乳腺癌患者血清中VEGF－C的表达及siRNA靶向抑制MCF－7细胞侵袭与转移的研究

目的：探讨VEGF－C（ vascular endothelial growth factor －C）在乳腺癌患者血清中的表达及与肿瘤转移的关系;VEGF－CsiRNA对MCF－7中VEGF－C表达及细胞侵袭与转移的影响。方法：ELISA法检测30例乳腺癌及10名健康者空腹血清VEGF－C浓度;合成VEGF－CsiRN并采用LipofectamineTM2000转染MCF－7细胞;RT－PCR法和免疫细胞化学检测VEGF－CmRNA和蛋白的表达;Transwell检测细胞侵袭情况。结果：乳腺癌患者血清中VEGF－C浓度明显升高,且发生淋巴结转移的VEGF－C浓度明显高于对照组（ P＜0.05）;转染VEGF－CsiRNA的细胞VEGF－CmRNA和蛋白表达明显降低（ P＜0.05）,侵袭能力也明显下降（P＜0.05）。结论：VEGF－CsiRNA不仅能特异性降低VEGF－CmRNA及蛋白的表达,且能抑制MCF－7的侵袭和转移。     

姜黄素对人乳腺癌细胞增殖的抑制效应及机制

目的 研究姜黄素对人乳腺癌细胞株增殖的抑制效应及其机制。方法 采用Northern印迹和Western印迹法观察人乳腺癌细胞mRNA及其蛋白表达;用CAT报告基因来研究人雌激素反应元件(ERE)的转录活性;用Matrigd侵袭池(Matrigel invasion chamber)研究细胞侵袭性。结果 姜黄素能够抑制雌激素受体(ER)阳性MCF—7及ER阴性MDA—MB—231两种人乳腺癌细胞的增殖。对MCF—7细胞,姜黄素调节ER下调基因pS2和TGF—α的表达及ERE的活性;对MDA—MB—231乳腺癌细胞,姜黄素通过下调MMP—2和上调TIMP—1的活性表现出很强的抗侵袭活性,其还能够抑制MDA—MB—231细胞两个主要的血管生成因子VEGF和b—FGF、在转录水平的表达。结论 姜黄素通过多种机制抑制乳腺癌细胞的生长,姜黄素的化学预防是多途径作用的结果。     

Grb2在MCF-7 乳腺癌细胞中的核定位研究

目的　构建Grb2的红色荧光蛋白融合表达质粒pDsRed1 C/Grb2 ,并观察Grb2在MCF 7乳腺癌细胞的核定位情况。方法　用PCR方法扩增Grb2cDNA ,与红色荧光蛋白表达质粒pDsRed1 C重组构建融合蛋白表达质粒pDsRed1 C/Grb2 ,酶切、测序鉴定后 ,瞬时转染MCF 7乳腺癌细胞株 ,用荧光显微镜观察Grb2的核定位情况。结果　pDsRed1 C/Grb2质粒有正确的阅读框 ,融合表达蛋白DsRed1 C/Grb2可定位于MCF 7乳腺癌细胞核内。结论　成功构建了Grb2的红色荧光蛋白融合表达质粒pDsRed1 C/Grb2 ,在MCF 7乳腺癌细胞株中 ,Grb2除存在于胞浆外 ,也可定位于细胞核内 ,可能与其参与核内基因的转录调控有关。     

SPAG5通过影响JAK2/STAT3信号通路磷酸化促进乳腺癌细胞的增殖和侵袭

目的：探究人类精子相关抗原5（sperm?associated antigen 5,SPAG5）对乳腺癌细胞增殖和侵袭的影响及分子机制。方法：采用qRT?PCR和Western blot筛选相应SPAG5高表达细胞株和低表达细胞株;分别设计并构建重组SPAG5敲减/过表达质粒（siRNA?SPAG5/OE?SPAG5）,通过慢病毒表达质粒系统,制备敲减/过表达SPAG5的稳定细胞株;采用CCK?8增殖实验和裸鼠成瘤实验,检测乳腺癌细胞增殖能力的变化;采用细胞划痕实验、Transwell细胞实验检测乳腺癌细胞迁移、侵袭能力的变化;采用qRT?PCR和Western blot检测JAK2/STAT3通路和下游蛋白的变化;采用WP1066抑制JAK2/STAT3通路,检测细胞增殖和侵袭能力的变化。结果：相较于各自对照组,SPAG5敲减的 si1?MDA?MB?231组细胞SPAG5 mRNA 和蛋白水平显著降低,SPAG5过表达的OE?MCF?12A组细胞SPAG5 mRNA 和蛋白水平显著升高（P＜0.05）;同时,si1?MDA?MB?231组细胞的增殖、迁移、侵袭能力受到抑制,OE?MCF?12A组细胞得到增强（P＜0.05）;si1?MDA?MB?231组细胞的JAK2/STAT3通路磷酸化水平和下游蛋白表达受抑制（P＜0.05）,OE?MCF?12A组细胞的JAK2/STAT3通路磷酸化水平和下游蛋白表达水平提高（P＜0.05）;抑制JAK2/STAT3通路后SPAG5对乳腺癌细胞增殖和侵袭的促进作用消失（P＜0.05）。结论：SPAG5通过JAK2/STAT3信号通路增强乳腺癌细胞的增殖和侵袭能力。     

转染BRMS1基因对乳腺癌MDA MB 231细胞体外侵袭力的影响

目的：探讨BRMS1基因对乳腺癌MDA MB 231细胞体外侵袭力的影响。方法：将携带BRMS1基因的真核表达载体pcDNA3.1/myc BRMS1通过脂质体介导稳定转染乳腺癌MDA MB 231细胞；RT PCR检测转染前后细胞中BRMS1mRNA的表达；扫描电镜观察细胞表面超微结构的改变；Boyden小室检测细胞体外侵袭力。结果：转染成功的乳腺癌MDA MB 231细胞其BRMS1mRNA呈高表达；细胞表面超微结构发生改变；体外侵袭力下降。结论:转染BRMS1基因可抑制乳腺癌MDA MB 231细胞的体外侵袭能力。     

外周血中乳腺癌细胞hMAM表达检测方法的建立及初步应用

目的建立反转录结合荧光定量PCR(FQ-PCR)检测外周血乳腺癌细胞hMAM基因表达方法,了解其监测乳腺癌细胞微转移的应用价值。方法克隆目的片段,制备标准品并做测序验证。培养乳腺癌MDA-MB453、MCF7细胞株,制成血液标本模型,GAPDH基因为内对照,反转录结合FQ-PCR检测乳腺癌细胞株血标本模型和10例乳腺癌患者外周血的hMAM表达。结果电泳和测序证实,克隆产物系预期的目的片段。FQ-PCR标准曲线的相关系数为-1.0;用103拷贝/微升标准品重复检测5次,x±s:718.9±120.5,CV值:16.7%;MDA-MB453细胞的血标本做灵敏度试验,相当于104个血细胞中含1个乳腺癌细胞可检出阳性。未检测到MCF7细胞的hMAM表达。10例乳腺癌患者,淋巴结转移的5例中,3例hMAM表达阳性、2例阴性;无淋巴结转移的5例中,1例hMAM表达阳性、4例阴性。结论本方法可有效监测外周血中微转移的乳腺癌细胞,发现不同乳腺癌细胞系hMAM表达程度差异大。     

RNA干扰下调midkine基因表达对人乳腺癌细胞粘附和侵袭力的影响

目的探讨中期因子（midkine,MK）基因siRNA对乳腺癌细胞生物学行为的影响。方法培养人乳腺癌Bcap-37、LCC1、MCF-7、MDA—MB-231、MDA—MB-435、MDA—MB-468及ZR75—1细胞株,以荧光实时定量PCR方法检测MK基因mRNA表达;筛选出MK表达最高者。采用MK siRNA转染乳腺癌细胞株,分别以荧光实时定量RT—PCR和免疫荧光方法观察MK基因mRNA和蛋白水平,然后以四唑蓝（MTT）比色法检测细胞粘附性,以Boyden小室方法检测癌细胞侵袭能力。结果7株乳腺癌细胞中,MK均有不同程度的表达,以MCF-7细胞最高;以MKsiRNA转染乳腺癌MCF-7细胞后,癌细胞MK基因mRNA和蛋白水平明显下降,且呈浓度依赖性;siRNA转染组细胞粘附数量明显下降,细胞侵袭力明显下降,均呈浓度依赖性（P〈0．01;P〈0．01）。结论MK基因在乳腺癌细胞粘附和侵袭中发挥着重要作用;以siRNA转染乳腺癌细胞,可抑制乳腺癌细胞粘附和侵袭能力。     

去磷酸化RB蛋白在乳腺癌细胞凋亡中的作用

目的 :研究去磷酸化RB蛋白表达在人乳腺癌细胞凋亡中的作用。　方法 :以乳腺癌细胞株MCF 7/S(化疗敏感细胞株 )和MCF 7/Adr(耐多柔比星细胞株 )为研究对象 ,采用免疫细胞化学法检测多柔比星 (ADR)作用前、后RB蛋白的表达状态 ;应用流式细胞术检测ADR诱导乳腺癌细胞凋亡程度。　结果 :加ADR前两种细胞胞核中的磷酸化RB蛋白均为阳性 ,去磷酸化RB蛋白均为阴性。经不同浓度ADR处理后 ,随着其浓度的增加 ,MCF 7/S细胞去磷酸化RB蛋白表达量逐渐增高 ,而MCF 7/Adr细胞去磷酸化RB蛋白表达量无明显增高。流式细胞术检测细胞凋亡率和细胞周期 ,结果表明ADR诱导MCF 7/S细胞凋亡作用比MCF 7/Adr强 (P <0 .0 1)。　结论 :去磷酸化RB蛋白表达与MCF 7/S细胞凋亡呈正相关 ,而与MCF 7/Adr细胞凋亡无明显相关性     

miR⁃588在乳腺癌中的表达及对乳腺癌细胞增殖和侵袭的作用

目的：探讨miR?588在乳腺癌中的表达及对乳腺癌细胞增殖、迁移和侵袭能力的影响。方法：用化学合成的miR?588成熟模拟物（miR?588 mimic）过表达miR?588,并验证miR?588过表达率;采用MTT、克隆形成实验检测miR?588对乳腺癌细胞MCF7和MDA?MB?231增殖的影响;采用划痕愈合实验和Transwell侵袭实验检测miR?588对MDA?MB?231细胞迁移和侵袭的影响。结果：miR?588在乳腺癌组织及细胞中的相对表达均低于癌旁正常组织及乳腺正常上皮细胞;过表达miR?588能明显抑制MCF7和MDA?MB?231细胞的增殖;上调miR?588可显著抑制MDA?MB?231细胞的迁移和侵袭能力。结论：miR?588在乳腺癌中低表达,过表达miR?588可抑制乳腺癌细胞的增殖和侵袭。     

MDA-MB-231细胞源exosome对人脐静脉内皮细胞(HUVEC)VEGF自分泌及体外成管作用的影响

 目的 研究人乳腺癌MDA MB 231细胞源exosome对人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)血管内皮生长因子(vascular endothelial growth factor,VEGF)自分泌及体外成管作用的影响,探讨肿瘤细胞源exosome在肿瘤微环境中对血管内皮细胞血管生成的调控作用。方法 低温超速离心及密度梯度离心法提取乳腺癌MDA MB 231细胞源exosome;酶联免疫吸附试验(ELISA)检测HUVEC与exosome共培养24 h后上清液中VEGF的变化水平;Western blot技术检测HUVEC与exosome共培养24 h后VEGF、VEGFR2及p VEGFR2的蛋白表达情况;RT PCR法检测HUVEC与exosome共培养24 h后VEGF的基因表达情况;观察HUVEC与exosome共培养24 h后的体外成管能力。 结果 HUVEC与exosome共培养24 h后上清液中VEGF为(110.851±18.404)pg/mL,与对照组相比差异具有统计学意义(P＜0.05);Western blot结果显示,HUVEC与exosome共培养24 h后VEGF和p VEGFR2的蛋白表达水平均增加(P＜0.05);RT PCR结果显示,HUVEC与exosome共培养24 h后VEGF的基因表达水平增加(P＜0.05);体外成管实验显示,exosome显著提高了HUVEC的管腔形成能力(P<0.05)。结论 乳腺癌MDA MB 231细胞源exosome促进了血管内皮细胞VEGF的表达及分泌,激活了血管内皮细胞VEGF/VEGFR2自分泌环并提高了血管内皮细胞的体外成管能力,对促肿瘤血管生成有一定的调控作用。     

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…