凋亡抑制蛋白家族在莱菔硫烷抑制腺样囊性癌ACC-M裸鼠移植瘤中作用的实验研究

发布时间：2018-05-04 20:16

本文选题：裸鼠 + 莱菔硫烷　；参考：《河北医科大学》2017年硕士论文

【摘要】：背景:涎腺腺样囊性癌(salivary adenoid cystic carcinoma,SACC)是一种恶性肿瘤,主要来源于涎腺上皮。具有如下特点:局部浸润性非常强,极易早期侵犯神经和血管,所以癌细胞容易沿血液扩散到远处。采用手术方法切除肿瘤后,仍然有较高的局部复发率和远处转移率,SACC的肺部转移率远远高于颌面部的其它肿瘤。截止目前,人类在SACC方面的相关研究成果还不能清楚地阐述它的发病及转移机制,所以我们要继续研究SACC的机制,争取从分子和蛋白的层面,搞清楚腺样囊性癌的作用机理,为早日在临床上应用新的抗癌方法或抗癌药物做出贡献。莱菔硫烷(sulforaphane,SFN)是从十字花科蔬菜中提取出来的一种植物性物质,它属于异硫氰酸盐(isothiocyanates,ITCS)类,这种物质有抗癌功效。我们前期关于莱菔硫烷的体外研究成果显示,莱菔硫烷可以提升半胱天冬酶(Caspase)的功能,加速了肿瘤细胞的凋亡,从而抑制了腺样囊性癌细胞系ACC-M,在这个过程中,Bcl-2家族成员也起到了重要作用。凋亡抑制蛋白家族(inhibitor of apoptosis protein,IAP)主要功能是调节细胞凋亡,可以通过抑制Caspase来抑制细胞凋亡,还能参与细胞周期与细胞分裂的调节等。其家族成员共有八位,分别是c-IAP1、c-IAP2、Survivin、Livin、X-IAP、NAIP、Bruce、ILP-2。c-IAP1、c-IAP2、X-IAP能够直接抑制Caspase-3、-7、-9,而Survivin不但能够抑制Caspase-3、-7,还能增强X-IAP等其它IAP家族成员的抗凋亡作用。Livin的作用是降低Caspase-3、-7、-9的活性,ILP-2可以调节Bcl-2的表达,并且使其升高,降低Caspase-9的活性,达到抑制细胞凋亡的目的。NAIP的研究主要集中在神经系统,Bruce可抑制Caspase-9及Smac的活性。在莱菔硫烷抑制腺样囊性癌细胞系ACC-M的增殖过程中,凋亡抑制蛋白家族成员是不是起到一定的作用,在经过SFN处理的人腺样囊性癌裸鼠移植瘤组织中,凋亡抑制蛋白是否表达异常?以往的研究成果尚不能合理的解释这一问题。目的:观察移植瘤被莱菔硫烷抑制的过程中,IAP家族成员的作用如何,是否受到抑制,家族中各个成员的表现是否一致,有没有其它蛋白分子干扰IAP蛋白的作用,研究其可能的分子作用机制,为日后临床应用莱菔硫烷抗癌提供理论依据。方法:1材料1.1人涎腺腺样囊性癌细胞株ACC-M购自上海交大颌面外科实验室,该细胞株建于1995年。1.2莱菔硫烷(SFN)购自美国LKT实验室,纯度≥99%。1.3裸鼠自北京军事医学科学院购买,雄性10只,4~6周龄,体重为16~20克。1.4抗体从英国Abcam公司购买的X-IAP多克隆抗体,c-IAP2多克隆抗体,c-IAP1多克隆抗体,Livin多克隆抗体,从美国AR公司购买Survivin多克隆抗体,自无锡傲锐东源生物科技有限公司购买二抗酶标羊抗小鼠/兔Ig G聚合物。2实验方法2.1细胞培养将冻存的人涎腺腺样囊性癌细胞复苏,然后在37度恒温培养箱中培养。细胞密度达到要求时,开始传代,最后收集细胞悬液。2.2移植瘤的建立碘酊消毒裸鼠的左前背部以及右后背部,然后将细胞悬液分别注射于上述部位,经口腔给药,实验组予以莱菔硫烷(6 mmol SFN/0.1ml PBS)口服,而对照组口服等量的PBS,每周分三次给药。2.3解剖瘤体三周以后开始将裸鼠处死,然后解剖瘤体,清除掉瘤体表面的血管、坏死组织。然后用石蜡包埋肿瘤组织块,蜡块准备完毕后行免疫组化实验。2.4免疫组化本实验采用免疫组化的方法,观察IAP家族成员中c-IAP1、c-IAP2、Survivin、Livin、XIAP五种蛋白在裸鼠移植瘤组织中的表达情况,并统计染色阳性细胞数目。2.5数据的统计处理本研究有实验、对照两个组,每组10个样本,统计每个样本的随机5个视野阳性细胞总数,用t检验的方法进行分析,最后得出结论。结果:1实验组中,五个视野中c-IAP1阳性细胞总数为1027.2±107.88个,与对照组的1210.6±108.31个相比,阳性细胞数明显减少,有统计学差异(P0.05);2实验组中,五个视野中c-IAP2阳性细胞总数为995.4±113.35个,与对照组的1140.30±122.35个相比,阳性细胞数减少明显,有统计学差异(P0.05);3实验组中,五个视野中Livin阳性细胞总数为1038±156.10个,与对照组的1163.5±85.21个相比,阳性细胞数减少明显,有统计学差异(P0.05);4实验组中,五个视野中Survivin阳性细胞总数为547.80±189.77个,与对照组的959.30±179.77个相比,阳性细胞数减少非常明显,有统计学差异(P0.01);5实验组中,五个视野中X-IAP阳性细胞总数为1072±86.95个,与对照组的1183.2±127.49个相比,阳性细胞数减少明显,有统计学差异(P0.05)。结论:口服莱菔硫烷能够使人涎腺腺样囊性癌ACC-M裸鼠移植瘤中IAP家族成员c-IAP1、c-IAP2、Survivin、Livin、XIAP的表达明显受到抑制。提示IAP家族成员在莱菔硫烷诱导人涎腺腺样囊性癌ACC-M移植瘤细胞凋亡的过程中起重要作用,这可能是莱菔硫烷诱导腺样囊性癌凋亡的机制之一。
[Abstract]:Background: salivary adenoid cystic carcinoma (SACC) is a malignant tumor, mainly from the salivary epithelium. It has the following characteristics: the local invasion is very strong, and it is very vulnerable to the early invasion of the nerve and blood vessels, so the cancer cells are easy to spread along the blood. After surgical resection of the tumor, there is still a higher position. The lung metastasis rate of SACC is far higher than that of other tumors in the maxillofacial region. So far, the related research results in SACC can not clearly explain its pathogenesis and metastasis mechanism, so we should continue to study the mechanism of SACC and strive for the understanding of adenoid cystic carcinoma from the molecular and protein level. Sulforaphane (SFN) is a plant substance extracted from the cruciferous vegetables, and it belongs to the isothiocyanates (ITCS) class. This substance has anti-cancer effect. The results show that sulforaphane can enhance the function of caspase (Caspase), accelerate the apoptosis of tumor cells and inhibit the adenoid cystic carcinoma cell line ACC-M. In this process, the members of the Bcl-2 family also play an important role. The main function of the apoptosis inhibitory protein family (inhibitor of apoptosis protein, IAP) is to regulate the main function. Apoptosis can be inhibited by inhibiting Caspase to inhibit cell apoptosis, and can also participate in the regulation of cell cycle and cell division. The family members have eight members, which are c-IAP1, c-IAP2, Survivin, Livin, X-IAP, NAIP, Bruce, ILP-2.c-IAP1, c-IAP2. The anti apoptotic effect of.Livin, such as X-IAP and other IAP family members, is to reduce the activity of Caspase-3, -7, -9. ILP-2 can regulate the expression of Bcl-2, and make it increase, reduce the activity of Caspase-9, and achieve the purpose of inhibiting the apoptosis of.NAIP mainly concentrated in the divine system, Bruce can inhibit Caspase-9 and activity. Does sulforaphane inhibit the proliferation of adenoid cystic carcinoma cell line ACC-M, does the member of the apoptosis inhibitory protein family play a certain role. Is the apoptosis inhibitory protein expression abnormal in the transplanted tumor tissue of human adenoid cystic carcinoma treated with SFN? The previous research results can not explain this problem reasonably. In the process of inhibition of sulforaphane, how is the role of IAP family members, whether it is inhibited, the expression of each member of the family is consistent, the role of other protein molecules interfering with IAP protein, and the possible molecular mechanism of its molecular action to provide a theoretical basis for the future clinical application of sulforaphane anticancer. Method: 1 material 1.1 The human salivary adenoid cystic carcinoma cell line ACC-M was purchased from the Shanghai Jiaotong maxillofacial surgery laboratory. The cell line was built in 1995.1.2 sulforaphane (SFN) purchased from the American LKT laboratory. The purity of the cell line was purchased from the Military Medical Science Academy of the PLA, the purity of the nude mice from the Military Medical Science Academy of the PLA, Beijing, and the male 10, 4~6 weeks old, and the weight of 16 ~20 grams of.1.4 antibody purchased from the UK Abcam company. C-IAP2 polyclonal antibody, c-IAP1 polyclonal antibody, Livin polyclonal antibody, Livin polyclonal antibody, purchased Survivin polyclonal antibody from American AR company, purchased two anti enzyme labelled Sheep anti mouse / rabbit Ig G polymer.2 experiment method and 2.1 cell culture, and then resuscitated the cryopreserved human salivary gland adenoid cystic carcinoma cells, and then, then the human salivary gland adenoid cystic carcinoma cells are resuscitated. In the 37 degree constant temperature incubator, the cell density reached the requirement, and the cell suspension.2.2 transplanted tumor was finally collected to establish iodine tincture to disinfect the left anterior back and right back of the nude mice. Then the cell suspension was injected into the above part, and the oral administration was given by the oral administration, and the experimental group was given oral sulforaphane (6 mmol SFN/0.1ml PBS), while the experimental group was given oral administration. After taking oral equal amount of PBS, three times a week, the nude mice were dissected three times a week for three weeks. Then the nude mice were executed, then the tumor bodies were dissected and the blood vessels and necrotic tissues were removed. Then the tumor tissue was embedded with paraffin. After the wax was prepared, the immuno histochemical method was used to observe the immunohistochemical method and observe the IA. The expression of five kinds of protein in P family members c-IAP1, c-IAP2, Survivin, Livin, XIAP in the transplanted tumor tissues of nude mice, and statistical processing of the number of.2.5 data for the number of positive cells were statistically analyzed. Compared with the two groups of 10 samples in each group, the total number of positive 5 visual field cells in each sample was statistically analyzed by t test. In the 1 experimental group, the total number of c-IAP1 positive cells in the five field of vision was 1027.2 + 107.88, and the number of positive cells decreased significantly compared with the control group. The number of positive cells decreased significantly (P0.05). In the 2 experimental group, the total number of c-IAP2 positive cells in five fields was 995.4 + 113.35, and 1140.30 + 122.3 of the control group. The number of positive cells decreased significantly in the 5 Comparison (P0.05); in the 3 experimental group, the total number of Livin positive cells in five fields of vision was 1038 + 156.10, compared with 1163.5 + 85.21 of the control group, the number of positive cells decreased significantly (P0.05); in the 4 experimental group, the total number of Survivin positive cells in five fields was 547.80 + 189.7. 7, compared with the 959.30 + 179.77 control group, the number of positive cells decreased very obviously (P0.01). In the 5 experimental group, the total number of X-IAP positive cells in five fields was 1072 + 86.95, compared with the 1183.2 + 127.49 control group, the number of positive cells decreased significantly (P0.05). Conclusion: oral sulforaphane is able to be taken orally. The expression of c-IAP1, c-IAP2, Survivin, Livin, XIAP in human salivary adenoid cystic carcinoma (ACC-M) xenografts in nude mice is obviously inhibited. It suggests that the members of IAP family play an important role in the apoptosis of adenoid cystic carcinoma of human salivary gland induced by sulforaphane, which may be the apoptosis of adenoid cystic carcinoma induced by sulforaphane. One of the mechanisms.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.8

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