本文選題：ER-α36 + 膠質(zhì)瘤��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：膠質(zhì)瘤是最為常見的腦部腫瘤,約占原發(fā)性神經(jīng)系統(tǒng)腫瘤的60%,其發(fā)病率存在明顯的性別差異,男性發(fā)病率是女性的1.85倍。膠質(zhì)瘤具有復(fù)發(fā)率高、治療效果不佳,預(yù)后極差等特點,多數(shù)患者的生存期一般小于一年。目前,針對膠質(zhì)瘤的治療方法主要是手術(shù)切除,然后輔助以放療、化療,但是三種方法結(jié)合也僅能極其有限地延長病人的生存期。對于膠質(zhì)瘤的治療,近年來越來越多的研究小組將研究方向轉(zhuǎn)向靶向藥物的研究,而靶向藥物的研究最根本的是尋找治療疾病的靶標(biāo)。因為膠質(zhì)瘤在男女中的發(fā)病率存在明顯差異,我們推測雌激素受體可能在膠質(zhì)瘤的發(fā)生發(fā)展中發(fā)揮特定作用。雌激素受體-α36(estrogen receptor-α36,ER-α36)是一種新型雌激素受體,其主要定位在細(xì)胞膜上,分子質(zhì)量為35.7 kD,介導(dǎo)由膜起始的雌激素信號通路。已證實ER-α36參與多種癌癥的發(fā)生發(fā)展,其在乳腺癌中的作用已經(jīng)研究得較為深入,但是ER-α36在膠質(zhì)瘤發(fā)生中的作用至今不清。本研究發(fā)現(xiàn)在臨床采集的膠質(zhì)瘤樣本中,腫瘤ER-α36的表達量明顯高于瘤旁。下調(diào)膠質(zhì)瘤細(xì)胞系中ER-α36的表達后,膠質(zhì)瘤細(xì)胞的增殖、遷移、侵襲能力明顯減弱。同時,荷瘤實驗結(jié)果證明,敲低ER-α36后,膠質(zhì)瘤細(xì)胞的成瘤能力顯著降低。因此,我們推測,ER-α36和膠質(zhì)瘤的發(fā)生和發(fā)展有密切關(guān)系,可能成為臨床治療膠質(zhì)瘤的新靶標(biāo)。背景膠質(zhì)瘤是最為常見的腦部腫瘤,發(fā)病率存在明顯的性別差異。ER-α36是一種新型的膜雌激素受體,其生物學(xué)作用正在引起越來越多的關(guān)注。因為膠質(zhì)瘤的發(fā)病存在顯著性別差異,所以我們推測激素受體可能在膠質(zhì)瘤的發(fā)生及發(fā)展中發(fā)揮作用。有文獻報道,ER-α36高表達顯著升高乳腺癌患病風(fēng)險,而且ER-α36介導(dǎo)的快速雌激素信號傳導(dǎo)通路與乳腺癌干/祖細(xì)胞功能密切相關(guān)。另有文獻報道,ER-α36被激活后能夠刺激子宮內(nèi)膜細(xì)胞的增生。有研究表明,ER-α36在胃癌、骨質(zhì)疏松等疾病發(fā)生中也發(fā)揮重要作用,ER-α36的表達量增高能夠削弱化療藥物對乳腺癌細(xì)胞的殺傷作用。ER-α36和多種腫瘤的發(fā)生和發(fā)展具有密切的關(guān)系,其有望成為腫瘤治療的新靶點。雖然已經(jīng)明確ER-α36參與了乳腺癌等若干種腫瘤的發(fā)生和發(fā)展,但是其在膠質(zhì)瘤發(fā)生發(fā)展中的作用目前尚未見報道。目的建立ER-α36敲低的膠質(zhì)瘤細(xì)胞株,用MTT實驗、劃痕實驗和Transwell實驗研究ER-α36敲低后對細(xì)胞的增殖、遷移、侵襲等造成的影響,并用荷瘤實驗進行在體驗證,明確ER-α36敲低后對膠質(zhì)瘤細(xì)胞的增殖、遷移、侵襲等的影響。在此基礎(chǔ)上,進一步研究介導(dǎo)上述作用的信號通路,初步探明膠質(zhì)瘤中ER-α36發(fā)揮作用的分子機制。方法臨床采集膠質(zhì)瘤及瘤旁組織,利用Western blot檢測ER-α36表達,觀察分析ER-α36在腫瘤組織和瘤旁組織的表達情況。分別采用ER-α36激動劑G1和抑制劑IC162處理膠質(zhì)瘤細(xì)胞株觀察細(xì)胞增殖的改變,初步明確ER-α36在膠質(zhì)瘤細(xì)胞增殖過程中的作用。將shER-α36質(zhì)粒轉(zhuǎn)入野生型膠質(zhì)瘤細(xì)胞U87和U251中,構(gòu)建ER-α36穩(wěn)定敲低的細(xì)胞株,MTT實驗檢測細(xì)胞增殖能力的改變并通過裸鼠荷瘤實驗進行在體驗證,進一步明確敲低ER-α36對膠質(zhì)瘤細(xì)胞增殖中的影響。通過劃痕實驗及Transwell等實驗檢測ER-α36敲低后,膠質(zhì)瘤細(xì)胞遷移以及侵襲能力的改變,同時Western blot檢測上皮以及間質(zhì)細(xì)胞標(biāo)志蛋白。隨后,檢測可能參與膠質(zhì)瘤增殖、遷移、侵襲作用的信號通路,初步探明ER-α36參與膠質(zhì)瘤增殖、遷移、侵襲等行為的可能機制。結(jié)果(1)Western blot檢測結(jié)果表明,在12對膠質(zhì)瘤樣本中,其中10對樣本的ER-α36在腫瘤組織中的表達量高于瘤旁組織。(2)MTT結(jié)果表明隨著ER-α36特異性激動劑G1濃度增加,膠質(zhì)瘤細(xì)胞的增殖能力增強,而ER-α36特異性抑制劑IC162可以顯著降低膠質(zhì)瘤細(xì)胞的增殖能力。(3)MTT檢測結(jié)果證實,穩(wěn)定敲低ER-α36的膠質(zhì)瘤細(xì)胞,其增殖能力較對照細(xì)胞減弱。裸鼠荷瘤實驗結(jié)果表明,膠質(zhì)瘤細(xì)胞中ER-α36敲低后,其體內(nèi)成瘤能力相較于對照組膠質(zhì)瘤細(xì)胞明顯降低。(4)劃痕實驗以及Transwell實驗結(jié)果表明,ER-α36敲低能夠降低膠質(zhì)瘤細(xì)胞的遷移和侵襲能力。Western blot檢測上皮細(xì)胞以及間質(zhì)細(xì)胞標(biāo)志性蛋白,ER-α36敲低能夠抑制膠質(zhì)瘤細(xì)胞的上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)過程。(5)Western blot 結(jié)果表明,ER-α36 敲低后 MAPK/ERK 和 PI3K/AKT 信號通路的激活減弱。結(jié)論ER-α36參與了膠質(zhì)瘤的增殖、遷移、侵襲等過程,并與膠質(zhì)瘤的EMT過程密切相關(guān),這些作用可能是通過MAPK/ERK和PI3K/AKT信號通路來實現(xiàn)的。
[Abstract]:Glioma is the most common brain tumor, accounting for about 60% of the primary nervous system tumors. The incidence of glioma has a significant sex difference, and the incidence of male is 1.85 times. The recurrence rate of the glioma is high, the treatment effect is poor, the prognosis is very poor and so on. Most of the patients are usually less than one year. At present, the treatment of glioma The main methods are surgical resection, and then adjuvant chemotherapy and radiotherapy, but the combination of the three methods can only prolong the patient's survival time. For the treatment of glioma, more and more research teams have turned the research direction to the target drug research in recent years, and the target drug research is the most important target for the target treatment of the disease. There is a significant difference in the incidence of glioma in men and women. We speculate that the estrogen receptor may play a specific role in the development of glioma. Estrogen receptor - alpha 36 (estrogen receptor- alpha 36, ER- alpha 36) is a new type of estrogen receptor, which is mainly located on the cell membrane, and the molecular mass is 35.7 kD, mediated by the membrane initiation. The estrogen signaling pathway has been confirmed that ER- alpha 36 has been involved in the development of various cancers, and its role in breast cancer has been studied in depth, but the role of ER- alpha 36 in the occurrence of glioma is not clear. This study found that the expression of ER- alpha 36 was significantly higher than that of the tumor in the clinical samples of glioma. After the expression of ER- alpha 36 in the cell line, the proliferation, migration and invasiveness of glioma cells decreased significantly. At the same time, the tumor bearing experimental results showed that the tumorigenesis ability of glioma cells decreased significantly after knocking down ER- alpha 36. Therefore, we speculate that ER- alpha 36 is closely related to the occurrence and development of glioma, and may become a new target for the clinical treatment of glioma. Background glioma is the most common brain tumor. There is a significant gender difference in the incidence of.ER- a 36 is a new type of membrane estrogen receptor. Its biological role is causing more and more attention. Because of the significant gender difference in the pathogenesis of glioma, we speculate that the hormone receptor may be in the development and development of glioma. It is reported that the high expression of ER- alpha 36 significantly increases the risk of breast cancer, and the ER- alpha 36 mediated rapid estrogen signaling pathway is closely related to the function of the stem / progenitor cells of breast cancer. It is also reported that the activation of ER- alpha 36 can stimulate the proliferation of endometrium cells. Studies have shown that ER- alpha 36 is in gastric cancer and bone thinning. It also plays an important role in the occurrence of pine and other diseases. The increase of ER- alpha 36 can weaken the killing effect of chemotherapeutic drugs on breast cancer cells..ER- alpha 36 is closely related to the occurrence and development of various tumors. It is expected to be a new target for cancer treatment. Although it has been clear that ER- alpha 36 has been involved in a number of cancers, such as breast cancer But its role in the development of glioma has not yet been reported. Objective to establish a glioma cell line of ER- alpha 36 knockout. The effects of ER- alpha 36 on the proliferation, migration and invasion of the cells were studied by MTT experiment, scratch test and Transwell test, and the tumor bearing experiment was used to identify the ER- alpha 36. On the basis of the effects of the proliferation, migration and invasion of glioma cells. On this basis, we further study the signal pathway which mediate the above action and preliminarily explore the molecular mechanism of ER- alpha 36 in glioma. Methods the clinical collection of glioma and para tumor tissue, the detection of ER- alpha 36 expression by using blot blot, and the observation and analysis of ER- alpha 36 in tumor tissue and tumor. ER- alpha 36 agonist G1 and inhibitor IC162 were used to observe the proliferation of glioma cell lines, and the role of ER- alpha 36 in the proliferation of glioma cells was preliminarily identified. ShER- alpha 36 plasmids were transferred into the wild glioma cells U87 and U251 to construct ER- alpha 36 with a stable low cell strain and MTT experimental examination. The changes in cell proliferation ability were measured and the effect of ER- alpha 36 on the proliferation of glioma cells was further clarified through the nude mice bearing experiment. The changes in the migration and invasion of glioma cells were detected by the scratch test and the Transwell and other tests. At the same time, the Western blot was used to detect the epithelium and the interstitial fine. Then, the possible mechanism of ER- alpha 36 involved in the proliferation, migration and invasion of glioma was detected. Results (1) the results of Western blot detection showed that in 12 pairs of glioma samples, the expression of ER- alpha 36 in 10 pairs of samples was high. (2) MTT results showed that the proliferation ability of glioma cells increased with the increase of ER- alpha 36 specific agonist G1 concentration, while ER- alpha 36 specific inhibitor IC162 could significantly reduce the proliferation ability of glioma cells. (3) MTT detection results confirmed that the glioma cells with low ER- alpha 36 were stable and the proliferation ability was weaker than that of the control cells. The tumor bearing test in nude mice showed that the tumorigenicity of ER- alpha 36 in the glioma cells was significantly lower than that of the control group. (4) the scratch test and the results of Transwell experiment showed that the ER- alpha 36 knock down could reduce the migration of glioma cells and the invasive energy.Western blot to detect epithelial cells and stromal cells. ER- alpha 36 knocks low to inhibit the epithelial mesenchymal transition (epithelial-mesenchymal transition, EMT) of glioma cells. (5) Western blot results show that the activation of MAPK/ERK and PI3K/AKT signaling pathway is weakened after ER- alpha 36 knocks. Conclusion ER- alpha 36 participates in the proliferation, migration, invasion and other processes of glioma and is associated with glioma. The EMT process is closely related, and these effects may be achieved through MAPK/ERK and PI3K/AKT signaling pathways.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.41

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