發(fā)布時間：2018-05-30 13:21

  本文選題：NF-κB信號通路 + 山楂酸��； 參考：《華東師范大學(xué)》2010年博士論文

【摘要】： 核因子NF-κB (Nuclear Factor-κB)作為一個關(guān)鍵的轉(zhuǎn)錄調(diào)節(jié)分子參與多種生物學(xué)功能。現(xiàn)在已經(jīng)證實其廣泛存在于真核細(xì)胞內(nèi),能與多種基因啟動子或增強子序列的特定位點結(jié)合而促進(jìn)轉(zhuǎn)錄和表達(dá),參與炎癥反應(yīng)、免疫應(yīng)答以及細(xì)胞的增殖、轉(zhuǎn)化和凋亡等重要的病理生理過程。NF-κB的活性受到嚴(yán)格精密的調(diào)控,是一個受到多因素共同維持的平衡,只有嚴(yán)格維系這種平衡,細(xì)胞才能正常生存和行使生理功能。一旦這種平衡被打破,就會導(dǎo)致生理功能紊亂,產(chǎn)生疾病。研究發(fā)現(xiàn)大部分腫瘤組織中NF-κB活性升高,另外,在一些炎性疾病、感染性疾病,如哮喘,各種關(guān)節(jié)炎,肩周炎,牙周炎以及艾滋病感染早期患者中也發(fā)現(xiàn)NF-κB的活性顯著升高,而在另外一些自身免疫性疾病中發(fā)現(xiàn)NF-κB活性降低。前期研究已經(jīng)證實維持NF-κB活性的平衡具有重要作用,其活性異常與眾多疾病密切相關(guān)。因此,NF-κB已經(jīng)成為治療包括癌癥在內(nèi)的多種疾病藥物作用和開發(fā)的分子靶點。 NF-κB的活化是一個多步驟的過程,當(dāng)激活因子從細(xì)胞表面進(jìn)入細(xì)胞內(nèi)后,首先激活上游激酶(IKK),再通過激酶磷酸化NF-κB的抑制亞單位IκBα,進(jìn)而導(dǎo)致NF-κB從細(xì)胞質(zhì)進(jìn)入到細(xì)胞核,與特定的靶標(biāo)結(jié)合,調(diào)控相關(guān)基因表達(dá),最后調(diào)控相關(guān)的生理過程。根據(jù)NF-κB這些特點,本課題進(jìn)行了以下方面的研究： 一、建立了NF-κB活性的各種檢測方法,包括：NF-κB報告基因分析方法,免疫熒光的方法(檢測NF-κB在細(xì)胞內(nèi)的定位),電泳遷移率實驗(EMSA-electrophoretic mobility shift assay),蛋白印跡的方法(分析抑制亞單位IκBα的降解情況和調(diào)控基因表達(dá)情況)和染色質(zhì)免疫共沉淀的方法(chromatin immunoprecipitation assay, ChIP)來研究分析NF-κB對疾病的調(diào)控作用和篩選發(fā)現(xiàn)調(diào)控NF-κB活性的藥物。另一方面,我們還建立了包括裸鼠荷瘤,腫瘤轉(zhuǎn)移和骨質(zhì)疏松的動物模型,以期望進(jìn)一步在體內(nèi)對NF-κB進(jìn)行機理和應(yīng)用方面的深入研究。 二、利用建好的NF-κB實驗?zāi)Ｐ?我們篩選得到了數(shù)個NF-κB信號通路的抑制劑,并選取了其中一個活性小分子——山楂酸,進(jìn)一步對山楂酸在抗腫瘤方面的機理進(jìn)行了研究。我們研究發(fā)現(xiàn)： 1、山楂酸具有抑制胰腺癌細(xì)胞增值的作用。在TNFα存在時,這種抑制效果更加明顯,統(tǒng)計表明顯著增強； 2、山楂酸與TNFα協(xié)同抑制胰腺癌細(xì)胞增殖的作用,是通過促進(jìn)細(xì)胞凋亡來實現(xiàn)的； 3、山楂酸是通過抑制NF-κB的活化來促進(jìn)胰腺癌細(xì)胞凋亡的； 4、在體外,山楂酸處理能夠抑制NF-κB下游的相關(guān)基因的表達(dá)； 5、裸鼠體內(nèi)實驗進(jìn)一步證明山楂酸處理能夠抑制胰腺癌腫瘤的生長,促進(jìn)胰腺腫瘤細(xì)胞凋亡。 這些結(jié)果提示山楂酸處理能夠分別在體內(nèi)和體外抑制腫瘤細(xì)胞的生長,他的抗癌機理是通過抑制NF-κB信號通路的活化和NF-κB下游相關(guān)基因的表達(dá)來實現(xiàn)的,這就提示,山楂酸和TNFα一起聯(lián)合使用可能對胰腺癌的治療有所幫助。 三、我們課題的另外一個部分研究發(fā)現(xiàn)山楂酸能夠抑制破骨細(xì)胞的分化和功能。這些研究結(jié)果包括： 1、研究發(fā)現(xiàn)山楂酸能夠抑制破骨細(xì)胞分化,而且具有時間和濃度劑量依賴性； 2、山楂酸還能夠抑制破骨細(xì)胞特殊的功能結(jié)構(gòu)肌動蛋白環(huán)(ACTIN-RING)的形成； 3、pits assay證實,在體外山楂酸能夠抑制破骨細(xì)胞對骨片的吸收；4、在體內(nèi),我們進(jìn)一步發(fā)現(xiàn),山楂酸能夠抑制由于卵巢切除導(dǎo)致的小鼠骨質(zhì)流失； 5、進(jìn)一步研究表明,山楂酸能夠抑制由于卵巢切除導(dǎo)致的破骨細(xì)胞活性升高。 這些現(xiàn)象提示,山楂酸能夠抑制破骨細(xì)胞的分化和功能,而且對卵巢切除導(dǎo)致的骨質(zhì)疏松有預(yù)防和治療作用。我們進(jìn)一步探討了這些現(xiàn)象的機理。研究發(fā)現(xiàn)： 1、山楂酸處理能夠抑制RANKL誘導(dǎo)引起的NF-κB信號通路的活化； 2、山楂酸處理能夠抑制RANKL誘導(dǎo)的引起的MAPK-AP1信號通路的活化； 3、山楂酸處理能夠抑制NFAT-c1的表達(dá),但是不影響其活性； 4、山楂酸處理能夠抑制破骨細(xì)胞分化和功能相關(guān)基因的表達(dá)； 5、山楂酸處理不影響RANKL誘發(fā)的細(xì)胞內(nèi)鈣流水平； 通過上述研究,我們初步證明了山楂酸能夠通過抑制NF-κB信號通路和MAPK-AP1信號通路的活化來抑制破骨細(xì)胞的分化和功能,而且我們在動物水平驗證了山楂酸能夠抑制由于激素水平下降導(dǎo)致的骨質(zhì)流失,這些結(jié)果為山楂酸在預(yù)防和治療破骨細(xì)胞相關(guān)疾病如骨質(zhì)疏松癥方面提供了理論支持。 綜上所述,我們成功的建立了在分子、細(xì)胞和動物水平的NF-κB信號通路研究模型,為下一步進(jìn)行跟NF-κB信號通路相關(guān)的機理研究和運用研究奠定了基礎(chǔ)。利用建立的模型,我們篩選獲得了數(shù)個NF-κB信號通路的小分子抑制劑。緊接著,我們對其中之一的山楂酸的功能和機理進(jìn)行了深入研究。我們發(fā)現(xiàn)：山楂酸和腫瘤壞死因子α聯(lián)合使用具有良好的抗腫瘤效果。而且我們的研究還發(fā)現(xiàn),山楂酸能夠抑制破骨前體細(xì)胞的分化和功能,在體內(nèi)能夠抑制由于卵巢切除導(dǎo)致的骨質(zhì)疏松。這些研究為以后山楂酸在腫瘤治療,破骨細(xì)胞相關(guān)疾病的預(yù)防和治療方面提供了理論支持。同時,我們的這部分研究也為以后利用NF-κB信號通路研究模型提供了有益探索和嘗試。
[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.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R346

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 高靜;龍建綱;劉健康;;骨質(zhì)疏松癥發(fā)生與防治的線粒體機制[J];航天醫(yī)學(xué)與醫(yī)學(xué)工程;2013年06期

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本文編號：1955445