萤光素酶报告基因检测法初步探究甲状腺肿瘤与女性体内孕激素水平关系

发布时间：2018-12-12 07:27

【摘要】：甲状腺肿瘤是一种常见的颈部肿瘤,其发病率女性明显高于男性,因而很多学者认为这种差异性很可能与男女体内性激素水平有关。孕激素是一种雌性激素,主要在生殖、发育系统上发挥作用。但是随着孕激素类药物被广泛应用在医疗、畜牧业和农业等上,给社会带来巨大贡献的同时,也给环境带来了严重的激素污染,已经开始影响到生物体自身的内分泌系统。孕激素受体(Progesterone receptor,PR)属于核受体,只有当PR被孕激素激活后才能结合到孕激素受体应答元件的DNA区域,从而启动受体应答元件下游靶基因的表达。本课题利用这种机制并将萤光素酶报告基因克隆到受体应答元件下游,从而建立了一种高灵敏度、高通量的可用于孕激素水平生物检测的稳转细胞株,并将其应用于甲状腺相关肿瘤病人血液中的孕激素类物质水平的检测分析,以期探究孕激素类物质与甲状腺肿瘤发生的关联性。本研究共构建了含有孕激素受体基因的3种不同表达载体和3种含有不同串联倍数的孕激素受体应答元件片段的载体,通过萤光素酶的活性分析,利用质粒共转染技术将活性最高的一对重组质粒pEGFP-N2-hPR/pGL4-PRE4转染到HeLa细胞中,然后经过抗生素筛选和有限稀释法挑选出10个单克隆细胞株。再利用分子克隆技术、亚细胞定位技术鉴定出阳性单克隆细胞株。从第5代到第23代细胞的萤光素酶活性的检测结果表明该细胞株具有较强的遗传稳定性和较高的灵敏度,其检出限可以达到10 pM。因此,该稳转细胞株与萤光素酶活性检测技术结合起来即建立孕激素检测分析系统。同时,还观察到细胞株的萤光素酶活性随着外界环境中孕激素浓度的升高而增强,但是浓度高于106 pM孕激素反而会降低萤光素酶活性甚至是引起细胞死亡,这也可以说明环境中孕激素长期污染能给动物和人类健康造成危害,人类应该更加高度重视环境激素污染问题。为了研究孕激素水平和女性甲状腺肿瘤高发率之间的关联性,本研究利用孕激素受体的稳转细胞株检测分析系统对甲状腺相关肿瘤疾病的女性患者血清中孕激素水平进行检测分析。经过生物统计学分析发现,甲状腺癌病人的血清中孕激素水平低于与正常人血清中孕激素水平,并且有26.7%甲状腺肿瘤病人血清种孕激素平均水平低于1 nM,而正常组血清孕激素平均水平都高于1 nM;正常组中有22.7%样品的孕激素水平超过50 nM,而甲状腺肿瘤组中却没有。通过差异性分析显示甲状腺肿瘤组和正常组的孕激素水平存在显著性差异。尽管女性体内孕激素水平的波动较大,但本研究发现甲状腺肿瘤患者血液中的孕激素水平明显低于正常人血液中孕激素水平,提示在一定范围内甲状腺肿瘤患者肿瘤的发生与体内孕激素水平呈现负相关,故深入研究女性血液中孕激素水平可能对于甲状腺肿瘤早期诊断具有一定的参考价值。
[Abstract]:Thyroid neoplasms are a common cervical tumor. The incidence of thyroid neoplasms is significantly higher in women than in men, so many scholars believe that this difference may be related to sex hormone levels in men and women. Progesterone is an estrogen that plays a major role in the reproductive and developmental system. However, progesterone drugs have been widely used in medical treatment, animal husbandry and agriculture, which have brought great contributions to the society, but also brought serious hormone pollution to the environment, which has begun to affect the endocrine system of the organism itself. Progesterone receptor (Progesterone receptor,PR) is a nuclear receptor. Only when PR is activated by progesterone can it bind to the DNA region of the progesterone receptor response element, thus initiating the expression of downstream target gene of the receptor response element. By using this mechanism and cloning the luciferase reporter gene downstream into the receptor response element, a highly sensitive and high-throughput stable cell line was established for bioassay at progesterone level. It was applied to detect and analyze the levels of progesterone in the blood of patients with thyroid associated neoplasms in order to explore the relationship between progesterone and thyroid tumorigenesis. In this study, three different expression vectors containing progesterone receptor gene and three vectors containing different tandem multiple progesterone receptor response element fragments were constructed, and the activity of luciferase was analyzed. A pair of recombinant plasmids pEGFP-N2-hPR/pGL4-PRE4 with the highest activity were transfected into HeLa cells by co-transfection technique, and then 10 monoclonal cell lines were selected by antibiotic screening and limited dilution method. The positive monoclonal cell lines were identified by molecular cloning and subcellular localization. The results of luciferase activity from passage 5 to passage 23 showed that the cell line had strong genetic stability and high sensitivity, and the detection limit could reach 10 pM.. Therefore, the stably transformed cell line was combined with luciferase assay to establish a progesterone assay system. It was also observed that the activity of luciferase increased with the increase of progesterone concentration in the environment, but the concentration of progesterone above 106 pM decreased the activity of luciferase and even led to cell death. This can also show that long-term pollution of progesterone in the environment can cause harm to animal and human health, and human should pay more attention to the problem of environmental hormone pollution. To study the relationship between progesterone levels and the high incidence of thyroid neoplasms in women, In this study, the serum progesterone levels in female patients with thyroid associated tumor diseases were analyzed by using a stable cell line analysis system of progesterone receptor. Biostatistical analysis showed that the serum progesterone level in thyroid cancer patients was lower than that in normal controls, and the average serum progesterone level was less than 1 nM, in 26.7% of thyroid cancer patients. The average serum progesterone level in normal group was higher than 1 nM;. Progesterone levels exceeded 50 nM, in 22.7% of normal controls and not in thyroid neoplasms. Difference analysis showed that there were significant differences in progesterone levels between thyroid tumor group and normal group. Although the levels of progesterone fluctuate in women, this study found that the levels of progesterone in patients with thyroid tumors were significantly lower than those in normal people. The results suggest that there is a negative correlation between tumor occurrence and progesterone level in patients with thyroid neoplasms in a certain range, so it may be valuable to study the serum progesterone levels in women for early diagnosis of thyroid neoplasms.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R736.1;R730.43

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