核因子NF-κB信号通路模型的建立及其小分子抑制剂山楂酸在肿瘤、免疫中的应用研究

发布时间：2018-05-30 13:21

本文选题：NF-κB信号通路 + 山楂酸　；参考：《华东师范大学》2010年博士论文

【摘要】： 核因子NF-κB (Nuclear Factor-κB)作为一个关键的转录调节分子参与多种生物学功能。现在已经证实其广泛存在于真核细胞内,能与多种基因启动子或增强子序列的特定位点结合而促进转录和表达,参与炎症反应、免疫应答以及细胞的增殖、转化和凋亡等重要的病理生理过程。NF-κB的活性受到严格精密的调控,是一个受到多因素共同维持的平衡,只有严格维系这种平衡,细胞才能正常生存和行使生理功能。一旦这种平衡被打破,就会导致生理功能紊乱,产生疾病。研究发现大部分肿瘤组织中NF-κB活性升高,另外,在一些炎性疾病、感染性疾病,如哮喘,各种关节炎,肩周炎,牙周炎以及艾滋病感染早期患者中也发现NF-κB的活性显著升高,而在另外一些自身免疫性疾病中发现NF-κB活性降低。前期研究已经证实维持NF-κB活性的平衡具有重要作用,其活性异常与众多疾病密切相关。因此,NF-κB已经成为治疗包括癌症在内的多种疾病药物作用和开发的分子靶点。 NF-κB的活化是一个多步骤的过程,当激活因子从细胞表面进入细胞内后,首先激活上游激酶(IKK),再通过激酶磷酸化NF-κB的抑制亚单位IκBα,进而导致NF-κB从细胞质进入到细胞核,与特定的靶标结合,调控相关基因表达,最后调控相关的生理过程。根据NF-κB这些特点,本课题进行了以下方面的研究：一、建立了NF-κB活性的各种检测方法,包括：NF-κB报告基因分析方法,免疫荧光的方法(检测NF-κB在细胞内的定位),电泳迁移率实验(EMSA-electrophoretic mobility shift assay),蛋白印迹的方法(分析抑制亚单位IκBα的降解情况和调控基因表达情况)和染色质免疫共沉淀的方法(chromatin immunoprecipitation assay, ChIP)来研究分析NF-κB对疾病的调控作用和筛选发现调控NF-κB活性的药物。另一方面,我们还建立了包括裸鼠荷瘤,肿瘤转移和骨质疏松的动物模型,以期望进一步在体内对NF-κB进行机理和应用方面的深入研究。二、利用建好的NF-κB实验模型,我们筛选得到了数个NF-κB信号通路的抑制剂,并选取了其中一个活性小分子——山楂酸,进一步对山楂酸在抗肿瘤方面的机理进行了研究。我们研究发现： 1、山楂酸具有抑制胰腺癌细胞增值的作用。在TNFα存在时,这种抑制效果更加明显,统计表明显著增强； 2、山楂酸与TNFα协同抑制胰腺癌细胞增殖的作用,是通过促进细胞凋亡来实现的； 3、山楂酸是通过抑制NF-κB的活化来促进胰腺癌细胞凋亡的； 4、在体外,山楂酸处理能够抑制NF-κB下游的相关基因的表达； 5、裸鼠体内实验进一步证明山楂酸处理能够抑制胰腺癌肿瘤的生长,促进胰腺肿瘤细胞凋亡。这些结果提示山楂酸处理能够分别在体内和体外抑制肿瘤细胞的生长,他的抗癌机理是通过抑制NF-κB信号通路的活化和NF-κB下游相关基因的表达来实现的,这就提示,山楂酸和TNFα一起联合使用可能对胰腺癌的治疗有所帮助。三、我们课题的另外一个部分研究发现山楂酸能够抑制破骨细胞的分化和功能。这些研究结果包括： 1、研究发现山楂酸能够抑制破骨细胞分化,而且具有时间和浓度剂量依赖性； 2、山楂酸还能够抑制破骨细胞特殊的功能结构肌动蛋白环(ACTIN-RING)的形成； 3、pits assay证实,在体外山楂酸能够抑制破骨细胞对骨片的吸收；4、在体内,我们进一步发现,山楂酸能够抑制由于卵巢切除导致的小鼠骨质流失； 5、进一步研究表明,山楂酸能够抑制由于卵巢切除导致的破骨细胞活性升高。这些现象提示,山楂酸能够抑制破骨细胞的分化和功能,而且对卵巢切除导致的骨质疏松有预防和治疗作用。我们进一步探讨了这些现象的机理。研究发现： 1、山楂酸处理能够抑制RANKL诱导引起的NF-κB信号通路的活化； 2、山楂酸处理能够抑制RANKL诱导的引起的MAPK-AP1信号通路的活化； 3、山楂酸处理能够抑制NFAT-c1的表达,但是不影响其活性； 4、山楂酸处理能够抑制破骨细胞分化和功能相关基因的表达； 5、山楂酸处理不影响RANKL诱发的细胞内钙流水平；通过上述研究,我们初步证明了山楂酸能够通过抑制NF-κB信号通路和MAPK-AP1信号通路的活化来抑制破骨细胞的分化和功能,而且我们在动物水平验证了山楂酸能够抑制由于激素水平下降导致的骨质流失,这些结果为山楂酸在预防和治疗破骨细胞相关疾病如骨质疏松症方面提供了理论支持。综上所述,我们成功的建立了在分子、细胞和动物水平的NF-κB信号通路研究模型,为下一步进行跟NF-κB信号通路相关的机理研究和运用研究奠定了基础。利用建立的模型,我们筛选获得了数个NF-κB信号通路的小分子抑制剂。紧接着,我们对其中之一的山楂酸的功能和机理进行了深入研究。我们发现：山楂酸和肿瘤坏死因子α联合使用具有良好的抗肿瘤效果。而且我们的研究还发现,山楂酸能够抑制破骨前体细胞的分化和功能,在体内能够抑制由于卵巢切除导致的骨质疏松。这些研究为以后山楂酸在肿瘤治疗,破骨细胞相关疾病的预防和治疗方面提供了理论支持。同时,我们的这部分研究也为以后利用NF-κB信号通路研究模型提供了有益探索和尝试。
[Abstract]:Nuclear factor NF- kappa B (Nuclear Factor- kappa B), as a key transcriptional regulator, participates in a variety of biological functions. It has been proved that it exists widely in eukaryotic cells and can be combined with specific loci of a variety of promoter or enhancer sequences to promote transcription and expression, participate in inflammatory response, immune response and cell proliferation. The activities of.NF- kappa B, which are important pathophysiological processes such as transformation and apoptosis, are regulated strictly and precisely. It is a balance maintained by multiple factors. Only by strictly maintaining this balance can the cells survive and exercise their physiological functions. Once the balance is broken, it will lead to physical dysfunction and disease. Studies have found large numbers of diseases. In some tumor tissues, the activity of NF- kappa B increases. In addition, the activity of NF- kappa B in some inflammatory diseases, such as asthma, all kinds of arthritis, periarthritis of shoulder, periodontitis, and early AIDS infection, is also found to be significantly increased, and the decrease of NF- kappa B activity is found in some other autoimmune diseases. The balance of the activity of NF- kappa B plays an important role, and its activity is closely related to many diseases. Therefore, NF- kappa B has become a molecular target for the treatment and development of various diseases, including cancer.
The activation of NF- kappa B is a multistep process. When the activating factor enters the cell from the cell surface, it activates the upstream kinase (IKK), and then inhibits the subunit I kappa B alpha by the kinase phosphorylation of NF- kappa B, and then leads to the NF- kappa B from the cytoplasm into the nucleus, and combines with a specific target to regulate the related gene expression and finally regulates the correlation. According to these characteristics of NF- kappa B, we have studied the following aspects:
First, various detection methods of NF- kappa B activity were established, including: NF- kappa B report gene analysis method, immunofluorescence method (detecting the localization of NF- kappa B in cell), electrophoresis mobility test (EMSA-electrophoretic mobility shift assay), Western blot method (segregation suppression subunit I kappa B alpha degradation and regulation gene expression) Chromatin immunoprecipitation assay (ChIP) was used to study the regulation of NF- kappa B on the disease and the screening and screening of drugs to regulate the activity of NF- kappa B. On the other hand, we also established animal models including nude mice bearing tumor, tumor metastasis and osteoporosis in order to expect to further be in vivo against NF- kappa. B carries out deep research on the mechanism and application.
Two, using the established NF- kappa B experimental model, we screened several inhibitors of the NF- kappa B signaling pathway and selected one of the small active molecules, haataegic acid, to further study the mechanism of crataegic acid in anti-tumor. We found that:
1, hawthorn acid can inhibit the proliferation of pancreatic cancer cells. When TNF alpha is present, this inhibition effect is more obvious, statistically significant enhancement.
2, the synergistic inhibition of hawthorn acid and TNF alpha on the proliferation of pancreatic cancer cells is achieved by promoting cell apoptosis.
3, hawthorn acid promotes apoptosis of pancreatic cancer cells by inhibiting the activation of NF- kappa B.
4, in vitro, hawthorn acid treatment can inhibit the expression of NF- kappa B downstream related genes.
5, nude mice experiment further proved that hawthorn acid treatment could inhibit the growth of pancreatic cancer and promote the apoptosis of pancreatic cancer cells.
These results suggest that crataegic acid can inhibit the growth of tumor cells in vivo and in vitro. His anticancer mechanism is achieved by inhibiting the activation of the NF- kappa B signaling pathway and the expression of the downstream related genes of NF- kappa B. This suggests that the combined use of hawthorn and TNF alpha may be helpful for the treatment of pancreatic cancer.
Three, another part of our research found that hawthorn acid could inhibit osteoclast differentiation and function.
1, it was found that hawthorn acid inhibited osteoclast differentiation in a dose and time dependent manner.
2, hawthorn acid can also inhibit the formation of specific functional structure of osteoclasts (actin ring) (ACTIN-RING).
3, pits assay confirmed that hawthorn acid could inhibit the absorption of osteoclasts to bone fragments in vitro; 4, in the body, we further found that crataegic acid could inhibit bone loss in mice caused by ovariectomy;
5, further studies showed that hawthorn acid could inhibit the increase of osteoclast activity due to ovariectomy.
These phenomena suggest that crataegic acid inhibits the differentiation and function of osteoclasts and has a preventive and therapeutic effect on ovariectomy induced osteoporosis. We further explored the mechanism of these phenomena.
1, hawthorn acid treatment could inhibit the activation of NF- kappa B signaling pathway induced by RANKL.
2, hawthorn acid treatment could inhibit the activation of MAPK-AP1 signaling pathway induced by RANKL.
3, hawthorn acid treatment could inhibit the expression of NFAT-c1, but did not affect its activity.
4, hawthorn acid treatment can inhibit the expression of osteoclast differentiation and function related genes.
5, hawthorn acid treatment did not affect the intracellular calcium flow induced by RANKL.
Through these studies, we have preliminarily demonstrated that crataegic acid can inhibit the differentiation and function of osteoclasts by inhibiting the activation of the NF- kappa B signaling pathway and the MAPK-AP1 signaling pathway, and we have demonstrated at animal levels that crataegic acid can inhibit the loss of bone caused by a decrease in hormone levels, which results in the prevention and treatment of hawthorn acid. It provides theoretical support for osteoclast related diseases, such as osteoporosis.
To sum up, we have successfully established the NF- kappa B signal pathway research model at molecular, cellular and animal levels, laying the foundation for the next step in the study and application of the mechanism related to the NF- kappa B signaling pathway. We have found that hawthorn acid and tumor necrosis factor alpha are combined with good antitumor effects. And our study also found that crataegic acid can inhibit the differentiation and function of osteoclast cells and inhibit the bone caused by ovariectomy in the body. These studies provide theoretical support for the prevention and treatment of Crataegus acid in cancer and osteoclast related diseases. Meanwhile, this part of our study also provides useful exploration and attempt for the future use of the NF- kappa B signaling pathway.
【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R346

【引证文献】