全反式维甲酸诱导荷瘤裸鼠A549肺腺癌细胞凋亡的机制

发布时间：2018-05-04 15:39

本文选题：ATRA + 顺铂　；参考：《郑州大学》2015年硕士论文

【摘要】：肺癌是中国最常见的恶性肿瘤之一,现今发病率、死亡率逐年升高,并呈年轻化趋向,严重威胁人类生命健康,已成为世界重大公共卫生问题之一。由于肺癌早期临床症状易被忽视,导致许多肺癌患者确诊时已至晚期,失去手术治疗机会,仅能保守治疗,并且即使早期确诊的肺癌患者,应用外科手术治疗完全切除原发病灶后,仍然有一定的肿瘤复发率。现如今,化疗作为肺癌的重要治疗方法,其对于根治术后患者也有一定价值。目前,铂类是非小细胞肺癌(Non-Small Cell Lung Cancer,NSCLC)的化疗方案的基础,其联合如紫杉醇、吉西他滨等其他化疗药物仍是晚期NSCLC标准一线化疗方案。随着化疗在临床的广泛应用,化疗药物的耐药现象也突显,多药耐药是成功化疗的主要障碍,化疗耐药主要是由于化疗药物诱导肿瘤细胞凋亡的能力降低,所以诱导肺癌细胞凋亡是其治疗的重要手段。全反式维甲酸(all-trans retinoic acid,ATRA)是一种维A酸类衍生产物,可调节细胞增殖、分化、凋亡等,其中对细胞的诱导分化作用最强,它应用于治疗急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)方面的临床效果显著,临床缓解率可达到85%以上;在其他实体肿瘤中的实验研究如在胃癌、乳腺癌、胰腺癌等方面的研究,也证实了ATRA应用于实体肿瘤治疗的潜力。研究发现肿瘤疾病的发生、进展以及其转归预后与细胞凋亡密切相关[24]。治疗性的细胞凋亡干预,是一种探索中的肿瘤疾病治疗手段,目前已成为全世界肿瘤治疗领域研究的一个热点。作为凋亡抑制基因家族的新成员survivin基因,于多种肿瘤组织、转化细胞中高表达,鉴于其在肿瘤组织中的表达特异性,以及它抑制细胞凋亡、影响细胞有丝分裂的作用,survivin基因于肿瘤方面的相关研究日渐深入。半胱氨酸天冬氨酸蛋白3(caspase-3)作为生物体发生细胞凋亡的关键因子,其于胞质中是以无活性的酶原形式存在,仅在细胞凋亡发生时被激活。近年来大量研究证实,ATRA可以诱导多种实体瘤细胞发生细胞凋亡,但其诱导凋亡的分子学机制还没有完全清楚。本实验研究中,我们运用ATRA药物作用于荷肺腺癌A549细胞移植瘤的裸鼠,采用RT-PCR方法检测裸鼠移植瘤组织中凋亡相关基因survivin和caspase-3信使核糖核酸(messenger RNA,m RNA)的表达情况,以探讨全反式维甲酸诱导肺腺癌A549细胞发生凋亡的可能机制。为肺癌发展新的治疗方法提供理论依据。目的:通过动物体内实验,探讨ATRA诱导荷瘤裸鼠肺腺癌A549细胞凋亡的可能机制。方法:1.首先培养人肺癌A549细胞,收集处于对数生长期的A549细胞,后将其接种于裸鼠背部皮下,构建裸鼠的肺腺癌A549细胞移植瘤动物模型。将荷瘤裸鼠动物模型随机分为模型组、ATRA组、顺铂组、ATRA+顺铂组4组,每组7只,隔日腹腔注射给药,共给药6次,计算各组荷瘤裸鼠移植瘤体积、体重。末次注射干预后次日脱颈处死裸鼠,取出裸鼠体内移植瘤瘤体。组织标本置于液氮中保存,每组取部分瘤体组织标本于10%甲醛溶液中固定,而后进行常规石蜡包埋及病理切片,以进行后续的实验检测。2.采用原位末端转移酶介导的缺口末端标记(Td T-Mediated d UTP-Biotin Nick End Labeling,TUNEL)染色法,检测4组裸鼠移植瘤组织中的细胞凋亡情况。3.采用逆转录-聚合酶链反应(Reverse Rranscriotion-Polymerase Chain Reaction,RT-PCR)法,检测各组裸鼠移植瘤组织中caspase-3和survivin信使核糖核酸m RNA的表达水平。结果:1.ATRA组、顺铂组及ATRA+顺铂组移植瘤体积、重量明显低于模型组,差异具有统计学意义(P0.05)。ATRA+顺铂组移植瘤体积、重量明显低于ATRA及顺铂单药组,差异具有统计学意义(P0.05)。2.ATRA组、顺铂组及ATRA+顺铂组A549细胞裸鼠移植瘤细胞凋亡率较模型组显著增加,差异具有统计学意义(P0.05);ATRA+顺铂组细胞凋亡率明显高于ATRA组与顺铂组,差异具有统计学意义(P0.05)。3.模型组移植瘤组织中caspase-3 m RNA的相对表达量为0.143±0.053,显著低于ATRA组、顺铂组及ATRA+顺铂联合用药组,差异具有统计学意义(P0.05);ATRA组与顺铂组移植瘤组织中caspase-3 m RNA相对表达量均明显低于联合用药组,差异具有统计学意义(均P0.05)。模型组移植瘤组织中survivin m RNA的相对表达量为0.623±0.125,显著高于ATRA组、顺铂组及ATRA+顺铂联合用药组,差异具有统计学意义(P0.05);ATRA组与顺铂组移植瘤组织中survivin m RNA相对表达量均明显高于联合用药组,差异具有统计学意义(均P0.05)。结论:1.ATRA与顺铂均能抑制人肺癌A549细胞裸鼠移植瘤的生长,促进移植瘤组织内细胞凋亡增加,且二者联合作用更强。2.ATRA可诱导荷瘤裸鼠肺腺癌A549细胞发生凋亡,其作用机制可能与上调caspase-3 m RNA的表达以及下调survivin m RNA的表达有关。
[Abstract]:Lung cancer is one of the most common malignant tumors in China. The incidence and mortality rate are increasing year by year, and the trend is young. It is a serious threat to human life and health. It has become one of the major public health problems in the world. Because of the early clinical symptoms of lung cancer, the clinical symptoms are easily ignored, which leads to the late diagnosis of many lung cancer patients and the loss of surgical treatment opportunities. It is only conservative treatment, and even the early diagnosis of lung cancer patients still have a certain tumor recurrence rate after complete resection of primary lesions. Now, chemotherapy, as an important treatment for lung cancer, is also of certain value for patients after radical resection. Before, platinum is Non-Small Cell Lung Can The basis of CER, NSCLC) chemotherapy regimen, combined with other chemotherapeutic drugs such as Taxol and gemcitabine, is still the advanced first-line chemotherapy regimen of advanced NSCLC. With the extensive use of chemotherapy, the drug resistance of chemotherapeutic drugs is also highlighted. Multidrug resistance is the main obstacle to successful chemotherapy. Chemotherapeutic drug resistance is mainly due to chemotherapy induced swelling. The apoptosis ability of tumor cells is reduced, so inducing apoptosis of lung cancer cells is an important means of treatment. All-trans retinoic acid (ATRA) is a derivative of vitamin A, which can regulate cell proliferation, differentiation, apoptosis and so on, which is the most effective in inducing differentiation of cells. It is used in the treatment of acute promyelocytic leukemia. The clinical effects of (acute promyelocytic leukemia, APL) are significant, and the clinical remission rate can be over 85%. Experimental studies in other solid tumors, such as gastric cancer, breast cancer, and pancreatic cancer, have also confirmed the potential of ATRA in the treatment of solid tumors. [24]. therapeutic intervention with apoptosis, which is closely related to apoptosis, is an exploration of tumor disease treatment. It has become a hot spot in the field of cancer treatment all over the world. As a new member of the family of apoptosis suppressor gene, survivin gene is highly expressed in many tumor tissues and transformed cells, in view of its tumor The expression in the tissue is specific, and it inhibits apoptosis and affects cell mitosis, and the related research of survivin gene in tumor is deepening. Cysteine aspartic acid protein 3 (caspase-3) is the key factor of cell apoptosis in organism. It exists in cytoplasm in the form of inactive zymogen, only in fine form. In recent years, a large number of studies have proved that ATRA can induce apoptosis in various solid tumor cells, but the molecular mechanism of its induction of apoptosis is not completely clear. In this experimental study, we used ATRA drugs in nude mice of A549 cell xenografts in the lung adenocarcinoma of the lung. The RT-PCR method was used to detect nude mice. The expression of apoptosis related genes survivin and caspase-3 messenger ribonucleic acid (messenger RNA, m RNA) in the tumor tissue, in order to explore the possible mechanism of apoptosis induced by all trans retinoic acid induced lung adenocarcinoma A549 cells, and to provide a theoretical basis for the development of new treatment methods for lung cancer. Objective: to explore the induced tumor bearing nude mice induced by ATRA through the animal experiments. The possible mechanism of apoptosis of lung adenocarcinoma A549 cells. 1. first, the human lung cancer A549 cells were cultured and the A549 cells in the logarithmic growth period were collected. Then they were inoculated into the nude mouse's back subcutaneous, and the nude mice were constructed with A549 cell xenografts. The tumor bearing nude mice were divided into model group, ATRA group, cisplatin group, and ATRA+ cisplatin group 4. 7 rats in each group were injected intraperitoneally for 6 times every other day to calculate the volume and weight of the transplanted tumor in nude mice. After the last injection, the nude mice were removed from the nude mice and removed from the nude mice on the next day. The tissue specimens were stored in the liquid nitrogen, and each group was fixed in 10% Formaldehyde Solution, and then the conventional stone was carried out. Paraffin embedding and pathological sections for subsequent experimental detection of.2. using in situ terminal transferase mediated nick end labeling (Td T-Mediated D UTP-Biotin Nick End Labeling, TUNEL) staining, detection of apoptosis in 4 groups of nude mice transplanted tumor tissue.3. using reverse transcriptase polymerase chain reaction (Reverse Rranscriotion-Polymerase) The expression of Caspase-3 and Survivin messenger RNA m RNA in the transplanted tumor tissues of nude mice was detected by Chain Reaction, RT-PCR. Results: the volume of the transplanted tumor in the group 1.ATRA, the cisplatin group and the ATRA+ cisplatin group was significantly lower than that in the model group. The difference was statistically significant (P0.05).ATRA+ cisplatin group, the weight of the transplanted tumor was significantly lower than that of ATRA. In the group of cisplatin, the difference was statistically significant (P0.05).2.ATRA, the apoptosis rate of transplanted tumor cells in nude mice of cisplatin group and ATRA+ cisplatin group increased significantly compared with that of model group, the difference was statistically significant (P0.05), and the apoptosis rate of ATRA+ cisplatin group was significantly higher than that in ATRA group and cisplatin group, and the difference was statistically significant (P0.05).3. model group shift. The relative expression of Caspase-3 m RNA in the tumor tissue was 0.143 + 0.053, significantly lower than that in the ATRA group. The difference was statistically significant (P0.05), and the relative expression of Caspase-3 m RNA in the transplanted tumor tissues of the ATRA group and the cisplatin group was significantly lower than that of the combined drug group, and the difference was statistically significant (P0.05). The relative expression of survivin m RNA in the transplanted tumor tissues of the group was 0.623 + 0.125, significantly higher than that in the ATRA group. The difference was statistically significant (P0.05), and the relative expression of survivin m RNA in the transplanted tumor tissues of the ATRA group and the cisplatin group was significantly higher than that in the combination group, and the difference was statistically significant (P0.05) Conclusion: both 1.ATRA and cisplatin can inhibit the growth of human lung cancer A549 cell xenografts in nude mice and increase the apoptosis in the transplanted tumor tissues, and the combination of two stronger.2.ATRA can induce apoptosis of A549 cells in lung adenocarcinoma of nude mice. The mechanism may be associated with up regulation of the expression of Caspase-3 M RNA and down regulation of the expression of survivin m RNA. Of

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2

【参考文献】