【作者】 王琳；

【导师】 吴逸明；

【作者基本信息】 郑州大学 ， 流行病与卫生统计学， 2008， 博士

【摘要】 肺癌的发病率和死亡率正在迅速上升,在很多发达国家,肺癌的发病率占男性常见恶性肿瘤的第一位,女性常见恶性肿瘤的第二、三位;肺癌的组织学类型也在发生显著变化,腺癌的发病率不断上升。虽然手术治疗、化学治疗、放射治疗、免疫治疗及综合治疗等都取得了长足发展,但在美国肺癌患者5年的生存率仅为15%,欧洲不过是10%,发展中国家则不到8.9%。化疗药物紫杉醇（TAX）抗肿瘤效果显著,取得了较好的临床疗效。肿瘤热疗在肿瘤综合治疗中显示了良好的效果,加热不会增加药物的细胞毒性效应,所以,热疗增加肿瘤细胞对化疗的敏感性受到越来越多的重视,因此,本研究旨在验证热疗促进TAX对肺肿瘤细胞抑制作用的发生及其机制。研究方法1.热疗与TAX联合应用对肺部细胞生物学特性的影响采用人肺腺癌A549细胞、人小细胞肺癌NCI-H446细胞和人呼吸道上皮BEAS-2B细胞,每种细胞分别采用3种方式处理,即对照组、TAX组和43℃+TAX组（见表1）;倒置显微镜观察细胞形态,照相记录,噻唑蓝比色法检测细胞增殖率;提取细胞总蛋白,按LDH Kit要求检测LDH活力,划痕实验检测细胞损伤修复能力,荧光Hoechst33342/PI双染法以及提取细胞DNA Ladder检测细胞凋亡。2.细胞内重要信号传导通路在热疗与TAX诱导肺肿瘤细胞增殖过程中的作用及其相互关系采用人肺腺癌A549细胞和人小细胞肺癌NCI-H446细胞,每种细胞分别采用6种方式处理,即对照组、TAX组、43℃+TAX组（见表1）和SP600125组（43℃+TAX组加入JNK信号转导通路抑制剂SP600125）、wortmannin组（43℃+TAX组加入Akt通路抑制剂wortmannin）、NAC组（43℃+TAX组加入ROS抑制剂NAC）;分别检测各组ROS、p-JNK、p-Akt以及Caspase-3等的表达变化,并用流式细胞术检测细胞凋亡率和细胞周期的变化,同时做体内实验的验证。3.HSP70在热疗与TAX抑制肺肿瘤细胞作用中的地位采用人肺腺癌A549细胞、人小细胞肺癌NCI-H446细胞和人呼吸道上皮BEAS-2B细胞。每种细胞分别采用4种方式处理,即对照组、TAX组、43℃+TAX组和43℃组（见表1）;检测各组HSP70的表达变化,同时做体内实验的验证。4.GTSP1在热疗与TAX抑制肺肿瘤细胞过程中的作用构建重组质粒pcDNA3.0-GSTP1,转染A549细胞,热疗联合TAX处理转染和未转染细胞,倒置显微镜观察细胞形态,照相记录,噻唑蓝比色法检测细胞增殖率,流式细胞术检测各组细胞周期的变化。5.统计学分析采用SPSS 12.0进行统计分析,统计学处理采用t检验、单因素方差分析,多重比较采用LSD法,构成比的比较采用卡方检验,以α=0.05为检验水准。结果1.热疗与TAX联合应用对肺部细胞生物学特性的影响1.1热疗可以减少TAX对正常细胞的损伤作用（P＜0.05）,明显增强TAX对肺肿瘤细胞的抑制作用（P＜0.05）;1.2热疗造成肺肿瘤细胞膜损伤,使得大量LDH外漏,所以检测细胞内LDH发现,43℃+TAX组的LDH较对照组和TAX组明显减少（P＜0.05）;1.3划痕后24h,对照组细胞开始向划痕区生长,其余各组划痕区中央均未见细胞生长,且有部分细胞死亡,TAX组细胞存活数目多于43℃+TAX组;划痕后48h,对照组细胞填满划痕区,TAX组细胞部分存活,存在较宽的划痕区,43℃+TAX组细胞大多死亡;1.4用Hoechst 33342结合PI染料对细胞进行双染色,在荧光显微镜下观察,呈现低绿色/低红色的是正常细胞,高绿色/低红色的是凋亡细胞,坏死细胞则为低绿色/高红色,对照组细胞呈低绿色和低红色,证明细胞并未发生坏死和凋亡,TAX组细胞则绿色数量增加,红色没有变化,证明已有凋亡发生,43℃+TAX组细胞呈现明显的亮绿色,证明细胞发生了明显的凋亡,而并没有发生坏死;1.5 43℃+TAX组细胞其DNA条带出现典型的DNA片段梯带现象（DNA ladder）,而对照组细胞未出现DNA梯状条带,TAX组细胞的DNA梯状条带不明显,说明热疗可以诱导肺肿瘤细胞发生凋亡。2.细胞内重要信号传导通路在热疗与TAX抑制肺肿瘤细胞过程中的作用及其相互关系2.1 ROS产生水平的变化荧光法检测各组细胞内ROS,证明热疗可以明显提高细胞内ROS的表达（P＜0.05）,ROS的增多可以改变细胞内氧化还原水平,损伤细胞膜,作为信号在细胞内传递,引起细胞内信号转导通路的一系列变化。2.2 MKK4表达和p-JNK水平的变化43℃+TAX组的JNK信号转导通路被激活,产生的MKK4和p-JNK高于对照组和TAX组（P＜0.05）,SP600125组的p-JNK水平被抑制（P＜0.05）,wortmannin组二者表达均未见明显变化（P＞0.05）,NAC组二者的水平均低于43℃+TAX组（P＜0.05）,说明热疗过程中产生的ROS可以激活JNK信号转导通路。2.3 p-Akt水平的变化43℃+TAX组的Akt信号转导通路被抑制,p-Akt的水平低于对照组和TAX组（P＜0.05）,wortmannin组的p-Akt水平完全被抑制（P＜0.05）,SP600125组的p-Akt水平低于43℃+TAX组（P＜0.05）,NAC组的p-Akt水平明显高于43℃+TAX组（P＜0.05）,说明热疗过程中产生的ROS可以抑制Akt通路的活化,同时,JNK信号转导通路的激活影响Akt通路的活化。2.4 Caspase-3表达水平的变化43℃+TAX组的Caspase-3表达水平高于对照组和TAX组（P＜0.05）,SP600125组的Caspase-3表达水平低于43℃+TAX组（P＜0.05）,wortmannin组的Caspase-3表达水平高于43℃+TAX组（P＜0.05）,NAC组的Caspase-3表达水平完全被抑制（P＜0.05）,说明热疗诱导的细胞凋亡是ROS通过JNK和Akt 2条通路传给caspase途径而引发的,且ROS、JNK信号转导通路对caspase有活化作用,Akt通路对其有抑制作用。2.5细胞凋亡率和细胞周期的变化43℃+TAX组G₀/G₁期和G₂/M期细胞增多,S期细胞减少（P＜0.05）,wortmannin组S期细胞更少（P＜0.05）,SP600125组和NAC组S期细胞数目增加（P＜0.05）,G₂/M期细胞减少（P＜0.05）;43℃+TAX组细胞凋亡率增加（P＜0.05）,wortmannin组细胞凋亡率进一步增加（P＜0.05）,而SP600125组和NAC组细胞凋亡率减少（P＜0.05）。3.HSP70在热疗与TAX抑制肺肿瘤细胞作用中的地位对照组和TAX组的HSP70表达水平几乎一致（P＞0.05）,43℃组和43℃+TAX组的HSP70表达水平均较前两组增加（P＜0.05）,且43℃+TAX组的HSP70表达水平低于43℃组（P＜0.05）,说明热疗诱导产生的HSP70,对不同性质的细胞产生不同的效应,热疗联合TAX可能激活和/或抑制其他通路的激活,减弱了HSP70对肺肿瘤细胞的保护作用,但这个现象在正常细胞中没有观察到。4.GTSP1在热疗与TAX抑制肺肿瘤细胞过程中的作用成功构建pcDNA3.0-GSTP1真核表达载体后,转染A549细胞,获得转染与未转染的两种A549细胞,MTT结果示,GSTP1本身对细胞生长没有影响,转染与未转染细胞增殖率一致（P＞0.05）,GSTP1能促进化疗药物的代谢,不同浓度TAX作用后,未转染组细胞增殖率低于转染组细胞（P＜0.05）,43℃热疗联合不同浓度TAX作用后,未转染组细胞增殖率高于转染组细胞（P＜0.05）;43℃热疗联合TAX作用于转染与未转染细胞,流式细胞仪检测发现,G₀/G₁期和G₂/M期细胞增多,S期细胞减少（P＜0.05）。结论及创新点1.在热疗的作用机制研究中,首次使用人呼吸道上皮BEAS-2B细胞作为研究对象,证明热疗可以减少化疗药物对正常细胞的损伤作用;热化疗联合应用可以明显增加化疗药物对肺肿瘤细胞生长的抑制作用,损伤肺肿瘤细胞膜,抑制肺肿瘤细胞的迁移,诱导肺肿瘤细胞凋亡的发生;2.通过使用通路抑制剂证明,热疗可以诱导ROS的产生,继而激活JNK信号转导通路和抑制Akt通路的活化,通过caspase途径诱导细胞凋亡的发生,热疗主要作用于肺肿瘤细胞的S期,并可使肺肿瘤细胞阻滞于G₂/M期,从而促进TAX对肺肿瘤细胞的抑制作用,同时发现,在热疗中,JNK信号转导通路的激活可以调节Akt通路的活化状态,以上结论在体内实验中得到证实;3.热疗诱导产生的HSP70,对不同性质的细胞产生不同的效应,热化联合的协同作用可能激活和/或抑制其他通路的激活,减弱了HSP70对肺肿瘤细胞的保护作用,因此比热疗能更好地抑制肺肿瘤细胞的生长,但在正常细胞中尚未观察到这个现象;4.首次构建了pcDNA3.0-GSTP1真核表达载体,并在A549细胞中进行表达;GSTP1能促进化疗药物的代谢,导致肺肿瘤细胞对化疗药物的抵抗性增高,热疗可以提高肺肿瘤细胞对化疗药物的敏感性。更多还原

【Abstract】 The incidence rate of lung cancer was increasing rapidly and in many developed countries,it had already been in the first place in male malignant tumor and the second or third place in female. The histological types of lung cancer changed significantly and the incidence rate of adenocarcinoma went on increasing. Though treatments of lung cancer developed very well, the survival rate of lung cancer was only 15% in USA, 10% in European countries and no more than 8.9% in developing countries. TAX had notable anti-tumor effect. Thermo-therapyplayed an important role in comprehensive treatments of cancer and could not increase cell tocoxic effect, so sensitivity of tumor cells to chemotherapy enhanced by thermotherapy gained more and more attention. This research aimed at enhancement of therapeutic effect of TAX on lung tumor cells and its underlying mechanisms.Methods1. Influence of Thermotherapy with TAX on Biological Characteristcs of Lung CellsA549, H446 and BEAS-2B cells were used and treated by three methods, which were control group, TAX group and 43℃+TAX group （Table 1）, then they were observed and recorded by microscope. MTT assay was adopted to check proliferation rates of cells. LDH checking and wounding-healing assay were carried out. Apoptosis was detected by Hoechst33342/PI and DNA ladder. 2. Function of Important Signal Transducting Pathways in Therapeutic Effect of Thermotherapy with TAX on Lung Tumor Cells and their RelationshipsA549 and H446 cells were used and treated by six methods, which were control group, TAX group, 43℃+TAX group （Table 1）, SP600125 group （43℃+TAX group with SP600-125）, wortmannin group （43℃+TAX group with wortmannin） and NAC group （43℃+TAX group with NAC）, and then ROS, Phosphorylation of JNK and Akt, Caspase-3 and MKK4 of each group were checked. FCM was used to check changes of apoptosis and cell cycle. Tests in vivo were carried out.3. Function of HSP70 in Therapeutic Effect of Thermotherapy with TAX on Lung Tumor CellsA549, H446 and BEAS-2B cells were used and treated by four methods, which were control group, TAX group, 43°C+TAX group and 43℃group （Table 1）. Western Blotting was used to check changes of HSP70 and tests in vivo were carried out at the same time.4. Function of GTSP1 in Therapeutic Effect of Thermotherapy with TAX on Lung Tumor CellsThe Recombinant Plasmid pcDNA3.0-GSTP1 was constructed and transfected into A549 cells. Transfected cells and non-transfected cells were treated by thermotherapy with TAX, and then observed and recorded by microscope. MTT assay was adopted to check proliferation rates of cells. FCM was used to check cell cycles.5. Statistics AnalysisSPSS 12.0 was adopted. T-Test, ANOVA, Chi-Square Test and LSD were used. P value less than 0.05 was considered statistically significant.Results1. Influence of Thermotherapy with TAX on Biological Characteristcs of Lung Cells1.1 Thermotherapy with TAX can reduce injury of TAX on BEAS-2B cells （P＜0.05） and enhance the therapeutic effect of TAX on A549 cells and H446 cells （P＜0.05）;1.2 Thermotherapy can injure cell membrane, which made LDH leak, so LDH in 43℃+TAX group was lower than that in control group and TAX group （P＜0.05）;1.3 24h after nicking, cells in control group began to grow to the nicking area while other groups’cells began to die. 48h after nicking, cells in control group filled in the nicking area, but the nicking areas in other groups’were still very large and cells had almostly died;1.4 Cells in control group showed dark green and dark red, cells in TAX group had more green, which told us that apoptosis happened, but in 43℃+TAX group, cells showed bright green to testify obvious apoptosis;1.5 DNA electrophoresis showed that DNA in 43℃+TAX group had classical DNA ladder and did not happen in control or in TAX group, which told us thermotherapy can induce cell apoptpsis.2. Function of Important Signal Transducting Pathways in Therapeutic Effect ofThermotherapy with TAX on Lung Tumor Cells and their Relationships2.1 Changes of ROSThe expression of ROS in the cells of 43℃+TAX group was higher than the others’ （P＜0.05）, which can change cell inner environment, injure cell brane and transduct as signal in cells to change signal trnasducting pathways in cells.2.2 Changes of MKK4 and p-JNKJNK signal transducting pathway was activated in 43℃+TAX group, so the expression of MKK4 and phosphorylation of JNK were higher than those in control group and TAX group （P＜0.05）; after SP600125 was added in, phosphorylation of JNK was inhibited （P＜0.05）; they had no change with wortmannin （P＞0.05）, after NAC was added in, they was lower than those in 43℃+TAX group （P＜0.05）, which told us that ROS induced in thermotherapy can activate JNK signal transducting pathway.2.3 Changes of p-AktAkt pathway was inhibited in 43℃+TAX group, so phosphorylation of Akt was lower than that in control group and TAX group （P＜0.05）; after wortmannin was added in, phosphorylation of Akt was inhibited completely （P＜0.05）, with SP600125, phosphorylation of Akt was lower than that in 43℃+TAX group （P＜0.05）, after NAC was added in, Phosphorylation of Akt was increased （P＜0.05）, which showed that ROS induced in thermotherapy can inhibit activation of Akt pathway and activation of JNK signal transducting pathway would regulate activation of Akt pathway.2.4 Changes of Caspase-3 The expression of Caspase-3 in 43℃+TAX group was higher than that in control group and TAX group （P＜0.05）; after SP600125 was added in, its expression was lower than that in 43℃+ TAX group （P＜0.05）, after wortmannin was added in, its expression was higher than that in 43℃+ TAX group （P＜0.05）, after NAC was added in, its expression was inhibited completely （P＜0.05）. So apoptosis induced by thermothrapy was triggered through JNK signal transducting pathway and Akt pathway to caspase channel and ROS, JNK signal transducting pathway can activate casapase channel, Akt pathway can inhibit its activation.2.5 Changes of Apoptosis and Cell CycleThe number of cells in G₀/G₁ phase and G₂/M phase increased and those in S phase decreased in 43℃+TAX group （P＜0.05）. The number of cells in S phase became less after wortmannin was added in （P＜0.05） and more after SP600125 and NAC was added in （P＜0.05）, while those of G₂/M phase in SP600125 group and NAC group became less （P＜0.05）. The rate of apoptosis in 43℃+ TAX group increased （P＜0.05）. The rate of apoptosis went on increasing in wortmannin group and decreased in SP600125 group and NAC group （P＜0.05）.3. Function of HSP70 in Therapeutic Effect of Thermotherapy with TAX on Lung Tumor CellsThe expression of HSP70 in control group and TAX group was almost identical （P＞0.05） and that in 43℃+TAX group and 43℃group was higher （P＜0.05）. Its expression in 43℃+TAX group was lower than that in 43℃group （P＜0.05）, which showed that HSP70 induced by thermotherapy can protect cells can be inhibited in lung tumor cells by activating or inhibiting some signal transducting pathways, but this phenomenon was not observed in normal cells.4. Function of GTSP1 in Therapeutic Effect of Thermotherapy with TAX on Lung Tumor CellsThe recombinant plasmid was successfully constructed and tramsfected into A549 cells. So transfected and non-transfected cells were obtained. The proliferation rates of transfected and non-transfected cells were consistent （P＞0.05） because GSTP1 had no effect on cell proliferation. Putting TAX in different concentrations on transfected and non-transfected cells, proliferation rates of non-transfected cells were lower than those of transfected cells （P＜0.05）. With thermotherapy, proliferation rates of non-transfected cells were higher than those of transfected cells （P＜0.05）. Under thermotherapy with TAX, FCM showed that the number of cells in G₀/G and G₂/M phase of transfected cells became more and those in S phase were lessConclusion and Innovetions1. BEAS-2B cells were used in the research of mechanisms of thermotherapy at the first time and it showed that thermotherapy can decrease injury of chemotherapy on normal cells. Thermotherapy can enhance therapeutic effect of themotherapy on lung tumor cells, injure cell membrane, change cells’ ability of migration and induce apoptosis;2. Thermotherapy can increase expression of ROS, then activated JNK signal transducting pathway and inhibited the activation of Akt pathway, then induced apoptosis by caspase channel; thermotherapy mainly effected S phase and blocked lung tumor cells in G₂/M phase; activation of JNK signal transducting pathway in thermotherapy can influence activation of Akt pathway, which were all authenticated in vivo;3. HSP70 induced by thermotherapy can protect cells, which can be inhibited in lung tumor cells by activating or inhibiting some signal transducting pathways, but this phenomenon was not observed in normal cells;4. The recombinant plasmid, pcDNA3.0-GSTP1 and a new cell line had been successfully constructed at the first time; GSTP1 can promote metabolism of chemotherapy drugs, which increased drug-resistence of lung tumor cells, but thermotherapy can increase the sensitivity of lung tumor cells to chemotherapy drugs.更多还原