發(fā)布時間：2018-06-26 14:04

  本文選題：膠質(zhì)瘤 + RACK1　； 參考：《中南大學(xué)》2014年博士論文

【摘要】：背景：腦膠質(zhì)瘤是中樞神經(jīng)系統(tǒng)中最常見的惡性腫瘤。手術(shù)徹底切除困難,且其生長迅速,術(shù)后易復(fù)發(fā),復(fù)發(fā)間隔時間短,預(yù)后差。高級別膠質(zhì)瘤平均生存期仍然在一年左右。膠質(zhì)瘤生物遺傳學(xué)及分子生物學(xué)的研究進展,揭示了膠質(zhì)瘤的發(fā)生發(fā)展同樣是一個復(fù)雜的生物學(xué)過程,涉及到許多相關(guān)基因的異常表達。因此積極地探索膠質(zhì)瘤的發(fā)生發(fā)展的分子生物學(xué)機制對指導(dǎo)臨床治療具有重要的意義。已有研究證實了RACK1基因在細胞的生長、分化、惡變、轉(zhuǎn)移、侵襲等方面發(fā)揮了關(guān)鍵的調(diào)節(jié)作用,但該基因如何調(diào)控并作用于神經(jīng)上皮組織,與膠質(zhì)瘤發(fā)生,發(fā)展的關(guān)系究竟如何,以及與膠質(zhì)瘤生物學(xué)行為的相關(guān)性等問題,目前國內(nèi)外尚未見系統(tǒng)的研究和報道。我們推測RACK1基因可能參與了膠質(zhì)瘤的發(fā)生發(fā)展過程并發(fā)揮了重要的調(diào)節(jié)作用,RACK1基因可能具有成為膠質(zhì)瘤靶向治療新靶點的應(yīng)用前景,可能給臨床基因診斷及預(yù)后評價提供參考。目的： 探討RACK1基因在人腦膠質(zhì)瘤組織中的表達情況及與病理分級關(guān)系。通過瞬時和穩(wěn)定RNA干擾技術(shù)在人膠質(zhì)瘤細胞株中下調(diào)RACK1的表達,探討RACK1對膠質(zhì)瘤細胞生長、增殖、遷移、凋亡等多種生物學(xué)特性的影響,并初步分析其發(fā)揮作用的分子生物學(xué)機制,結(jié)合臨床病理初步評價其臨床應(yīng)用前景。方法： 對收集的45例各級別臨床膠質(zhì)瘤及10例正常腦組織標本以及U87-MG, CHG-5細胞株進行Real-Time PCR和Western-Blot分別檢測RACK1mRNA和蛋白的表達水平,證實了RACK1在人腦膠質(zhì)瘤中的異常高表達,然后用siRNA瞬時干擾U87-MG, CHG-5細胞株中RACK1的基因,觀察RACK1基因干擾前后膠質(zhì)瘤細胞株生物學(xué)特性(增殖,侵襲,凋亡等)的改變,再通過慢病毒為載體穩(wěn)定轉(zhuǎn)染U87-MG細胞株后,RACK1表達下調(diào)后進行裸鼠成瘤試驗,于體內(nèi)進一步證實RACK1對膠質(zhì)瘤增殖的調(diào)控作用,最后初步探討其調(diào)控膠質(zhì)瘤的發(fā)生發(fā)展的機制,并結(jié)合臨床病理資料,來評價RACK1用于人腦膠質(zhì)瘤中基因診斷及治療的潛在價值。 結(jié)果： 結(jié)果顯示低級別膠質(zhì)RACK1表達較正常腦組織增高有統(tǒng)計學(xué)差異(P0.05),高級別膠質(zhì)瘤和正常腦組織統(tǒng)計學(xué)差異更為顯著(P0.01),低級別與高級別膠質(zhì)瘤之間亦有統(tǒng)計學(xué)差異(P0.05)RACK1的表達增高和膠質(zhì)瘤病理級別正相關(guān)。U87-MG和CHG-5細胞株中RACK1的表達也較正常明顯升高。RACK1經(jīng)RNAi后,細胞的抗凋亡及遷移侵襲能力形成明顯受到抑制(P0.01),并降低了細胞株的增殖率(P0.05),裸鼠成瘤試驗中,經(jīng)慢病毒介導(dǎo)的shRNA干擾后,U87-MG細胞株成瘤與對照組同時期內(nèi)瘤體相比明顯減小,成瘤速度也明顯減慢,結(jié)果有統(tǒng)計學(xué)差異(P0.01)。RACK1經(jīng)siRNA干擾后,無論U87-MG還是CHG-5其Bcl-2的表達均明顯下降,Bax的表達顯著升高(P0.01), Bcl-2/Bax比值也明顯下降,siRNA下調(diào)了RACK1的表達后檢測Src/Akt磷酸化,兩者在兩組細胞株中均顯著的表達下調(diào)(P0.01和P0.05)。同時證實RACK1可能與膠質(zhì)瘤的大小,血運,累及部位有一定的相關(guān)性(P0.05) 結(jié)論： 1. RACK1在膠質(zhì)瘤中高表達； 2. RACK1在體外及體內(nèi)試驗中均能正向調(diào)控膠質(zhì)瘤的增殖,侵襲及 抗凋亡；3. RACK1可能通過Bcl-2/Bax, Src/Akt相關(guān)的信號傳導(dǎo)通路發(fā)揮作用。
[Abstract]:Background: glioma is the most common malignant tumor in the central nervous system. It is difficult to be excised thoroughly, and its growth is rapid. The recurrence of glioma is easy after operation. The recurrence interval is short and the prognosis is poor. The average survival time of the high grade glioma is still about one year. The progress of the biological genetics and subbiology of glioma has revealed the hair of glioma. It is also a complex biological process that involves the abnormal expression of many related genes. Therefore, it is of great significance to actively explore the molecular mechanism of the development of glioma to guide clinical treatment. The research has proved that the RACK1 gene plays a role in cell growth, differentiation, malignant transformation, metastasis, invasion and so on. It is a key regulatory role, but how the gene regulates and regulates the relationship between the neuroepithelial tissue, the relationship with the occurrence of glioma, the relationship between the development of glioma, and the correlation with the biological behavior of glioma. There is no systematic research and report at home and abroad. We speculate that the RACK1 gene may be involved in the development of glioma. The RACK1 gene may have the prospect of becoming a new target for targeting glioma, and may provide a reference for clinical gene diagnosis and prognosis evaluation.
To investigate the expression of RACK1 gene in human glioma tissue and its relationship with pathological classification. Through transient and stable RNA interference technique, the expression of RACK1 in human glioma cell lines is downregulated, and the effects of RACK1 on the growth, proliferation, migration and apoptosis of glioma cells are discussed, and the molecules that play a role are preliminarily analyzed. Biological mechanism, combined with clinical pathology, preliminary evaluation of its clinical application prospects.
The expression levels of RACK1mRNA and protein were detected in 45 cases of clinical gliomas and 10 normal brain tissue specimens and U87-MG, CHG-5 cell lines, Real-Time PCR and Western-Blot respectively, which confirmed the abnormal high expression of RACK1 in human glioma, and then interfered with U87-MG, RACK1 gene in CHG-5 cell lines by siRNA transient. The changes of the biological characteristics (proliferation, invasion, apoptosis, etc.) of glioma cell lines before and after the interference of RACK1 gene were observed, and then the U87-MG cell line was transfected steadily through the lentivirus as the carrier, and the expression of RACK1 was downregulated after the downregulation of the tumor. The regulation of RACK1 on the proliferation of glioma was further confirmed in the body. Finally, the regulation of glioma was preliminarily discussed. To evaluate the potential value of RACK1 in gene diagnosis and treatment of human gliomas by combining the mechanism of occurrence and development with clinicopathological data.
Result:
The results showed that the expression of low grade glial RACK1 was significantly higher than that of normal brain tissue (P0.05). The statistical difference between high grade glioma and normal brain tissue was more significant (P0.01), and there was a statistically significant difference between low grade and high grade glioma (P0.05), the higher expression of RACK1 and the positive correlation between.U87-MG and CHG-5 cells in the pathological grade of glioma. The expression of RACK1 in the plant was also significantly higher than that of the normal.RACK1. After RNAi, the anti apoptosis and migration and invasion ability of the cells were obviously inhibited (P0.01), and the proliferation rate of cell lines was reduced (P0.05). In the nude mouse tumor test, after the slow virus mediated shRNA interference, the adult tumor of U87-MG cell line was significantly reduced compared with the control group in the same period. After siRNA interference, the expression of Bcl-2 in U87-MG or CHG-5 decreased significantly, the expression of Bax increased significantly (P0.01) and Bcl-2/Bax ratio decreased significantly (P0.01) and siRNA decreased after siRNA interference. SiRNA decreased the RACK1 expression and detected Src/Akt phosphorylation. Both were significant in the two groups of cell lines. The expression of P0.01 (P0.05 and RACK1) was down regulated. It was also confirmed that RACK1 may be related to the size, blood flow and location of glioma (P0.05).
Conclusion:
1. RACK1 was highly expressed in glioma.
2. RACK1 in vitro and in vivo can positively regulate the proliferation and invasion of glioma.
Anti apoptosis; 3. RACK1 may play a role through Bcl-2/Bax and Src/Akt related signal transduction pathways.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.41

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本文編號：2070620