【作者】 杨春旭；

【导师】 谢丛华；

【作者基本信息】 武汉大学 ， 肿瘤学， 2014， 博士

【摘要】 第一部分：人非小细胞肺癌获得性TRAIL耐药细胞株H460-R的构建目的：构建获得性TRAIL耐药的人非小细胞肺癌细胞株H460-R,并验证该耐药模型构建成功。检测外源性凋亡通路中死亡受体及诱骗受体的变化,并检测凋亡通路中相关蛋白的表达量,以明确获得性耐药过程中凋亡通路相关信号分子的改变。方法：通过特定浓度的TRAIL选择性杀伤H460敏感细胞,而后由低浓度逐渐诱导至高浓度,最终以低于IC50值的浓度培养,维持细胞的耐药性。通过乳酸脱氢酶法(LDH)检测亲本株与耐药株对TRAIL的细胞药物毒性反应。流式细胞术检测凋亡水平的改变。通过RT-PCR检测死亡受体DR4、DR5,诱骗受体DcR1、DcR2的基因表达水平。通过Western blot检测死亡受体DR4、DR5,诱骗受体DcR1、DcR2,死亡诱导信号复合体中相关蛋白FADD、cFLIP、caspase8的表达,以及检测凋亡下游效应蛋白caspase3的表达,并检测了Ⅰ型信号复合体中凋亡相关蛋白TRADD、TRAF2的表达。结果：获得性耐药细胞株H460-R的药物半数抑制浓度较亲本株明显升高(P<0.05)。流式细胞术结果显示给予相同浓度的TRAIL处理后,与H460细胞相比,H460-R细胞凋亡率显著下降,差异有统计学意义(P<0.05)。然而,亲本株和耐药株之间死亡受体DR4、DR5,诱骗受体DcR1、DcR2的基因表达水平及蛋白表达水平均未见明显差异(P>0.05)。死亡诱导信号复合体(DISC)中相关蛋白的表达,FADD、caspase8等在亲本株与耐药株之间无明显差异(P>0.05)。caspase8家族中凋亡下游效应蛋白caspase3的表达在两株细胞之间无明显差异(P>0.05)。在耐药细胞株中凋亡拮抗因子cFLIP的表达较亲本株明显增加(P<0.05)。Ⅰ型复合体中TRADD、TRAF2的表达均无明显变化(P>0.05)结论：成功构建人非小细胞肺癌获得性TRAIL耐药细胞株,命名为H460-R。验证了耐药株对TRAIL的敏感性降低。在亲本细胞与耐药细胞中,死亡受体DR4、DR5的基因及蛋白表达水平均无差异,诱骗受体DcR1、Dc12的基因及蛋白表达水平均无差异。提示在构建获得性耐药细胞株的过程中,并未引起受体总体表达水平的改变,为下一步研究TRAIL耐药机制奠定了重要的基础。在DISC中相关蛋白的表达检测中,我们发现,FADD及caspase8表达无差异。在耐药细胞中,cFLIP的表达量较亲本细胞中明显增加,可能是其耐药的重要原因,这为后续研究耐药相关机制以及寻找增敏途径提供了方向。Ⅰ型复合体中拮抗凋亡的重要的蛋白TRADD、TRAF2的表达未见明显变化,这两种蛋白主要在NF-κB信号的激活中发挥重要作用,提示在获得性耐药中该通路无明显改变,为后续实验研究做好基础准备。第二部分：放射线逆转获得性TRAIL耐药的相关研究目的：利用放射线照射获得性耐药细胞株H460-R,探讨放射线对逆转获得性TRAIL耐药的作用及机制。初步分析亲本株与耐药株放射生物学行为的变化,并明确在放射线处理后,凋亡相关的重要蛋白表达量以及相互作用的改变。方法：2Gy放射线照射获得性耐药细胞株H460-R后,通过乳酸脱氢酶(LDH)法检测对TRAIL的细胞毒性变化。通过流式细胞术检测凋亡率的变化。RT-PCR检测放射线处理前后死亡受体DR4、DR5及诱骗受体DcR1、DcR2的基因表达量。Western blot法检测死亡受体、诱骗受体的蛋白表达。并检测凋亡通路的下游相关蛋白的表达量的变化。细胞放射生物学方面,通过流式细胞术检测亲本株与耐药株放射线处理前后细胞周期的变化。并通过克隆形成实验检测两株细胞的放射敏感性。分别构建携带FADD、cFLIPL基因的重组表达质粒pCMV-C-HA-FADD、PCMV-C-Tag2C-Flag-cFLIPL。上述质粒转染人胎肾HEK293T细胞,经Western blot检测验证FADD、cFLIPL过表达效果。收集转染过表达质粒的HEK293T细胞,提取蛋白,经免疫共沉淀(co-IP)检测FADD与cFLIPL之间存在相互作用；接下来检测亲本株与耐药株在相同浓度的TRAIL处理后,FADD与cFLIPL之间的相互作用；最后检测耐药细胞株H460-R在放射线处理前后FADD与cFLIPL之间相互作用的变化。结果：经2Gy X线照射后,H460-R细胞株对TRAIL的毒性反应较未处理组明显增加。由结果可知,TRAIL联合射线后能显著增加获得性耐药细胞株对TRAIL的敏感性(P<0.05)。流式细胞检测凋亡的结果提示放射线联合TRAIL后,获得性耐药细胞株的早期凋亡率增加,与单纯给予TRAIL组及单纯放射线组比较,其凋亡率差异有统计学意义(P<0.05)。凋亡相关蛋白表达量检测结果表明：放射线能够使H460-R对’TRAIL敏感性增加,这种增敏作用并不是由放射线直接诱导的细胞凋亡。细胞周期检测结果中,我们发现,两株细胞的周期分布比例无明显差异,我们给予放射线处理后再次检测结果提示：两株细胞的细胞周期中G2/M比例同时增高,其差异无统计学意义(P>0.05)。克隆形成实验结果显示两株细胞的放射敏感性无明显差异。我们成功构建了真核表达载体pCMV-C-HA-FADD, pCMV-C-Tag2C-Flag-cFLIPL重组表达质粒,经DNA测序验证正确后,转染人胚胎肾细胞HEK293T,经Western blot验证FADD、cFLIPL较对照组表达显著增加(P<0.05)。免疫共沉淀检测发现FADD与cFLIPL之间存在相互作用。分别检测了亲本株与耐药株中两者FADD与cFLIPL的相互作用。结果显示：相同浓度的TRAIL处理细胞后,利用免疫共沉淀检测,在亲本细胞株中未见两者相互作用,而耐药细胞株H460-R中则存在着两者的相互作用。在获得性耐药细胞株给予2GyX线预处理前后,再次检测FADD与cFLIPL之间的相互作用,双向免疫共沉淀结果提示,放射线使两者之间的相互作用减弱或者消失。死亡诱导信号复合体中,caspase8的活化的片段P43/41的表达在放射线联合TRAIL处理后较单纯TRAIL组、单纯射线组及阴性对照组高(P<0.05)。结论：放射线能够增加获得性耐药细胞H460-R对TRAIL的敏感性,但不改变细胞的死亡受体的总体表达量。亲本株与耐药株的细胞周期分布比例无明显差异,放射线处理后两株细胞的周期分布也无明显差异,提示TRAIL并未改变细胞周期的分布。克隆形成结果提示耐药株与亲本株的放射敏感性无差异,耐药株H460-R仍然是放射敏感细胞。在获得性耐药细胞株H460-R中FADD的表达与H460无差异,而cFLIPL的表达较亲本株增加,从而使获得性耐药细胞株中FADD被cFLIPL竞争性结合,无法活化caspase8,进而使凋亡信号无法向下传递。放射线能在一定程度逆转耐药细胞株H460-R对TRAIL的耐药性,其作用机制可能与放射线处理后蛋白FADD与cFLIPL之间相互作用发生改变相关。凋亡通路下游相关蛋白表达量的改变,进一步说明放射线能够一定程度上逆转TRAIL耐药,从而引起下游凋亡的发生。第三部分：放射线改变死亡受体状态对TRAIL敏感性影响的相关研究目的：探讨人非小细胞肺癌获得性耐药细胞株H460-R经放射线处理后,细胞膜表面死亡受体定位、表达量的变化以及相关功能的改变,初步探讨放射线逆转TRAIL耐药的相关机制。方法：获得性耐药细胞株H460-R经2GyX线预处理后,通过间接免疫荧光法分别标记细胞内死亡受体DR4、DR5,使用激光共聚焦检测其在细胞内的定位。通过死亡受体与高尔基体荧光双标记的方法,检测两者的细胞内定位。流式细胞术检测死亡受体细胞膜表面的表达量。利用western blot法检测放射线处理后不同时间点细胞膜表面死亡受体的表达量。通过O-糖苷酶的酶切水解蛋白后,行western blot法检测死亡受体DR5糖基化蛋白的变化。结果：免疫荧光法标记死亡受体DR4、DR5后,激光共聚焦检测结果发现,在耐药细胞株H460-R中,死亡受体DR4、DR5均匀分布于细胞浆中,放射线预处理细胞3小时后再次行免疫荧光标记,激光共聚焦检测发现死亡受体大量定位于细胞膜。而放射线处理6小时后细胞膜表面死亡受体较3小时表达量降低。我们通过流式细胞术检测细胞膜表面死亡受体的表达,与共聚焦结果一致,放射线处理组死亡受体的表达量与单纯TRAIL组、单纯射线组以及阴性对照组相比,差异具有统计学意义(P<0.05)。在细胞膜表面蛋白定量检测结果中,我们发现,放射线处理3小时后,细胞膜表面死亡受体DR4、DR5的表达量均较单纯TRAIL组、单纯射线组增加,其差异有统计学意义(P<0.05)。共聚焦检测结果中还显示：在H460-R细胞中,放射线处理后死亡受体DR5形成了浓聚。结合细胞超微结构分析,我们再次进行了死亡受体DR5与高尔基体免疫荧光双标。激光共聚焦检测结果显示：死亡受体DR5在放射线预处理后一定时间内,定位于高尔基体,而DR4无此现象。随后我们通过糖苷酶水解蛋白后,检测死亡受体DR5的蛋白表达,发现死亡受体DR5在放射线处理后,发生了O-糖基化。结论：放射线能够瞬时增加获得性耐药细胞株H460-R细胞膜表面死亡受体DR4、DR5表达,从而增加该细胞株对TRAIL的敏感性。在免疫荧光检测结果中,我们发现,放射线处理细胞后,死亡受体DR5在细胞内核旁浓聚,根据细胞器分布,我们推测该部位可能在高尔基体或者内质网。免疫荧光标记DR5与高尔基体narker Giantin后,我们发现,放射线能够诱导细胞内死亡受体DR5在高尔基体定位。结合细胞器功能我们分析,死亡受体DR5在高尔基体上完成蛋白质的糖基化修饰,我们通过O-糖苷酶水解后的蛋白检测结果证实了DR5的O-糖基化修饰。而死亡受体的糖基化能够增加肿瘤细胞对TRAIL的敏感性。由此我们推断,糖基化后的死亡受体DR5能够增加H460-R对TRAIL的敏感性,从而逆转获得性TRAIL耐药,是放射线增加获得性耐药细胞株H460-R对TRAIL敏感性的重要原因。更多还原

【Abstract】 Part I The establishment of the acquired TRAIL resistant human non-small cell lung cancer cell line H460(H460-R)Objective To construct the acquired-TRAIL resistant human non-small cell lung cancer cell line H460(H460-R) and verify the efficiency. To detect the expression of the extrinsic apoptotic pathways related protein. In order to make clear the change of apoptotic signal molecular protein in the process of acquired drug resistance.Methods Firstly, the medium concentration for killing the sensitive cells was selected. Then the cells were gradually induced by low concentration to high concentration, and eventually, maintained the selected-cells with the concentration below IC50. LDH kit was used to testify the toxicity in H460-R and its parent cell line H460. The level of apoptosis was tested through flow cytometry, the mRNA expression of death receptors and death decoy receptors were tested through RT-PCR assay and the western blot assay was used to test the protein expression of death receptors, decoy receptors, the protein related to death induced signaling complex and complex I.Results The IC50concentration was obviously increased in the acquired-TRAIL resistant H460-R than its parent cell line H460. After pretreating with TRAIL, the cell apoptosis rate was significantly statistical changed between the two cell lines in the flow cytometry testing apoptosis(P<0.05). However, the expression of TRAIL receptors was not remarkabry changed in mRNA and protein level(P>0.05). The expression of the anti-apoptosis regulator cFLIP was dramatically increased in H460-R(P<0.05), while there was no difference in TRADD and TRAF2which belong to complex I (P>0.05).Conclusion The acquired-TRAIL resistant H460(H460-R) was successfully established and the difference expression of TRAIL death receptors and decoy receptors were verified. According to the expression of protein in the death inducing signaling complex, there were no difference in FADD and caspase8.But in the acquired resitant cell line H460-R, the expression of cFLIP was obviously higher than in H460.The difference protein expression of the death-inducing signaling complex (DISC) was identified, so that the target we aimed to investigate in the mechanism of TRAIL-resistance was clear enough for the next step. Part Ⅱ The study on radiation reversing the acquired drug resistance to TRAILObjective To verify the function of ironing radiation in reversing the acquired TRAIL resistant human non-small cell lung cancer cell line H460(H460-R).Methods The acquired drug resistant cell line was treated with2Gy X Ray ionizing radiation, and LDH kit was used to detect the cell toxicity changes, while the changes in the rate of apoptosis was detected by flow cytometry.We applied RT-PCR and Western blot to detect the gene and protein expressions of death receptor after ironing radiation. Western blot was used to detect the expressions of apoptotic downstream related proteins. We tested the cell cycle of H460and H460-R before and after ironing radiation by flow cytometry. Then we tested the radiosensitivity of H460and H460-R by Colony-formation assay.After restriction enzyme digestion of FADD and cFLIPL cDNA plasmids and eukaryotic expression plasmids pCMV-C-HA and pCMV-C-Tag2C-Flag according to the graph provided by enzyme loci enzyme respectively, we built the recombinant pCMV-C-HA-FADD and pCMV-C-Tag2C-Flag-cFLIPL expression plasmids. The recombinant expression plasmids were transformed into E.coli cells DH5alpha, then we screened the positive clones through kanamycin. The positive clones were amplified and collected. Then the plasmids were extracted from the collected bacteria liquid, and the DNA sequencing identificated the expression of plasmids being built successfully. Then we transfected the human embryonic kidney293T (HEK293T) cells using the above plasmids, the total protein was collected to verify the over expression effect of FADD and cFLIPL by Western blot. And then we did the co-immunecoprecipitation (Co-IP) to definite the change of protein interaction between FADD and cFLIPL.Results The cell toxicity of H460-R to TRAIL after2Gy ionizing radiation was obviously changed(P<0.05). And the results about apoptosis showed that ionizing radiation joint TRAIL could increase the early apoptosis rate of H460-R.The cycle of both the two cell lines had no differences(P>0.05).After radiation,the rate of G2/M was dramatically increased in both the cell lines.But there was no difference between H460and H460-R(P>0.05). The clone formation assay verified that we successfully constructed the eukaryotic expression vector pCMV-C-HA-FADD and pCMV-C-Tag2C-Flag-cFLIPL, and verified by DNA sequencing. Then we did the transfection of human embryonic kidney cell293T using the overexpression plasmids and the overexpression effect was verified by western blot assay. Total protein was extracted to verify the interaction between FADD and cFLIPL by the test of co-immunecoprecipitation (Co-IP). After ionizing radiation, the interaction was changed according to the H460-R.We found that the downstream apoptotic related protein caspase8expression level was in some degree changed before and after the radiation treatment.Conclusion The cause of TRAIL resistant of H460-R is that the FADD is being competed combination by cFLEP with the consequence of apoptosis signaling being disturbed. Radiation can partly alleviate the resistance of H460-R. The mechanism may due to the change of the interaction between proteins after radiation. The quantitative analysis of expression of the apoptotic pathways downstream related protein further clarified that radiation could change the TRAIL resistance. Part III The function and translocation of death receptors influence TRAIL sensitivity in acquired-TRAIL resistant H460(H460-R)Objective With the radiation treatment to TRAIL-resistant cell line, we aimed to focus on the translocation of death receptors on the cell membrane and the related functional changes, which elaborated the mechanism of radiation reversing the TRAIL sensitivity in acquired TRAIL resistant H460-R.Methods After2Gy ionizing radiation pretreatment to H460-R, cell death receptors DR4, DR5were marked respectively by indirect immunofluorescence method, using laser scanning confocal microscope to detect their intracellular localization; as the same, the co-localization of death receptors and Golgi apparatus were determinate by the double-immunofluorescent labeling method. The death receptors expressed on the cell membrane surface were detected by the flow cytometry and western blot methods. With the hydrolysis of glycosidase enzymes O protein, western blot was performed to detect the glycosylation of DR5protein.Results In acquired-TRAIL resistant H460(H460-R), death receptors DR4, DR5were evenly distributed in the cytoplasm, while radiation pretreatment to H460-R three hours later, death receptors were translocation to the cell membrane. Meanwhile, the expression of death receptors on the cell membrane surface detected by the flow cytometry was the same as the results by the immunofluorescence method mentioned above. After the radiation pretreatment from3hours to6hours, the expression of death receptors on the cell membrane surface were decreased. An interesting phenomenon was detected by the laser scanning confocal microscope, which was after radiation treatment, death receptors5not death receptor4formed a thick poly in cytoplasm. By analysis the cell ultrastructure, we speculated that the thick parts might be in the Golgi apparatus. As a result, by double-immuno fluorescent labeling the death receptors and the Golgi apparatus, laser confocal detection showed that in a short period of time after radiation pretreatment, DR5translocated to the Golgi apparatus. By the hydrolysis of glycosidase enzymes0protein to DR5, death receptor was found to be glycosylated after radiation treatment. Conclusion Radiation augments the expression of death receptors DR4, DR5on the cell membrane surface in H460-R, as a result of the sensitivity of TRAIL increased. On the other hand, radiation induced the intracellular DRS transbcation to Golgi apparatus, with O-glycosylation modification. The glycosylation of death receptor increased the sensitivity to TRAIL, paralleling the reversing of sensitivity in acquired-TRAIL resistant cells.更多还原