大鼠咽肌前运动神经元的跨突触定位

支配咽肌的运动神经元位于疑核，控制凝核的前运动神经元位于孤束核。孤束核中的前运动神经元在控制吞咽运动中起着重要作用。但支配咽肌的前运动神经元位于孤束核的什么部位还不清楚。文内用假狂犬病毒研究字大鼠咽肌前运动神经元在孤束核中的定位。     

Encephalomyocarditis (EMC) virus-induced sialodacryoadenitis in mice

The mode of occurrence of the D variant of encephalomyocarditis (EMC-D) virus-induced acute sialodacryoadenitis was investigated using three strains of mice differing in their sensitivity to EMC-D virus-induced diabetes (C57BL/6: resistant; BALB/c: moderately sensitive; DBA/2: highly sensitive). Mice were intranasally inoculated with high (10(5) PFU/mouse) or low dose (10(2) PFU/mouse) of EMC-D virus. Although there were individual differences, the blood virus titer generally reached the peak earlier in the high-dose group than in the low-dose group. Signals of viral RNA and histopathological changes were seen in parotid glands and intraorbital and extraorbital lachrymal glands. In these glands, signals of viral RNA and histopathological changes were detected only in acinar cells and initial lesions were characterized by pyknosis of acinar cells. Coagulative necrosis with interstitial inflammatory cell infiltration developed later in parotid glands of BALB/c mice of the high-dose group and in intraorbital and extraorbital lachrymal glands of all groups except for C57BL/6 mice of the low-dose group. Such changes were not observed in epithelial cells of the ductal system. The present results indicate that EMC-D virus shows clear tissue and cell tropism within the salivary and lachrymal glands, probably due to the distribution of receptors for EMC virus.     

逆转录病毒载体介导的HBsAg抗原表达及其稳定性研究

目的 探讨逆转录病毒载体介导的HBsAg抗原的表达及其稳定性。方法 将HBsAg基因插入逆转录病毒载体PLXSN中，构建成重组逆转录病毒载体。用电穿孔法转染PA317细胞，包装成假病毒颗粒，在不同温度下冻存。于不同时间用假病毒颗粒感染HepG2、NIH3T3及293细胞，用RT—PCR及ELISA法检测HBsAg表达；以感染后G418抗性克隆形成数确定假病毒颗粒的活力。结果 在各个时间段HBsAg表达量均差异无显著性。在-20℃冻存时，6个月后克隆数即下降过半，12个月后仅形成少数克隆，24个月后无克隆形成；在-40℃冻存时12个月后HepG2、NIH3T3、293形成的克隆数分别为121、332和89，24个月后分别为42、137和43，与冻存前比较差异有显著性；-70℃冻存时，24个月后克隆数分别为159、463和112，与冻存前比较差异无显著性。结论 HBsAg重组逆转录病毒颗粒在-70℃保存2年后，病毒活力未受明显影响，HBsAg表达量亦无明显改变。     

丙型肝炎病毒核心蛋白在HepG2细胞中的表达及其对端粒酶活性的影响

目的 建立HCV核心蛋白细胞表达模型，并探讨其对细胞端粒酶活性的影响。方法 用PCR法扩增出HCV核心基因cDNA，将其插入真核表达载体pBK-CMV的HindⅢ和BamHⅠ位点间，构建重组质粒pBK-HCVc。再将重组质粒pBK-HCVc和空载体分别导入肝癌细胞株HepG2中，G418筛选，RT－PCR、免疫组化和蛋白印迹鉴定HCV核心蛋白表达。PCR－ELISA法检测端粒酶活性。结果 构建的pBK-HCVc质粒在HepG2细胞中有稳定表达。表达HCV核心蛋白的细胞HepG2-C的端粒酶活性较转染空载体的细胞HepG2-CMV明显升高。结论 HCV核心蛋白上调了端粒酶活性，可能是HCV诱发肝细胞癌的一种途径。     

一个能诱生戊型肝炎病毒中和抗体的ORF2编码蛋白短片段

目的：比较戊型肝炎病毒（HEV）第4基因型ORF2编码蛋白片段pN472-C617（aa472～617）和pN477-C613（aa477～613）的免疫原性，找到能诱生HEV中和抗体的ORF2编码蛋白的更短片段。方法：表达和纯化pN472-C617和pN477-C613，分别免疫BALB/c小鼠，以间接ELISA检测免疫血清的抗体效价，并以基于PCR的体外中和试验检测免疫血清的中和活性。结果：pN472-C617和pN477-C613可在大肠杆菌高效可溶性表达，纯化后的重组蛋白能在小鼠体内诱导出高效价的抗体。基于PCR的体外中和试验显示pN472-C617免疫血清可有效中和HEV，阻断其在敏感细胞表面的吸附和细胞内复制；而两端仅比pN472-C617各短5个氨基酸的pN477-C613的免疫血清不具有中和HEV的活性。结论：重组蛋白pN472-C617具有良好的抗原性和诱生中和抗体的能力，是目前文献报道中含有HEV中和抗原表位的最短ORF2编码蛋白片段，可作为重组亚单位候选疫苗用于HEV疫苗的开发。     

慢性乙肝患者血清趋化因子RANTES水平与肝功能生化指标等的相关性研究

Objective To investigate the level of the serum chemokine RANTES and its correlation with serum biochemical indices of liver function test, HBeAg and HBV DNA load in patients with chronic hepatitis B.Methods 144 patients with chronic hepatitis B (observed group) and 18 normal cases (control group) were enrolled in this study. The serum level of chemokine RANTES was detected with an ABC-ELISA assay. Statistical analysis was performed on the software of SPSS13.0. Results The serum chemokine RANTES level in the observed group (3930.12 ng/ml±2856.96) ng/ml was significantly higher than that in the control group (329.46 ng/ml±152.23) ng/ml. The results from the observed group indicated the positive correlation of serum RANTES level with indices of liver function test, including ALT (r=0. 197, P=0.018), AST(r=0.239, P=0.004) and TBil (r=0.316, P=0.001), but did not with PTA (r=-0.078, P=0.357). Neither difference of serum chemokine RANTES level between HBeAg-positive group and HBeAg-negative group nor that between high HBV DNA load group (≥105 copies/ml) and low HBV DNA load group (< 105 copies/ml) were statistically significant (P=0.407 and 0.185, respectively). Conclusions Serum chemokine RANTES level in patients with chronic hepatitis B elevates significantly and is not affected by HBeAg or HBV DNA load. Its positive correlation with indices of liver function test indicates that RANTES might play an important rule in the pathogenesis of chronic hepatitis B.     

血清乙型肝炎病毒DNA水平对HBV全基因组克隆效率的影响

目的：通过比较血清HBV DNA水平对一片段PCR法和两片段PCR法这两种不同方法的HBV全基因组克隆效率的影响，选择出合适的HBV全基因组克隆技术，供后续的HBV的基础和临床研究。 方法：采集了慢性乙型肝炎（CHB）患者85份血清，分别用本研究建立的一片段PCR法及两片段PCR法扩增HBV全基因组，经双酶切鉴定后将PCR产物克隆入载体，进行HBV全基因组序列测定。同时用实时荧光定量PCR法检测上述85份血清的HBV DNA滴度。 结果： 本研究建立的一片段PCR法扩增成功需要的血清HBV DNA浓度高于两片段PCR法,两种方法扩增的效率比较为：一片段PCR法扩增的阳性率低于两片段PCR法（P<0.01）。一片段PCR法的保真性和灵敏度分析发现： 保真性：核苷酸的人为突变率为1.13 bp/kb；灵敏度：为102个初始模板。浓度大于103的重组质粒DNA80%可以成功扩增，浓度低于该值的扩增效率比较低。结论： 血清HBV DNA水平对两种扩增方法的阳性率有一定影响，滴度高低是选择合适PCR扩增方法的依据。HBV DNA滴度大于106copies/L的样本，可选用一片段PCR法的全基因组扩增、克隆技术，而对HBV DNA滴度小于106copies/L样本，则可选用两片段PCR法的全基因组扩增、克隆技术。本研究建立的一片段PCR法扩增HBV全基因组克隆的技术是一种值得推荐的好方法，但还需进一步优化。     

戊型肝炎病毒Ⅰ、Ⅱ与Ⅲ、Ⅳ基因型B细胞抗原表位的异同

目的：制备戊型肝炎病毒(HEV)缅甸株和墨西哥株ORF2重组蛋白(p166Bur和p166Mex)的单克隆抗体(McAbs)，用于分析HEV不同基因型B细胞抗原表位的特点。方法：将免疫BALB/C小鼠脾细胞与SP2/0骨髓瘤细胞融合，获得分泌抗-p166Bur和抗-p166Mex McAbs的杂交瘤细胞株，然后采用ELISA和免疫印迹法测定McAbs与不同基因型HEV ORF2编码蛋白p166的免疫反应性。结果：获得4株杂交瘤细胞株，即分泌抗-p166Bur McAbs的2G2、2B1以及分泌抗-p166Mex McAbs的D8G10和E5E12，其中2B1分泌的McAb仅能与第Ⅰ、Ⅱ基因型编码的重组蛋白结合，而其余3株分泌的McAbs既能与Ⅰ、Ⅱ基因型HEV的p166重组蛋白发生反应，也能与Ⅲ、Ⅳ基因型的p166蛋白反应。结论：HEV第Ⅰ、Ⅱ基因型与Ⅲ、Ⅳ基因型ORF2编码蛋白既有共同又有不同的B细胞抗原表位。     

RNAi抑制XJ-160病毒复制的研究

目的通过建立RNA干扰(RNA interference，RNAi)抑制XJ-160病毒复制的模型，在序列特异性、位置效应和量效关系等方面，探讨RNAi对XJ-160病毒复制的影响，为进一步研究RNAi的抗病毒作用和机制奠定基础。方法按照siRNA(short interferencing RNA)的设计要求合成XJ-160病毒特异siRNA，脂质体法转染BHK-21细胞后感染XJ-160病毒，通过测定病毒滴度、蛋白表达量及XJ-160病毒：RNA合成研究siRNA对XJ-160病毒复制的抑制。结果成功建立了RNAi抑制XJ-160病毒复制的模型，探讨了RNAi抑制该病毒复制作用的特点。结论外源导入的siRNA能够抑制XJ-160病毒的复制，并且这种抑制作用具有明显的序列特异性、位置效应和存在量效关系等特点；siRNA是通过降解病毒RNA实现抑制病毒复制作用的。     

1Alpha,25-dihydroxyvitamin D3 restores thymocyte apoptosis sensitivity in non-obese diabetic (NOD) mice through dendritic cells

AIMS/HYPOTHESIS: Resistance of NOD thymocytes to apoptosis-inducing signals is restored by 1alpha,25-dihydroxyvitamin D3 (1alpha,25OH2D3), a therapy preventing diabetes in NOD mice. We studied whether modulation of thymocyte apoptosis is due to direct effects on thymic T lymphocytes or indirect effects via thymic dendritic cells, since both cell types constitute known targets for 1alpha,25OH2D3. METHODS AND RESULTS: Female NOD mice were treated with 1alpha,25OH2D3 (5microg/kg/2d) from 21 to 70 days. Vehicle-treated NOD and NOR mice served as controls. Analysis of thymic T lymphocytes from 1alpha,25OH2D3)-treated mice revealed a decrease in number of apoptosis-resistant CD4+CD8+ and CD4+CD8-HSA(high) T lymphocyte subsets, higher pro-apoptotic IL-2 and FasL, and lower anti-apoptotic Bclx-L mRNA expression levels. Thymic dendritic cells from 1alpha,25OH2D3-treated NOD mice had increased CD8alpha+FasL+ and CD80+/86+ expression compared to control NOD mice. In a syngeneic co-culture system of thymocytes and thymic dendritic cells, apoptosis levels were 20% higher only in co-cultures where both T cell- and dendritic cell-compartments originated from 1alpha,25OH2D3-treated mice. Activation-induced cell death-sensitivity in peripheral T lymphocytes was comparable to levels present in NOR mice, confirming better thymic selection in 1alpha,25OH2D3-treated mice. CONCLUSION/INTERPRETATION: We conclude that 1alpha,25OH2D3 needs both thymic T cell- and dendritic cell-compartments to exert its apoptosis-restorative effects in NOD thymocytes.