本文選題：ATRA + 順鉑　； 參考：《鄭州大學》2015年碩士論文

【摘要】：肺癌是中國最常見的惡性腫瘤之一,現(xiàn)今發(fā)病率、死亡率逐年升高,并呈年輕化趨向,嚴重威脅人類生命健康,已成為世界重大公共衛(wèi)生問題之一。由于肺癌早期臨床癥狀易被忽視,導致許多肺癌患者確診時已至晚期,失去手術治療機會,僅能保守治療,并且即使早期確診的肺癌患者,應用外科手術治療完全切除原發(fā)病灶后,仍然有一定的腫瘤復發(fā)率。現(xiàn)如今,化療作為肺癌的重要治療方法,其對于根治術后患者也有一定價值。目前,鉑類是非小細胞肺癌(Non-Small Cell Lung Cancer,NSCLC)的化療方案的基礎,其聯(lián)合如紫杉醇、吉西他濱等其他化療藥物仍是晚期NSCLC標準一線化療方案。隨著化療在臨床的廣泛應用,化療藥物的耐藥現(xiàn)象也突顯,多藥耐藥是成功化療的主要障礙,化療耐藥主要是由于化療藥物誘導腫瘤細胞凋亡的能力降低,所以誘導肺癌細胞凋亡是其治療的重要手段。全反式維甲酸(all-trans retinoic acid,ATRA)是一種維A酸類衍生產物,可調節(jié)細胞增殖、分化、凋亡等,其中對細胞的誘導分化作用最強,它應用于治療急性早幼粒細胞白血病(acute promyelocytic leukemia,APL)方面的臨床效果顯著,臨床緩解率可達到85%以上;在其他實體腫瘤中的實驗研究如在胃癌、乳腺癌、胰腺癌等方面的研究,也證實了ATRA應用于實體腫瘤治療的潛力。研究發(fā)現(xiàn)腫瘤疾病的發(fā)生、進展以及其轉歸預后與細胞凋亡密切相關[24]。治療性的細胞凋亡干預,是一種探索中的腫瘤疾病治療手段,目前已成為全世界腫瘤治療領域研究的一個熱點。作為凋亡抑制基因家族的新成員survivin基因,于多種腫瘤組織、轉化細胞中高表達,鑒于其在腫瘤組織中的表達特異性,以及它抑制細胞凋亡、影響細胞有絲分裂的作用,survivin基因于腫瘤方面的相關研究日漸深入。半胱氨酸天冬氨酸蛋白3(caspase-3)作為生物體發(fā)生細胞凋亡的關鍵因子,其于胞質中是以無活性的酶原形式存在,僅在細胞凋亡發(fā)生時被激活。近年來大量研究證實,ATRA可以誘導多種實體瘤細胞發(fā)生細胞凋亡,但其誘導凋亡的分子學機制還沒有完全清楚。本實驗研究中,我們運用ATRA藥物作用于荷肺腺癌A549細胞移植瘤的裸鼠,采用RT-PCR方法檢測裸鼠移植瘤組織中凋亡相關基因survivin和caspase-3信使核糖核酸(messenger RNA,m RNA)的表達情況,以探討全反式維甲酸誘導肺腺癌A549細胞發(fā)生凋亡的可能機制。為肺癌發(fā)展新的治療方法提供理論依據(jù)。目的:通過動物體內實驗,探討ATRA誘導荷瘤裸鼠肺腺癌A549細胞凋亡的可能機制。方法:1.首先培養(yǎng)人肺癌A549細胞,收集處于對數(shù)生長期的A549細胞,后將其接種于裸鼠背部皮下,構建裸鼠的肺腺癌A549細胞移植瘤動物模型。將荷瘤裸鼠動物模型隨機分為模型組、ATRA組、順鉑組、ATRA+順鉑組4組,每組7只,隔日腹腔注射給藥,共給藥6次,計算各組荷瘤裸鼠移植瘤體積、體重。末次注射干預后次日脫頸處死裸鼠,取出裸鼠體內移植瘤瘤體。組織標本置于液氮中保存,每組取部分瘤體組織標本于10%甲醛溶液中固定,而后進行常規(guī)石蠟包埋及病理切片,以進行后續(xù)的實驗檢測。2.采用原位末端轉移酶介導的缺口末端標記(Td T-Mediated d UTP-Biotin Nick End Labeling,TUNEL)染色法,檢測4組裸鼠移植瘤組織中的細胞凋亡情況。3.采用逆轉錄-聚合酶鏈反應(Reverse Rranscriotion-Polymerase Chain Reaction,RT-PCR)法,檢測各組裸鼠移植瘤組織中caspase-3和survivin信使核糖核酸m RNA的表達水平。結果:1.ATRA組、順鉑組及ATRA+順鉑組移植瘤體積、重量明顯低于模型組,差異具有統(tǒng)計學意義(P0.05)。ATRA+順鉑組移植瘤體積、重量明顯低于ATRA及順鉑單藥組,差異具有統(tǒng)計學意義(P0.05)。2.ATRA組、順鉑組及ATRA+順鉑組A549細胞裸鼠移植瘤細胞凋亡率較模型組顯著增加,差異具有統(tǒng)計學意義(P0.05);ATRA+順鉑組細胞凋亡率明顯高于ATRA組與順鉑組,差異具有統(tǒng)計學意義(P0.05)。3.模型組移植瘤組織中caspase-3 m RNA的相對表達量為0.143±0.053,顯著低于ATRA組、順鉑組及ATRA+順鉑聯(lián)合用藥組,差異具有統(tǒng)計學意義(P0.05);ATRA組與順鉑組移植瘤組織中caspase-3 m RNA相對表達量均明顯低于聯(lián)合用藥組,差異具有統(tǒng)計學意義(均P0.05)。模型組移植瘤組織中survivin m RNA的相對表達量為0.623±0.125,顯著高于ATRA組、順鉑組及ATRA+順鉑聯(lián)合用藥組,差異具有統(tǒng)計學意義(P0.05);ATRA組與順鉑組移植瘤組織中survivin m RNA相對表達量均明顯高于聯(lián)合用藥組,差異具有統(tǒng)計學意義(均P0.05)。結論:1.ATRA與順鉑均能抑制人肺癌A549細胞裸鼠移植瘤的生長,促進移植瘤組織內細胞凋亡增加,且二者聯(lián)合作用更強。2.ATRA可誘導荷瘤裸鼠肺腺癌A549細胞發(fā)生凋亡,其作用機制可能與上調caspase-3 m RNA的表達以及下調survivin m RNA的表達有關。
[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

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R734.2

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本文編號：1843568