【作者】 杨艳明；

【导师】 曲雅勤；

【作者基本信息】 吉林大学 ， 放射医学， 2010， 博士

【摘要】 乏氧／辐射双敏感启动子介导shTRAIL联合放射治疗肺癌的体外抑瘤效应研究肿瘤的治疗通常采用手术、放疗和化疗,但是都存在局限性。肿瘤基因治疗的提出为肿瘤治疗提供了新的思路,研究者试图将基因治疗与手术、放疗和化疗中的一种或多种联合应用,提高治疗的效果。基因治疗是针对癌基因的激活和抑癌基因的失活,采用已知的基因片段导入、转移和重组等技术来消除激活的癌基因和增殖肿瘤中抑癌基因的功能,以此改变细胞的生物学特性,提高细胞对药物或者其他因素的敏感性,增强宿主抗肿瘤特性,控制肿瘤的生长。由于肿瘤部位为乏氧区域,肿瘤细胞辐射抗性较强,放射治疗方案受限。近年来,研究者考虑将基因治疗和放疗联合应用,进行肿瘤基因-放射治疗。主要是通过辐射敏感启动子介导下游基因在辐射条件下高效表达,既可以达到辐射杀伤,又可以实现基因治疗,一般辐射敏感启动子应用较多的是早期生长反应因子1(early growth response genel, Egrl)。为了提高乏氧条件下Egrl的辐射诱导增强,可以将缺氧反应元件(hypoxia response elements, HREs)与其构建嵌合性启动子,增加Egr-1乏氧条件下的辐射诱导增强特性。本实验利用Egrl启动子的辐射诱导增强特性,HREs启动子在乏氧条件下增强下游基因表达特性和TRAIL基因强烈诱导肿瘤细胞凋亡,而正常组织细胞却对其不敏感特性,将三者有机结合起来,构建双启动子介导的表达载体,并联合电离辐射探讨对肺腺癌A549细胞的抑制作用。本实验结果为肿瘤治疗提供新的思路。1重组质粒的构建及鉴定本实验共构建了5个重组表达质粒,分别为pcDNA3.1-Egrl-EGFP、pcDNA3.1-HRE/Egr-EGFP、pcDNA3.1-shTRAIL、pcDNA3.1-Egrl-shTRAIL和pcDNA3.1-HRE/Egrl- shTRAIL。将李艳博赠予的pcDNA3.1+质粒、pshuttle-EGFP质粒及pMD19T-Egrl质粒分别酶切得到pcDNA3.1载体片段、EGFP和Egr1片段,顺序连接构建pcDNA3.1-Egrl-EGFP (pcEE),PCR、酶切及测序鉴定；由上海生物工程公司合成HRE序列,将其连接到pcDNA3.1-Egr1-EGFP质粒Egr1的上游,构建pcDNA3.1-HRE/Egr-EGFP (pcHEE),经过PCR、酶切和测序鉴定；将pshuttle- shTRAIL酶切得到shTRAIL,连接到pcDNA3.1载体上,构建pcDNA3.1-shTRAIL (pcshT), PCR、酶切及测序鉴定；将pcDNA3.1-CMV-shTRAIL酶切去CMV后,用Egrl代替,构建pcDNA3.1-Egr1-shTRAIL (pcEshT),经过PCR、酶切和测序鉴定；pcDNA3.1-Egr1-shTRAIL再次酶切,将HRE连接于Egr1上游,构建pcDNA3.1-HRE/Egr1-shTRAIL(pcHEshT),经PCR、酶切和测序鉴定。经PCR、酶切和测序将定,本实验构建的5个重组质粒与预测的完全一致,说明构建正确。2 Egr-1辐射诱导特性将pEE质粒通过脂质体转染到肺腺癌A549细胞后,在常氧和乏氧条件下,进行0、2、4、6、8和10GyX射线照射,0、3、8、12、24和36 h后流式细胞术检测绿色荧光蛋白的表达,结果显示常氧条件时,转染pcEE质粒的A549细胞中绿色荧光蛋白表达在不同剂量X射线照射,随着时间的延长和剂量的增加表达量逐渐增加,照射后除了8Gy照射后12h达到峰值,24h后开始下降,其他剂量组都是8h时达到峰值,12h后开始降低。乏氧条件时,不同乏氧浓度条件下相同剂量照射对绿色荧光蛋白表达抑制比较明显,当氧浓度低于2.5%时,随着氧浓度的降低抑制的越多,而当氧浓度高于2.5%时绿色荧光蛋白被抑制的与氧浓度差别不是很大,与常氧条件下不同剂量照射后8h表达比较差异具有显著性(P<0.05,P<0.01)。这说明Egr1具有辐射诱导增强特性,而乏氧能够抑制Egr1启动子的特性。3 HRE对Egr-1辐射诱导特性的影响将pEE质粒通过脂质体转染到肺腺癌A549细胞后,在常氧和乏氧条件下,进行0、2、4、6、8和10 Gy X射线照射,0、3、8、12、24和36 h后流式细胞术检测绿色荧光蛋白的表达,结果显示常氧条件时,各剂量照射后不同时间绿色荧光蛋白表达在pEE质粒转染后的表达情况基本一致。而乏氧条件时,0.1%-2.5%的氧浓度条件下,EGFP表达明显增加,且随着氧浓度和剂量的增加而逐渐增加,在1%氧浓度时表达量最大,与常氧条件下不同剂量照射后8h表达比较差异具有显著性(P<0.05,P<0.01)；在5%-10%氧浓度条件下，EGFP表达与常氧条件下基本一致。这说明HRE具有乏氧条件下增强Egr1辐射诱导特性,而在常氧条件下则无此功能。4重组质粒在A549细胞中的辐射诱导表达规律转染pcshT、pcEshT和pcHEshT3种质粒进入A549细胞后,在常氧和／或1%氧浓度乏氧后给予6GyX射线照射后,检测TRAIL mRNA和shTRAIL蛋白的表达,结果显示转染pcshT和pcEshT质粒的A5459细胞在照射与乏氧+照射条件下都能增强shTRAIL mRNA表达,与对照组比较差异具有显著性,而且二者联合表达增强更明显(P<0.05)；而转染pcHEshT质粒的A549细胞在乏氧、照射及乏氧+照射条件下都能增强shTRAIL mRNA的表达,与对照组比较差异具有显著性(P<0.05),而且乏氧比单纯照射表达增多,二者联合表达增强更明显(P<0.05)。而shTRAIL蛋白表达的规律性与mRNA表达的规律性基本一致。这说明辐射能够诱导转染3种质粒的A549细胞shTRAIL表达增强,而乏氧条件下辐射增强效果更明显。5重组质粒对A549细胞的抑制作用5.1重组质粒抑制A549细胞增殖转染pcshT、pcEshT和pcHEshT3种质粒进入A549细胞后,在常氧和／或1%氧浓度乏氧后给予6GyX射线照射后,利用台盼兰染色分别于0,2,4,6和8d利用台盼兰染色计数活细胞数,绘制细胞生长曲线,结果显示1%氧浓度乏氧对3种质粒转染的A549细胞生长有抑制作用,其中转染pcHEshT质粒的细胞抑制最明显；6 Gy X射线照射对3种质粒转染的A549细胞生长有抑制作用,其中转染pcEshT和pcHEshT质粒的细胞抑制最明显；而1%氧浓度和6GyX射线照射联合作用对3种质粒转染的细胞抑制作用都很强,尤其以pcHEshT质粒转染细胞抑制作用最强。同时利用MTT法检测3种质粒转染细胞后,常氧和／或1%氧浓度乏氧后给予6GyX射线照射后0～72h细胞吸光度值的变化,结果显示1%氧浓度乏氧后3种质粒转染的A549细胞吸光度值降低,其中转染pcHEshT的细胞降低最大；而6 Gy照射后3种质粒转染的A549细胞吸光度值降低,其中转染pcEshT的细胞降低最大；1%氧浓度和6 Gy X射线照射联合作用后转染pcHEshT质粒的细胞在48 h时降低最大。以上结果提示1%氧浓度和6GyX射线照射对pcHEshT质粒转染的细胞抑制能力最强。5.2重组质粒对细胞周期和凋亡的影响转染pcshT、pcEshT和pcHEshT3种质粒进入A549细胞后,在常氧和／或1%氧浓度乏氧后给予6 Gy X射线照射后,PI单染后流式细胞术检测细胞周期和凋亡的变化,结果显示1%氧浓度乏氧和6GyX射线照射使得3种质粒转染的细胞Go／G1期百分比增加,G2/M期百分比降低,而S期百分比基本不变,、细胞的凋亡百分比增加。同时,以TUNEL方法检测细胞凋亡的变化,乏氧和辐射对3种质粒转染的细胞凋亡都具有促进作用,乏氧对pcshT和pcEshT转染的细胞凋亡百分比促进作用不是太明显,而对pcHEshT转染细胞较明显；辐射对3种质粒转染的细胞凋亡百分比促进作用都很明显,以pcEshT和pcHEshT转染的细胞促进作用最明显；而乏氧和辐射对pcshT转染细胞凋亡百分比促进作用一般,对pcEshT转染细胞凋亡百分比促进作用较强,而对pcHEshT转染细胞凋亡百分比促进作用最强。这和流式细胞术结果基本一致,说明3种质粒转染后,乏氧和辐射能有诱导细胞Go／G1期阻滞,凋亡增加。5.3重组质粒对细胞辐射敏感性的影响正常细胞和转染pcshT、pcEshT和pcHEshT质粒的A549细胞在常氧和1%氧浓度乏氧6h后,分别进行2～10Gy X射线的照射,培养14d后计数克隆数,计算存活分数,按照多击单靶模型进行曲线拟合计算出Do值,分析Normal、转染pcshT质粒、转染pcEshT质粒和转染pcHEshT质粒的4种细胞放射敏感性的差异。结果显示常氧条件下,4种细胞的D0值分别为3.26、2.12、1.91和1.89,说明转染了pcEshT和pcHEshT质粒的细胞放射敏感性最大；而在乏氧条件下,4种细胞的D0值分别为5.81、6.12、4.24和1.13,说明乏氧能够降低Normal、转染pcshT质粒、转染pcEshT质粒细胞的敏感性,而能增强转染了pcHEshT质粒的细胞放射敏感性。5.4重组质粒对DR4、DR5和caspase-3表达的影响pcshT、pcEshT和pcHEshT质粒转染A549细胞后进行1%氧浓度乏氧和6GyX射线照射,利用RT-PCR的方法检测DR4和DR5 mRNA的表达,结果显示单纯乏氧对pcshT和pcEshT质粒转染的细胞中DR4 mRNA表达影响不大,而能够诱导pcHEshT质粒转染的细胞DR4 mRNA表达增加,但是能够诱导3种质粒转染细胞DR5 mRNA表达增加,其中诱导pcHEshT质粒转染的细胞DR5mRNA表达增加最明显；单纯辐射能够诱导3种质粒转染细胞DR4 mRNA和DR5 mRNA表达增强；而乏氧和辐射联合作用都能诱导DR4 mRNA和DR5mRNA表达增强,其中诱导pcHEshT质粒转染的细胞DR5 mRNA表达增加最明显。另外,利用Western blot检测DR4、DR5和caspase-3蛋白的表达,结果显示DR4和DR5蛋白表达的情况与mRNA表达的情况基本相似,说明转染3种质粒的细胞在辐射或／和乏氧的条件下能够诱导DR4和DR5表达增加。3种质粒转染细胞后,乏氧或／和辐射对caspase-3蛋白的表达具有诱导作用,以转染了pcHEshT质粒的细胞在乏氧和辐射条件下增加最明显,caspase-3蛋白表达规律与细胞凋亡规律基本一致。更多还原

【Abstract】 Research on anti-tumor effect of Hypoxia/radiation dual-sensitive promoter-mediated shTRAIL combined with radiation on lung cancer cells in vitroCancer therapy usually applys operation, radiotherapy and chemotherapy, but they have limitation. Gene therapy provides a new way for cancer therapy, some researchers attempt to combine gene therapy with operation, radiotherapy and chemotherapy in one or more of them which can improve the therapy effctiveness. Gene therapy aims at the activation of oncogenes and inactivation of tumor suppressor genes, using known gene fragments introduction, transfer and recombination techniques to eliminate the proliferation of activated oncogenes and tumor suppressor gene function, thereby changes the cell biological character to enhance cell sensitivity to drugs and other factors, and enhances the host anti-tumor properties, holds tumor growth.. Because of the lack of oxygen in the tumor area, tumor cells have strong radiaton resistance, radiotherapy regimen is limited. In recent years, gene therapy was combined with radiotherapy to apply cancer gene-radiotherapy. It mainly utilize radiatoin sensitive promoter to mediate downstream genes high expression under the radiation conditions, both achiving radiation breakdown effect, in generally, radiation sensitive promoter early growth response genel(Egrl) is applied commonly. In order to improve the radiation inducing effects enhancement of Egrl under hypoxic conditions, hypoxia-responsive elements (hypoxia response elenments, HREs) is consider to construct chimeric promoter with Egrl, to increase radiation inducing effects of Egrl under hypoxic conditions. In the study, using radiation enhancing effect property of Egrl promoter, the enhancing downstream gene expression character of HREs promoter under hypoxic condition and strongly inducing cancer cell apoptosis of TRAIL gene, while normal cells are not sensitive to its characteristics, to combine these three together and to construct dual-sensitive promoter mediated expression vector, and to combine the ionizing radiation to approach the inhibition effect on lung adenocarcinoma A549 cells in vitro, The results provide new ideas for cancer treatment.1 Construction and identification of reconminant plasmidsIn this experiment, five plasmids were constructed, they are pcDNA3.1-Egr1-EGFP、pcDNA3.1-HRE/Egr-EGFP, pcDNA3.1-shTRAIL, pcDNA3.1-Egr1-shTRAIL and pcDNA3.1-HRE/Egr1-shTRAIL, respectively. pcDNA3.1, pshuttle-EGFP and pMD19T-Egr1 plasmids were endowed by Li Yanbo doctor, they were digested by different restriction enzymes, then pcDNA3.1 vector, EGFP fragement and Egr1 fragement were gotten respectively, ligating them in order to construct pcDNA3.1-Egr1-EGFP plasmid, call it pcEE, they were identificated by PCR, restriction enzyme digestion and sequencing; HRE sequences synthesized by Shanghai bio-engineering company, to connect it to upstream of Egr1 in pcDNA3.1-Egr1-EGFP and construct pcDNA3.1-HRE/Egr1-EGFP, and call it pcHEE, it was identificated by PCR, restriction enzyme digestion and sequencing; shTRAIL were gotten from pshuttle-shTRAIL which digested by restrition enzyme, inserted it to pcDNA3.1 vector and construct pcDNA3.1-shTRAIL plasmid, and call it pshT, it was identificated by PCR, restriction enzyme digestion and sequencing; CMV promoter was cut from pcDNA3.1-CMV-shTRAIL, then to replace it with Egr1, to construct pcDNA3.1-Egr1-shTRAIL and call it pcEshT, it was identificated by PCR, restriction enzyme digestion and sequencing; pcDNA3.1-Egr1-shTRAIL plasmid was digested by restriction enzyme again, conneting HRE upstream to Egr1 and construct pcDNA3.1-HRE/Egr1-shTRAIL, and call it pcHEshT, it was identificted by PCR, restriction enzyme digestion and sequencing. In the study, the five recombinant plamid is fully consistent with the predication, indicates that construction is right.2 Radiation inducing character of Egr1pEE plasmid was transfected into lung adenocarcinoma A549 cells by liposome, under nomaxic and hypoxic conditions, these cells were radiated by 1,2,4,6,8 and 10 Gy X-rays,0,3,8,12,24 and 36 h later, the green fluorescent protein expression were detected by flow cytometry. The results show that the normal oxygen conditions, after pcEE plamid transfection into A549 cells, the green fluorescent protein expression increase with time prolongation and dose increasing, and it reachs peak 12 h after 8 Gy irradiation, but they reach peak 8 h after other dose X-rays irradiation, then they all begin to fall after 24 h. Under hypoxic condition, when oxygen concentration were different, green fluorescent protein expression was inhibited by the same dose irradiation, inhibition was obvious when the oxygen concentration was less than 2.5%, with the lower oxygen concentration the more inhibition, and then the oxygen concentration is higher than 2.5%, green fluorescent protein expression inhibtion is not large with the relationship between oxygen concentration. The expression difference 8 h after different dose irradiation between normoxic condition and hypoxic condition was signifinant (P<0.05, P <0.01). This shows that Egrl has radiation inducing expression enhancement character, and hypoxia can inhibit Egrl pormoter features.3 The influence of HRE on Egrl radiation inducing enhancement characterAfter pEE plasmid was transfected into lung adenocarcinoma A549 cells, under normaxic and hypoxic conditions, these cells were irradiated by 0,2,4,6,8 and 10 Gy, after 0,3,8,12,24 and 36 h, green fluorescent protein expression was measured by flow cytometry, results showed that green fluorescent protein expression in cells transfected by pEE plasmid at different time after various dose irradiation was basically same under the normoxic condition. But under hypoxic condition of 0.1%-2.5% of the oxygen concentration, EGFP expression significantly increased, and increased with oxygen concentration and radiation dose increase, and gradually reached the maximum in 1% oxygen concentration, the expression has significant difference with those in cells 8 h after different dose irradiation under normaxiccondition (P< 0.05, P<0.01). under the condition of 5%-10% oxygen concentration. EGFP expression was basically similar with the expression under normaxic condition. This indicated that HRE has the feature to enhance radiation inducing properties of Egrl under hypoxic condition, but has not under normoxic condition.4 Radiation inducing expression regularity of the recombinant plasmid in A549 cellsAfter pcshT, pcEshT and pcHEshT three kinds of plasmid transfected into A549 cells, under normoaxic and/or hypoxic condition of 1% oxygen concentration, cells irradiated by 6 Gy. then TRAIL mRNA and TRAIL protein expression were measured, results showed that the TRAIL mRNA expression increased in A549 cells transfected by pcshT and pcEshT plasmid after 6 Gy irradiation and 6 Gy irradiation + hypoxia of 1% oxygen concentration, and had significant difference with expression in control cells, but the expression was more under 6 Gy irradiation + hypoxia of 1% oxygen concentration condition (P<0.05); and the TRAIL mRNA expression increased in A549 cells transfected by pcHEshT plasmid after 1% oxygen concentration hypoxia,6 Gy irradiation and 6 Gy irradiation +1% oxygen concentration hypoxia, it had significant difference compared with control group(P <0.05), and expression was more under radiation condiation than hypoxic condition, and the expression was more obvious under radiation and hypoxic condition (P <0.05). The shTRAIL protein expression regularity was basically same with mRNA expression regularity, those indicated that radiation could induce shTRAIL expression enhancement in A549 cells transfected by 3 plasmids. and under hypoxic condition the enhancement effect was more obvious.5 Inhibition effect of recombinant plasmid on A549 cells5.1 The recombinant plasmid inhibited A549 cell proliferationAfter pcshT, pcEshT and pcHEshT three kinds of plasmid transfected into A549 cells, under normoaxic and/or hypoxic condition of 1% oxygen concentration, A 549 cells were irradiated by 6Gy, then counted the living cells number by trypan blue dye at 0,2,4,6 and 8 d respectively and ploted the cell growth curve, the results showed that the hypoxia of 1% oxygen concentration inhibited A549 cells growth. in which A549 cells transfected by pcHEshT plasmid were inhibited most:and 6 Gy X-rays also inhibited A549 cells growth transfected by 3 plasmid,which cells transfected by pcEshT and pcHEshT plasmid were inhibited most obviously; cells transfected by 3 plasmids all were inhibited very strongly under 6 Gy and 1% oxygen concentration, particularly cells transfected by pcHEshT were inhibited most strongly. At the same time, After pcshT, pcEshT and pcHEshT three kinds of plasmid transfected into A549 cells, under normoaxic and/or hypoxic condition of 1% oxygen concentration. A 549 cells were irradiated by 6 Gy, the cell obsorbance value (A) at 0,12,24,48 and 72 h was detected by MTT, results showed that the A value of A549 cells transfected by 3 plasmids decreased after 1% oxygen concentration hypoxia, in which A value in the cells transfected by pcHEshT decreased greatest; but the A value of A549 cells transfected by 3 plasmids decreased after irradiation by 6 Gy decreased, in which A value in cells transfected by pcEshT decreased greatest; the A value in cells transfected by pcHEshT after 1% oxygen concentration and 6 Gy irradiation decreased greastest at 48 h. These results showed that the inhibition was most strong in cells transfected by pcHEshT after 1% oxygen concentration and 6 Gy X-rays irradiation.5.2 Effect on cell phage and apoptosis of recombinant plasmidAfter pcshT, pcEshT and pcHEshT three kinds of plasmid transfected into A549 cells, under normoaxic and/or hypoxic condition of 1% oxygen concentration, A 549 cells were irradiated by 6Gy, cell apoptosis was detected by flow cytometry with PI sigle staining, results showed that after transfected 3 plasmids cell percentage of G0/G1 phase increased under hypoxia of 1% oxygen concerntration and 6 Gy irradiation condition, and cell percentage of G2/M phage decreased, but cell percentage of S phage did not change basically, at same time, and cell percentage of apoptosis increased.At same time, cell apoptosis was measured by TUNEL, cell apoptosis was enhanced after 3 plasmids transfection from radiation and hypoxia. apoptosis change of cell transfected by pcshT and pcEshT plasmid from hypoxia was not obvious, and apoptosis change of cell transfected by pcHEshT was obvious: apoptosis change of cell transfected by pcshT and pcEshT plasmid from hypoxia was obvious, in which apoptosis change of cell transfected by pcEshT and pcHEshT was most obvious, hypoxia and radiation had common promotion on apoptosis change of cell transfected by pcshT, and had more promotion on apoptosis change of cell transfected by pcEshT, and had most promotion on apoptosis change of cell transfected by pcHEshT. This is basically the same as the results of flow cytometry. indicating after transfection by three kinds of plasmids. hypoxia and radiation can induce G0/G1 cell cycle arrest and apoptosis increase.5.3 Effect on radiation sensitivity of recombinant plasmidNormal A549 cells and cells transfected by pcshT, pcEshT and pcHEshT plasmid irradiated by 0,2,4,6.8 and 10 Gy under nomoxic and hypoxic condition, cells were cultured for 14 days, then counted the clones and calculated survival fraction, in accordance with multi-hit single-target model for curve fitting to calculate the Do value, analysised the radiation sensitivity difference among normal cells, cells transfected by pcshT, pcEshT and pcHEshT plasmid. Results showed that under normoxic condition. Do value of 4 cells was 3.26,2.12,1.91 and 1.89 respectively, this indicated that radiation sensitivity in cells transfected by pcEshT and pcHEshT plasmid was maximum;but under hypoxic condition, the Do value was 5.81.6.12,4.24 and 1.13 respectively, this indicated that hypoxia could decrease radiation sensitivity of normal cell and cell transfected by pcshT and pcEshT. and enhance radiation sensitivity of cell transfected by p cHEshT plasmid.5.4 Effect on DR4, DR5 and caspase-3 of recombinant plasmidAfter pcshT, pcEshT and pcHEshT three kinds of plasmid transfected into A549 cells, under normoaxic and/or hypoxic condition of 1% oxygen concentration, cells irradiated by 6 Gy. then DR4 and DR5 mRNA were measured by RT-PCR, results showed that hypoxia effect little on DR4 mRNA in cell transfected by pcshT and pcEshT plasmid, but could induce DR4 mRNA increase in cell transfected by pcHEshT plasmid; but hypoxia could induce DR5 mRNA increase, in which the increase of DR5 mRNA in cell transfected by pcHEshT was most obvious:6 Gy radiation could induce DR4 and DR5 mRNA increase in cell transfected by 3 plasmids;hypoxia and radiation combination could induce increased DR4 and DR5 mRNA, in which DR5 mRNA increase in cells transfected by pcHEshT was most obvious. In addition, DR4, DR5 and caspase-3 protein expression was detected by Western blot, results showed that DR4 and DR5 protein expression regularity were similar with their mRNA, this indicated that cells transfected by 3 plasmids could induce DR4 and DR5 increase under hypoxia and/or radiation.After 3 plasmids transfection, hypoxia and radiation could induce caspase-3, caspase-3 expression in cells transfected by pcHEshT was most obvious under hypoxia and randiation combination, caspase-3 expression regularity was similar with apoptosis regularity.更多还原