泪腺腺样囊性癌中Survivin，Livin，Caspase-3基因表达及银杏叶提取物的抑癌作用

发布时间：2018-08-26 10:03

【摘要】：背景腺样囊性癌是一种恶性肿瘤,它起源于腺体,在颜面部的涎腺中发生最多,其次是泪腺。泪腺腺样囊性癌是泪腺上皮性肿瘤中最为常见并且恶性程度最高的肿瘤,其发生率居于第二位,仅次于多形性腺瘤。泪腺腺样囊性癌进展快,预后差,且其发病机制不明,除了手术外尚无有效治疗方法。从基因和蛋白质水平探讨泪腺腺样囊性癌的发生机制,寻找与其发生相关的生物学标记物成为研究的一个热点。分子生物学方面的研究认为,肿瘤是一类由于某些染色体上的DNA损伤引起细胞内某些基因异常表达,以致细胞生长失去控制、缺乏分化及异常增生的一类复杂的基因疾病。现代医学认为,在肿瘤的发生发展过程中,与其相关的特征基因在不同组织(如癌、瘤、正常组织)的表达具有差异性。探索发现这些表达差异的特征基因,对进一步了解肿瘤的发病机制、提高治疗的针对性、做好早期预防及预后有着重大意义。Survivn和Livin是新近发现的IAPs家族中两种最具有表达特异性的凋亡抑制蛋白,能够直接或间接抑制Caspase-3凋亡信号转导过程中相关因子的活性,成为研究的重点。另外,在现代抗肿瘤药物中,中药占居重要的地位,其中银杏叶提取物有明显的抗肿瘤活性及辅助化疗作用,其能够通过调节凋亡基因的表达来抑制肿瘤细胞增殖和加速细胞凋亡,且对正常组织细胞的不良反应较少,近年来在临床上的应用越来越广泛。然而,目前Survivin、Livin和Caspase-3在泪腺腺样囊性癌中的表达分析,及银杏叶提取物在泪腺腺样囊性癌发生发展中的作用很少有报道。目的通过比较Survivin、Livin和Caspase-3在泪腺腺样囊性癌、泪腺多形性腺瘤和正常泪腺组织中的表达差异,探讨其在泪腺腺样囊性癌发生、发展中的作用及与临床预后之间的关系；探讨中药银杏叶提取物对人泪腺腺样囊性癌细胞株增殖、凋亡及Survivin、Livin和Survivin Caspase-3表达的影响,为明确泪腺腺样囊性癌的发病机理及探索新的治疗方向提供一定的理论基础。方法 1.采用RT-PCR、免疫印迹及免疫组化的方法检测Survivin、Livin和Caspase-3在泪腺腺样囊性癌、泪腺多形性腺瘤及正常泪腺组织中的表达情况,利用数学算法探讨基因间表达的相关性。 2.通过体外培养人泪腺腺样囊性癌ACC-2细胞株,应用MTT法检测细胞增殖,Annexin V/PI双染色流式细胞仪检测细胞凋亡和细胞周期；免疫印迹法检测蛋白表达来分析银杏叶提取物(EGB)对泪腺腺样囊性癌ACC-2细胞增殖、凋亡及Survivin、Livin和Caspase-3蛋白表达的影响。结果 1. Livin和Survivin在正常泪腺组织中低表达或者不表达,在多形性腺瘤和泪腺腺样囊性癌中高表达,并且随着肿瘤恶性程度的增加表达量不断增加。Caspase-3在正常泪腺组织中高表达,在泪腺多形性腺瘤和泪腺腺样囊性癌中的表达量随肿瘤恶性程度的增加而下降。 2. Livin和Survivin在泪腺腺样囊性癌中的表达均与Caspase-3呈负相关,但是二者之间没有相关性。Caspase-3和Livin在泪腺腺样囊性癌中的表达与其临床分期、病理类型和转移有关系,Survivin仅与肿瘤的大小及转移有关系。 3.不同浓度的EGB对人泪腺腺样囊性癌ACC-2细胞的体外增殖均有一定的抑制作用,并且随着EGB浓度的升高,抑制作用增强,量效关系显著(P0.01)。EGB的加入能够使ACC-2细胞Go-G1期逐渐增加,G2-M期和S期逐渐减少,并且随着剂量的增加,ACC-2细胞凋亡率明显增加。EGB不同浓度对ACC-2细胞Livin和Survivin基因均有一定的抑制作用,并且随着EGB浓度的升高,抑制作用增强。与之相反,Caspase-3基因的相对含量则随着EGB给药剂量的加大而逐渐增高。结论 1.泪腺腺样囊性癌的发病可能与Livin和Survivin基因的表达异常上调及Caspase-3基因的表达下调有关。Caspase-3、Livin的表达与泪腺腺样囊性癌的临床分期、病理类型和转移有关,Survivin与肿瘤大小及是否转移有关。 2. Livin和Survivin在泪腺腺样囊性癌中的表达均与Caspase-3呈负相关,但是二者之间没有相关性。 3.银杏叶提取物能够抑制人腺样囊性癌ACC-2细胞的体外增殖、促进细胞凋亡,对Caspase-3基因的表达有一定的促进作用,而对Livin和Survivin基因的表达有一定的抑制作用,并且随剂量的增加,其促进或抑制效应增强。
[Abstract]:background
Adenoid cystic carcinoma (ACC) is a malignant tumor originating from the gland and occurring most frequently in the salivary glands of the face, followed by the lacrimal gland. The pathogenesis of adenoid cystic carcinoma of the lacrimal gland is unknown, and there is no effective treatment except surgery. To explore the mechanism of the occurrence of adenoid cystic carcinoma of the lacrimal gland from the level of gene and protein, and to find the biological markers related to the occurrence of adenoid cystic carcinoma of the lacrimal gland has become a hot research topic. A complex genetic disease characterized by abnormal expression of certain genes in the cells leading to uncontrolled cell growth, lack of differentiation and dysplasia. Modern medicine considers that the expression of characteristic genes associated with tumors is different in different tissues (such as cancer, tumor, normal tissue) during the development of tumors. Survivn and Livin are two of the most specific inhibitors of apoptosis in IAPs family, which can directly or indirectly inhibit caspase-3 apoptosis signal transduction. In addition, in modern anti-tumor drugs, traditional Chinese medicine occupies an important position, Ginkgo biloba extract has obvious anti-tumor activity and adjuvant chemotherapy effect, which can inhibit the proliferation of tumor cells and accelerate apoptosis by regulating the expression of apoptotic genes, and is harmful to normal tissue cells. However, the expression of Survivin, Livin and Caspase-3 in lacrimal adenoid cystic carcinoma and the role of Ginkgo biloba extract in the development of lacrimal adenoid cystic carcinoma are rarely reported.
objective
To compare the expression of Survivin, Livin and Caspase-3 in adenoid cystic carcinoma of lacrimal gland, pleomorphic adenoma of lacrimal gland and normal lacrimal gland tissues, and to explore the role of Survivin, Livin and Caspase-3 in the occurrence, development and clinical prognosis of adenoid cystic carcinoma of lacrimal gland. And the effects of Survivin, Livin and Survivin Caspase-3 expression on lacrimal adenoid cystic carcinoma provide a theoretical basis for clarifying the pathogenesis of lacrimal adenoid cystic carcinoma and exploring new therapeutic directions.
Method
1. The expressions of Survivin, Livin and Caspase-3 in lacrimal adenoid cystic carcinoma, pleomorphic adenoma and normal lacrimal gland tissues were detected by RT-PCR, Western blotting and immunohistochemistry. The correlation of gene expression was studied by mathematical algorithm.
2. Human lacrimal adenoid cystic carcinoma ACC-2 cell line was cultured in vitro, cell proliferation was detected by MTT method, cell apoptosis and cell cycle were detected by Annexin V/PI double staining flow cytometry, and the proliferation, apoptosis and Survivin, Livin and Casp of lacrimal adenoid cystic carcinoma ACC-2 cells were analyzed by immunoblotting. The effect of ase-3 protein expression.
Result
1. Livin and Survivin were low or non-expressed in normal lacrimal gland tissues, high expression in pleomorphic adenoma and lacrimal adenoid cystic carcinoma, and increased with the increase of malignancy. Caspase-3 was high expression in normal lacrimal gland tissues and high expression in pleomorphic adenoma and lacrimal adenoid cystic carcinoma. The degree of malignancy increases and decreases.
2. The expression of Livin and Survivin in lacrimal adenoid cystic carcinoma was negatively correlated with Caspase-3, but there was no correlation between them.
3. Different concentrations of EGB inhibited the proliferation of ACC-2 cells in vitro, and with the increase of EGB concentration, the inhibition was enhanced. The dose-effect relationship was significant (P 0.01). The addition of EGB could gradually increase the Go-G1 phase of ACC-2 cells, decrease the G2-M phase and S phase, and with the increase of dose, ACC-2 cells withered. Different concentrations of EGB inhibited Livin and Survivin genes in ACC-2 cells, and the inhibition increased with the increase of EGB concentration. On the contrary, the relative content of Caspase-3 gene increased gradually with the increase of EGB dosage.
conclusion
1. The pathogenesis of lacrimal adenoid cystic carcinoma may be related to the abnormal expression of Livin and Survivin genes and the down-regulation of Caspase-3 genes. The expression of Caspase-3 and Livin is related to the clinical stage, pathological type and metastasis of lacrimal adenoid cystic carcinoma. Survivin is related to tumor size and metastasis.
2. The expression of Livin and Survivin in lacrimal adenoid cystic carcinoma was negatively correlated with Caspase-3, but there was no correlation between them.
3. Ginkgo biloba extract can inhibit the proliferation of human adenoid cystic carcinoma ACC-2 cells in vitro, promote cell apoptosis, promote the expression of Caspase-3 gene to a certain extent, but inhibit the expression of Livin and Survivin gene to a certain extent, and with the increase of dosage, its promotion or inhibition effect is enhanced.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.7;R285

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