MuSK-mCherry融合荧光蛋白的构建及对重症肌无力患者MuSK抗体的检测

目的：构建肌肉特异性激酶（MuSK）与红色荧光蛋白（mCherry）的重组融合蛋白（MuSK-mCherry）,并作为抗原用于重症肌无力（ MG）患者血清中的MuSK抗体（ MuSKAb）的检测。方法：应用PCR技术,从含有mCherry 基因的载体pRSET-B扩增mCherry基因,经T载体（pGEM-T Easy Vector）克隆至含有MuSK细胞外区第22-452位氨基酸肽段基因的载体pMT/BiP/V5-His（ MuSK）上,构建MuSK-mCherry融合荧光蛋白基因。将重组载体转染果蝇S2细胞,表达产物以共聚焦显微镜检查。以MuSK-mCherry融合蛋白作为抗原,应用荧光免疫沉淀试验检测MG患者MuSKAb。结果：成功地构建了MuSK-mCherry融合蛋白基因,并得到了表达。经对已知MuSKAb阳性的MG患者血清中的MuSKAb的检测证实,构建的MuSK-mCherry融合蛋白在荧光免疫沉淀试验中可以检测到MuSKAb。结论：以MuSK细胞外肽段与mCherry构建的融合荧光蛋白作为抗原,可以用于MG患者血清中的MuSKAb的检测。     

心脏起搏器综合征患者心室失同步化和收缩功能的变化

目的研究心脏起搏器综合征（PMS）患者心室失同步化和收缩功能的变化。方法14例心房颤动患者为对照组;因慢室率心房颤动安置心室按需型永久起搏器（VVI）的患者中,16例安置后出现PMS的患者为PMS组,18例未出现PMS的患者为无PMS组。用组织多普勒影像技术测定左、右心室侧壁和室间隔的收缩峰值速度、加速度和达峰时间。结果观察组左、右心室侧壁收缩期峰值速度、加速度较对照组明显下降（P〈0．05）;左心室侧壁与室间隔的收缩达峰时间间隔较对照组显著延长（P〈0．01）,右心室侧壁与室间隔的收缩达峰时间间隔较对照组显著缩短（P〈0．01）。PMS组与无PMS组比较,左、右心室侧壁室间隔的收缩期峰值速度、加速度、左室侧壁与室间隔的收缩达峰时间间隔差异无统计学意义（P〉0．05）,而右室侧壁与室间隔的收缩达峰时间间隔差异有统计学意义（P〈0．01）。结论右室失同步化是PMS中的一个重要因素,而心室收缩功能的变化和左室失同步化并不重要。     

活细胞内筛选地塞米松衍生物作用靶蛋白的研究

背景与目的：地塞米松衍生物（9-氟-16α-甲基11β，17-二羟基-3-氧-1，4-雄二烯-17β-羧酸）具有优于地塞米松的抗肿瘤活性，为探讨其抗肿瘤机制，本研究利用酵母三杂交技术在活细胞内筛选与之相互作用的靶蛋白。方法：构建诱饵质粒pGBKT7-GRα-LBD，利用酵母三杂交技术从人K562细胞cDNA文库中筛选与地塞米松衍生物相互作用的靶蛋白。结果：诱饵质粒成功构建，经Western blot分析可表达约31ku的诱饵蛋白，且诱饵蛋白没有毒性、渗漏和自激活现象。利用酵母三杂交技术从人K562细胞cDNA文库中筛选到37个能与地塞米松衍生物相互作用的蛋白质，并经酵母回转实验验证，得到20个真阳性克隆。结论：通过酵母三杂交技术在活细胞内筛选到20个与地塞米松衍生物有相互作用的蛋白。     

磷酸组织胺诱导的Ⅰ型超敏反应对肝损伤作用的研究

目的:通过观察与Ⅰ型超敏反应相关的生物活性介质--组织胺对家兔肝脏有无直接损伤作用,进一步论证Ⅰ型超敏反应对肝脏的损伤作用.方法:选择34只家兔随机分为对照组、实验Ⅰ组和实验Ⅱ组3组;对照组只进行正常饲料喂养,实验Ⅰ组在正常饲料喂养的同时每天给予0.4μg/kg耳静脉注射磷酸组胺注射液,实验Ⅱ组在正常饲料喂养的同时每天给予0.08 μg/kg耳静脉注射磷酸组胺注射液;动态观察以上3个组的血清谷丙转氨酶(ALT)及血清谷草转氨酶(AST)变化;利用光学显微镜观察以上3个组肝组织的病理变化.结果:无论是实验Ⅰ组或实验Ⅱ组,经过一段时间的观察,发现血清内ALT和AST含量均显着高于对照组(P＜0.01),但Ⅰ、Ⅱ组之间无显着性差异(P＞0.05);实验Ⅰ组和实验Ⅱ组在显微镜下观察,其肝脏均有不同程的损伤和病理改变,且实验Ⅱ组的损伤和变化大于实验Ⅰ组,而对照组的肝脏则无明显的病理变化.结论:组织胺对家兔肝脏确实有一定的损伤作用,而且随着投予剂量和时间的增加,肝脏的损伤和病理变化也越显著;通过本研究,可以得出Ⅰ型超敏反应导致肝脏病理变化及损伤的见解.     

超声测量颈动脉内膜中层厚度与颈动脉斑块的关系

为了探讨颈动脉内膜中层厚度与局限性颈动脉斑块的联系 ,对 91名受试对象的颈总动脉内膜中层厚度及颈内动脉和颈动脉分叉处的斑块进行超声检测 ,并将颈动脉内膜中层厚度进行分级。结果观察到有斑块者较无斑块者其颈动脉内膜中层厚度明显增加 ( 0 .83± 0 .16mm比 0 .6 4± 0 .12mm ,P <0 .0 1) ,且随斑块的严重程度增加 ,其内膜中层厚度呈增厚趋势。该结果支持颈总动脉内膜中层厚度与颈动脉局限性动脉粥样硬化斑块明显相关 ,提示颈动脉内膜中层厚度增厚可能是颈动脉粥样硬化的早期表现     

Development of an oral DNA vaccine against MG7－Ag of gastric cancer using attenuated salmonella typhimurium as carrier

AIM: To develop an oral DNA vaccine against gastric cancer and evaluate its efficacy in mice. METHODS: The genes of the MG7-Ag mimotope and a universal Th epitope (Pan-DR epitope, PADRE) were included in the PCR primers. By PCR, the fusion gene of the two epitopes was amplified. The fusion gene was confirmed by sequencing and was then cloned into pcDNA3.1(+) plasmid. The pcDNA3.1 (+)-MG7/PADRE was used to transfect an attenuated Salmonella typhimurium. C57BL/6 mice were orally immunized with 1X10(8) cfu Salmonella transfectants. Salmonella harboring the empty pcDNA3.1(+) plasmid and phosphate buffer saline (PBS) were used as negative controls. At the 6th week, serum titer of MG7-Ag specific antibody was detected by ELISA. At the 8th week cellular immunity was detected by an unprimed proliferation test of the spleenocytes by using a ((3)H)-thymidine incorporation assay. Ehrlich ascites carcinoma cells expressing MG7-Ag were used as a model in tumor challenge assay to evaluate the protective effect of the vaccine. RESULTS: Serum titer of antibody against MG7-Ag was significantly higher in mice immunized with the vaccine than that in control groups (0.841 vs 0.347, P<0.01; 0.841 vs 0.298, P<0.01), while in vitro unprimed proliferation assay of the spleenocytes showed no statistical difference between those three groups. Two weeks after tumor challenge, 2 in 7 immunized mice were tumor free, while all the mice in the control groups showed tumor formation. CONCLUSION: Oral DNA vaccine against the MG7-Ag momitope of gastric cancer is immunogenic. It can induce significant humoral immunity against tumor in mice, and the vaccine has partially protective effects.     

小儿输液治疗中发生护患纠纷的原因与防范

由于社会、家长、患儿及护理人员各方面的原因,在输液过程中常会产生护患纠纷.防范护患纠纷,首先要做好患儿及家长的心理疏导,同时要对护士进行评估与沟通方面的进行培训,并努力提高护士自身的技术水平和心理素质.     

Oral attenuated Salmonella typhimurium vaccine against MG7-Ag mimotope of gastric cancer

AIM: To develop an oral attenuated Salmonella typhimurium vaccine against gastric cancer and to evaluate its efficacy in mice. METHODS: A complementary sequence of Nco Ⅰ site and a sequence coding for MG7-Ag mimotope were designed at the 5'terminus of forward primer. Using p1.2 Ⅱ-HBCAg plasmid as template, PCR was performed to get a fusion gene of the mimotope and a HBcAg gene. The fusion gene was then subcloned into the plasmid pYA3341 complementary to Salmonella typhimurium X4550, and the recombinant plasmid was then transformed into attenuated Salmonella typhimurium X4550. Balb/c mice were orally immunized with the recombinant Salmonella typhimurium X4550. The mice were immunized every 2 wk to reinforce the immunity. At the 6th wk, serum titer of antibody was detected by ELISA, and at the 8th wk, cellular immunity was detected by 51Cr release test. Ehrlich ascites carcinoma cells expressing MG7-Ag were used in tumor challenge assay as a model to evaluate the protective effect of the vaccine. RESULTS: Serum titer of antibody against MG7-Ag was significantly higher in mice immunized with the vaccine than in control groups (0.9538±0.043 vs 0.6531±0.018, P<0.01; 0.9538±0.043 vs 0.6915±0.012, P<0.01), while in vitro 51Cr release assay of the splenocytes showed no statistical difference in the three groups. Two weeks after tumor challenge, 1 in 5 immunized mice was tumor free, while all the mice in the control group presented tumor. CONCLUSION: Oral attenuated Salmonella typhimurium vaccine against the MG7-Ag mimotope of gastric cancer is immunogenic. It can induce significant humoral immunity against tumors in mice, and has some protective effects.     

重症肌无力治疗性单价IgG抗体基因的构建

目的:构建一株对重症肌无力(MG)具有特异性免疫治疗作用的单价IgG类抗体基因.方法:应用定点突变技术,将致病性抗乙酰胆碱受体(AChR)抗体IgG637的重(H)链第322位氨基酸进行K322A突变,获得的突变型抗体IgG637/K322A基因再进行H链互补决定区3(CDR3)缺失突变,获得突变型抗体IgG637/K322A/CDR3ΔPLKP基因.经转化大肠杆菌XL1-Blue进行增殖后,转染哺乳类细胞CHO-k1进行表达,表达产物经ELIAS检测与补体C3的结合活性,经RIA检测与特异性抗原人AChR的结合活性.突变抗体H链再经杵(T366Y)臼(Y407T)突变,以利于异源H链的配对.结果:突变型抗体IgG637/K322A丧失了与补体C3结合的能力,突变型抗体IgG637/K322A/CDR3ΔPLKP丧失了与人AChR结合的能力.测序证实已经获得了预想的杵臼突变序列.结论:已经成功的制备了无补体激活能力的单价IgG类抗AChR抗体基因.