AJAP1在腦膠質(zhì)瘤中異常表達(dá)及抑制細(xì)胞增殖與侵襲的分子機(jī)制研究
發(fā)布時(shí)間：2018-07-14 11:00
【摘要】：腦膠質(zhì)瘤是最常見(jiàn)的顱內(nèi)原發(fā)性腫瘤，其中2/3以上為惡性膠質(zhì)瘤，占成人全身腫瘤約2%，膠質(zhì)母細(xì)胞瘤中位生存期僅1年，5年的生存期幾乎為零[1-2]。手術(shù)切除，術(shù)后化療和放療綜合療效仍不是很理想，目前尚無(wú)根治方法。腦膠質(zhì)瘤侵襲性生長(zhǎng)的特性是膠質(zhì)瘤難以根治和復(fù)發(fā)的根本原因。因此膠質(zhì)瘤治療策略的優(yōu)化不僅應(yīng)著眼在原發(fā)部位腫瘤的治療，還應(yīng)著力控制腫瘤細(xì)胞向正常腦組織的侵襲和轉(zhuǎn)移。尋找具有抑制膠質(zhì)瘤細(xì)胞侵襲、轉(zhuǎn)移特性的相關(guān)基因，深入揭示其作用機(jī)制，對(duì)膠質(zhì)瘤治療具有重要意義。 越來(lái)越多的證據(jù)表明在人類染色體1p36區(qū)域編碼著許多類似腫瘤抑制基因[3-4]。最近研究報(bào)告發(fā)現(xiàn)粘附連接相關(guān)蛋白-1(Adherens Junctional AssociatedProtein-1，AJAP1,也稱為Shrew-1)作為另一個(gè)類似抑癌基因定位于人類染色體1p36區(qū)域。Radlwimmer研究組對(duì)膠質(zhì)母細(xì)胞瘤患者進(jìn)行基因組雜交和微陣列分析比較，AJAP1首先被描述為一種與膠質(zhì)母細(xì)胞發(fā)生的生物標(biāo)記物[5]。隨后,Zhang課題組證實(shí)在SVHVC, LOVO, SW480,MCF-7等多種細(xì)胞系中，AJAP1啟動(dòng)子區(qū)存在著高度甲基化[6]。但是，在膠質(zhì)瘤細(xì)胞中改變AJAP1的表達(dá)水平影響細(xì)胞侵襲和增殖的機(jī)制仍然不清楚。 本課題重點(diǎn)研究AJAP1在膠質(zhì)瘤中的異常表達(dá)情況，探討其與膠質(zhì)瘤級(jí)別之間的關(guān)系，應(yīng)用AJAP1重組腺病毒轉(zhuǎn)染技術(shù)改變膠質(zhì)瘤細(xì)胞中AJAP1的表達(dá)水平，初步探討研究AJAP1對(duì)膠質(zhì)瘤生物學(xué)表型的影響及其潛在機(jī)制。 方法 第一部分應(yīng)用免疫組織化學(xué)染色以及免疫熒光共聚焦成像方法檢測(cè)48例膠質(zhì)瘤標(biāo)本、5例正常腦組織標(biāo)本中AJAP1的表達(dá)水平，采用Western blot方法對(duì)8個(gè)膠質(zhì)瘤細(xì)胞系、1個(gè)正常細(xì)胞系HEK293中AJAP1蛋白表達(dá)水平進(jìn)行檢測(cè)，初步研究分析AJAP1與膠質(zhì)瘤級(jí)別之間的關(guān)系。 第二部分側(cè)重于研究人腦膠質(zhì)瘤細(xì)胞轉(zhuǎn)染AJAP1重組腺病毒后對(duì)膠質(zhì)瘤細(xì)胞惡性生物學(xué)表型及對(duì)細(xì)胞骨架影響的體外研究，并初步探討其潛在機(jī)制。U87、U251細(xì)胞轉(zhuǎn)染AJAP1重組腺病毒質(zhì)粒后，Transewell實(shí)驗(yàn)檢測(cè)膠質(zhì)瘤細(xì)胞侵襲能力的改變；平板克隆形成試驗(yàn)檢測(cè)細(xì)胞的增殖能力；劃痕實(shí)驗(yàn)評(píng)價(jià)細(xì)胞遷移能力。Western blot方法檢測(cè)膠質(zhì)瘤細(xì)胞增殖、侵襲等相關(guān)指標(biāo)的變化，同時(shí)應(yīng)用免疫熒光方法檢測(cè)過(guò)表達(dá)AJAP1后對(duì)細(xì)胞骨架的變化。 第三部分則利用顱腦立體定向技術(shù)將同時(shí)轉(zhuǎn)染AJAP1重組腺病毒和熒光素酶慢病毒的U87膠質(zhì)瘤細(xì)胞接種在5周大小的BALB/c-nu（SPF）雌性裸鼠尾狀核建立顱內(nèi)膠質(zhì)瘤模型，設(shè)空白對(duì)照組，利用活體生物發(fā)光成像系統(tǒng)（IVISLumina Imaging System）動(dòng)態(tài)觀察腫瘤生長(zhǎng)情況及裸鼠的生存期的改變，應(yīng)用組織病理學(xué)方法檢測(cè)兩組動(dòng)物腫瘤標(biāo)本中增殖、侵襲等蛋白表達(dá)水平的變化。結(jié)果 通過(guò)免疫組化對(duì)石蠟切片檢測(cè)顯示：膠質(zhì)瘤樣本中AJAP1的表達(dá)水平顯著低于正常腦組織，并且隨著膠質(zhì)瘤級(jí)別的增高，AJAP1的表達(dá)隨著降低。組織免疫熒光試驗(yàn)也同樣證實(shí)在高級(jí)別的膠質(zhì)瘤標(biāo)本中，AJAP1的表達(dá)明顯低于低級(jí)別膠質(zhì)瘤標(biāo)本，與免疫組化結(jié)果相符，AJAP1定位于胞漿。因此AJAP1的表達(dá)水平與膠質(zhì)瘤病理分級(jí)呈負(fù)相關(guān)。在膠質(zhì)瘤細(xì)胞系中，Western blot檢測(cè)顯示在人胚腎上皮細(xì)胞HEK293中呈高表達(dá)，而在大部分膠質(zhì)瘤細(xì)胞系中顯著低表達(dá)，以A172、TJ905、TJ899低表達(dá)最為顯著。隨后，利用AJAP1重組腺病毒轉(zhuǎn)染U87、U251膠質(zhì)瘤細(xì)胞，通過(guò)提高細(xì)胞中AJAP1的蛋白表達(dá)水平，AJAP1組與空白對(duì)照組及轉(zhuǎn)染空載體vector組相比較，平板克隆形成試驗(yàn)顯示AJAP1組腫瘤細(xì)胞的克隆數(shù)形成減低，transwell試驗(yàn)顯示AJAP1組細(xì)胞的侵襲能力受到明顯抑制，劃痕試驗(yàn)顯示細(xì)胞的遷移能力下降，Western Blot檢測(cè)Ki-67、MMP-9蛋白的表達(dá)水平均有明顯的下調(diào)。在細(xì)胞免疫熒光試驗(yàn)中，過(guò)表達(dá)AJAP1后，，U87、U251細(xì)胞骨架結(jié)構(gòu)發(fā)生改變，AJAP1組與vector組相比較，細(xì)胞生長(zhǎng)模式發(fā)生改變，由原來(lái)的球狀聚集生長(zhǎng)形態(tài)變?yōu)槠瑺钌L(zhǎng)，絲狀偽足明顯減少。 在體內(nèi)動(dòng)物實(shí)驗(yàn)中，活體動(dòng)物生物熒光成像結(jié)果證實(shí)U87-AJAP1組成瘤明顯小于U87-vector組，且vector組于32d內(nèi)全部死亡，AJAP1組32d內(nèi)僅死亡4只。免疫組化結(jié)果表明AJAP1的表達(dá)明顯上調(diào)，而Ki-67、MMP-9的表達(dá)明顯下調(diào)，與細(xì)胞實(shí)驗(yàn)結(jié)果相符。 結(jié)論 1.以正常腦組織做對(duì)照，AJAP1基因在膠質(zhì)瘤標(biāo)本和惡性膠質(zhì)瘤細(xì)胞系中存在低表達(dá)，與腫瘤級(jí)別呈負(fù)相關(guān)，提示AJAP1可能作為抑癌基因參與膠質(zhì)瘤的發(fā)生與發(fā)展。 2.應(yīng)用AJAP1重組腺病毒載體轉(zhuǎn)染人腦膠質(zhì)瘤U87和U251細(xì)胞后，可有效抑制腫瘤細(xì)胞的增殖、侵襲和遷移能力，細(xì)胞免疫熒光提示在人腦膠質(zhì)瘤細(xì)胞中過(guò)表達(dá)AJAP1后，可能通過(guò)對(duì)細(xì)胞骨架的重建、絲狀偽足的減少，從而降低膠質(zhì)瘤細(xì)胞侵襲、遷移能力。 3.成功建立U87細(xì)胞顱內(nèi)膠質(zhì)瘤模型，在體內(nèi)進(jìn)一步證實(shí)了AJAP1的抑瘤作用，與體外實(shí)驗(yàn)結(jié)果相符。 4.通過(guò)對(duì)AJAP1對(duì)膠質(zhì)瘤增殖、侵襲、遷移等抑制作用的分子機(jī)制的初步研究，表明AJAP1可作為膠質(zhì)瘤基因治療的潛在靶點(diǎn)。AJAP1與細(xì)胞骨架關(guān)系密切，值得進(jìn)一步深入探討。
[Abstract]:Glioma is the most common primary intracranial tumor, of which more than 2/3 is malignant glioma, which accounts for about 2% of adult tumor, the median survival time of glioblastoma is only 1 years, the survival time of 5 years is almost zero [1-2]., and the comprehensive curative effect of postoperative chemotherapy and radiotherapy is still not very ideal. There is no radical cure. The long characteristic is the root cause of glioma which is difficult to cure and relapse. Therefore, the optimization of the treatment strategy of glioma should not only focus on the treatment of primary tumor, but also focus on controlling the invasion and metastasis of tumor cells to normal brain tissue. To find the related genes that can inhibit the invasion and metastasis of glioma cells, and to reveal its role in depth. The mechanism is of great significance for the treatment of glioma.
More and more evidence suggests that a number of recent studies in the human chromosome 1p36 region encode a number of tumor suppressor genes, [3-4]., the adhesion associated protein -1 (Adherens Junctional AssociatedProtein-1, AJAP1, also known as Shrew-1) as another similar tumor suppressor gene located in the human chromosome 1p36 region.Radlwimmer study The group of patients with glioblastoma were compared with genomic hybridization and microarray analysis. AJAP1 was first described as a biomarker [5]. associated with glioblastoma. The Zhang topic group proved that the AJAP1 promoter region was highly methylated [6]. but in the various cell lines, such as SVHVC, LOVO, SW480, MCF-7, but in glioma cells. The mechanism of changing the expression level of AJAP1 on invasion and proliferation of cells is still unclear.
This topic focuses on the abnormal expression of AJAP1 in glioma, discusses its relationship with the grade of glioma, and changes the expression level of AJAP1 in glioma cells by AJAP1 recombinant adenovirus transfection technology, and preliminarily studies the effect of AJAP1 on the biological phenotype of glioma and its potential mechanism.
Method
In the first part, immunohistochemical staining and immunofluorescence confocal imaging were used to detect the expression level of AJAP1 in 48 specimens of glioma and 5 normal brain tissue specimens. The level of AJAP1 protein expression in 8 glioma cell lines and 1 normal cell lines HEK293 was detected by Western blot method, and AJAP1 was preliminarily studied and analyzed. The relationship between the grade of glioma.
The second part focuses on the study of the human glioma cells transfected with AJAP1 recombinant adenovirus to the malignant biological phenotype of glioma cells and the effect of the cytoskeleton on the cytoskeleton in vitro. The potential mechanism.U87, U251 cells transfected with AJAP1 recombinant adenovirus plasmid, the Transewell test was used to detect the invasion ability of glioma cells. The proliferation ability of the cells was detected by the plate cloning test, and the scratch test was used to evaluate the cell migration ability.Western blot method to detect the changes of the proliferation and invasion of glioma cells, and the changes of cytoskeleton after the expression of AJAP1 were detected by immunofluorescence.
In the third part, the U87 glioma cells transfected with AJAP1 recombinant adenovirus and luciferase lentivirus were injected into the caudate nucleus of BALB/c-nu (SPF) female nude mice at the size of 5 weeks to establish the intracranial glioma model, and a blank control group was set up with the dynamic bioluminescence imaging system (IVISLumina Imaging System) dynamics. The growth of tumor and the change of the survival period of nude mice were observed. Histopathological methods were used to detect the changes in the expression of proliferation and invasion of the two groups of animal tumor specimens.
The detection of paraffin section by immunohistochemistry showed that the expression level of AJAP1 in the glioma samples was significantly lower than that of the normal brain tissue, and the expression of AJAP1 decreased with the increase of glioma level. The tissue immunofluorescence test also confirmed that the expression of AJAP1 in the high grade glioma specimens was significantly lower than that of the low grade glioma. In accordance with the immunohistochemical results, AJAP1 was located in the cytoplasm. Therefore, the expression level of AJAP1 was negatively correlated with the pathological grade of glioma. In the glioma cell line, the Western blot detection showed high expression in the human embryonic renal epithelial cell HEK293, but in most glioma cell lines, the low expression was the most low expression of A172, TJ905, TJ899. Then, U87, U251 glioma cells were transfected with AJAP1 recombinant adenovirus. By improving the protein expression level of AJAP1 in the cells, the AJAP1 group was compared with the blank control group and the empty carrier vector group. The clone formation test showed that the number of clones in the AJAP1 group was reduced, and the Transwell test showed the invasion of the AJAP1 group. The attack ability was obviously inhibited, the scratch test showed that the cell migration ability decreased, Western Blot detected Ki-67, and the expression level of MMP-9 protein decreased obviously. In the cell immunofluorescence test, after overexpression of AJAP1, the structure of U87 and U251 cytoskeleton changed, and the cell growth pattern of AJAP1 group was compared with the vector group, and the cell growth pattern changed. The original globular growth morphology changed to lamellar growth and filamentous foot decreased significantly.
In vivo animal experiments, the results of biological fluorescence imaging of living animals confirmed that the U87-AJAP1 formation tumor was significantly less than the U87-vector group, and the vector group died within 32D, and only 4 in AJAP1 group 32D died. The immunohistochemical results showed that the expression of AJAP1 was obviously up-regulated, and the table of Ki-67 and MMP-9 decreased obviously, which was in accordance with the results of cell experiment.
conclusion
1. the low expression of AJAP1 gene in glioma specimens and malignant glioma cell lines is negatively correlated with normal brain tissue, suggesting that AJAP1 may be used as a tumor suppressor gene to participate in the occurrence and development of glioma.
2. transfection of AJAP1 recombinant adenovirus vector to human glioma U87 and U251 cells can effectively inhibit the proliferation, invasion and migration of tumor cells. Cell immunofluorescence suggests that after overexpression of AJAP1 in human glioma cells, it may be possible to reduce the invasion and migration of glioma cells by reconstructing the cytoskeleton and reducing the filamentous pseudo foot. Ability to move.
3. the U87 cell glioma model was successfully established, which further confirmed the inhibitory effect of AJAP1 in vivo.
4. the preliminary study of the molecular mechanism of AJAP1 on the proliferation, invasion and migration of glioma shows that AJAP1 can be a potential target of gene therapy for glioma,.AJAP1 is closely related to cytoskeleton, and it is worth further exploring.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41
本文編號(hào)：2121429
[Abstract]:Glioma is the most common primary intracranial tumor, of which more than 2/3 is malignant glioma, which accounts for about 2% of adult tumor, the median survival time of glioblastoma is only 1 years, the survival time of 5 years is almost zero [1-2]., and the comprehensive curative effect of postoperative chemotherapy and radiotherapy is still not very ideal. There is no radical cure. The long characteristic is the root cause of glioma which is difficult to cure and relapse. Therefore, the optimization of the treatment strategy of glioma should not only focus on the treatment of primary tumor, but also focus on controlling the invasion and metastasis of tumor cells to normal brain tissue. To find the related genes that can inhibit the invasion and metastasis of glioma cells, and to reveal its role in depth. The mechanism is of great significance for the treatment of glioma.
More and more evidence suggests that a number of recent studies in the human chromosome 1p36 region encode a number of tumor suppressor genes, [3-4]., the adhesion associated protein -1 (Adherens Junctional AssociatedProtein-1, AJAP1, also known as Shrew-1) as another similar tumor suppressor gene located in the human chromosome 1p36 region.Radlwimmer study The group of patients with glioblastoma were compared with genomic hybridization and microarray analysis. AJAP1 was first described as a biomarker [5]. associated with glioblastoma. The Zhang topic group proved that the AJAP1 promoter region was highly methylated [6]. but in the various cell lines, such as SVHVC, LOVO, SW480, MCF-7, but in glioma cells. The mechanism of changing the expression level of AJAP1 on invasion and proliferation of cells is still unclear.
This topic focuses on the abnormal expression of AJAP1 in glioma, discusses its relationship with the grade of glioma, and changes the expression level of AJAP1 in glioma cells by AJAP1 recombinant adenovirus transfection technology, and preliminarily studies the effect of AJAP1 on the biological phenotype of glioma and its potential mechanism.
Method
In the first part, immunohistochemical staining and immunofluorescence confocal imaging were used to detect the expression level of AJAP1 in 48 specimens of glioma and 5 normal brain tissue specimens. The level of AJAP1 protein expression in 8 glioma cell lines and 1 normal cell lines HEK293 was detected by Western blot method, and AJAP1 was preliminarily studied and analyzed. The relationship between the grade of glioma.
The second part focuses on the study of the human glioma cells transfected with AJAP1 recombinant adenovirus to the malignant biological phenotype of glioma cells and the effect of the cytoskeleton on the cytoskeleton in vitro. The potential mechanism.U87, U251 cells transfected with AJAP1 recombinant adenovirus plasmid, the Transewell test was used to detect the invasion ability of glioma cells. The proliferation ability of the cells was detected by the plate cloning test, and the scratch test was used to evaluate the cell migration ability.Western blot method to detect the changes of the proliferation and invasion of glioma cells, and the changes of cytoskeleton after the expression of AJAP1 were detected by immunofluorescence.
In the third part, the U87 glioma cells transfected with AJAP1 recombinant adenovirus and luciferase lentivirus were injected into the caudate nucleus of BALB/c-nu (SPF) female nude mice at the size of 5 weeks to establish the intracranial glioma model, and a blank control group was set up with the dynamic bioluminescence imaging system (IVISLumina Imaging System) dynamics. The growth of tumor and the change of the survival period of nude mice were observed. Histopathological methods were used to detect the changes in the expression of proliferation and invasion of the two groups of animal tumor specimens.
The detection of paraffin section by immunohistochemistry showed that the expression level of AJAP1 in the glioma samples was significantly lower than that of the normal brain tissue, and the expression of AJAP1 decreased with the increase of glioma level. The tissue immunofluorescence test also confirmed that the expression of AJAP1 in the high grade glioma specimens was significantly lower than that of the low grade glioma. In accordance with the immunohistochemical results, AJAP1 was located in the cytoplasm. Therefore, the expression level of AJAP1 was negatively correlated with the pathological grade of glioma. In the glioma cell line, the Western blot detection showed high expression in the human embryonic renal epithelial cell HEK293, but in most glioma cell lines, the low expression was the most low expression of A172, TJ905, TJ899. Then, U87, U251 glioma cells were transfected with AJAP1 recombinant adenovirus. By improving the protein expression level of AJAP1 in the cells, the AJAP1 group was compared with the blank control group and the empty carrier vector group. The clone formation test showed that the number of clones in the AJAP1 group was reduced, and the Transwell test showed the invasion of the AJAP1 group. The attack ability was obviously inhibited, the scratch test showed that the cell migration ability decreased, Western Blot detected Ki-67, and the expression level of MMP-9 protein decreased obviously. In the cell immunofluorescence test, after overexpression of AJAP1, the structure of U87 and U251 cytoskeleton changed, and the cell growth pattern of AJAP1 group was compared with the vector group, and the cell growth pattern changed. The original globular growth morphology changed to lamellar growth and filamentous foot decreased significantly.
In vivo animal experiments, the results of biological fluorescence imaging of living animals confirmed that the U87-AJAP1 formation tumor was significantly less than the U87-vector group, and the vector group died within 32D, and only 4 in AJAP1 group 32D died. The immunohistochemical results showed that the expression of AJAP1 was obviously up-regulated, and the table of Ki-67 and MMP-9 decreased obviously, which was in accordance with the results of cell experiment.
conclusion
1. the low expression of AJAP1 gene in glioma specimens and malignant glioma cell lines is negatively correlated with normal brain tissue, suggesting that AJAP1 may be used as a tumor suppressor gene to participate in the occurrence and development of glioma.
2. transfection of AJAP1 recombinant adenovirus vector to human glioma U87 and U251 cells can effectively inhibit the proliferation, invasion and migration of tumor cells. Cell immunofluorescence suggests that after overexpression of AJAP1 in human glioma cells, it may be possible to reduce the invasion and migration of glioma cells by reconstructing the cytoskeleton and reducing the filamentous pseudo foot. Ability to move.
3. the U87 cell glioma model was successfully established, which further confirmed the inhibitory effect of AJAP1 in vivo.
4. the preliminary study of the molecular mechanism of AJAP1 on the proliferation, invasion and migration of glioma shows that AJAP1 can be a potential target of gene therapy for glioma,.AJAP1 is closely related to cytoskeleton, and it is worth further exploring.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
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2 宿華威;崔云甫;吳德全;韓德恩;;活體生物螢光成像技術(shù)新進(jìn)展[J];世界華人消化雜志;2006年24期
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