肺腺癌表皮生长因子受体T790M突变裸鼠模型的建立与评价

目的 建立表皮生长因子受体（EGFR）T790M突变的肺腺癌裸鼠模型,为探讨肺腺癌EGFR-TKIs 的继发性耐药机制提供工具。方法 建立对吉非替尼耐药的人肺腺癌细胞株RPC-9,以皮下异位移植法建立EGFR T790M突变肺腺癌裸鼠模型。进行组织病理学分析,以基因测序法检测EGFR基因突变;以Western blot法检测EGFR及磷酸化EGFR表达水平。结果 吉非替尼浓度增到10μmol/L时,基因测序法检测显示PC-9细胞EGFR基因E20发生突变,其氨基酸变化为790T>T/M。在4周内,PC-9移植组及RPC移植组肿瘤体积明显差异;移植后第5周时,RPC移植组肿瘤体积为944±20mm³,PC移植组肿瘤体积为878±8mm³,RPC移植组肿瘤体积显著大于PC-9移植组（P<0.05）。PC-9移植及RPC移植组肿瘤组织病理均为低分化腺癌。RPC-9移植组肿瘤组织EGFR基因存在E20发生突变,其氨基酸变化为790T>T/M。PC-9移植组及RPC移植组肿瘤组织EGFR及Tyr992磷酸化的EGFR表达水平差异均无统计学意义（P>0.05）。结论 通过皮下移植对吉非替尼继发性耐药的人肺腺癌细胞RPC-9成功建立EGFR T790M 突变的肺腺癌裸鼠模型,为研究肺腺癌EGFR-TKIs 的继发性耐药提供了有力工具。     

苦参对耐吉非替尼人肺腺癌PC9/ZD细胞上皮间质转化及吉非替尼药物敏感性的影响

目的探讨苦参对耐吉非替尼人肺腺癌PC9/ZD细胞上皮间质转化及吉非替尼药物敏感性的影响。方法选取对细胞无毒性作用的苦参浓度,采用体外苦参作用于耐吉非替尼人肺腺癌PC9/ZD细胞;分为对照组、吉非替尼组、苦参联合吉非替尼组。分别通过MTT法、Transwell实验检测各组人肺腺癌PC9/ZD细胞增殖及侵袭能力,Western blot实验检测EMT相关蛋白表达,流式细胞术检测细胞凋亡率。结果 MTT法检测结果显示,苦参联合吉非替尼组人肺腺癌PC9/ZD细胞增殖抑制率(74.59±3.21)%,明显高于吉非替尼组(33.46±0.84)%(P0.05);Western blot实验结果显示,在苦参作用下,吉非替尼诱导的人肺腺癌PC9/ZD细胞波形蛋白及N钙黏蛋白表达下调,E钙黏附分子表达上调;Transwell实验检测结果显示,苦参联合吉非替尼组人肺腺癌PC9/ZD细胞侵袭率(48.59±7.26)%,较吉非替尼组(64.23±8.04)%和对照组(91.03±11.02)%明显降低(P均0.05);流式细胞术检测结果显示,对照组人肺腺癌PC9/ZD细胞凋亡率(31.14±5.26)%,吉非替尼组(56.48±8.77)%,苦参联合吉非替尼组(76.25±10.28)%,三组间人肺腺癌PC9/ZD细胞凋亡率比较,差异均有统计学意义(P均0.05)。结论苦参可逆转人肺腺癌PC9/ZD细胞上皮间质转化,增加对吉非替尼药物敏感性。     

吗啡联合厄洛替尼对肺腺癌裸鼠成瘤的抑制作用及机制

目的构建人HCC827肺腺癌细胞裸鼠成瘤模型,并探讨吗啡联合厄洛替尼对肿瘤的抑制作用及其机制。方法裸鼠前腋皮下注射HCC827细胞悬液,成瘤后随机数字表法分为对照组、吗啡组、厄洛替尼组、吗啡联合厄洛替尼组(联合组),各6只;连续观察3周测量并计算各组大鼠肿瘤体积,流式细胞术检测细胞凋亡率,Western blot方法检测各组肿瘤组织中Bcl-2、Bax和Caspase-3蛋白的表达。结果与对照组比较,吗啡组肿瘤体积、肿瘤细胞凋亡率及凋亡相关蛋白Bcl-2、Bax和Caspase-3的表达差异均无统计学意义(P> 0. 05),厄洛替尼组及联合组肿瘤体积明显缩小(P=0.000),细胞凋亡率增加(t/P=20.455/<0.001,t/P=24.954/<0.001),凋亡相关蛋白Bcl-2表达下调、Bax和Caspase-3表达增加,差异均具有统计学意义(t=101.555、161.308、96. 757,P均=0. 000);与厄洛替尼组比较,联合组在肿瘤体积、肿瘤细胞凋亡率及凋亡相关蛋白Bcl-2、Bax和Caspase-3的表达差异比较均无统计学意义(P>0. 05)。结论吗啡与厄洛替尼联合应用时,不影响厄洛替尼诱导的凋亡相关性抗肿瘤效应。     

ABT737通过激活JNK/c-Jun通路上调Bim诱导宫颈癌细胞凋亡

【目的】探讨JNK/c-Jun信号通路激活Bim在ABT737诱导宫颈癌细胞凋亡中的作用。【方法】用MTT法检测ABT-737对人宫颈癌Hela细胞的生长抑制作用;用流式细胞术检测细胞凋亡率;用Western blot方法检测JNK、phospho-JNK、c-Jun、phospho-c-Jun以及Bim蛋白的表达;用RT-PCR检测Bim在mRNA水平的变化;用JNK特异性抑制剂SP600125和siRNA瞬时转染抑制JNK及c-Jun的活性。 【结果】ABT-737能抑制Hela细胞的生长,诱导HeLa细胞发生凋亡。ABT737可激活JNK激酶活性其下游靶分子c-Jun,凋亡相关基因Bim在mRNA水平和蛋白水平的表达也随之上调。应用JNK特异性抑制剂SP600125和靶向JNK及c-Jun的siRNA抑制JNK或c-Jun的活性或表达后,ABT737诱导的Bim上调及细胞凋亡亦被有效阻断。【结论】ABT737通过JNK/c-Jun信号通路上调凋亡相关基因Bim的表达,诱导HeLa细胞发生凋亡。     

抑制STAT3增强对吉非替尼耐药的非小细胞肺癌药物敏感性的研究

目的:探讨非小细胞肺癌(NSCLC)中表皮生长因子受体T790M突变导致的吉非替尼耐药与信号转导及转录激活因子3(STAT3)的相关性。方法:不同浓度STAT3特异性抑制剂JSI-124处理NSCLC H1975细胞和PC9细胞后,CCK-8法检测细胞抑制率,Western blot法检测蛋白水平表达,RT-PCR法检测相关基因mRNA水平的表达。结果:NSCLC中EGFR T790M突变的H1975细胞中STAT3的表达明显高于PC9细胞(P0.01);JSI-124可剂量和时间依赖性地抑制H1975突变细胞中STAT3的活性,并能增强其对吉非替尼的敏感性。且此现象未在对吉非替尼敏感的PC9细胞中观察到。结论:STAT3因子与表皮生长因子受体T790M突变导致的吉非替尼耐药机制相关,抑制STAT3活性可逆转吉非替尼耐药。     

肺金生方抗非小细胞肺癌EGFR-TKIs耐药性的分子机制研究

目的探讨肺金生方抗非小细胞肺癌(NSCLC)酪氨酸磷酸酶抑制剂(TKIs)靶向药物耐药的作用及分子机制。方法选用NSCLC细胞系HCC827,按0.01、0.05、0.10、0.50和1.0μM浓度梯度诱导构建吉非替尼耐药细胞株,采用CCK8法检测耐药株是否构建成功,采用荧光定量PCR(q-PCR)检测耐药株c-MET扩增水平。20只BALB/c裸鼠按随机数字表法分成空白对照组、吉非替尼组、肺金生方组和肺金生方+吉非替尼组,每组5只。按每只4×10~7/200μL将吉非替尼耐药细胞株注射于裸鼠后肢皮下,构建皮下移植瘤模型。四组均腹腔注射生理盐水,吉非替尼组给予吉非替尼50mg/kg灌胃;肺金生方组给予肺金生方剂量53.6g/kg灌胃;肺金生方+吉非替尼组给予吉非替尼50mg/kg+肺金生方剂量53.6g/kg联合灌胃给药。给药频次每2天1次,连续给药4周。CCK8法检测耐药HCC827 GR细胞增殖率,流式细胞技术检测耐药HCC827 GR细胞凋亡率,Western blot检测耐药HCC827 GR细胞p-EGFR、p-MET、p-AKT、p-ERK1/2表达水平。结果与空白对照组比较,吉非替尼组裸鼠皮下移植瘤瘤重无差异[(1.34±0.16)g比(1.41±0.13)g,P>0.05],肺金生方组裸鼠皮下移植瘤瘤重明显减轻[(0.77±0.28)g比(1.41±0.13)g,P<0.01],吉非替尼+肺金生方组裸鼠皮下移植瘤明显小于肺金生方组和吉非替尼组[(0.34±0.16)g比(0.77±0.28)g、(1.34±0.16)g,P均<0.01];肺金生方单用或与吉非替尼联用能够明显抑制耐药HCC827 GR细胞的增殖能力[(43.45±4.47)%、(24.25±5.21)%比(100.00±2.14)%,P均<0.01];肺金生方组、吉非替尼+肺金生方组细胞凋亡率显著高于空白对照组[(7.40±0.56)%、(12.80±0.72)%比(3.05±0.54)%,P均<0.01];肺金生方组、吉非替尼+肺金生方组c-MET和p-c-MET表达水平明显降低,并明显抑制下游AKT/ERK1/2磷酸化水平。结论肺金生方或和吉非替尼联用能有效抑制吉非替尼耐药皮下移植瘤的生长,抑制耐药细胞的增殖并促进凋亡,其作用机制可能与下调c-MET蛋白表达以及下游AKT/ERK1/2磷酸化水平有关。     

清肺化瘀解毒方对肺癌靶向耐药细胞的逆转作用及机制研究

目的:观察清肺化瘀解毒方对肺癌吉非替尼获得性耐药的调节作用及其机制。方法:采用大剂量冲击和逐渐增加剂量相结合的方法诱导建立人肺腺癌A549细胞株吉非替尼获得性耐药系,清肺化瘀解毒方含药血清及吉非替尼干预后,WST-1法检测细胞增殖率,流式细胞术检测细胞凋亡率,Western-blot、Real-time PCR技术检测CMET、Integrinβ1、IGF-1R蛋白及mRNA的表达。结果:清肺化瘀解毒方、清肺化瘀解毒方联合吉非替尼对A549/R细胞的增殖有明显抑制作用,且呈一定时效及量效关系;清肺化瘀解毒方能明显促进A549/R细胞的凋亡,与吉非替尼联用后促进作用更为明显(P0.05);清肺化瘀解毒方组、清肺化瘀解毒方联合吉非替尼组CMET、IGF-1R蛋白及mRNA表达与空白血清组及吉非替尼组相比明显下调(P0.05),Integrinβ1蛋白及mRNA表达差异不明显。结论:清肺化瘀解毒方有逆转A549吉非替尼获得性耐药的作用,其机制可能与下调CMET、IGF-1R表达有关。     

非小细胞肺癌表皮生长因子受体P848L突变及其功能研究

目的:检测一组中国非小细胞肺癌(non-small-cell lung cancer,NSCLC)患者的表皮生长因子受体(epidermal growth factor receptor,EGFR)突变情况,探讨检出的1例EGFR突变类型P848L的功能特征。方法:收集55例手术或活检确诊的NSCLC标本,抽提细胞DNA,对EGFR的1~27号外显子进行筛查以检出突变情况;利用EGFR野生型全长基因载体,通过定点诱变技术构建出P848L突变型,进一步构建出P848L突变与T790M突变的双重突变体,分别转入293T细胞,观察细胞EGFR磷酸化活性及EGFR靶向抑制剂吉非替尼对其磷酸化的抑制作用。结果:共发现8例NSCLC携带EGFR突变,均为腺癌患者,占非吸烟腺癌患者的34.78%(8/23);其中1例NSCLC患者携带P848L突变体的细胞对吉非替尼的抑制反应与野生型相近,低于经典型突变体;当引入T790M形成双重突变后,对吉非替尼的反应性显著下降。结论: EGFR激活突变在非吸烟的肺腺癌患者具有较高的发生率,而在鳞状细胞癌等类型未发现;P848L突变不能增强吉非替尼对EGFR受体酪氨酸磷酸化的抑制效应,在携带P848L突变的EGFR基因上,引进耐药的T790M突变,能完全抑制其对吉非替尼的敏感性。     

RNA干扰及厄洛替尼阻断EGFR信号途径对A549细胞抗增殖的影响

目的 研究RNA干扰（RNAi）及厄洛替尼阻断表皮生长因子受体（EGFR）信号途径对肺腺癌A549细胞增殖的影响。方法 肺腺癌A549细胞分为对照组、RNAi组、厄洛替尼组。采用倒置相差显微镜观察EGFR信号阻断前后细胞形态的变化,RT-PCR检测EGFR mRNA表达,流式细胞术检测细胞凋亡,Western blot检测EGFR及Bcl-2、Bax表达。结果 RNAi阻断EGFR mRNA表达下调。与对照组相比,RNAi组及厄洛替尼组A549细胞凋亡增加, EGFR表达降低,Bcl-2水平表达降低,Bax蛋白表达增高, Bcl-2/Bax比值降低。结论 阻断EGFR信号途径可促进肺腺癌A549细胞凋亡,其机制可能与降低Bcl-2/Bax比值水平相关。     

塞来昔布联合厄罗替尼对人肺癌裸鼠移植瘤生长及血管生成的影响

目的 探讨厄洛替尼和塞来昔布联合阻断表皮生长因子受体(EGFR)和环氧合酶-2(COx-2),对人肺腺癌裸鼠移植瘤生长及血管形成的影响.方法 人肺癌细胞A549接种于裸鼠皮下,成瘤后随机分为4组:对照组、厄洛替尼组、塞来昔布组、塞来昔布和厄洛替尼联合用药组,给予灌胃处理,观察裸鼠生长状况,每周两次测量肿瘤大小,绘制肿瘤生长曲线.40 d后处死裸鼠,称取移植瘤质量.采用免疫组化检测肿瘤微血管密度(MVD),ELISA法检测裸鼠血清中血管内皮生长因子(sVEGF)的含量,Western Blot法检测Bcl-2、Bax蛋白的表达情况并计算Bcl-2/Bax的值.结果 联合用药组肿瘤明显小于塞来昔布组、厄洛替尼组和对照组(P＜0.05).联合用药组微血管数目、sVEGF的含量明显小于塞来昔布组、厄洛替尼组和对照组(P＜0.05).厄洛替尼组Bcl-2的表达明显小于对照组(P＜0.05),塞来昔布组Bcl-2的表达与对照组相比差异无统计学意义(P＞0.05),联合用药组Bcl-2的表达与对照组、塞来昔布组、厄洛替尼组相比均明显降低(P＜0.05).Bax的表达在各组之间差异无统计学意义(P＞0.05).联合用药组Bcl-2/Bax较塞来昔布组、厄洛替尼组明显降低(P＜0.05).结论 塞来昔布联合厄洛替尼相对厄洛替尼或塞来昔布明显抑制人肺癌移植瘤的生长.其中减少微血管生成、增加肿瘤细胞的凋亡可能为其抗肿瘤机制.     

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…