华南HCV 1b亚型NS5A插入式复制子重组质粒的构建和序列分析

目的构建来自华南不同慢性丙型肝炎(CHC)患者的HCV NS5A的复制子重组质粒并测序分析,探索HCV NS5A在抗病毒应答中生物学特性。方法以能够高效复制的1b型HCV复制子质粒为骨架,构成带有MIuⅠ和BclⅠ双酶切位点的开关质粒,采用逆转录-聚合酶链反应从不同CHC患者血清中扩增获得HCV 1b亚型NS5A全长片段,克隆到pMD-18载体中,测序分析其中的PKRBD、ISDR、V3和IRRDR区域的变异情况。扩增产物中无MIuⅠ和BclⅠ酶切序列的,在引物中引入相关酶切序列后再扩增,双酶切后将NS5A区段置换入HCV 1b复制子骨架中。结果成功扩增出NS5A全长片段并克隆测序,将NS5A区ISDR-V3区域置换入复制子中,测序结果正确。序列比对显示应答株的ISDR和PKRBD氨基酸变异数目比无应答株高;V3和IRRDR区域存在较高氨基酸突变率。结论成功构建包含华南HCV 1b亚型NS5A核心区域的复制子插入式重组质粒,为研究HCV NS5A生物学特性、干扰素抵抗的机制以及难治性CHC患者的个体化抗病毒治疗分析奠定了基础。     

慢性丙型肝炎和丙型肝炎肝硬化患者HCV NS5A和NS5B抑制剂天然耐药变异分析

目的探讨慢性丙型肝炎和丙型肝炎肝硬化患者丙型肝炎病毒(HCV)NS5A和NS5B抑制剂天然耐药变异的差异。方法收集158例广州医科大学附属市八人民医院住院或门诊的慢性丙型肝炎和丙型肝炎肝硬化患者的血清标本,使用Illumina二代测序平台进行测序,比较两组患者不同基因型HCV NS5A和NS5B抑制剂相关天然耐药变异的差异。结果共纳入72例慢性丙型肝炎和86例丙型肝炎肝硬化患者,除HCV1b基因型外,其他基线资料差异无统计学意义(P0.05)。慢性丙型肝炎患者存在A30K、L31M、P58S和Y93H等HCV NS5A抑制剂耐药相关变异,丙型肝炎肝硬化患者存在Q24R、A30K、L31M、P/T58S/A和Y93H等HCV NS5A抑制剂耐药相关变异,各基因型两组患者的HCV NS5A抑制剂耐药相关变异差异无统计学意义(P0.05)。慢性丙型肝炎患者存在L159F、T179A、I293L和C316N等HCV NS5B抑制剂耐药相关变异,丙型肝炎肝硬化者存在L159F、T179A、S282T、I293L、C316N和L320F等HCV NS5B抑制剂耐药相关变异,各基因型两组患者HCV NS5B抑制剂耐药相关变异差异亦无统计学意义(P0.05)。结论慢性丙型肝炎和丙型肝炎肝硬化患者的HCV NS5A和NS5B抑制剂相关天然耐药变异差异无统计学意义,丙型肝炎肝硬化患者较低的持续病毒学应答率可能与丙型肝炎肝硬化患者天然耐药变异无关。     

华南HCV 1b亚型NS5A插入式复制子重组质粒的构建和序列分析

摘要：目的构建来自华南不同慢性丙型肝炎（CHC）患者的HCV NS5A的复制子重组质粒并测序分析,探索HCV NS5A在抗病
毒应答中生物学特性。方法以能够高效复制的1b型HCV复制子质粒为骨架,构成带有MIuⅠ和BclⅠ双酶切位点的开关质
粒,采用逆转录-聚合酶链反应从不同CHC患者血清中扩增获得HCV 1b亚型NS5A全长片段,克隆到pMD-18载体中,测序分
析其中的PKRBD、ISDR、V3和IRRDR区域的变异情况。扩增产物中无MIuⅠ和BclⅠ酶切序列的,在引物中引入相关酶切序
列后再扩增,双酶切后将NS5A区段置换入HCV 1b复制子骨架中。结果成功扩增出NS5A全长片段并克隆测序,将NS5A区
ISDR-V3区域置换入复制子中,测序结果正确。序列比对显示应答株的ISDR和PKRBD氨基酸变异数目比无应答株高;V3和
IRRDR区域存在较高氨基酸突变率。结论成功构建包含华南HCV 1b亚型NS5A核心区域的复制子插入式重组质粒,为研究
HCV NS5A生物学特性、干扰素抵抗的机制以及难治性CHC患者的个体化抗病毒治疗分析奠定了基础。
     

HCV1b亚型ISDR变异影响PEG-IFN/RBV抗病毒疗效的新特点分析

目的 探讨HCV干扰素敏感决定区(ISDR)氨基酸(aa)序列变异度对聚乙二醇干扰素(PEG-IFN)联合利巴韦林(RBV)治疗1b亚型慢性丙型肝炎(CHC)疗效的影响.方法 58例HCV 1b亚型慢性感染者采用PEG-IFN/RBV联合方案治疗48周,并随访24周.定量检测血清HCV RNA,逆转录-聚合酶链反应扩增治疗前血标本中HCV ISDR片段并测序,MEGA 4分析氨基酸序列变异度;二分类Logistic回归分析各变量与持续病毒学应答(SVR)之间的关系.结果 治疗前血清HCV的ISDR氨基酸序列与HCVJ株比较,15例为野生型(未突变),42例为中间型(1-3个突变),1例为突变型(≥4个突变).其中2218位点突变最多,约为60.3％(35/58).dSDR aa突变数目与SVR关系密切(P=0.000),ISDR aa突变数≥2的CHC组所获得的EVR和SVR明显高于aa突变数＜2的CHC组(P=0.041/P=0.012).结论 华南HCV 1b亚型ISDR突变型(氨基酸变异≥4)极少.ISDR aa变异能够预测SVR;采用PEG-IFN/RBV联合方案治疗,对SVR有预测价值的ISDR aa突变数可由4个减少为2个.     

丙型肝炎病毒NS5B RNA聚合酶抑制剂研究进展

丙型肝炎病毒(hepatitis C virus, HCV)是引起慢性肝炎并进而发展为肝硬化和肝细胞癌的主要病原体之一。目前,临床上采用α-干扰素(IFN-α)和利巴韦林(RBV)联合用药治疗丙型肝炎,但有效率仅为40%~50%。寻找HCV特定靶向抗病毒治疗药物是抗HCV研究的重要方向,相应靶点包括NS2 和 NS3 蛋白酶,NS4A、NS4B、NS5A 和NS5B,其中以NS5B RNA依赖性RNA聚合酶(NS5B RdRp)为靶标的抗HCV药物研究近年来颇受关注。本文在介绍NS5B及NS5B RdRp结构和功能的基础上,总结归纳以NS5B RdRp为靶点的HCV特定靶向抗病毒治疗药物研究的主要策略,以及近年来相关NS5B RdRp抑制剂的研究进展。     

索磷布韦、达拉他韦联合或不联合利巴韦林对基因1b型慢性丙肝患者HCVRNA转阴率的影响

目的 分析索磷布韦、达拉他韦联合或不联合利巴韦林对基因1b型慢性丙型肝炎(CHC)患者丙型肝炎病毒(HCV)-RNA(HCV RNA)转阴率的影响。方法 将本院收治的82例基因1b型CHC患者作为研究对象,根据患者治疗方法的不同,分为A组(36例)给予达拉他韦+索磷布韦+利巴韦林治疗,B组(46例)给予达拉他韦+索磷布韦治疗。比较两组患者治疗后HCV RNA转阴率,治疗结束后第4、12周HCV RNA不可测(SVR4、SVR12)及不良反应发生情况等差异。结果 在治疗4、8周时,两组患者HCV RNA转阴率有所增加;但治疗后12周时,两组患者HCV RNA转阴率并未继续升高,与治疗后8周时的转阴率持平。结果显示,两组患者在治疗4、8、12周时HCV RNA转阴率的比较,均无显著性差异(P>0.05)。两组患者SVR4、SVR12的比较,亦无显著性差异(P>0.05)。A组治疗期间出现贫血、皮疹、头痛、疲劳、恶心及心悸等不良反应发生率较B组明显升高(P<0.05)。结论 达拉他韦、索磷布韦联合或不联合利巴韦林对基因1b型CHC患者的临床疗效相当,均具有较高的HCV RNA转阴率,但联合利巴韦林可能存在不良反应增加的风险,且达拉他韦+索磷布韦联合治疗基因1b型CHC患者可尝试将疗程缩短为8周。     

成人社区获得性肺炎降钙素原与中医证型相关性分析

[目的]探讨成人社区获得性肺炎(community-acquired pneumonia,CAP)患者降钙素原(procalcitonim,PCT)与中医证型之间的相关性、中医辨证分型的微观结构物质。[方法]回顾2012年09月1日至2014年12月31日于广东省中医院呼吸内科住院治疗并确诊CAP、年龄≥18岁的患者236例,获得降钙素原(PCT)及中医证型等资料,分析PCT与中医证型的相关性。[结果]成人CAP患者PCT在各中医证型中的分布存在差异,由高到低顺序分别为:肺胃热盛(0.44ng/m L)、痰热壅肺(0.20ng/m L)、风热犯肺(0.18ng/m L)、正虚邪恋(0.10ng/m L),其中肺胃热盛与痰热壅肺、风热犯肺、正虚邪恋比较,均具有统计学差异(P0.05)。[结论]CAP患者随炎症轻重的不同而呈现不同的结果,说明PCT可以指导CAP中医的辨证分型以及辨证治疗,并用作判别疾病虚实的参考指标。     

基于因子分析新型冠状病毒感染恢复期证候与证素学研究

目的 分析湖北省400例新型冠状病毒感染恢复期患者半年随访的后遗症特征、证候分布规律及中医证素学特点。方法 通过对760名新型冠状病毒感染恢复期患者的前瞻性随访研究,采集到出院半年后仍有明显后遗症状患者的人口学特征数据与中医四诊资料,提取其中医证候要素,应用频数统计、因子分析、聚类分析等统计学方法,确定其后遗症的存在情况,中医证候、证素及证型的分布规律。结果 随访760例新型冠状病毒感染恢复期患者,其中有400例患者在出院半年后仍有后遗症症状,恢复期最常见的病位证素主要涉及肺、肝、脾、肾等;病性证素主要涉及气虚、阳虚、阴虚、痰、血虚、湿、气滞等。结论 新型冠状病毒感染患者进入恢复期半年后,仍普遍存在多种后遗症症状和诸多身心健康问题。其核心病机可概括为“正虚邪恋”,此时“正虚”以肺、脾、肾三脏亏虚,气阴两虚为主;“邪恋”可表现为肝郁气滞与湿、痰、瘀、热等邪留滞体内。     

丙型肝炎病毒NS3抗原检测现状分析

丙型肝炎是一种由丙型肝炎病毒(hepatitis C virus,HCV)感染引发的危害严重的传染病,慢性HCV感染影响全球约1.8亿人[1],在中国的感染率为3.2％,约80％的HCV感染者会转化为慢性丙型肝炎(chronic hepatitis C,CHC,简称丙肝),使罹患肝癌的风险增加了6倍.1989年,Choo等[2]成功克隆出HCV的基因组.HCV属于黄病毒科,肝病毒属,单股正链RNA病毒,病毒颗粒直径55～65 nm,基因组大小约为9 600 bp,可编码一个含有3010或3011个氨基酸的多聚蛋白前体.多聚蛋白前体被宿主和病毒的蛋白酶共同切割成10个具有独立功能的HCV蛋白,分别为衣壳蛋白C、包膜蛋白E1和E2、p7蛋白、非结构蛋白NS2、NS3、NS4A、NS4B、NS5A和NS5B[3].目前HCV感染的主要诊断指标为抗HCV和HCV RNA,但二者存在一定局限性.HCV-NS3抗原(HCV NS3 antigen,HCAg-NS3)检测具有早期诊断HCV的价值,并可能作为HCV治疗预后监测的新指标,现将HCAg-NS3检测等相关研究进展综述如下.     

Megsin基因E1-5’UTR区A267G与免疫球蛋白A型肾病阴虚证的相关性

目的：观察增生为主的原发性免疫球蛋白A型肾病（immunoglobulin A nephropathy,IgAN）患者阴虚证（气阴两虚证、肝肾阴虚证）与megsin基因E1-5’UTR区A267G的相关性,寻找原发性IgAN中医证型微观辨证可能的物质基础。方法：筛选符合标准的原发性IgAN患者120例,进行megsin基因E1-5’UTR区A267G的单核苷酸多态性（single nucleotide polymorphism,SNP）测序,观察中医证型与SNP的相关性。 结果：120例患者中GG型83例,GA型34例,AA型3例。肝肾阴虚证在AA与GA型IgAN患者中所占比例较高,气阴两虚在GG型中所占比例较高（Pd0．01）;GG型与GA型＋AA型比数比（oddsratio,OR）为9．800,95％可信区间（confidence interval,CI）为3．969～24．199。这样的差异同样存在于不同性别和不同年龄之间。 结论：Megsin基因E1-5’UTR区A267G可能是区分原发性IgAN肝肾阴虚证和气阴两虚证的物质基础之一。     

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.     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

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.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.     

Scientific principles and rigorous processes should be followed in developing clinical guidelines for therapeutic interventions of integrative medicine

A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care（~＇4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.