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Bcl-2家族成员在莱菔硫烷抑制人涎腺腺样囊性癌移植瘤中的作用

发布时间:2018-06-28 10:07

  本文选题:裸鼠 + 莱菔硫烷 ; 参考:《河北医科大学》2014年硕士论文


【摘要】:目的:裸鼠是一种全身没有毛发,缺乏胸腺的动物。胸腺具有制造T细胞的功能。裸鼠缺乏T细胞的原因是由于其一基因的突变,这种突变基因被称为nu基因,属于隐性遗传基因,只有当nu/nu结合时,才能出现表现型。T细胞具有免疫的功能,能够辅助机体抑制以及杀伤进入体内的外源物质。没有胸腺,不能产生T细胞的裸鼠,其免疫力很低,当外源物质进入体内时,不能够及时的被抑制或者消除。基于裸鼠的这些特征,其就成为了移植瘤模型的一种理想动物。通常,裸鼠经常被用来研究恶性肿瘤的治疗。根据裸鼠的这些特征,裸鼠移植瘤模型就成为本次实验研究Bcl-2家族成员在莱菔硫烷抑制人涎腺腺样囊性癌移植瘤中的作用的理想模型。 涎腺腺样囊性癌(salivary adenoid cystic carcinoma,SACC)是一种恶性程度较高的口腔颌面部肿瘤。其临床特征为转移性强,浸润周围神经生长等。细胞学特征主要是基底细胞样肿瘤,由上皮细胞和肌上皮细胞组成并排列成类管状,类筛网状,和实性巢样等不同的结构。 根据我们前期实验,莱菔硫烷(Sulforaphane,SFN)能够在体外抑制涎腺腺样囊性癌ACC-M细胞的增殖并诱导细胞凋亡。SFN诱导涎腺腺样囊性癌凋亡的过程是由Caspase介导的。另外,在体外莱菔硫烷诱导人涎腺腺样囊细胞凋亡的过程中,Bcl-2家族成员起到了不可忽视的作用:莱菔硫烷可以引起促凋亡蛋白因子Bax、Bak的表达增加,而使抗凋亡蛋白因子Bcl-2和Bcl-xl的表达下降。我们刚刚进行的莱菔硫烷抑制人腺样囊性癌裸鼠移植瘤的体内实验研究显示,体内环境中,莱菔硫烷可抑制腺样囊性癌ACC-M裸鼠移植瘤的增殖生长,并能诱导腺样囊性癌ACC-M在裸鼠移植瘤中发生细胞凋亡。在这个过程中,Bcl-2蛋白家族是否也和体外实验一样,起到重要作用,国内外尚未见报道。本研究的目的,是利用人涎腺腺样囊性癌细胞构建的裸鼠移植瘤模型,通过免疫组织化学方法来验证Bcl-2家族蛋白的表达是否与体外实验中莱菔硫烷诱导人腺样囊性癌ACC-M细胞凋亡的情况一致。 方法: 1细胞培养:复苏冻存的人涎腺腺样囊性癌细胞,在37度,5%CO2的恒温培养箱中进行贴壁培养。当细胞达到一定浓度时,用0.25%的胰酶进行消化传代,收集并调整细胞密度为2×107/ml的细胞悬液。 2建立移植瘤模型:将预备好的细胞悬液注射于裸鼠的左前背部以及右后背部,并分组(给药组、对照组)经口腔给药,给药组给予莱菔硫烷(6mmol SFN/0.1ml PBS),对照组给予等量的PBS,每周给药3次。观察。 3解剖瘤体:三周后,采用断颈的方法处死裸鼠,通过外科手术的方法剥离瘤体,,去除瘤体表面的坏死组织,以及小血管组织等。剥离下来的肿瘤组织经过石蜡的包埋,处理,制成蜡块,备用。 4免疫组织化学染色:将制成的蜡片,采用免疫组织化学染色的方法,在显微镜下观察Bcl-2家族成员中Bcl-2、Bax、Bcl-xl、Bak四种蛋白的表达情况,并记分。 5统计学分析:采用秩和检验的方法对等级资料结果进行统计计算分析,并得出结论。 结果: 与对照组相比,实验组抑制凋亡蛋白Bcl-2、Bcl-xl表达显著减少,有统计学差异(P<0.05);促凋亡蛋白Bax、Bak蛋白的表达略有增加,但无统计学差异(P>0.05)。 结论: 在体内用莱菔硫烷诱导人涎腺腺样囊性癌细胞ACC-M移植瘤的凋亡过程中,Bcl-2家族成员起重要作用。
[Abstract]:Objective: nude mice are a kind of animals without hair and thymus. The thymus has the function of producing T cells. The reason for the lack of T cells in nude mice is due to the mutation of one gene. This mutant gene is called the Nu gene, which belongs to the recessive gene. Only when nu/nu is combined, can the expressive.T cells have immune function and can be immune. It is sufficient to assist the body to inhibit and kill the exogenous substances that enter the body. Without thymus, nude mice that do not produce T cells have low immunity and can not be suppressed or eliminated in time when exogenous substances enter the body. Based on these characteristics of nude mice, it is an ideal animal for the transplanted tumor model. To study the treatment of malignant tumors. Based on these characteristics of nude mice, the transplanted tumor model in nude mice is an ideal model for the study of the role of Bcl-2 family members in sulforaphane suppressing human salivary adenoid cystic carcinoma.
Salivary adenoid cystic carcinoma (SACC) is a high malignancy of oral and maxillofacial tumors. Its clinical features are strong metastatic and infiltrating peripheral nerve growth. The cytological features are mainly basal cell like tumors, composed of epithelial and myoepithelial cells and arranged into tubular, sieved reticulation, and solid. Different structures such as sex nests.
According to our previous experiments, Sulforaphane (SFN) can inhibit the proliferation of ACC-M cells in salivary adenoid cystic carcinoma in vitro and induce apoptosis of adenoid cystic carcinoma of salivary gland induced by.SFN. In addition, in the process of apoptosis of human salivary gland like sac cells induced by sulforaphane in vitro, the Bcl-2 family is formed. The role played an important role: sulforaphane can cause an increase in the expression of apoptotic protein factor Bax, Bak, and the decrease of the expression of anti apoptotic factor Bcl-2 and Bcl-xl. The proliferation and growth of adenoid cystic carcinoma ACC-M nude mice can induce the apoptosis of the adenoid cystic carcinoma ACC-M in nude mice. In this process, whether the Bcl-2 protein family also plays an important role as in vitro, has not been reported at home and abroad. The purpose of this study is to construct human salivary gland adenoid cystic carcinoma cells. The model of xenograft in nude mice was used to verify whether the expression of Bcl-2 family protein was consistent with the apoptosis of human adenoid cystic carcinoma ACC-M cells induced by sulforaphane in the experiment in vitro.
Method:
1 cell culture: resuscitation of frozen human salivary gland adenoid cystic carcinoma cells, adherent culture at 37 degrees, 5%CO2 constant temperature incubator. When cells reached a certain concentration, the cells were digested with 0.25% pancreatin, and the cell density of cell density of 2 x 107/ml was collected and adjusted.
2 the model of transplanted tumor was established: the prepared cell suspension was injected into the left anterior back and right back of the nude mice. The group (drug group, control group) was given oral administration by oral administration, 6mmol SFN/0.1ml PBS was given to the drug group, the control group was given the same amount of PBS, and the drug was given 3 times a week.
3 anatomic body: after three weeks, the nude mice were killed by the method of breaking the neck. The tumor body was stripped by surgical procedure, the necrotic tissue on the surface of the tumor and the small vascular tissue were removed. The dissected tumor tissue was embedded and processed by paraffin.
4 immunohistochemical staining: the prepared wax tablets, using immunohistochemical staining method, under the microscope observation of the Bcl-2 family members Bcl-2, Bax, Bcl-xl, Bak four kinds of protein expression, and score.
5 statistical analysis: rank sum test was used to analyze and calculate the results of grade data and draw conclusions.
Result:
Compared with the control group, the experimental group inhibited the apoptosis protein Bcl-2, the expression of Bcl-xl decreased significantly (P < 0.05), and the expression of apoptotic protein Bax and Bak protein increased slightly, but there was no statistical difference (P > 0.05).
Conclusion:
Bcl-2 family members play an important role in the apoptosis of human salivary adenoid cystic carcinoma ACC-M cells induced by sulforaphane in vivo.
【学位授予单位】:河北医科大学
【学位级别】:硕士
【学位授予年份】:2014
【分类号】:R739.87

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