环介导等温扩增法检测孕妇血清中单纯疱疹病毒Ⅰ型方法的建立

目的建立稳定、可靠的环介导等温扩增法（LAMP）快速检测孕妇血清中单纯疱疹病毒Ⅰ（HSV-Ⅰ）DNA的方法。方法通过对影响LAMP反应体系的几个主要因素进行优化,确定最佳反应条件,并对其特异性、灵敏度及可靠性进行评判。结果 LAMP检测HSV-Ⅰ DNA的最佳反应条件：甜菜碱和Mg2＋的终浓度分别为1.0mol/L和10mmol/L,于62℃恒温反应1h。扩增产物可被Hinf Ⅰ消化,理论上检测下限可达10个拷贝,与定量PCR相比符合率达94.5%。结论本实验建立的LAMP检测HSV-I的方法具有灵敏度高、特异性强、操作简便等特点,适合基层用于HSV-Ⅰ筛查。     

小鼠冷冻胚胎、精子常见病原菌快速检测方法的建立

目的建立实验动物冷冻资源(胚胎、精子等)常见病原菌的微量、快速监测方法。方法以实验动物三个常见病原菌(金黄色葡萄球菌、肺炎克雷伯氏菌和乙型溶血性链球菌)为研究对象,分别提取三个阳性菌株的DNA,通过引物设计和相应PCR条件的优化,建立三种阳性菌株的PCR检测方法。然后提取冷冻资源(胚胎、精子等)中的DNA,按照三个阳性菌的PCR条件进行检测,并将一部分样品DNA用荧光定量PCR方法进行检测。结果 1成功建立三个阳性菌株的PCR检测方法。三种病原菌的最低检测浓度,金黄色葡萄球菌为4.19×10~(-5)ng/μL,肺炎克雷伯氏菌为1.98×10~(-5)ng/μL,乙型溶血性链球菌为1.07×10~(-3)ng/μL.2通过普通PCR和荧光定量PCR方法均未检测出样品中三种阳性菌的存在。结论建立了小鼠冷冻胚胎、精子常见病原菌微量、快速检测方法。     

兔斯氏艾美耳球虫环介导等温扩增技术检测方法的建立

建立检测兔斯氏艾美耳球虫(E.stiedai)环介导等温扩增技术(LAMP)的方法。方法根据E.stiedai的ITS1-5.8s RNA-ITS2基因序列设计特异引物,通过优化反应条件,建立E.stiedai的LAMP检测方法。结果 25μL的反应体系中Bst聚合酶的最优添加量是1.5μL,DNA的最优添加量是75 ng,环引物、内引物和外引物的最优浓度依次是0.8μmol/L、1.6μmol/L和0.15μmol/L,在63℃反应1 h后能特异性地扩增目的基因,并且具有较高的灵敏度。结论建立了E.stiedai的LAMP检测方法,为E.stiedai感染的快速检测提供了新思路。     

红花SRAP扩增体系的建立和优化

目的:探讨影响红花SRAP扩增的各种因素,建立能够稳定扩增红花基因组的体系,为研究红花重要性状的遗传基础及建立分子辅助标记育种的技术平台奠定基础.方法:用CTAB法提取红花DNA,设计Taq酶浓度(0.02、0.04、0.06 U/μl)、dNTP浓度(0.15、0.25、0.30 mmol/L)、Primer浓度(0.15、0.30、0.45 μmol/L)3因素3水平27次实验和Mg2 浓度(0.5、1.0、1.5、2.0、2.5、3.0、3.5、4.0 mmol/L)8水平单因素实验.在25 μl体系中加入模板DNA 20 ng.对体系进行优化,用琼脂糖进行检测.结果:本研究建立了适合红花的SRAP体系,Taq酶浓度为0.02 U/μl,dNTP浓度为0.25 mmol/L,Primer 浓度为0.30 μmol/L,Mg2 的浓度为3.0 mmol/L,优化后的体系目标条带增多,重现性好,得到了较好的扩增效果.结论:本研究建立的反应体系适合红花SRAP的研究.     

金黄色葡萄球菌反向线性杂交探针检测方法的建立

目的建立一种检测金黄色葡萄球菌的简单、快速、灵敏、准确的方法。方法根据金黄色葡萄球菌的耐热核酸酶nuc 基因,设计一对通用引物及两条特异性探针,用生物素标记通用引物的5′端,将两条特异性探针固定于硝酸纤维膜上,使PCR产物与探针杂交。结果建立的反向线性杂交探针方法,其检测限为2 ng/μL,检测特异性和准确性均为100%。结论建立的反向线性杂交检测方法具有较高的敏感性和特异性,可用于实验动物金黄色葡萄球菌的快速检测。     

LAMP技术对耐甲氧西林金黄色葡萄球菌的快速鉴定

目的建立基于环介导等温扩增(LAMP)技术进行快速鉴定耐甲氧西林金黄色葡萄球菌(MRSA)的方法。方法采集自鼻拭子样品的MRSA菌株,针对MRSA耐药因子mec A所在基因组岛SCCmec,挑选具有高保守性的边界末端开放阅读框,设计特异性引物,进行LAMP快速检测,优化和确定LAMP反应体系和条件。结果以PCR法作为对照,LAMP检测MRSA的灵敏度为8×10~3 CFU/ml,PCR的灵敏度为8×10~5 CFU/ml。同时,LAMP快速检测法的检出率与特异性分别达92.7%与100.0%。结论 LAMP是可应用于MRSA检测的灵敏和特异性方法。     

LAMP技术检测弓形虫感染方法的建立

目的建立一种快速、简便的检测弓形虫感染的环介导等温扩增方法(LAMP)。方法根据弓形虫RH株SAG1基因序列合成2对特异性LAMP引物,优化扩增体系与参数。通过与常规PCR方法的比较,评估优化后的LAMP反应的特异性和灵敏性。利用建立的LAMP初步检测人工感染弓形虫4 d后白兔与大鼠的肌肉组织。结果 LAMP法检测弓形虫基因组DNA的灵敏度是传统PCR方法的100倍。LAMP法检测与其他常见寄生原虫、寄生虫无非特异性阳性结果。LAMP法检测人工接种动物的肌肉组织,阳性率达到100%,对照组为0,差异有统计学意义(P0.01)。结论 LAMP法检测弓形虫感染快速简便、敏感性高、设备要求低,具有较好的应用前景。     

金黄色葡萄球菌nuc基因特异性核酸探针的制备及应用

目的　建立核酸探针检测实验动物金黄色葡萄球菌的方法。方法　将重组质粒pGEM NUC中金黄色葡萄球菌nuc基因特异性片段酶切回收 ,经生物素标记后作为核酸斑点杂交试验探针 ,对细菌菌株及临床样品进行了检测。结果　核酸探针与金黄色葡萄球菌DNA呈杂交阳性 ,而与表皮葡萄球菌、化脓性链球菌、大肠埃希菌等其他细菌DNA呈杂交阴性 ,斑点杂交检测的灵敏度为 1pg基因组DNA。用斑点杂交试验和PCR对 32 2份SPF小鼠的临床样品进行了检测 ,检出率为 3 1% (10 /32 2 ) ,符合率为 10 0 % ,与细菌学检查的结果相一致。结论　建立的DNA探针检测金黄色葡萄球菌方法具有高度的特异性和敏感性 ,且较快速 ,可用于实验动物金黄色葡萄球菌的监测。     

树鼩粪便中沙门氏菌LAMP检测方法建立及应用

目的建立树鼩粪便沙门氏菌的环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法,并对此方法进行初步应用。方法根据沙门氏菌种属特异性基因inv A(侵袭蛋白基因A)的保守序列,设计合成特异性LAMP引物。分别设定十个反应温度即57℃~66℃和十个反应时间即24~42 min进行优化,并测定本方法的特异性和灵敏度;同时进行普通PCR实验,作为本方法的验证和比较。用建立的LAMP法对91份野生来源的树鼩稀便样品进行检测。结果优化反应条件选出反应温度为62℃,反应时间34 min;该方法对沙门氏菌的灵敏度可达3.36×101CFU/m L,比普通PCR方法高10~100倍;对10种树鼩来源的肠道细菌进行LAMP检测,只有沙门氏菌属的肠炎沙门氏菌和乙型副伤寒沙门氏菌检测为阳性,其余为阴性;对91份野生树鼩粪便样品进行LAMP检测,阳性检出率为20.88%;LAMP法的所有反应可在40 min内完成。结果可通过肉眼观察颜色变化直接判定。结论所建立的LAMP方法具有便捷、快速、灵敏、特异等特点,可用于树鼩粪便中沙门氏菌的大规模快速检测。     

金黄色葡萄球菌、绿脓杆菌和肺炎克雷伯杆菌多重PCR方法的建立与初步应用

目的建立金黄色葡萄球菌、绿脓杆菌和肺炎克雷伯杆菌多重PCR检测方法,为实验动物细菌检测提供快速、灵敏、简便的方法。方法根据金黄色葡萄球菌nuc基因、绿脓杆菌Las I基因、肺炎克雷伯杆菌Pho E基因和细菌通用16S rRNA基因设计出特异性引物;经过多重PCR引物浓度和退火温度的优化,特异性和敏感性的检测,建立多重PCR体系。应用该PCR体系对人工感染标本、实验动物粪便样本进行验证检测,与传统检测方法对比。结果多重PCR分别扩增出金黄色葡萄球菌(153 bp)、绿脓杆菌(600 bp)、肺炎克雷伯杆菌(368 bp)和通用(520 bp)的目的条带。多重敏感性为10 pg,特异性检测未从其他细菌中检测出目的条带。应用建立的多重PCR体系检测出不同组合的人工感染标本,从76只粪便检测出绿脓杆菌阳性1例,与传统检测方法结果一致。结论本文建立的多重PCR方法具有特异、灵敏、简便、快速等优点,为实验动物微生物的快速检测提供了可靠的方法。     

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…     

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.     

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.     

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.     

猪肺磷脂注射液联合经鼻持续气道正压通气对呼吸衰竭早产儿的临床疗效及肌酸激酶同工酶活性的影响

目的 探讨猪肺磷脂注射液联合经鼻持续气道正压通气(NCPAP)对呼吸衰竭早产儿的临床疗效及肌酸激酶同工酶活性(CK-MB)的影响.方法 选取呼吸衰竭早产儿80例,分为观察组和对照组各40例.对照组采用NCPAP给氧治疗,观察组给予NCPAP给氧联合猪肺磷脂气管内给药.观察两组患儿治疗前及治疗12h、24 h后PaO2、PaCO2、血氧饱和度(SaO2)、pH的变化情况,检测治疗前及治疗5d后血清CK-MB水平;评估两组患儿的临床治疗效果.结果 两组患儿PaO2、PaCO2、SaO2、pH比较,差异均有统计学意义(P＜0.05),其中观察组治疗后的PaO2、SaO2、pH均高于对照组,PaCO2则低于对照组.两组的PaO2、SaO2、pH均随观察时间延长而升高(P＜0.05),PaCO2均随观察时间的延长而降低(P＜0.05).观察组治疗有效率为87.5％,显著高于对照组的70.0％ (P ＜0.05).治疗5d后两组患儿血清CK-MB水平均较前降低(P＜0.05),且观察组明显低于对照组(P＜0.05).结论 猪肺磷脂注射液气管内给药联合NCPAP可以显著降低呼吸衰竭早产儿CK-MB的含量,提高治疗有效率,起到很好的呼吸循环支持作用.     

Survey and practice of reporting quality of randomized controlled clinical trials on traditional Chinese medicine

Evidence obtained from randomized controlled trials （RCTs） has been generally accepted as the gold standard in the evaluation of clinical effectiveness. Readers need to understand the trial design, implementation, results, analysis and interpretation, so as to fully Jnderstand the results of RCTs. Thus, the investigators of RCTs have to report these items in a complete, accurate and clear manner. Since 1998, we have conducted several evaluations on the reporting quality of RCTs published in Chinese journals on traditional Chinese medicine （TCM） and results have shown that there is an urgent need for higher quality RCTs on TCM.     

TCM Treatment of Ankylosing Spondylitis by Tonifying the Kidney and Strengthening the Governor Vessel

Ankylosing spondylitis is a chronic and progressive disorder with inflammation mainly involving the central axis joints. It mainly affects the cervical spine and the lumbosacral area, with the pathogenesis closely related to the kidney and the Governor Vessel (GV). TCM holds that the syndrome is deficiency in origin and excess in superficiality, which is due to insufficiency of the kidney, deficiency of GV, and blocking of the channels with the invasion of exogenous evil, leading to poor circulation of qi and blood and malnutrition of the bones, muscles and joints. The TCM method of tonifying the kidney and strengthening GV to regulate circulation of qi and blood and check the arthralgia pain should be adopted, with the Kidney-Tonifying and GV Strengthening Decoction (益肾强督汤) prescribed.     

IMPROVING P-gp EXPRESSION IN HUMAN MONONUCLEAR CELLS IN VITRO TRANSFECTED BY MULTIDRUG RESISTANCE-1 mRNA

CHEMOTHERAPY playsa greatrolein the treat- ment of malignanttumors,especiallyingynecolo- gicalones.But inanticancerchemotherapy,leuko-cytopeniaisfrequentlytheprimarydose-limitingsideeffect factor.Moreover,cancersarefrequentlychemoresistantbe-causeof overexpressionof P-glycoprotein(P-gp), which isencodedby multidrugresistancegene (MDR1 ) and detectableinup to50% ofhuman cancersand renderscellsresistancetoanticancerdrugs.The safetyand potentialtherapeuticbenefitof mdr1 gene transferredto h…