乙型肝炎病毒攻击肝细胞对β-干扰素表达的影响

目的探讨乙型肝炎病毒（hepatitisBvirus,HBV）攻击肝细胞对B一干扰素表达的影响。方法构建B一干扰素启动子调控的荧光素酶报告载体,与参比质粒共同转染细胞,用双荧光素酶报告系统检测poly（I：c）和血清中HBV攻击HepG2和L-O2两种肝细胞对细胞中B一干扰素转录活性的影响,并结合PCR技术检测HBV攻击肝细胞后B一干扰素的转录变化。结果血清中HBV攻击肝细胞能够引起细胞中β-干扰素的转录,但转录水平较低。此外,ItBV对肝细胞中β-千扰素的转录有促进作用。结论自然感染状态下,HBV能引起肝细胞中以β一千扰素表达为代表的天然免疫反应．．     

E-钙黏蛋白和N-钙黏蛋白启动子的构建及活性测定

目的：构建含有E-钙黏蛋白（cadherin）基因和N-钙黏蛋白基因启动子的荧光素酶报告基因载体,并检测E-钙黏蛋白启动子和N-钙黏蛋白启动子的活性。方法利用PCR技术从乳腺癌细胞ZR75-1基因组中扩增出E-钙黏蛋白和N-钙黏蛋白启动子片段,插入到荧光素酶报告基因载体pGL4-basic中,确定所扩增的DNA序列后,将其转染至乳腺癌细胞ZR75-1和肝癌细胞HepG2中,检测启动子的活性。结果测序结果表明,扩增的E-钙黏蛋白启动子序列正确;荧光素酶活性实验表明,E-钙黏蛋白启动子和N-钙黏蛋白启动子在乳腺癌细胞ZR75-1和肝癌细胞HepG2中表达都具有活性。结论成功构建了E-钙黏蛋白启动子和N-钙黏蛋白启动子报告基因表达载体,为进一步筛选调节E-钙黏蛋白和N-钙黏蛋白表达的转录因子提供了重要基础。     

人Puma启动子荧光素酶报告基因的构建和鉴定

目的：克隆人Puma基因的启动子,插入荧光素酶报告基因载体中,并在细胞内检测其活性。方法：采用PCR技术,从人HepG2细胞中扩增出Puma启动子,插入荧光素酶报告基因载体pGL3-basic中,测序所扩增的DNA序列,并将其转染入H1299细胞中检测其活性。结果：测序结果表明,扩增的Puma启动子序列正确;双报告基因实验检测荧光素酶活力表明,构建的报告基因具有启动子活性。结论：Puma启动子的克隆及人Puma启动子报告基因的成功构建,为p53家族凋亡通路的功能研究提供了必要的实验材料。     

核因子κB降低小鼠胚胎成纤维细胞中Lmp2基因转录

目的 研究在小鼠胚胎成纤维细胞(MEF)中,核因子κB(NF-κB)对Lmp2基因转录的影响.方法 利用NF-κB荧光素酶报告基因检测MEF和c-abl/arg缺失株(DKO)中内源性NF-κB活性;构建包含Lmp2启动子序列和NF-κB结合序列突变体的荧光素酶报告基因,通过双荧光素酶报告系统,研究NF-κB对Lmp2启动子转录活性的调控,并应用定量PCR、Western印迹比较Lmp2基因在MEF和DKO细胞中表达水平.结果 DKO细胞中内源性NF-κB活性上调;DKO细胞中Lmp2基因启动子的转录活性发生下调,并且NF-κB结合序列突变后,其转录活性升高;定量PCR和Western印迹结果表明,DKO细胞中Lmp2基因mRNA和蛋白表达水平比较MEF细胞均发生下调.结论 MEF中内源性NF-κB可负调控Lmp2基因转录,并降低其蛋白表达水平.     

人IP-10启动子的克隆和转录活性的检测

目的 克隆干扰素诱导蛋白10（IP-10）5’非编码区（NCR）片段，检测克隆的IP-10启动子驱动的荧光素酶报告基因在LPS激活的人脐静脉内皮细胞（HUVEC）中的转录活性。方法 提取人外周血淋巴细胞基因组DNA，以其为模板，利用巢式PCR扩增人IP-10启动子片段，测序正确后克隆入荧光素酶报告基因表达载体pGL3；将重组质粒pGL3／IP-10P导入HUVEC细胞，检测LPS刺激下荧光素酶活性。结果 正确克隆出人IP-10启动子（-2028～-1）片段，成功构建了pGL3／IP-10P报告基因表达载体；证实了LPS可诱导HUVEC细胞中IP-10的高表达。结论 成功克隆出人的IP-10启动子片段，并利用荧光素报告基因证实了在HUVEC细胞中LPS可诱导IP-10高转录表达，为深入研究LPS诱导IP-10转录表达的调控机制提供了基础。     

p53与HBV相互作用对7721细胞凋亡及p21启动子的影响

为了观察HBV与p53是否存在相互作用的关系，以进一步揭示与原发性肝细胞肝癌发生发展的相关机制，选用7721肝癌细胞系（表达野生型p53，HBV阴性）为靶细胞，应用磷酸钙转染法，将pCMVp53单独或者与野生型HBV质粒（pCMVHBVa）、突变型HBV质粒（pCMVHBVb）共转染细胞，用annexin-V-FITC标记及流式细胞仪检测细胞凋亡的变化。各组另与含有p21基因启动子的荧光素酶报告基因质粒p21-luc共转染细胞，通过检测荧光毒酶的表达，了解p21启动子的活化情况。结果表明，单独的pCMVp53质粒转染7721细胞系能够促进p21报告基因的表达，细胞凋亡率升高；pCMVp53与pCMVHBV。共同转染时，对p21基因启动子的激活作用增强、细胞凋亡率明显增加，而与pCMVHBVb共转染时则否。提示HBV在该细胞内的复制能够诱导增强p53的作用并对其下游基因p21的转录有促进作用，导致细胞凋亡的增强。     

内质网应激对ATF6调控XBP1启动子转录的影响

目的 确定XBP1启动子的转录核心区域,探讨激活转录因子6(ATF6)在内质网应激(ERS)与非ERS状态下对XBP1启动子转录活性的调控作用.方法 采用基因重组技术构建ATF6基因及两个缺失体ATF6s1p、ATF6s2p的真核表达载体pcDNA3.1 (-)-ATF6、pcDNA3.1 (-)-s1p、pcDNA3.1(-)-s2p,以及XBP1启动子报告基因载体pGL3-XBP1.针对XBP1启动子的各个组成元件设计和构建XBP1一系列截短体的报告基因载体.采用双荧光素酶报告基因法检测并确定XBP1启动子的核心启动子区,Western blotting检测ATF6及缺失体s2p、s1p在ERS与非ERS状态下对XBP1核心启动子区转录的调控作用.结果 XBP1启动子的-407bp-+133bp区域是转录活性调控的核心区域,-2000bp--407bp区域不具有转录活性,该区域可能含有抑制转录活性的组成元件.非ERS状态时,ATF6及两个缺失体s2p、s1p可明显上调XBP1启动子的转录活性及XBP1蛋白表达;而在ERS状态时,ATF6及两个缺失体s2p、s1p下调了XBP1启动子的转录活性和XBPl蛋白表达.结论 ATF6对XBP1的转录调控作用在ERS状态和非ERS状态下存在差异.非ERS状态时,ATF6上调XBP1的转录和表达,而ERS状态时,ATF6下调XBP1的转录和表达.     

大鼠糖皮质激素受体变异体cDNA序列克隆和表达及功能研究

目的：构建大鼠糖皮质激素受体变异体表达载体，研究其表达和功能。方法：运用RT—nested PCR扩增糖皮质激素受体不含有编码LBD结构域的cDNA序列，将PCR产物定向克隆至pEGFP—N2质粒载体，测序筛选重组质粒；荧光显微镜观察重组质粒转染后的表达情况；相对荧光素酶法检测转录激活活性。结果：扩增出长1612bp的cDNA序列，测序证实：克隆片段与Genebank该基因序列同源性为100％；重组质粒表达产物定位于细胞核；转染组细胞转录激活活性增强。结论：成功构建糖皮质激素受体变异体的表达载体，该变异体具有不依赖激素的转录激活活性。     

ST8SiaV基因启动子的克隆及功能鉴定

目的：克隆ST8SiaV基因启动子及构建ST8SiaV基因启动子-pGL3-Basic-GFP表达栽体，并对该栽体进行功能鉴定。方法：①基于NCBI公布的大鼠基因组ST8SiaV基因序列，以SD大鼠肝基因组DNA为模板，采用PCR方法，扩增ATG起始密码子上游1．7kb片段。②分离、纯化PCR产物，与pMD 18-T栽体相连，构建克隆栽体，转化大肠杆菌(DH5α)，筛选阳性克隆并测序。③启动子片段进行SacⅠ和Bg2Ⅱ双酶切，克隆进pGL3-Basic质粒载体。为探索该基因在神经干细胞分化过程的活体表达，将pGL3-Basic的luciferase报告基因替换为GFP，构建成promoter-pGL3-Basic-GFP表达栽体。④将构建的表达载体电转染SD大鼠神经干细胞，对该表达载体进行生物学功能鉴定。结果与结论：成功地克隆到1．7kb的具有生物学活性的ST8SiaV基因启动子，并构建了适于活体研究的promoter-pGL3-Basic-GFP表达栽体，为进一步研究该基因的启动子区转录及调控作用奠定了基础。     

G1764A/C1766T/T1768A三联突变对HBV核心启动子活性的上调作用研究

目的 构建乙型肝炎病毒(HBV)核心启动子(CP)荧光素酶表达载体,探讨HBV CP变异对下游基因转录活性的影响.方法 2005年5月至2008年3月于解放军302医院就诊的慢性乙型肝炎(CHB)及慢加急性肝衰竭(ACLF)患者各40例,采集血清提取HBV DNA,采用巢式PCR法扩增HBV CP片段,克隆至pGEM-T Easy载体.挑选含有CP相关变异位点的质粒,经Kpn Ⅰ/Bgl Ⅱ双酶切后,构建pGL3-CP荧光素酶真核报告质粒,并采用定点突变方法获得野生型质粒,将两者同时转染肝癌细胞系HepG2,48h后进行荧光素酶检测.结果 患者临床资料经统计分析后显示,CHB患者HBeAg阳性率和HBV DNA载量均高于ACLF患者(P<0.01),而TBIL含量则低于ACLF患者(P<0.01);对CP区热点突变频率分析后发现:G1764A/C1766T/T1768A三联突变在CHB患者中为0.0%(0/40),在ACLF患者中为12.5%(5/40,P<0.05).细胞转染结果显示:典型CP双联突变A1762T/G1764A病毒株的启动子活性为相应野生株的1.67倍,而G1764A/C1766T/T1768A三联突变病毒株的启动子活性为相应野生株的1.43～1.80倍.结论 HBeAg阳性率、TBIL水平、HBV DNA载量与乙型肝炎重症化相关,HBV CP中存在的A1762T/G1764A、G1764A/C1766T/T1768A突变有顺式激活下游基因转录活性的作用.     

Knee injury and Osteoarthritis Outcome Score (KOOS) - validation of a Swedish version

The Knee injury and Osteoarthritis Outcome Score (KOOS) is a self-administered instrument measuring outcome after knee injury at impairment, disability, and handicap level in five subscales. Reliability, validity, and responsiveness of a Swedish version was assessed in 142 patients who underwent arthroscopy because of injury to the menisci, anterior cruciate ligament, or cartilage of the knee. The clinimetric properties were found to be good and comparable to the American version of the KOOS. Comparison to the Short Form-36 and the Lysholm knee scoring scale revealed expected correlations and construct validity. Item by item, symptoms and functional limitations were compared between diagnostic groups. High responsiveness was found three months after arthroscopic partial meniscectomy for all subscales but Activities of Daily Living.     

Endovascular management of carotid-cavernous fistulas

Objective To investigate endovascular treatment of traumatic direct carotid-cavernous fistulas （CCF） and their complications such as pseudoaneurysms. Methods： Over a five-year period, 22 patients with traumatic direct CCFs were treated endovascularly in our institution. Thirteen patients were treated once with the result of CCF occluded, 8 twice and 1 three times. Treatment modalities included balloon occlusion of the CCF, sacrifice of the ipsilateral internal carotid artery with detachable balloon, coll embolization of the cavernous sinus and secondary pseudoaneurysms, and covered-stem management of the pseudoaneurysms. Results All the direct CCFs were successfully managed endovascularly. Four patients developed a pseudoaneurysm after the occlusion of the CCF with an incidence of pseudoaneurysm formation of 18.2% （4/22）. A total number of 8 patients experienced permanent occlusion of the ICA with a rate of ICA occlusion reaching 36.4% （8/22）. Followed up through telephone consultation from 6 months to 5 years, all did well with no recurrence of CCF symptoms and signs. Conclusion Traumatic direct CCFs can be successfully managed with endovascular means. The pseudoaneurysms secondary to the occlusion of the CCFs can be occluded with stent-assisted coiling and implantation of covered stents.     

The acutely limping child

Acute limping may be the result of multiple pathologies in children. The differential diagnosis varies based on the age of the child. Irrespective of age, the initial imaging work-up includes AP and frog leg radiographs of the pelvis and ultrasound; MRI may sometimes be helpful. In children less than 3 years, infections and trauma are most frequent. MRI is the imaging modality of choice when osteomyelitis is clinically suspected. Between the ages of 3 and 10 years, transient synovitis of the hip and Legg-Calvé-Perthes disease are main considerations but infection, inflammation and focal bony lesions are also considered. In children over 10 years, slipped capital femoral epiphysis also is considered.     

The role of interventional radiology in trauma

Introduction Interventional Radiology has evolved into a specialty having enormous input into the care of the traumatized patient.In all hospitals,regardless of size,the Interventional Radiologist must consider their relationships with the trauma service in order to     

人体中的镭-226、镭-228、钋-210、铅-210

本文报道了广东阳江高本底地区6名、对照地区8名人尸体的骨²²⁶Ra、²²⁶Ra的浓度以及部分居民内脏器官中。²¹⁰Po、²¹⁰Pb的浓度。结果轰明阳江高本底地区和对照地区居民骨镭-226、镭-228的浓度分别为29.9pCi/kg, 26.9pCi/kgl 8.7pCi/kg, 8.2pCi/kg.由此估算出阳江高本底地区屠民骨中²²⁶Ra、²²⁸Ra的负薄璧及对骨衬、骨髓所产生的剂量当量分别为对照地区民民的3.4倍, 3.3倍。两地区居民内脏器官中^{210Po、210Pb的测定分析铡数较少但仍看出, 高本底地区均明显高于对照地区.}     

Do Balance Board Training Programs Reduce the Risk of Ankle Sprains in Athletes?

Introduction Ankle sprains are the most common musculo-skeletal injury that occurs in athletes,particularly in sports that require jumping and landing on one foot such as soccer,and basketball(1-4).These injuries often result in significant time loss from participation,long-term disability,and have a major impact on health care costs and resources(5-8).