【作者】 黄利敏；

【导师】 齐元富；

【作者基本信息】 山东中医药大学 ， 中西医结合临床， 2012， 博士

【摘要】 目的：通过体外实验观察六神丸含药血清对人肺癌A549细胞增殖、凋亡、细胞形态、生长状态、VEGF和VEGFmRNA、K-RAS、ERK1、ERK2、p-ERK蛋白表达的影响，K-RAS、ERK1、ERK2、p-ERK蛋白表达与VEGF表达的相关性，临床观察六神丸联合化疗治疗未经任何治疗ⅢB-IV期或不能手术ⅢA期肺癌患者的临床疗效和血清CEA、CYFRA21-1、VEGF的变化，探讨六神丸抗血管生成是否通过激活Ras→Raf→MEK→MAPK信号转导途径及血清VEGF是否可作为临床观察疗效指标之一。方法：1.实验研究采用体外培养人肺癌A549细胞，以不同浓度六神丸含药血清与A549共同孵育并分组观察，MTT法检测六神丸含药血清对人肺癌细胞系A549的增殖、光学显微镜下观察细胞形态、倒置相差显微镜下观察生长状态、流式细胞仪检测六神丸含药血清对细胞凋亡的影响、免疫细胞化学法测定VEGF、K-RAS、ERK1、ERK2、p-ERK蛋白表达情况，逆转录-聚合酶链反应法（RT-PCR）检测VEGFmRNA的影响。2.临床研究观察六神丸联合化疗及化疗组对非小细胞肺癌患者的临床疗效及血清CEA、CYFRA21-1、VEGF表达水平的影响。全部病例均来自山东中医药大学附属医院，2009年12月至2011年12月肿瘤内科住院病人。制定病例纳入标准，纳入研究病人60例，随机分为观察组30例，对照组30例，对照组采用TP化疗方案，观察组在化疗的同时及化疗期间，同时给予六神丸。收集所有患者治疗前及治疗两周期后外周血5ml，酶联免疫吸附测定法(ELISA)检测VEGF水平，放射免疫分析法检测CYFRA21-1、CEA表达水平，观察治疗前后的临床疗效。结果：1.实验研究（1）六神丸含药血清对体外培养A549细胞具有明显细胞毒作用，且随着含药血清浓度的增加OD值逐渐降低，有显著性差异。（2）各含药血清组均可破坏人肺癌A549细胞的正常形态，阻碍其增殖生长；六神丸含药血清对人肺癌A549细胞的增殖抑制作用与含药血清的浓度呈正相关；六神丸与DDP联合用药有协同作用。（3）各含药鼠血清组、DDP单药组以及DDP+10%含药鼠血清组的早期凋亡率明显高于对照组；随着含药浓度的不断升高，早期凋亡率也明显增加；联合组的早期凋亡率明显高于各含药鼠血清组和DDP单药组。（4）各含药鼠血清组、DDP单药组以及DDP+10%含药鼠血清组的VEGF、ERK1、ERK2、p-ERK、K-RAS表达量及VEGFmRNA相对表达量均明显低于10%胎牛血清组，有显著性差异；与药物浓度呈负比。（5）统计结果显示六神丸含药血清对A549细胞降低VEGF的表达与其对ERK-1、ERK-2、p-ERK、K-ras表达的影响呈显著正相关(r=0.887，0.835，0.952，0.866)。2.临床研究（1）两组化疗完成情况：观察组2周期化疗完成率93.3%（28/30），高于对照组86.7%（26/30）；（2）近期客观疗效：经χ2检验，两组有效率和病灶稳定率比较均无显著性差异；（3）两组治疗前后血清VEGF、CEA、CYFRA21-1水平的比较：两组治疗后VEGF、CEA、CYFRA21-1表达水平均较治疗前明显下降；与对照组相比，观察组VEGF、CEA表达水平更低，幅度下降更大，具有显著统计学差异。与对照组相比，观察组CYFRA21-1表达水平更低，但两组比较无统计学差异。（4）两组患者血清CEA、CYFRA21-1水平与VEGF水平行相关性分析：结果呈明显正相关(r=0.399、0.275，P<0.01)。结论：六神丸通过抑制Ras→Raf→MEK→MAPK信号转导途径降低肺癌A549细胞VEGF蛋白的表达从而发挥抗血管生成的作用，具有抑制肺癌A549细胞生长、促进凋亡的作用，可降低肺癌患者血清VEGF的水平，同时六神丸联合化疗效果更好，二者具有协同作用。临床联合检测肺癌患者血清VEGF、CEA、CYFRA21-1的变化具有观察疗效及监测病情的作用。更多还原

【Abstract】 Objective：to observe1.the effect of Six Spirits Pill medicated serum roling in the cells for theproliferation，apoptosis，cells form and growth condition of the lung cancer cell A549，and for the protein expression changes of VEGF，K-RAS，ERK1，ERK2，p-ERK andVEGFmRNA，the correlation between K-RAS， ERK1，ERK2，p-ERK protein expressionand of VEGF expression.2.the clinical effects and the changes of serum CEA，CYFRA21-1and VEGF for the lung cancer patients without any treatment IIIB-IV stage or IIIA stagepatients who can’t be operated on by Six Spirits pill combination chemotherapy treatment；The paper discusses whether Six Spirits Pill antiangiogenic is achieved by activating theRAS→Raf→MEK→MAPK signal transduction pathways and Whether serum VEGF canbe used as one of clinical observation curative effect.Methods：1. Experimental studyCulture the lung cancer A549cells in vitro utilizing the method of serumpharmacolog,incubate different concentration of serum which included Six Spirits Pilltogether with A549cells and observe them in group.Detect the effect of the serum with pillto proliferation by the use of the MTT method，observe cells’form with optical microscopeand growth state with inverted phase contrast microscope,detect cell apoptosis with theFlow cytometric，determine the protein expression changes of the VEGF，K-RAS，ERK1，ERK2，p-ERK in immunocytochemical method，test VEGFmRNA by means of retrovirus--polymerase chain reaction method. 2.Clinical studyObserve the clinical effects for the non-small cell lung cancer patient and the effectfor the expression changes of serum CEA，CYFRA21-1，VEGF from the six Spirits pillcombination chemotherapy treatment and chemotherapy treatment group.All case were thein-patients who were from shandong university of traditional Chinese medicine hospital，from May2010to December2011tumor. According to the criteria, there are60cases inall，some were divided into observation group of30cases and the other control group of30cases randomly.The control group uses the TP chemotherapy，the observation group wereadded Six Spirits Pill during chemotherapy.Before the treatment and after the two cycletreatment, Collect5ml peripheral blood of all patients，observe the VEGF level by the useof enzyme-linked immunosorbent assay (ELISA) test method，detect CYFRA21-1，CEAexpression level by radiation immune analysis method，and observe the clinical curativeeffect before and after treatment.Results：1.Experimental study(1)Utilizing medicated serum of Six Spirits Pill to A549cells has obvious cytotoxiceffects，and with the increase of medicated serum concentrations the OD value graduallyreduced，there are obviously differences.(2)The medicated serum group all can destroy thenormal form of A549cells，hinder the growth of proliferation，and have positivecorrelation with serum concentration. Six Spirits Pill and DDP have the synergisticeffect.(3)Early apoptosis rate of medicated serum groups was higher than the controlgroup；As the drug concentration rises，early apoptosis rate obviously increased；The jointgroup’s was higher than medicated serum group’s and DDP single drug group’s.(4)Theexpression amount of VEGF，ERK1，ERK2，p-ERK，K-RAS and the relative amountVEGFmRNA of medicated serum group were significantly lower than the control group，there are significantly differences；Drug concentration was negatively.(5)The statisticalresults show that the Six Spirits Pill medicated serum for A549cells reducing VEGFexpression and it’s roling in ERK1，ERK-2，p-ERK，K-ras expression has significantcorrelation (r=0.887，0.835，0.952，0.866).2.Clinical study(1) The chemotherapy accomplishment circumstances of the two groups：the2cycle chemotherapy completion rate of the observation group is93.3%(28/30)，higher than86.7%of the control group(26/30)；(2)the recent clinical curative effect：through thechi-square test，there are no significant difference between the effective rate and stabilityrate of two groups.(3)the comparison of serum VEGF，CEA，CYFRA21-1level of twogroups before and after treatment：after treating, the expression levels of VEGF，CEA，CYFRA21-1both descend obviously,compared with those before treatment. Comparedwith the control group，the serum VEGF，CEA expression levels of the observation groupare lower，their ranges decrease more，and they have statistically significant differences.Atthe same time，the CYFRA21-1expression level of observation group is more low too，butthe comparison of two groups is no statistical difference.(4)The correlation analysisbetween serum CEA，CYFRA21-1level and VEGF of two groups：The result is obviouslypositive correlation(r=0.399，0.275，P<0.01).ConclusionSix Spirits Pill plays the role of anti-angiogenesis through inhibition ofRas→Raf→MEK→MAPK signal transduction pathway to reduce lung cancer A549cellexpression of VEGF protein，to inhibit lung cancer A549cell growth and promoteapoptosis，to reduce the serum VEGF levels of lung cancer patients，at the same time,theeffect of Six Spirits Pill combination chemotherapy is better，the two have synergisticeffect.Clinical joint detection of lung cancer patients’s serum VEGF，CEA，CYFRA21-1changes plays the role in the observing efficacy and monitoring the patient condition.更多还原