte beads and cartilage entities were incubated in vitro for 48 h in the presence of rhIL-1β with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [Na₂³⁵SO₄] and NO production by cumulated nitrite release during the period of study.
3. Chondrocytes and patellae, as well as femoral head caps, responded concentration-dependently to IL-1β challenge (0 to 250 U ml⁻¹ and 0 to 15 U ml⁻¹ respectively) by a large increase in nitrite level and a marked suppression of proteoglycan synthesis. Above these concentrations of IL-1β (2500 U ml⁻¹ and 30 U ml⁻¹ respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration-response curves.
4. When studying the effect of NOS inhibitors (1 to 1000 μM) on NO production by cartilage cells stimulated with IL-1β (25 U ml⁻¹ or 5 U ml⁻¹), we observed that: (i) their ability to reduce nitrite level decreased from chondrocytes to cartilage samples, except for L-NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU>L-NMMA⩾SMT>AG⩾L-NAME and (iii) AETU was cytotoxic when used in the millimolar range.
5. When studying the effect of NOS inhibitors on proteoglycan synthesis by cartilage cells treated with IL-1β, we observed that: (i) they had more marked effects on proteoglycan synthesis in chondrocytes than in cartilage samples; (ii) they could be roughly classified in the following rank order of potency: L-NAME⩾L-NMMA>>AG>SMT>>AETU and (iii) potentiation of the IL-1 effect by AETU was consistent with cytotoxicity in the millimolar range.
6. D-isomers of L-arginine analog inhibitors (1000 μM) were unable to correct nitrite levels or proteoglycan synthesis in IL-1β treated cells. L-arginine (5000 μM) tended to reverse the correcting effect of L-NMMA (1000 μM) on proteoglycan synthesis, thus suggesting a NO-related chondroprotective effect. However, data with L-NAME and SMT argued against a general inverse relationship between nitrite level and proteoglycan synthesis.
7. Dexamethasone (0.1 to 100 μM) (i) failed to inhibit NO production in femoral head caps and chondrocytes beads whilst reducing it in patellae (50%) and (ii) did not affect or worsened the inhibitory effect of IL-1β on proteoglycan synthesis. Such results suggested a corticosteroid-resistance of rat chondrocyte iNOS. Data from patellae supported a possible contribution of subchondral bone in NO production.
8. In conclusion, our results suggest that (i) NO may account only partially for the suppressive effects of IL-1β on proteoglycan synthesis, particularly in cartilage samples; (ii) the chondroprotective potency of NOS inhibitors can not be extrapolated from their effects on NO production by joint-derived cells and (iii) L-arginine analog inhibitors are more promising than S-substituted isothioureas for putative therapeutical uses.

     

In vitro effects of monophthalates on cytokine expression in the monocytic cell line THP-1 and in peripheral blood mononuclear cells from allergic and non-allergic donors

It has recently been shown that plasticizers are present in indoor air dust, which may lead to human exposure via the inhalation route. Moreover, studies have indicated that plasticizers may possess adjuvant effects increasing the health damaging potential of allergens. The aim of this study was to investigate the in vitro effect of metabolites of phthalate plastisizers, such as whether an adjuvant effect is paralleled by changes of the cytokine expression in the monocytic cell line THP-1 and in peripheral blood mononuclear cells (PBMCs) from allergics and non-allergics. The toxicity monitored by cell viability was determined by incubating THP-1 cells with a 10-fold dilution series of monophthalates for 24 h. At different points in time cytokine expression (IL-1β, IL-6, IL-12 (p35)) in THP-1 cells incubated with non-toxic concentrations of monophthalate (2–20 μg/ml)±LPS (1 μg/ml) were determined using Quantitative Competitive RT-PCR. PBMCs from allergics and non-allergics were incubated with monophthalate 220 μg/ml) for up to 48 h and cytokine expression (IL-4, IL-5, IFN-γ) was measured using real-time PCR. The cytotoxic level of monophthalates is 20–200 μg/ml, depending on the individual monophthalate. There seems to be a correlation between increasing side-chain length and toxicity. Monophthalates did not induce changes in cytokine expression in THP-1 cells, though there is an increase when co-incubating with LPS. Cytokine expression in PBMC seems virtually unchanged when co-incubated with monophthalate, though mono-n-butyl phthalate (MBUP) tends to increase the level of IL-4 in PBMCs from allergic individuals. The two cellular models demonstrated the dynamics of regulated cytokine mRNA and are applicable for in vitro immunotoxicological investigations. The results regarding monophthalates suggest these to have a limited effect on cytokine expression in the monocytic cell line THP-1 and weak effect on cytokine expression in PBMCs from allergic and non-allergic individuals.