Osteopontin特异性siRNA真核表达载体的构建及其在GC9811胃癌细胞中沉默效应的鉴定

目的：构建骨桥蛋白(OPN)特异性siRNA(small interfere RNA)真核表达载体,体外观察对OPN基因的沉默作用．方法：采用基因克隆技术,将合成的特异性OPNRNA干扰寡核苷酸序列插入真核表达载体mU6pro,构建OPN siRNA真核表达载体．以脂质体法将mU6pro空载体和2个(mU6pro／OPN—siRNA1和mU6pro／OPN—siRNA2)重组质粒分别导入具有高转移特性的GC9811胃癌细胞系．72h后用RT—PCR和Western blotting技术检测各实验组胃癌细胞内OPN mRNA及蛋白水平的表达情况．结果：成功构建OPN siRNA真核表达载体．转染OPN—siRNA的胃癌细胞,72h后OPN mRNA及蛋白表达下调,而以mU6pro／OPN-siRNA1的抑制效果更为明显．结论：构建的RNA干扰真核表达载体能明显干扰OPN mRNA及蛋白的表达,为将其进一步应用于胃癌的治疗研究奠定了基础。     

hnRNP K siRNA真核表达载体的构建及其在A549肺癌细胞中沉默效应的研究

目的 构建hnRNP K特异性siRNA真核表达载体,体外观察对hnRNP K基因的沉默作用.方法 采用基因克隆技术,将合成的特异性hnRNP K RNA干扰寡核苷酸序列插入真核表达载体pSUPER,构建hnRNP K siRNA真核表达载体.采用Lipofect AMINE2000将pSUPER空载体和3个重组质粒(pSUPER/hnRNP K siRNAa,pSUPER/hnRNP K siRNAc和pSUPER/siRNAn)分别导入A549肺癌细胞(a、c、n分别代表hnRNP K编码序列中的A链和c链,以及无意义的对照序列Non链).24 h后用RT-PCR和Western blot技术检测各实验组肺癌细胞内hnRNP K mRNA及蛋白水平的表达情况.结果 成功构建了hnRNP K siRNA真核表达载体.转染hnRNP K siRNAa、hnRNP K siRNAc的肺癌细胞24 h后hnRNP K mRNA相对表达量分别为0.24±0.53和0.28±0.57,较对照组显著降低(P均<0.01);hnRNP K蛋白灰度值分别为0.23±0.11和0.28±0.09,较对照组显著降低(P均<0.05).结论 构建的RNA干扰真核表达载体能明显干扰A549细胞hnRNP K mRNA及蛋白的表达,为进一步研究hnRNP K基因的功能并应用于肺癌的治疗研究奠定了基础.     

糖皮质激素受体αsiRNA真核表达载体的构建与鉴定

目的 构建糖皮质激素受体α(GRα)小干扰RNA(siRNA)的真核表达载体,并检测其抑制结肠癌细胞株Lovo中糖皮质激素受体α表达的效果.方法 根据基因文库中GRα的cDNA序列,设计并合成两段siRNA,分别插入到pGenesil-1质粒中,并对重组质粒(命名为pGenesil-1/GRα1和pGenesil-1/GRα2)进行测序鉴定.脂质体介导下转染Lovo细胞株,RT-PCR和Western Blot检测转染后GRα的表达.结果 成功构建GRαsiRNA 真核表达载体(pGenesil-1/GRα1和pGenesil-1/GRα2);pGenesil-1/GRα2转染(脂质体法)Lovo细胞后,RT-PCR和Western blot检测细胞内GRα的表达显著降低.结论 GRαsiRNA真核表达载体的成功构建为进一步研究GR在结肠癌化疗中的作用奠定了重要的实验基础.     

Mob1a真核表达载体构建方法及对MEN通路基因表达的 影响研究

目的 探讨Mobla真核表达载体构建方法 及对MEN通路基因表达的影响.方法 利用PCR扩增技术扩增出Mobla编码基因,将其构建到真核表达Mobla载体上,利用脂质体将构建的真核表达载体瞬时转染干细胞,采用Western blot检测Mobla蛋白是否表达,采用荧光素梅报告基因实验对MEN通路基因表达活性的影响.结果 本研究成功构建Mobla真核载体,经过小鼠肝脏细胞转染后能对其蛋白质进行提取,Western blot显示:Mobla质粒在小鼠肝脏细胞中表达水平未见异常.荧光素酶报告基因试验进结果 显示:Mobla能抑制MEN通路基因信号,能抑制TNF-a刺激引起的MEN通路转录活性.结论 采用PCR扩增技术能成功扩增、构建出Mobla真核表达载体,并且对MEN通路基因表达存在一定的影响.     

RNA干扰技术特异性抑制骨肉瘤survivin的表达

目的:体外转录合成survivin siRNA,观察其在骨肉瘤细胞株U2-OS中抑制survivin的表达情况.方法:体外转录合成survivin序列特异性双链RNA(dsRNA),转染U2-OS细胞株中,用RT-PCR和Western blot法检测转染前后survivin基因的mRNA和蛋白的表达,MTF法检测细胞增殖,观察U2-OS细胞生物学特性的改变.结果:转染特异性siRNA的细胞其survivin mRNA及蛋白表达均下调,干涉后细胞的增殖受到抑制.结论:体外转录合成特异性survivin siRNA能有效抑制骨肉瘤细胞株U2-OS中survivin的表达,抑制细胞的增殖,RNA干扰技术为骨肉瘤的治疗提供了一种新策略.     

CENP-I表达下调对HEK293细胞生长及染色体分离的影响

目的 构建CENP-Ⅰ特异性RNA干扰真核表达载体,体外观察抑制CENP-Ⅰ基因表达对HEK293细胞生长、细胞周期和染色体数目异常率的影响.方法 构建CENP-Ⅰ siRNA真核表达载体,脂质体法将pGenesil-1空载体和pGene-sil-1/CENP-I-siRNA-1、pGenesil-1/CENP-Ⅰ-siRNA-2、pGenesil-1/CENP-Ⅰ-siRNA-3真核表达载体分别导入人胚肾HEK293细胞.转染后24、48、72 h收集细胞用Western blot和FQ-PCR检测HEK293细胞内CENP-Ⅰ蛋白及mRNA水平的表达情况,以确定干扰效果及最佳干扰时间.MTT法检测各实验组细胞生长情况,流式细胞术检测各组细胞的细胞周期分布,Giemsa染色法计数细胞分裂指数,常规法制备染色体标本.结果 成功构建CENP-Ⅰ siRNA真核表达载体,筛选出具有干扰效果的质粒载体即pGenesil-1/CENP-Ⅰ-siRNA-3,转染人胚肾HEK293细胞72 h后可使CENP-Ⅰ蛋白及mRNA表达显著下调.CENP-Ⅰ表达降低的HEK293细胞生长速度明显减慢(P<0.05),G2/M期细胞增加,分裂期细胞比例增大.结论 RNA干扰有效地特异性抑制着丝粒特异性蛋白质CENP-Ⅰ的表达.CENP-Ⅰ的抑制可使人胚肾HEK293细胞生长减慢,增殖能力减弱,使细胞分裂期延长.     

RNA干扰技术特异性抑制肾癌survivin表达的实验研究

目的:体外转录合成survlvin siRNA(Small interference RNA,siRNA),观察其在肾癌786-O细胞株中抑制survivin的表达情况.方法:体外转录合成3组survivin序列特异性双链RNA(Double-stranded RNA,dsRNA),转染786-O细胞株中,用RT-PCR和Western blot检测转染后survivin基因的mRNA和蛋白的表达,MTT法检测细胞活性.结果:有2组转染特异性siRNA的786-O细胞表达survivin mRNA及蛋白均下调.活性受到抑制,另一组效果不明显.结论:体外转录合成特异性survivin siRNA能有效抑制肾癌786-O细胞中survivin的表达,抑制细胞的活性,RNA干扰(RNA interfering,RNAi)技术可能为肾癌的基因治疗提供一种新策略.     

CENP-Ⅰ表达下调对HEK293细胞生长及染色体分离的影响

目的 构建CENP-Ⅰ特异性RNA干扰真核表达载体,体外观察抑制CENP-Ⅰ基因表达对HEK293细胞生长、细胞周期和染色体数目异常率的影响.方法 构建CENP-Ⅰ siRNA真核表达载体,脂质体法将pGenesil-1空载体和pGene-sil-1/CENP-I-siRNA-1、pGenesil-1/CENP-Ⅰ-siRNA-2、pGenesil-1/CENP-Ⅰ-siRNA-3真核表达载体分别导入人胚肾HEK293细胞.转染后24、48、72 h收集细胞用Western blot和FQ-PCR检测HEK293细胞内CENP-Ⅰ蛋白及mRNA水平的表达情况,以确定干扰效果及最佳干扰时间.MTT法检测各实验组细胞生长情况,流式细胞术检测各组细胞的细胞周期分布,Giemsa染色法计数细胞分裂指数,常规法制备染色体标本.结果 成功构建CENP-Ⅰ siRNA真核表达载体,筛选出具有干扰效果的质粒载体即pGenesil-1/CENP-Ⅰ-siRNA-3,转染人胚肾HEK293细胞72 h后可使CENP-Ⅰ蛋白及mRNA表达显著下调.CENP-Ⅰ表达降低的HEK293细胞生长速度明显减慢(P<0.05),G2/M期细胞增加,分裂期细胞比例增大.结论 RNA干扰有效地特异性抑制着丝粒特异性蛋白质CENP-Ⅰ的表达.CENP-Ⅰ的抑制可使人胚肾HEK293细胞生长减慢,增殖能力减弱,使细胞分裂期延长.     

siRNA沉默HBx基因对肝癌细胞YKL-40蛋白表达影响的研究

目的 观察HepG2.2.15细胞中乙型肝炎病毒X(HBx)基因沉默对酿酒酵母YKL-40蛋白表达的影响.方法 构建靶向HBx基因的siRNA真核表达载体pRNAT-HBx和阴性对照质粒,同时设空白对照组,采用Lipofectinamine 2000脂质体转染法转染人肝癌细胞系HepG2.2.15细胞.采用RT-PCR检测各组中HBx和YKL-40的mRNA水平,Western blot、细胞免疫荧光检测各组中HBx蛋白和YKL-40蛋白的表达.结果 pRNAT-HBx明显抑制HepG2.2.15细胞中HBx基因的表达,同时YKL-40的mRNA和细胞内蛋白表达水平也随之相应下调.结论 抑制HBx基因的表达能下调HepG2.2.15细胞中YKL-40蛋白的表达,提示HBx蛋白对YKL-40蛋白的表达具有一定的激活作用.     

靶向β-catenin的shRNA真核表达质粒的构建及鉴定

目的:针对β-连环蛋白(β-catenin)基因的不同部位,构建靶向β-catenin的shRNA真核表达质粒,鉴定并筛选出最佳抑制效率的表达质粒.方法:针对β-catenin基因的不同部位设计3对短发卡RNA(shRNA)的寡核苷酸片段,克隆到真核表达载体pGPU6中,构建靶向β-catenin的shRNA真核表达质粒pGPU6-β-catenin-shRNA-1、2、3.利用脂质体转染人胃癌细胞株AGS,Western blot法检测并筛选最佳抑制效率的shRNA表达质粒.结果:3个靶向β-catenin的shRNA真核表达质粒pGPU6-β-catenin-shRNA-1、2、3经限制性酶切和测序确实基因插入正确,Western blot法证实pGPU6-B-catenin-shRNA-3明显降低细胞内β-catenin蛋白的表达.结论:成功构建了靶向β-catenin的shRNA真核表达质粒pGPU6-β-catenin-shRNA-1、2、3,并筛选出最佳抑制效率的表达质粒,为进一步研究Wnt/β-catenin信号通路在胃癌中的作用奠定了基础.     

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…