应用RNA干扰治疗小鼠急性乙型肝炎病毒感染

目的建立小鼠急性乙型肝炎病毒感染的动物模型,在此基础上观察小干扰RNA(siRNA)对HBV复制和表达的影响。方法通过尾静脉注射1.3倍的HBV真核表达质粒pHBV1.3建立小鼠急性乙型肝炎感染模型,再将其与针对乙型肝炎病毒核心区的siRNA表达载体(pSI C)共注射,于注射后第6天取血测血清HBsAg(ELISA),做肝组织HBcAg免疫组化,通过RT PCR检测肝组织HBVC区mRNA转录子的水平。同时设立PBS对照组、无关干扰组与突变干扰组。结果转染后第6天血清HBSAg在pHBV1.3感染组中高表达(A值为4.08～9.04,平均值为5.07±1.07,A值≥2.1为阳性),肝内HBcAg阳性率为5%～10%;pSI C干扰组HBSAg,HBcAg的表达均受到抑制,血清HBsAg阴性,A值为0.04～1.5,平均值为0.62±0.59,与感染组相比有显著性差异(P<0.01),肝内HBcAg几乎无表达,RT PCR示肝内HBVCmRNA水平明显降低。而无关干扰pGFP及突变的干扰序列pSI Cmut则无此作用。结论RNAi技术能特异,高效地抑制小鼠体内HBV的复制和表达,提示siRNA作为一种新型抗病毒药物的巨大潜能,而通过水动力法建立的小鼠急性乙型肝炎病毒感染的动物模型,一定程度上解决了长期以来缺乏合适的HBV感染动物模型的问题,为今后进行药物筛选及抗病毒治疗的研究拓展了新的思路。     

小干扰RNA(siRNA)对小鼠乙型肝炎病毒复制和表达影响的实验研究

目的建立小鼠急性乙型肝炎病毒感染的动物模型,并在此基础上观察小干扰RNA(siRNA)对小鼠乙型肝炎病毒复制和表达的影响。方法用水动力学方法,向小鼠尾静脉注射1.5倍的HBV真核表达质粒pHBV1.5建立小鼠急性乙型肝炎病毒感染模型,明确感染后,再将其与针对乙型肝炎病毒核心区的siRNA表达载体(psiHBV4)共注射,于注射后第6天取血测血清HBsAg,HBeAg(MEIA),通过RT-PCR检测肝组织HBVC区mRNA转录子的水平。同时设立PBS对照组,空载体对照组和无关干扰对照组。结果注射后第6天血清中HBsAg,HBeAg在pHBV1.5感染组中高表达。psiHBV4干扰组中HBeAg的表达受到了明显的抑制,与感染组比较差异有显著性(P<0.01),RT-PCR显示肝内HBVCmRNA水平亦明显降低。而无关干扰对照组则无相应的干扰作用。结论RNAi能特异,高效的抑制小鼠体内HBV的复制和表达,同时通过水动力学方法,成功建立了小鼠急性乙型肝炎病毒感染的动物模型,为进一步研究乙型肝炎病毒奠定了基础。     

S区和C区小干扰RNA在小鼠体内抗乙型肝炎病毒的作用

目的:以乙型肝炎病毒(HBV)S区和C区为靶位,观察小干扰RNA(siRNA)在小鼠体内抗HBV的作用.方法:以流体动力学法建立HBV感染的动物模型,将pcDNA3.1-HBV和细胞体外实验证明有效的S区和C区siRNA尾静脉注射BALB/c小鼠,用时间分辨免疫荧光分析法(IFMA)检测小鼠血清中乙型肝炎表面抗原(HBsAg),用定量PCR法(FQ-PCR)检测血清HBV DNA,用RT-PCR法检测HBV S-mRNA和C-mRNA. 结果:在小鼠体内,和无关siRNA相比,S区和C区siRNA均能有效抑制血清中HBsAg的分泌(第1天的抑制率分别为96%和90%),降低HBV DNA的滴度(第1天分别减少47%和71%),HBV S-mRNA和C-mRNA的条带灰度明显减少,干扰效果至少持续3 d. 结论:在小鼠体内靶向HBV S区和C区的siRNA均能有效特异抑制HBV.     

乙型肝炎急性感染小鼠模型的建立

目的 通过尾静脉注射感染性小鼠血清的方法 建立一种简单复制乙型肝炎急性感染的小鼠模型.方法 收集通过水压法复制第3天的小鼠血清,使用不同剂量尾静脉注射感染同系小鼠.分别在不同的时间阶段检测血清中的HBsAg和HBeAg,肝组织中的HBcAg和血清中HBV的DNA.结果 注射0.1 ml血清的感染组的检测结果 均高于0.2 ml组.结论 通过注射感染血清可以成功地复制急性小鼠感染模型.该模型复制方法 中,感染的结果 与注射感染血清的剂量无关.     

HBsAg和HBcAg在慢性乙肝病毒携带者肝组织中的表达

目的 观察慢性乙肝病毒携带者血清标志物与肝组织HBcAg表达的相关性.方法 对80例慢性乙肝病毒携带者行快速经皮肝穿刺术取肝组织,免疫组化染色检查HBsAg,HBcAg.荧光定量PCR法检测血清HBV DNA.ELISA法检测血清HBsAg、抗-HBs、HBeAg、抗-HBe和抗-HBc. 结果 血清HBsAg均阳性,血清HBeAg阳性者48例、HBeAg阴性者32例,血清HBV DNA阳性者50例、HBV DNA阴性者30例.肝组织HBsAg阳性者68例、肝组织HBcAg阳性者43例. 结论 慢性乙肝病毒携带者肝组织HBcAg表达与血清标志物HBV DNA、HBeAg有相关性.     

shRNA表达质粒对转基因小鼠HBV的抑制作用

目的观察shRNA表达质粒对转基因小鼠HBV的抑制作用。方法将质粒DNA采用Hydrodynamic技术注入HBV转基因小鼠体内,观察血清ALT和AST水平、血清HBsAg和肝组织HBcAg表达和HBVmRNA的变化。结果在注射质粒后,ALT和AST呈一过性升高,3天后降至正常;血清HBsAg和肝细胞内HBcAg表达减少,蛋白抑制至少持续14天;质粒注射后第5天,实验组肝脏HBVmRNA与注射前相比明显减少,而对照组HBVmRNA信号无明显变化。结论shRNA表达质粒对HBV转基因小鼠HBV有抑制作用。     

抑制LSD1酶活性对HBV模型小鼠的作用

目的研究组蛋白赖氨酸去甲基化酶1（LSDl）酶活性下降对乙型肝炎病毒（HBV）模型小鼠的作用。方法尾静脉高压注射重组HBVl．3质粒建立HBV小鼠模型;将雌性BALB／c小鼠随机分为正常组、模型组、LSDl小干扰RNA（siRNA）处理组和反苯环丙胺（TCP）治疗组。取各组小鼠外周血,通过酶联免疫吸附试验、全自动微粒子化学发光法和Real—TimePCR检测小鼠体内乙肝表面抗原（hepatitis B surface antigen,HBsAg）和HBVDNA的表达;免疫组织化学法检测HBsAg在肝脏中的分布;Western blotting检测小鼠脾淋巴细胞LSDl的表达。结果HBV模型小鼠造模成功;LSDlsiRNA处理组和TCP治疗组小鼠HBsAg、HBVDNA和LSDl的表达水平均较模型组明显下降（P〈0．01）,肝组织中HBsAg的分布也明显减少。结论抑制LSDl酶活性对HBV模型小鼠体内HBV的清除有一定的作用。     

乙型肝炎转基因小鼠品系C57-TgN(HBV adr2.0)SMMU的生物学特征

目的：评价乙肝转基因小鼠品系C57－TgN(HBV adr2.0)SMMU生物学特征的稳定性。方法：以F6代乙肝转基因小鼠C57－TgN(HBV adr2.0)SMMU为研究对象，采用基因组DNA PCR，血清ELISA检测，Western印迹分析，免疫组织化学，血清DNA PCR，透射电镜和H－E染色的方法分析HBV基因在转基因小鼠中的整合，表达，复制和组织这变化。结果：F6代乙肝转基因小鼠基因组中稳定整合有HBV基因，肝组织中可检测用HBsAg,HBcAg和X蛋白3种病毒蛋白，血清中HBsAg和HBeAg的表达率分别为19．54％和3．39％，且在血清和肝组织中存在病毒NA和病毒样颗粒；长期的病毒DNA整合，表达和复制可以引起转基因小鼠肝，肺等组织的病理性损伤。结论：乙肝转基因小鼠品系C57－TgN(HBV adr2.0)SMMU具有基因组中稳定整合病毒DNA，血清和肝组织中有病毒蛋白表达和病毒复制的特征，并具有一定的组织这变化，作为生物医药研究的实验动物模型具有推广应用价值。     

HBV感染对人体T细胞免疫功能影响的研究

目的:通过检测乙型肝炎病毒(HBV)感染后外周血及感染肝组织内CD4 T和CD8 T细胞的数量及功能,探讨HBV对人体T细胞免疫的影响及其可能机制.方法:(1)用流式细胞仪检测36例HBV感染者和20例正常人外周血中T细胞亚群的数量.(2)用免疫组化S-P法检测64例慢性肝病的肝脏标本的HBsAg和HBcAg,将HBsAg和(或)HBcAg阳性的50例作为HBV感染组,而将肝组织HBsAg和HBcAg及血清病原学检查均为阴性的10例作为非HBV感染组(HBV阴性组).检测其中CD4、CD8、CD25表达情况.结果:(1)HBV感染组外周血T细胞亚群测定值中,CD4 T细胞和CD4 T/CD8 T比值明显低于对照组(Pb＜0.05);CD8 T细胞升高,明显高于对照组(Pb＜0.05);(2)肝组织内浸润的淋巴细胞主要聚集在汇管区和小叶坏死区,免疫组化结果显示HBV感染组肝组织内的CD8 T和CD4 T细胞数量多于非HBV感染组,统计学上其差异有非常显著意义(Pc＜0.01).HBV感染组肝组织内与T细胞活性有关的CD25 T细胞数量与对照组相比有所减少,但统计学上无显著意义.结论:乙型肝炎病毒感染者外周血和肝内的T细胞亚群紊乱,且活性可能有下降,免疫功能受损.     

双表达小干扰RNA抑制小鼠体内HBV感染

目的运用水压法复制HBV小鼠急性模型,用于研究双表达siRNA在小鼠体内对HBV复制的抑制作用。方法使用HBV质粒DNA对BALB/c小鼠进行尾静脉大剂量快速推注,获得急性HBV体内表达模型。同时使用相同方法对推注后的小鼠进行RNAi干预。对不同处理组动物的HBsAg、HBeAg、HBcAg和HBV DNA进行ELISA、免疫组织化学和实时定量RT-PCR检测。结果水压法复制HBV小鼠急性模型与HBV DNA感染剂量无明显相关。与单表达siRNA相比较,双表达siRNA对HBV抗原表达抑制较为明显。结论双表达siRNA不失为一种体内抑制HBV感染的一种策略。     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

安心颗粒对急诊经皮冠状动脉介入术后生活质量影响的研究

目的：评价使用安心颗粒对急诊经皮冠状动脉介入术（PPCI）术后生活质量的影响.方法：将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组（术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次）和对照组（仅接受基础药物治疗）.所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表（MIDAS）、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果：入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异（P＞0.05）;出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低（P＜0.05）;组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低（P＜0.05）.两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血（P＞0.05）.两组发生轻度血小板减少症的患者数比较无差异（P＞0.05）.结论：PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.     

The comparative studies of the influences of Urapidil and Nicardipine on sino-atrial node function, atrio-ventricular node function and hemodynamics

Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P＜0.01);the MAP decreased(P＜0.01)and the HR increased drastically(P＜0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.     

Expression of OPGmRNA and ODFmRNA in the patients of hip fracture due to osteoporosis

Objective：To investigate the gene expression of osteoprotegerin（OPG） and osteoclast differentiation factor（ODF） in the bone tissue of patients with hip fracture due to osteoporosis. Methods：OPGmRNA and ODFmRNA in the bone tissue in 50 cases of osteoporosis sufferers（over 50 years old） with hip fracture（Observer Group） and 30 cases of hip facture sufferers with no osteoporosis（Control group） were analyzed with the Semi-Quantitative RT-PCR method. Results：The mRNA expressed of ODF, OPG were both high in the patients with hip fracture. In the control group, the expression of OPG mRNA was observed, while the expression of ODF mRNA was very slight. Conclusion：Aged patients contained all signals including OPG, ODF that are essential for inducing osteoclastogenesis and promoting bone resorption.     

The clinicopathological and immuohistochemical analysis of solid-pseudopapillary tumor of the pancreas： report of 9 cases

Objective：To investigate the clinical features, pathological characteristics and immunophenotype of solid-pseudopapillary tumor of the pancreas（SPTP）. Methods：Nine surgically treated cases of SPTP were retrospectively reviewed. Hematoxylin and Eosin（HE） staining and immunohistochemical staining were used to analyze all cases, and the general clinical data was collected. Results：Six patients were asymptomatic except for a palpable mass. Two patients complained of vague-epigastric pain. One patient appeared jaundice. The tumor was encapsulated and solid tissues alternately with cystic tissues. Histologically, the histological structure of solid portion was pseudopapillary with a fibrovascular core. Tumor cells were uniform and medium-sized which were arranged in sheets ets or nests or pseudopapillary patterns. Immunohistochemical studies demonstrated that SPTP proved positive in vimentin（9/9 cases）, AAT（9/9 cases）, NSE（9/9 cases）, ACT（7/9 cases）, CK20（2/9 cases）, CgA（1/9 cases）, S-100（3/gcases）, PR（4/gcases）, Syn（3/9 cases） and CD56（5/9cases）, negative in CEA and ER. Conclusion：SPTP is a tumor predominantly occurring in young women frequently without special symptoms. This tumor has various characteristical histological patterns with different immunophenotype.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.     

Luo Lingjie's Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.     

Dr.Zhang Ren's Experience in the Acupuncture Treatment of Different Diseases with the Same Therapeutic Principle

Dr.Zhang Ren,the chief physician,is the chairman of Shanghai Acupuncture and Moxibustion Association.Having been engaged in medicine for about 40 years,he is experienced in treating various intractable diseases.In his long years of clinical practice,he advocates taking the TCM differentiation as the basis to seek for the acupuncture method for treatment of modern intractable diseases.The author of this essay had the fortune to follow Dr.Zhang in study.The following is a summary of Dr.Zhang's experience in the acupuncture treatment for different intractable diseases with the same therapeutic principle.