人vWF-A1区蛋白的表达及其对血小板聚集的抑制作用

目的:进一步研究血栓形成的机制,开发抗血栓药物。方法: 应用基因重组技术在大肠杆菌中表达人vWF-A1区蛋白,经过纯化、复性,获得重组蛋白(rvWF-A1),同时用流式细胞术检测rvWF-A1与血小板膜糖蛋白血小板膜糖蛋白(glycoprotein, GP)Ib的结合能力,应用血小板聚集仪测定rvWF-A1对瑞斯托霉素(ristocetin)诱导的血小板聚集抑制作用。结果: 重组表达载体pQE-31-vWF-A1在大肠杆菌M15中得到高效表达,表达的重组蛋白量占菌体总蛋白的30%,Ni-NTA agrose柱纯化后,其纯度为95%,经复性的rvWF-A1蛋白具有良好的生物学活性。它可与血小板模糖蛋白血小板膜糖蛋白GPIb结合,阳性率为78.6%;它可以抑制ristocetin诱导的血小板聚集,抑制率为84.7%。结论: 在原核细胞中可以成功地高效表达人vWF-A1区蛋白,该重组蛋白有可能开发为有效的抗血栓药物。     

关于研究生课程"生物医学工程前沿"建设的意见

"生物医学工程前沿"是国家所规定的生物医学工程专业研究生的课程之一.它以极广的知识领域,最新的学术动态,最先进的医疗技术等为主要内容,展示该专业的研究范围、前沿动态、发展趋向及未来前景,是学生们全面了解本专业的最好窗口,也是获取专业知识的最丰富园地.     

Interaction of Escherichia coli heat-stable enterotoxin B with cultured human intestinal epithelial cells.

Binding of Escherichia coli heat-stable enterotoxin B (STb) to the human intestinal epithelial cell lines T84 and HT29 and to polarized T84 cells was studied to define the initial interaction of this peptide toxin with target cells. Equilibrium and competitive binding isotherms showed that 125I-STb bound specifically to T84 and HT29 cells; however, the toxin-epithelial cell interactions could be characterized by low-affinity binding (< or = 10(5) M(-1)) to a high number of binding sites (> or = 10(6) per cell). STb binding to T84 and HT29 cells as a function of 125I-STb concentration did not approach saturation at levels well above the effective biological concentration of STb for fluid secretion. Treatment of the 125I-STb-bound T84 and HT29 cells with an acidic saline solution to remove surface-bound toxin revealed that only approximately 55% +/- 10% of 125I-STb could be removed by this treatment at 4 degrees C, suggesting that approximately half of the bound STb was stably associated with the plasma membrane and/or internalized into the cytoplasm. Similar results were obtained when binding and internalization experiments were conducted at 22 and 37 degrees C. Immunofluorescence studies demonstrated that the strongest signal for STb appeared in the plasma membrane even after acid treatment. Toxin-treated cells also displayed diffuse cytoplasmic staining, indicating that once cell bound, STb did not appear to preferentially associate with membrane vesicles or cellular organelles. Binding and subsequent internalization of 125I-STb were not affected by treatment of the cells with trypsin, endoglycosidase F/peptide N-glycosidase F, Vibrio cholerae neuraminidase, tunicamycin, or 5 mM sodium chlorate, which blocks sulfation of surface proteoglycans. In addition, the internalization process was not altered by preincubation of the cells with the cytoskeleton inhibitors cytochalasin D and colchicine or cellular perturbants (i.e., 0.45 M sucrose and 5 mM sodium azide), indicating that cell surface proteins or carbohydrates did not function as STb receptors. The binding of 125I-STb to polarized T84 cells was also examined, and the total and nonspecific binding isotherms were found to overlap, indicating that the apical surface of polarized T84 cells did not contain a specific receptor for STb. In comparison to undifferentiated cells, twice the amount of bound STb (approximately 80% +/- 10%) was removable from polarized T84 cells after treatment with acidic solution. The percentage of surface-bound STb to polarized T84 cells did not vary significantly with the transepithelial electrical resistance of the cells or when STb was applied basolaterally. Together, our results indicate that STb binds with relatively low affinity to the plasma membrane of cultured intestinal epithelial cells and polarized T84 cells, probably to membrane lipids, and becomes stably associated with the lipid bilayer. The fact that a significant portion of the bound STb becomes free in the cytoplasm, even at a low temperature, suggests that the bound toxin may directly traverse the membrane bilayer.     

中国北方人群系统性红斑狼疮患者KIR基因多态性的研究

目的:检测和分析系统性红斑狼疮患者的杀伤细胞免疫球蛋白样受体(KIR)基因的多态性, 探讨系统性红斑狼疮(SLE)从基因到临床表达的发病机制.方法:应用PCR-SSP技术检测北方62例确诊SLE患者和61例同一区域同一民族正常对照者的KIR基因位点多态性.结果:SLE 患者KIR基因表型分布较常见的为KIR3DP1、 2DL1、 2DP1、 3DL1、 2DL3及1D, 其次为2DS4、 2DL5、 3DS1、 2DS2、 2DS5和2DL2, 较低者为2DS1、 2DS3及3DP1V, SLE病例组KIR3DS1, 2DL2、 2DL5和2DL3基因型频率比对照组显著降低(P<0.01).结论:中国北方人群的SLE的发生可能与多个KIR基因等位基因有相关性.     

森林脑炎病毒prM-E蛋白在昆虫细胞中的表达及免疫活性测定

目的为了表达森林脑炎病毒prME蛋白,为森林脑炎快速诊断试剂的研制奠定基础。方法经过RTPCR扩增、重组转移载体构建、细菌内转座和昆虫细胞转染,以杆状病毒昆虫细胞表达系统成功地表达了森林脑炎病毒MDJ01株prME蛋白。结果从感染细胞上清中电镜观察到重组蛋白形成的球型颗粒,说明重组病毒感染细胞后产生病毒样表达颗粒(viruslikeparticlesVLPs),并且分泌至细胞外。免疫印迹试验和间接免疫荧光试验表明,表达的重组蛋白能够与抗森林脑炎病毒抗体特异结合,具有良好的抗原性。ELISA和间接免疫荧光染色证实,重组prME蛋白可以作为抗原用于检测患者血清特异性抗体。结论在昆虫细胞中表达的prME具有良好的抗原性,本研究为森林脑炎快速特异诊断试剂研制奠定了基础。     

MERKEL CELL TUMOR COEXPRESSING CYTOKERATIN AND NEUROFILAMENT PROTEINS

Whorled filaments 10 nm in width were identified by anti-intermediate filaments antibodies in a Merkel cell tumor from a 52-year-old man. Immunohistochemlcal tests revealed that the tumor was stained with anti-keratin antibody and antibodies against the 68-kd and 200-kd subunits of neurofilament proteins but not antibody against the 150-kd subunlt. This is the first reported case of Merkel cell tumor expressing a 200-kd subunit of neurofilament proteins.     

血清抗甲状腺球蛋白抗体和抗甲状腺过氧化物酶抗体检测在甲状腺疾病诊断及甲亢预后判断中的价值

采用放免法检测甲状豚疾病患者抗甲状腺球蛋白抗体（ＴＧＡｂ）和抗甲状腺过氧化物酶抗体（ＴＰＯＡｂ）、并对部分Ｇｒａｖｅｓ病患者停药后随诊一年的结果进行分析。结果显示：（１）自身免疫性甲状腺疾病患者ＴＧＡｂ和ＴＰＯＡｂ活性及阳性率明显高于非ＡＩＴＤ，尤以桥本甲状腺炎为然。（２）ＧＤ治疗前及停药时ＴＧＡｂ和ＴＰＯＡｂ均阴性者与均阳性者停药一年内的复发率分别为０．５８３和０．２３１。（３）ＴＧＡｂ和ＴＰＯＡｂ均阴性，而停药时甲状腺刺激抗体（ＴＳＡｂ）阳性者，停药时ＧＤ复发的机率最大（０．９０９），提示ＴＧＡｂ和ＴＰＯＡｂ检测在ＡＩＴＤ诊断，鉴别诊断以及ＧＤ预后判断中具有重要的临床意义。     

人前磨牙牙髓牙本质复合体层粘连蛋白的免疫组织化学研究

目的　研究层粘连蛋白在牙髓牙本质复合体内的表达 ,探讨其分布与功能的关系。　方法　收集不同年龄新鲜健康人前磨牙 6 0颗 ,固定 ,脱钙 ,石蜡包埋 ,层粘连蛋白免疫组织化学染色。　结果　层粘连蛋白免疫反应性 ( IR)普遍存在于牙髓、前期牙本质和成熟牙本质小管与成牙本质细胞突起之间 ,以牙髓的神经与血管周围最为丰富 ,冠髓较根髓密集。　结论　层粘连蛋白 IR不仅见于牙髓与前期牙本质 ,而且也见于成熟牙本质 ,它对支持、固定牙髓牙本质复合体各结构的正常位置、形态可能发挥了重要作用     

hIL-2分泌肽序列在HepG2细胞中对hEndostatin表达和分泌差异的影响

目的:了解分泌肽序列在HepG2细胞中对人内皮抑素基因(hEndostatin)的cDNA表达和分泌差异的影响。方法:构建不含hIL-2分泌肽序列的真核表达载体pBlast-hEndo质粒, 转染人的HepG2细胞株, 用RT-PCR的方法检测HepG2(pBlast-hIL2-hEndo), HepG2(pBlast-hEndo), HepG2(pBlast-Mcs)和HepG2中hEndostatin的mRNA表达水平, 及制备4种细胞的总蛋白和收集各自的培养上清, 进行Westernblot分析蛋白表达和分泌的差异。结果:发现HepG2(pBlast-hIL2-hEndo)中hEndostatin的mRNA的水平显著高于HepG2(pBlast-hEndo)。而仅在HepG2(pBlast-hIL-2-hEndo)的细胞总蛋白和上清, 及HepG2(pBlast-hEndo)的细胞总蛋白中检测到hEndostatin表达, 在其它各株细胞总蛋白及上清中, 未检测到hEndostatin。结论:hIL-2分泌肽序列能促进hEndostatin基因在HepG2细胞中表达及分泌。