兔关节软骨细胞的分离、培养和形态学特征

[目的]探讨兔关节软骨细胞的分离、培养方法,观察单层高浓度培养时细胞表型表达情况.[方法]无菌条件下,从 2周龄新西兰白兔的颞颌关节及四肢关节髁突面削取软骨片,采用机械-酶消化法分离软骨细胞,经台盼蓝拒染计数,将细胞按 1× 106个 /孔接种于 6孔培养板,传代培养,描绘生长曲线.利用相差显微镜及透射电镜观察细胞形态.应用甲苯胺蓝及Ⅱ型胶原免疫组化对细胞进行鉴定.[结果]每克软骨能获取 1 5× 106个软骨细胞,活性率为 95%.培养 2～ 3 d,细胞贴壁、变形,呈多角形; 8 d左右,细胞融合成层.透射电镜观察显示细胞核圆形,有丰富的粗面内质网、高尔基体及分泌的基质成分.甲苯胺蓝及Ⅱ型胶原染色阳性.细胞传至 5代后,出现"成纤维细胞样".[结论]本研究建立了简单易行的软骨细胞分离、培养方法;初代、第 2代细胞生长良好,适合于实验研究;软骨细胞 5代培养后,细胞表型发生改变.     

烧伤后早期应用中/长链甘油三酯对患者免疫功能的影响

目的　探讨烧伤后早期肠内喂养中链甘油三酯 (MCT) /长链甘油三酯 (LCT)对机体免疫功能的影响及其可能机制。　方法　选取 30例烧伤面积 >30 %TBSA的患者 ,随机分为两组 (每组 15例 )。F组 ,饲以含MCT/LCT的肠内营养制剂Fresubin 75 0MCT ;N组 ,饲以只含LCT的肠内营养制剂Nutrison。于伤后 2 4h内进行完全肠内营养支持 ,共持续 10d。于伤后 1、4、7、10d检测两组患者血浆白细胞介素 (IL) 2、IL 4、前列腺素 (PG)E2 及外周血T淋巴细胞转化率的改变。　结果　伤后各时相点F组患者血浆IL 2水平与N组相比 ,无明显差异 (P >0 .0 5 ) ;伤后 4dF组PGE2 水平较N组明显降低 (P <0 .0 1) ;伤后 4、7、10dF组IL 4水平及外周血T淋巴细胞转化率均明显高于N组 (P <0 .0 5～ 0 .0 1)。　结论　在改善烧伤后机体的免疫功能方面 ,含MCT/LCT的肠内营养制剂较单纯LCT制剂更具优势。     

细胞内游离Ca2+在发育中大鼠皮层神经元无镁诱导惊厥性损伤中的作用

目的: 观察细胞内游离Ca2+([Ca2+]i)在培养的不同发育阶段皮层神经元无镁诱导惊厥性损伤中的作用,探讨惊厥性脑损伤年龄依赖性的可能机制.方法:体外培养6 d、17 d的胚胎大鼠皮层神经元用无镁细胞外液处理3 h,或于无镁处理前用NMDA(N-甲基-D-门冬氨酸)受体拮抗剂或Ca2+通道阻滞剂预处理,用MTT代谢率测定的方法检测神经元损伤,以Fluo-3作标记用激光共聚焦显微镜扫描的方法检测[Ca2+]i.结果:体外培养6 d、17 d的神经元单纯无镁组MTT代谢率较同期对照组降低.应用MK-801 10 μmol*L-1、AP-5 50 μmol*L-1、尼莫地平10 μmol*L-1预处理后再给无镁处理,培养6 d、17 d的神经元MTT代谢率均不同程度高于同期单纯无镁组.培养6 d、17 d的神经元相对荧光强度之间差异有显著性,两者与基线荧光强度比较差异亦有显著性.应用上述各种拮抗剂后,[Ca2+]i改变的峰值均明显低于同期单纯无镁组.结论: 在体外不同发育阶段的神经元,短暂无镁处理诱导惊厥样放电所引起的神经元线粒体功能损伤以及[Ca2+]i改变程度不同.这种[Ca2+]i改变的年龄依赖性可能是惊厥导致神经元损伤的年龄依赖性的机制之一.NMDA受体-Ca2+通道激活是导致这种[Ca2+]i改变及神经元损伤的关键环节.     

Lethal effect of mononuclear cells derived from human umbilical cord blood differentiating into dendritic cells after in vitro induction of cytokines on neuroblastoma cells

BACKGROUND: Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granulocyte-macrophage colony stimulating factor (rhG-MCSF) and recombinant human interleukin-4(rhIL-4) can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that whether the lethal effect is associated with the most proper concentration of dendritic cells. OBJECTIVE: To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range. DESIGN: Open experiment. SETTING: Department of Pediatrics, the Medical School Hospital of Qingdao University. MATERIALS: The study was carried out in the Shandong Provincial Key Laboratory (Laboratory for the Department of Pediatrics of the Medical School Hospital of Qingdao University) during September 2005 to May 2006. Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were voluntarily donated by the puerperas. Main instruments: type 3111 CO2 incubator (Forma Scientific, USA), type 550 ELISA Reader (Bio-Rad, USA). Main reagents: neuroblastoma cell line SK-N-SH (Shanghai Institute of Life Science, Chinese Academy of Sciences), RPMI-1640 culture fluid and fetal bovine serum (Hyclone), rhIL-4 (Promega, USA), rhG-MCSF (Harbin Pharmaceutic Group Bioengineering Co.Ltd), rat anti-human CD1a monoclonal antibody and FITC-labeled rabbit anti-rat IgG (Xiehe Stem cell Gene Engineering Co.Ltd). METHODS: ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of dendritic cells after in vitro induction by rhG-MCSF and rhIL-4. ② Different concentrations of dendritic cells[ dendritic cells: neuroblastoma cells=20∶1,50∶1,100∶1（2×108 L-1，5×108 L-1，1×109 L-1）], 1×109 L-1 T cells and 1×107 L-1 neuroblastoma cells were added in the experimental group. 1×109 L-1 T cells and 1×107 L-1 neuroblastoma cells were added in the control group. ③ Main surface marker CD1a molecules of dendritic cells were detected with indirect immunofluorescence, and the percent rate of dendritic cells was counted with ultraviolet light and expressed as the expression rate of CD1a+ cells. ④ Single effector cells and target cells were respectively set in the experimental group and control group to obtain the lethal effect. The lethal effect of dendritic cells on neuroblastoma cells was indirectly evaluated by detecting cellular survival with MTT assay. The lethal effect(%)=（1-A experimental well-A effector cell well/A target cell well）×100%.⑤The experimental data were presented as Mean ±SD, and paired t test was used. MAIN OUTCOME MEASURES: ① Morphological characters of dendritic cells in the process of induction and differentiation. ②CD1a+ cellular expression rate. ③Lethal effect of dendritic cells on neuroblastoma cells. RESULTS: ①Morphological characters of dendritic cells in the process of induction and differentiation: On the 15th day after human umbilical cord blood mononuclear cells were induced by rhG-MCSF and rhIL-4, typical morphology of dendritic cells could be seen under an inverted microscope. ②Expression rate of CD1a+ cells was （43.12±5.83）%. ③Lethal effect of dendritic cells on neuroblastoma cells: Lethal effect of dendritic cells stimulated T cells in each experimental group ( dendritic cells: neuroblastoma cells=100∶1,50∶1,20∶1 respectively) on neuroblastoma cells was significantly higher than that in control group[（31.00 ±4.41）%，（30.92±5.27）%，(33.57±5.35)%,(26.23±5.20)%, t=3.51，2.98，4.24, P < 0.01）; But the lethal effect of dendritic cells on neuroblastoma was significantly lower when their ratio was 100∶1 and 50∶1 in comparison with 20:1 （t=2.01，2.36, P < 0.05）, and no significant difference in lethal effect existed between the ratio at 100∶1 and 50∶1（t=0.06,P > 0.05）. CONCLUSION: Dendritic cells differentiated from human umbilical cord blood mononuclear cells after in vitro induction of cytokines can promote the lethal effect of T cells on neuroblastoma cells. The lethal effect is associated with the concentration of dendritic cells within some range.     

吸入性损伤犬降钙素的变化

目的单因素观察吸入性损伤或烧伤后血清免疫反应性降钙素(iCT)的变化,分析其诊断意义。方法将24只犬随机分为单纯吸入性损伤后中度(A)、重度(B)、特重度损伤(C)组及单纯重度烧伤(D)组,每组6只。吸人性损伤犬均在伤后6 h行纤维支气管镜检查,以明确其损伤程度。分别于不同时相点抽取犬静脉血检测iCT含量,抽取动脉血做血气分析。结果(1)经纤维支气管镜检查,证实A、B、C组犬符合吸入性损伤的预期程度。(2)与伤前值(38±22)ng／L比较,各组吸人性损伤犬iCT含量在伤后1 h均明显升高(P<0．05),伤后4 h明显高于D组(P(0．05),其中A组于24 h达峰值(453±224)ng／L,B、C组在48 h内呈进行性升高。D组犬iCT含量在伤后2 h开始持续升高,至伤后48 h为(125±41)ng／L。(3)血气分析结果显示,与伤前值(109±8)mm Hg (1 mm Hg=0．133 kPa)比较,A、D两组氧分压(PaO2)伤后各时相点无明显差异(P>0．05),B、C组犬从伤后8 h和伤后4 h开始持续下降,分别为(65±6)、(71±9)mm Hg。与二氧化碳分压(PaCO2)的伤前值(38±5)mm Hg比较,C组犬伤后24 h PaCO2开始升高[(52±11)mm Hg]。结论在吸入性损伤后8 h内,iCT的变化明显早于血气分析指标,其诊断意义接近纤维支气管镜检查。     

诱生型一氧化氮合酶特异性抑制肽的制备

目的：通过噬菌体展示技术筛选iNOS特异性抑制肽。方法：将iNOSFAD结合区及其附近序列的基因片段装入pET-28A( )，在大肠杆菌BL21中表达，His.bind^TM亲和层析柱纯化目的蛋白，使用纯化蛋白筛选Ph.D.-12^TM噬菌体库，筛选iNOS活性抑制作用较高的噬菌体克隆，测序并合成其中具有一致序列的短肽。结果：得到具有较高表达量的目的蛋白，经His.Bind^TM柱亲和层析纯化后纯度大于95％，以纯化蛋白筛选Ph.D.-12^TM噬菌体库，经4轮筛选获得10株iNOS活性抑制作用较高的噬菌体克隆，测序发现其中5株序列完全相同，合成该12肽，初步研究表明其对iNOS表现为高浓度抑制，低浓度兴奋的作用，而对nNOS及eNOS则没有影响。结论：以iNOSFAD片段蛋白为靶蛋白筛选得到的克隆对iNOS活性具有特异性影响，可根据这些特征设计合成小分子前导药物，创造新的活性药物。     

基于实验均方误差F值的基因表达数据最佳聚类评判方法研究

目的 近年来产生了一些用于分析基因表达数据的聚类算法，却很少有关于评价聚类算法方法的研究。本研究的目的是尝试建立一个定量的评价基因表达数据聚类结果的方法。方法 本研究提供了一个系统的评价聚类结果的方法，利用我们提出的实验均方误差F值对几个常见的聚类算法进行比较。结果 利用F值对类质量的评价和利用已有的生物学知识对类进行分析的结果一致。结论 实验均方误差F值可以定量地评判用于基因表达数据的聚类算法。     

重组血红素加氧酶-2在大肠杆菌中表达与纯化方法的研究

目的　确定重组血红素加氧酶 2在大肠杆菌中表达与纯化方法。方法　将已构建的血红素加氧酶 2(HO 2 )的重组质粒pMW1 72a HO 2转入大肠杆菌BL 2 1 ,分析不同温度及摇床转速对HO 2表达的影响 ,确定可溶性表达最佳条件。使用超声波、超速离心、分级盐析、分子筛层析等方法纯化HO 2。检测酶活性。结果　确定了HO 2可溶性表达最佳条件为 37℃ ,(85± 5 )r min ;确定了HO 2具体纯化路线。得到蛋白纯度为 95 .0 % ,纯化倍数为 2 .9倍 ,收得率为 4 0 .9%的活性HO 2蛋白。结论　获得了纯度高、具有活性的HO 2蛋白 ,为进一步进行HO 2结构与功能之间关系的研究提供了可行的纯化路线     

肝细胞癌SCT表现特征与Rb,PCNA的关系

目的　探讨肝细胞癌 (HCC)SCT (spiralCT)表现特征与癌细胞中视网膜母细胞瘤基因(Rb)、增殖细胞核抗原 (PCNA)表达的关系。方法　对 39例 (41个病灶 )经手术病理证实且行SCT动、静脉双期增强扫描的肝细胞癌病例 ,观察每个病灶的SCT表现特征 :肿瘤的大小、包膜、侵袭危险性、强化类型和肝硬化情况。用免疫组化法检测癌组织中Rb ,PCNA表达 ,分析评价SCT表现特征与蛋白之间的关系。结果　HCCSCT图像中肿瘤大小、包膜形成、侵袭危险性、强化类型和周围肝组织有无硬化与Rb蛋白之间无统计学意义 (P >0 .0 5 )。肿瘤大小、侵袭危险性、强化类型与PCNA之间有统计学意义 (P <0 .0 5 )。结论　肝细胞癌的SCT表现特征与PCNA的表达相关 ,根据其表现特征 ,可在一定程度上推测肿瘤组织PCNA表达情况 ,这有助于HCC的早期诊断及治疗方案的选择