杆状病毒介导LacZ基因转染体外培养的人虹膜色素上皮细胞

目的：研究重组杆状病毒(baculovinus)载体介导β—半乳糖苷酶基因(LacZ)对体外培养虹膜色素上皮(IPE)细胞的转染和表达情况。方法：将质粒pCMV—β中的CMV—LacZ表达盒插入杆状病毒表达栽体pFast BacI的Eco RI／Hind Ⅲ位点，构建重组质粒pBac—β，并利用Bac—to—Bac表达系统在Sf9细胞中产生出重组杆状病毒BV—β。将MOI为200的BV—β感染经酶消化加显微法分离培养的IPE细胞，24h后进行X-gal染色。在显微镜下观察IPE中LacZ表达阳性的情况，记录阳性细胞所占的百分比。结果：限制性酶切分析及测序结果表明，我们已成功构建表达质粒pBac—β及重组杆状病毒BV—β。X-gal染色证实杆状病毒介导LacZ在细胞中的表达呈蓝色，弥漫于整个胞浆。感染后24h时表达阳性率达85％。结论：重组杆状病毒可有效介导报告基因在体外培养的IPE细胞中表达，为利用杆状病毒构建可供移植的基因工程化虹膜色素上皮细胞提供了实验依据。     

Electrical impedance scanning in breast tumor imaging: correlation with the growth pattern of lesion

Background This study researched the electric impedance properties of breast tissue and demonstrated the differentcharacteristic of electrical impedance scanning (EIS) images.Methods The impedance character of 40 malignant tumors, 34 benign tumors and some normal breast tissue from 69patients undergoing breast surgery was examined by EIS in vivo measurement and mammography screening, with aseries of frequencies set between 100 Hz-100 kHz in the ex vivo spectroscopy measurement.Results Of the 39 patients with 40 malignant tumors, 24 showed bright spots, 11 showed dark areas in EIS and 5showed no specific image. Of the 30 patients with 34 benign tumors there were almost no specific abnormality shown inthe EIS results. Primary ex vivo spectroscopy experiments showed that the resistivity of various breast tissue take thefollowing pattern: adipose tissue＞cancerous tissue＞mammary gland and benign tumor tissue.Conclusions There are significant differences in the electrical impedance properties between cancerous tissue andhealthy tissue. The impedivity of benign tumor is lower, and is at the same level with that of the mammary glandulartissue. The distinct growth pattern of breast lesions determined the different electrical impedance characteristics in theEIS results.     

大鼠脂肪干细胞复合胶原-壳聚糖-硫酸软骨素三维支架构建组织工程软骨

Objective To evaluate the character of the collagen-chitosan-chondroitin sulfate scaffold seeded with rat adipose tissue-derived stromal cells. Methods A dipose tissue were harvested from 6 weeks old Wistar rats and the stromal cells were harvested by type Ⅰ collagenase and then cultured in vitro. Type Ⅰ collagen was fully mixed with chitosan, freeze-dried and cross-linked with chondroitin sulfate, then freeze-dried again and sterilized by ethylene oxide. The pore diameter, water content, porosity of the scaffold were tested. The adipose tissue-derived stromal cells were digested, seeded into the plates, scaffold, and cen-trifuged into pellet, and then induced into cartilage. MTT detection for cell proliferation was done. After 3 weeks, the cell morphology, and cell proliferation and adhesion were observed, and chondrngenic differenti-ation was also analyzed. Results The pore diameter, water content, porosity tested for the scaffold showed an appropriate form. Cell proliferation showed faster in the scaffold and pellet culture system after 5 day, there was still cell proliferation in the scaffold system after 14 days but no obvious changes in the pellet cul-ture system; ceils on the scaffold proliferated densely showed by histological staining, but there was a scaf-fold structure residues in the inner layer. The finding of type Ⅱ immunohistochemistry stain showed that cells express strong positive for type Ⅱ collagen in the scaffold and pellet culture system whereas it was weakly positive in the plate culture system; the specific mRNA for cartilage, type Ⅱ collagen, aggrecan and SOX-9 were expressed in all three systems showed by RT-PCR, but type X collagen was expressed continu-ously in the plate culture system and expressed after 21 days in the pellet culture system, whereas it was not detected in the collagen-chitosan-chondroitin sulfate scaffold system. Conclusion The parameters of the collagen-chitosan-chondroitin sulfate scaffold were suitable in our study. The results suggested that it can promote the adipose tissue-derived stromal cells proliferation and chondrogenic differentiation better than the plate and pellet culture systems and maintain the phenotype of chondrocytes well; it is the optimal choice for cartilage tissue engineering in the future.     

脑机接口中一种改进的模式识别方法

为提高脑机接口中脑电识别率，分析了特征提取方面时频特征组合法的缺点，探讨了一种改进的模式识别方法。该方法以样本类平均距离为判据，采用滑动窗优化技术，获取时域均值的最佳时间段和频域功率谱均值的最佳频率段。用经过优化的时域均值和功率谱均值组合作为特征，形成特征向量。基于该特征向量，用神经网络对脑电信号进行分类。以识别正确率为指标，将改进方法与原方法进行对比，实验结果表明改进方法能够提高脑电识别率，具有应用价值。     

中性粒细胞分布数据序列小波变换研究

目的　研究小波分析结合复倒谱处理血膜中中性粒细胞分布数据信号的新方法及临床意义。方法　利用“虚拟盒计数法”方法采集血膜中的细胞分布数据信号 ,然后进行小波变换结合复倒谱分析方法处理。结果　提出血膜中细胞分布状态的“密集子”新概念。计算出小波变换复倒谱主极大值 ,并以此作为不同情况下血膜中中性粒细胞分布“密集子”间距的准确参数。结论　方法简单 ,能较好的反映血膜中细胞分布状态的微观组织结构特征 ,是血膜中细胞“密集子”表达定征的一种有效方法     

表达Cys548雌激素受体突变型cDNA的真核载体构建及其促转录活性分析

目的 考察一个548位点C→T单核苷酸突变的雌激素受体(ER)在体外条件下,对乳腺癌细胞株(MCF-7)信号通路的影响,以探讨ER单核苷酸突变引起非雌激素依赖性性早熟的可能机制.方法 用重叠延伸PCR定点诱变技术对548位点的碱基进行定点突变.构建定点突变表达载体pSG5-MuER.构建含雌激素受体反应元件(ERE)的萤光素酶报告基因载体pGL3-ERE-Luc.将野生和突变ER质粒分别和pGL3-ERE-Luc共转染MCF-7细胞,观察萤光素酶的变化,以检测突变ER反应活性的变化.结果 成功构建ER突变质粒psG5-MuER和ER报告质粒pGL3-ERE-Luc,psG5-MuER较pSG5-ER能够增加萤光素酶的产生.结论 该突变雌激素受体在体外具有高促转录活性特征,构建的载体可用于进行进一步的相关研究.     

慢性乙型肝炎患者外周血T细胞KIR表达研究

目的：检测慢性乙型肝炎患者外周血T细胞表面KIR的表达情况。方法：采用三色流式细胞术检测慢性乙型肝炎患者外周血CD4^＋T细胞和CD8^high T细胞表面KIR分子表达，并与正常外周血比较。结果：慢性乙型肝炎患者外周血中CD8^high T细胞KIR表达明显高于对照组CD8^high T细胞。正常外周血CD4^＋T细胞几乎不表达KIR，慢性乙型肝炎患者外周血中CD4^＋T细胞表达KIR。结论：慢性乙型肝炎患者T细胞表面KIR表达明显增加。     

甘肃省SARS抗体血清流行病学调查

目的 了解甘肃省严重急性呼吸综合征(SARS)患者、密切接触者和正常人群血清SARS冠状病毒特异性IgG抗体水平。方法 利用酶联免疫吸附试验(ELISA)法检测SARS病毒IgG抗体水平。检测对象包括甘肃省9例SARS患者的急性期和(或)恢复期系列血清，l109例直接护理SARS患者的医生、护士、实验室检测人员、疾控人员和曾与患者有接触的人员以及978例正常人血清。结果 9例临床诊断病例SARS冠状病毒特异性IgG抗体中6例为阳性，疾病恢复后12个月血清抗体仍为阳性；密切接触者1例特异性IgG抗体阳性；正常人3例特异性IgG抗体阳性。结论 病例抗体阳性符合临床诊断，密切接触者和正常人的抗体阳性数较低，提示可能不存在隐性感染。     

GKT原理的模拟犯罪测试范式实验研究

目的:本实验旨在以实际犯罪较接近的实验场景验证GKT的测谎机制,并探讨其对罪犯以及其他嫌疑人的判定有效性。方法:以72名健康大学生为被试,让被试在模拟犯罪的背景下采用三种包含不同说谎和认知成分的回答方式进行测谎测试,采用Limestone测谎仪测量被试皮肤电反应。结果:回答方式与角色两因子在判定分数上的主效应均显著,交互作用不显著。结论:在模拟犯罪测试范式下,GKT模式中认知与说谎机制是共存的,其中认知成分不占主要地位,说谎成分占主要地位,GKT模式无法兼顾有效地判定"犯罪"和"知情无辜"角色,需进一步改进。     

Cx43在造血调控及重建中的作用

目的:采用条件性基因敲除技术构建造血系统间隙连接蛋白43(Cx43)基因敲除(Cx43~(-/-))小鼠模型,并探讨Cx43在维持造血细胞自我更新及功能稳定中的作用。方法:将引进的2对转基因小鼠Cx43 loxP/loxP和Lyz-Cre/+杂交,选取F1雌性子代Cx43 loxP/-_Lyz-Cre/+与雄性Cx43 loxP/loxP合笼回配,提取所获得子代小鼠鼠尾组织基因组DNA,采用PCR方法鉴定小鼠基因型,RT-PCR方法筛选Cx43~(-/-)小鼠,同时分析小鼠不同器官中Cx43基因的表达差异;该类小鼠经5-氟尿嘧啶(5-FU;125 mg/kg)处理,在化疗前及化疗后第5、10和15天经眼球取血分析其血象变化。Cx43~(-/-)及Cx43~(+/+)小鼠予7.5 Gy(~(60)Co-γ)的致死量照射,剂量率1 Gy/min,照射后6 h分别给予事先准备就序的骨髓细胞,每只3×10~6细胞于尾静脉注入,2周后处死小鼠检测造血是否重建:分离股骨切片后,收集骨髓细胞进行细胞表型分析(选用的单抗为CD45R、Gr-1、CD4、 CD8a、TCRαβ、Mac-1、抗sIgM、TER119、Sca-1及CD117);同时进行体外造血细胞集落实验观察造血细胞的体外增殖能力。结果:本研究通过2种转基因小鼠间杂交和回交,成功获得造血系统选择性Cx43基因敲除小鼠;该类小鼠骨髓及外周血细胞无Cx43表达,参与造血的组织,如肝脏和脾脏中Cx43表达也显著下调(P0.01),而心脏和肾脏的Cx43表达则无影响,小鼠成年后外周血象分析并无明显异常,但应急代偿能力下降,经5-FU处理后,其造血功能恢复显著减缓,处理15 d后,Cx43~(+/+)小鼠造血功能已接近正常水平,而Cx43~(-/-)小鼠仍无明显的恢复迹象,血红蛋白、白细胞及血小板仍处低位,2者差别有统计学显著性(P0.01);体外集落试验也证实Cx43~(-/-)小鼠造血干/祖细胞的增殖能力下降,其CFU-GM或CFU-E集落数均明显少于Cx43~(+/+)小鼠(P0.01),但流式细胞术结果显示,Cx43~(-/-)小鼠骨髓中Lin~-/c-Kit~+/Sca-1~+细胞亚群数量与Cx43~(+/+)小鼠相比差异并无统计学显著性;Cx43~(-/-)小鼠在化疗或移植后其骨髓造血功能重建均延迟,且化疗15 d后骨髓切片及涂片均证实其骨髓中造血细胞增生程度明显降低,脂肪组织显著增多,而且T、B细胞发育也有异常。此外,其外周血中CD4~+CD8~+细胞比例比野生型小鼠增多(P0.05),但CD4~+T细胞显著减少(P0.01),尤其是TCRαβ亚群细胞减少最为明显(P0.01)。同样,Cx43~(-/-)小鼠外周血中CD45R~+sIgM~-细胞亚群比例与野生型小鼠相比显著减少(P0.01)。结论:骨髓中Cx43基因表达在造血干/祖细胞发育(尤其是应急状态时)具重要作用,敲除Cx43基因后造血干/祖细胞增殖减缓,造血及免疫重建功能受损。