【作者】 陈晓春；

【导师】 马海涛；

【作者基本信息】 苏州大学 ， 胸心血管外科学， 2015， 硕士

【摘要】 目的：通过测定RMP在人非小细胞肺癌组织和细胞系中的表达水平,来研究RMP与肿瘤病人临床病理特征的联系,同时探索其对NSCLC细胞生物学功能的影响,为NSCLC的基因靶向治疗提供强有力的依据。方法：1.通过qRT-PCR检测RMP在NSCLC细胞系和正常细胞系中的mRNA表达水平,同时用western blot检测RMP相应的蛋白表达水平。2.通过qRT-PCR检测RMP在人非小细胞肺癌组织和相应癌旁组织中的mRNA表达水平,同时用western blot检测RMP相应的蛋白表达水平。3.通过qRT-PCR检测RMP在不同特征NSCLC组织样本中的mRNA表达水平,分析其与肿瘤临床病理特征的联系,同时用表格统计病人的相关临床信息。4.荧光显微镜检测各组RMP质粒在NSCLC细胞A549中的瞬时转染情况,同时用qRT-PCR和western blot检测转染效率。5.通过CCK-8试验检测RMP对A549细胞增殖的影响。6.用不同浓度的吉非替尼处理转染了各组不同RMP质粒的A549细胞,通过CCK-8试验、流式细胞术检测RMP对A549细胞化疗敏感性的影响。7.通过CCK-8试验、流式细胞术检测RMP对A549细胞放疗敏感性的影响,同时用western blot检测细胞凋亡相关基因的蛋白表达水平。8.通过流式细胞术检测RMP对于A549细胞周期的影响,同时用western blot检测周期相关基因的蛋白表达水平。9.通过迁移和侵袭试验检测RMP对A549细胞迁移和侵袭能力的改变。10.通过裸鼠皮下移植瘤实验观察RMP对移植瘤生长情况的影响,同时用免疫组化检测RMP、Bax、Bcl-2和capase-3的蛋白表达情况。结果：1.qRT-PCR和western blot实验结果显示：无论是mRNA和蛋白表达水平,RMP在NSCLC细胞系中均呈相应的高表达状态。2.qRT-PCR和,western blot实验结果表明：与癌旁组织相比,RMP在人非小细胞肺癌组织中的：mRNA和蛋白的表达水平均明显提高。同时发现RMP的表达与肿瘤组织的淋巴结和T-stage状态有关,与病人的年龄、性别和肿瘤类型无关。3.荧光显微镜、qRT-PCR和western blot实验表明：各组RMP质粒在A549细胞中瞬时转染顺利,mRNA和蛋白表达水平呈相应的提高。4.CCK-8试验表明：RMP明显促进A549细胞的增殖。5.CCK-8试验表明：在化疗药物剂量逐渐增加的作用下,RMP仍能够减缓A549细胞的死亡；流式细胞术检测表明：RMP能够减少化疗引起的细胞凋亡6.CCK-8试验表明：在辐射剂量逐渐增加的作用下,RMP仍能够减缓A549细胞的死亡；流式细胞术检测表明：RMP能减少放疗引起的细胞凋亡；western blot实验检测表明：在放疗后,RMP能够抑制A549细胞中凋亡相关基因蛋白的表达,促进抗凋亡相关基因蛋白的表达。7.流式细胞术检测表明：RMP能够减少由放疗引起的A549细胞周期G2期的阻滞,同时western blot实验表明：放疗后,RMP能够调节A549中细胞周期相关基因蛋白的表达。8.迁移和侵袭试验表明：RMP能够促进A549细胞的迁移和侵袭能力。9.裸鼠皮下移植瘤实验表明：RMP能够促进移植瘤的生长,同时免疫组化检测表明：RMP能够抑制凋亡相关基因蛋白的表达,促进抗凋亡相关基因蛋白的表达,从而促进肿瘤的生长。结论：1.RMP在人非小细胞肺癌中呈高表达的状态,结合临床病人信息发现它的表达与非小细胞肺癌病人的淋巴结和T-stage状态有关,说明RMP可能与NSCLC的生长和恶性程度有着密切的联系,是潜在的一种NSCLC相关促癌基因。2.RMP的过表达能够改变A549如细胞增殖、凋亡、周期、迁移和侵袭等各项生物学功能,促进移植瘤的生长,进一步表明RMP在NSCLC中扮演着癌基因的角色,为我们对肺癌的基因靶向治疗又提供一个强有力的目标。更多还原

【Abstract】 Objective:In our research, we study the relationship between RMP and the clinical pathological features of NSCLC patients by measuring the expression level of RMP in human non-small lung cancer tissues and cell lines. At the same time, we explore the influence of RMP on carcinoma’s biological functions to provide powerful supports for gene targeting treatment of NSCLC.Methods:1. q RT-PCR and western blot were used to detect the m RNA and protein expression in NSCLC cell lines and the human normal bronchial epithelial cells.2. q RT-PCR and western blot were used to detect the m RNA and protein expression in NSCLC tissues and adjacent non-tumor tissues.3. q RT-PCR was applied to detect the expression of RMP in NSCLC tissues which have different characteristics. Then we analyzed the relationship between RMP and the tumors. At the same time, we used the table to summarize the relevant clinical information about the patients.4. Fluorescence microscope was used to observe the different RMP plasmids in A549 cells which belong to NSCLC. And we also successfully detected the transfection efficiency by using the q RT-PCR and western blot.5. CCK-8 assay was applied to the influence of RMP on cell proliferation of A549.6. A549 cells transfected with different groups of RMP plasmids were dealt with Gefitinib and then we used the CCK-8 assay and Flow cytometry to detect the effect of RMP on the sensitivity of A549 cells to chemotherapy.7. CCK-8 assay and Flow cytometry were applied to observe the effect of RMP on the sensitivity of A549 cells to radiotherapy. At the same time, we used western blot to detect the protein expression of apoptosis related genes.8. Flow cytometry was used to detect the effect of RMP on the cell cycle of A549 cells. At the same time, western blot was applied to observe the protein expression of cell cycle related genes.9. The migration and invasion assay was used to detect the effect of RMP on the ability of migration and invasion in A549 cells.10. The xenografts in nude mices were used to observe the influence of RMP on tumor formation and we also applied the immunohistochemical staining to detect the protein expression of RMP、Bax、Bcl2 and capase-3.Results1. q RT-PCR and western blot showed that both m RNA and protein level of RMP were relatively high expressed in NSCLC cells.2. q RT-PCR and western blot showed that both m RNA and protein level of RMP were distinctly high expressed in human non-small cell lung cancer tissues compared with corresponding normal tissues. At the same time, we found that the expression of RMP was associated with the status of lymphonodus and T-stage in carcinoma tissues. In addition, there was nothing to do with the patients’ age、gender and cancer forms.3. Fluorescence microscope、q RT-PCR and western blot showed that each group of RMP plasmids were transfected into A549 cells successfully. As a result, the m RNA and protein level were relatively improved.4. CCK-8 assay showed that RMP could distinctly promote the proliferation of A549 cells.5. CCK-8 assay showed that RMP could still slow down the death induced by different dose of chemotherapy agents. Apart from that, the Flow cytometry showed that RMP could decrease the apoptosis induced by chemotherapy.6. CCK-8 assay indicated that RMP could still inhibit the death induced by different dose of radiation. Additionally, the Flow cytometry showed that RMP could decrease the apoptosis induced by radiotherapy in A549 cells. What’s more, western blot demonstrated that RMP could suppress the protein expression of apoptosis related genes while improve that of anti-apoptosis related genes after the radiotherapy.7. The Flow cytometry showed that RMP could decrease the G2 phase arrest induced by the radiotherapy in A549 cells. What’s more, western blot demonstrated that RMP could regulate the protein expression of cell cycle related gene after the radiotherapy.8. The migration and invasion assay showed that RMP could improve the ability of migration and invasion in A549 cells.9. The xenografts in nude mices indicated that RMP could promote the growth of xenografts. At the same time, immunohistochemical staining showed that RMP could improve the tumor growth by restraining the protein expression of apoptosis related genes while promoting that of anti-apoptosis related genes.Conclusions1. RMP was highly expressed in non-small cell lung cancer. In addition, we found that the RMP expression was associated with the status of lymphonodus and T-stage by combining the patients’ relevant clinical information. All these indicated that RMP may be closely related to the growth and malignant degree of NSCLC. Furthermore, RMP would be a potential NSCLC related cancer-promoting gene.2. Overexpression of RMP could change a variety of biological functions such as cell proliferation、apoptosis、cell cycle、migration and invasion in A549 cells leading to promote the growth of xenografts in nude mices. All these further showed that RMP would play a cancer-promoting gene role in NSCLC which may provide us a powerful goal for gene targeting treatment of lung cancer.更多还原