ABCG2基因促进胶质瘤干细胞增殖与侵袭的实验研究

发布时间：2018-03-10 00:06

本文选题：ABCG2　切入点：胶质瘤干细胞　出处：《苏州大学》2015年博士论文　论文类型：学位论文

【摘要】：目前研究表明,恶性神经胶质瘤(glioma)简称胶质瘤,是中枢神经系统最常见的原发性恶性肿瘤。胶质瘤的发病率约占所有脑部原发性肿瘤的50%。广义的胶质瘤包含所有神经上皮性的肿瘤,狭义的定义仅包含胶质细胞来源的颅内肿瘤。胶质瘤细胞具有多源性,包含星形细胞瘤、少支胶质瘤和室管膜瘤等十余种类型。来源不同,肿瘤生物学行为亦相异。星形胶质细胞瘤是最常见的胶质瘤类型,发病率约占中枢神经系统肿瘤的31%,占胶质瘤的78%左右。此病男性多见,高发年龄为30~40岁。发病部位以大脑颞叶和额叶较为多见。星形胶质细胞瘤亦具有多形性,根据国际卫生组织(WHO)的分级标准,肥胖型星形细胞瘤为Ⅱ~Ⅲ级,间变型星形细胞瘤为Ⅲ级,多形性胶质母细胞瘤(GBM)则为Ⅵ级。分级愈高,侵袭性和浸润性愈强。目前,胶质瘤的外科治疗得到显著提高,并配合相应的放疗和化疗药物靶向作用提升,胶质瘤的治愈水平已经显著改善。但是由于其具有高度的侵袭性和浸润性,胶质瘤患者的总体治愈率仍差强人意。因此,有必要深入的了解胶质瘤细胞恶性肿瘤学生物行为的相关分子学机制,为今后的靶向治疗奠定一定的理论基础。(ATP-binding cassette sub-family G member 2,ABCG2)亦被称为乳腺癌抵抗蛋白或者palacental ABC protein/mitoxantrone抵抗蛋白。ABCG2蛋白隶属于ATP结合的cassette转运子家族。这一家族是成员最多和最为古老的转运子组成单位,具有高度的进化保守性和物种间同质性。现已证实:包含胰腺、肝脏、小肠和直肠在内的多种组织内均存在ABCG2蛋白的表达。此外,ABCG2蛋白在神经胶质瘤细胞、乳腺癌细胞和肺癌细胞表面存在过表达现象。新近,有研究报道:在多种干细胞表面观测到有ABCG2蛋白的表达,并具有高度相似性;此外,ABCG2蛋白亦被认为是一种调控干细胞发生“side-population”行为的关键分子。因此,不少研究者认为:ABCG2蛋白可能是一种非常有效的肿瘤干细胞标志物。通过调控ABCG2基因的表达,也许能够影响肿瘤细胞的生物学行为。已有研究表明,下调ABCG2的表达能够抑制结直肠癌细胞和肾母细胞瘤细胞的增殖、迁移和浸润能力。但是,abcg2基因调控和神经胶质细胞瘤的恶性生物学行为之间的关系,尚未得到充分阐明。基质金属蛋白酶(matrixmetalloproteinases,mmps)隶属于锌依赖性内肽酶家族。目前,在人体内,已被证实存在23位成员。mmps是促进细胞外基质(ecm)和细胞基底膜降解的主要酶类。ecm和细胞基底膜是调控单个细胞同细胞周围环境相互作用,以及多细胞和多组织间协同发育的重要媒介。因此,mmps在多种细胞生物学过程中均发挥了重要作用,这其中就包括肿瘤细胞的迁移和浸润。现已证实:mmp9活性增高与gbm细胞迁移和浸润性增强密切相关。新近,一项关于mmp9活性抑制剂,marimastat,联合化疗的临床研究已在gbm患者中开展。因此,有理由认为进一步深入了解mmps促进gbm发病机制和研制针对于mmps活性的靶向治疗技术,将可能有效提高gbm治疗水平。目的:1.研究abcg2基因对cd133阳性的u251干细胞增殖、迁移和侵袭性的影响,为胶质瘤的治疗提供理论依据。2.探讨abcg2蛋白表达水平与mmps的表达及其活性的关系,进一步揭示下调abcg2蛋白表达抑制gbm侵袭的分子学机制。方法:1.培养u251细胞系,免疫磁珠法分离cd133阳性细胞。分别使用abcg2-sirna和abcg2过表达质粒转染u251细胞,构建abcg2蛋白低表达和过表达模型。2.在给予cd133阳性u251细胞abcg2-sirna和abcg2过表达质粒转染后,观察abcg2蛋白低表达和高表达情况下,分别使用mtt、transell法和细胞划痕法测定细胞增殖、侵袭和迁移的变化。3.在给予cd133阳性u251细胞abcg2-sirna和abcg2过表达质粒转染后,abcg2蛋白低表达和高表达情况下,elisa法和pcr法分别测定mmp9蛋白和mrna的表达,采用明胶酶谱法测定mmp9的活性的改变。结果:1.在给予cd133阳性u251细胞abcg2-sirna后,abcg2蛋白和mrna表达明显减少。而给予cd133阳性u251细胞abcg2过表达质粒转染后,可以发现ABCG2蛋白和mRNA表达明显增加。2.在给予CD133阳性U251细胞ABCG2-siRNA后,伴随ABCG2蛋白表达减少,U251细胞的增殖、浸润和迁移受到明显抑制。而给予CD133阳性U251细胞ABCG2过表达质粒转染后伴随ABCG2蛋白表达增加,U251细胞的增殖、浸润和迁移显著增强。3.给予CD133阳性U251细胞ABCG2-siRNA和ABCG2过表达质粒转染,虽然能够改变ABCG2蛋白的表达,但是并不影响MMP9蛋白和mRNA的合成。在给予CD133阳性U251细胞ABCG2-siRNA后,伴随ABCG2蛋白表达减少,U251干细胞MMP9活性显著降低。在给予CD133阳性U251细胞过表达质粒后,伴随ABCG2蛋白表达增加,U251干细胞MMP9活性显著增强。结论:1.下调U251干细胞ABCG2蛋白的表达,能够有效抑制U251干细胞增殖、浸润和迁移。2.ABCG2基因表达调控对U251干细胞侵袭分子机制可能是由于其对MMP9活性的影响。
[Abstract]:The present study showed that malignant glioma (referred to as glioma) is the central nervous system of glioma, the most common primary malignant tumors. Glioma incidence of all brain glioma primary tumor of the generalized 50%. contains all neural epithelial tumors, narrow definition contains only glial origin intracranial tumors. Gliomas are heterogeneous, including astrocytomas, OLS and ependymoma. Ten types of different sources, the biological behavior of the tumor is different. Astrocytoma glioma is the most common type, incidence of tumors of the central nervous system in 31%, accounting for glioma 78%. The male, age 30~40 years old. The lesion on temporal lobe and frontal brain is more common. Astrocytoma is pleomorphic, according to the World Health Organization (WHO) classification standard, obesity Astrocytoma II ~ III anaplastic astrocytoma grade III, glioblastoma multiforme (GBM) for grade vi. Higher grade, invasive and invasive stronger. At present, surgical treatment of glioma is significantly improved, and the radiotherapy and chemotherapy drugs to enhance the role of glioma, the cure level has been significantly improved. But because of its highly invasive and invasive glioma patients, the overall cure rate is just passable. Therefore, it is necessary to further understand the molecular biological behavior of malignant glioma cells oncology learning mechanism, lay a theoretical foundation for future target to treat (ATP-binding cassette sub-family G. Member 2, ABCG2) also known as breast cancer resistance protein or palacental ABC protein/mitoxantrone resistance protein.ABCG2 protein belonging to cassette transporter family. This combination of ATP A family member is the largest and oldest transporter unit, are highly conserved between species and nature. It has been proved that contains the pancreas, liver, intestine and rectum, the expression of ABCG2 protein were found in other tissues. In addition, ABCG2 protein in glioma cells, the surface of breast cancer cells and lung cancer over expression of the cells. Recently, studies have reported that in a variety of stem cell surface observed the expression of ABCG2 protein, and a high degree of similarity; in addition, ABCG2 protein is also considered to be a key molecular regulation of stem cells "side-population" behavior. Therefore, many researchers believe that ABCG2 protein may be: a very effective tumor stem cell marker. By regulating the expression of ABCG2 gene, may influence the biological behavior of tumor cells. Studies have shown that can down regulate the expression of ABCG2 Inhibition of colorectal cancer cells and Wilms tumor cell proliferation, migration and invasion ability. However, the relationship between ABCG2 gene and the biological behavior of malignant glial cell tumors, has not been fully elucidated. Matrix metalloproteinases (matrixmetalloproteinases, MMPs) belong to the zinc dependent peptidase family. At present, in the human body that has been confirmed the existence of 23 members of.Mmps is to promote the extracellular matrix (ECM) cells and basement membrane degradation enzyme.Ecm and cell membrane is the regulation of individual cells with cell environment interactions, as well as an important medium and multi Organization collaborative multicellular development. Therefore, MMPs in a variety of cell biology plays an important role, including the migration and infiltration of tumor cells. It has been proved that the activity of MMP9 increased the migration and invasive enhancement is closely related to GBM cells. Recently, a On the activity of MMP9 inhibitor, Marimastat, clinical study of combination chemotherapy has been carried out in GBM patients. Therefore, there is reason to believe that further in-depth understanding of the pathogenesis and development of MMPs GBM in the MMPs activity of targeted therapy could effectively promote technology, improve the management level of GBM. Objective: 1. to study the ABCG2 gene on the proliferation of CD133 cells U251 positive effects of migration and invasion of the relationship, and provide a theoretical basis for the expression and activity of.2. on the expression level of ABCG2 protein and MMPs for the treatment of malignant glioma, further reveal the molecular mechanism of inhibiting the down-regulation of ABCG2 protein expression in GBM invasion. Methods: 1. U251 cells, CD133 positive cells were isolated by immunomagnetic beads. Using abcg2-sirna and ABCG2 expression plasmids were transfected into U251 cells, the construction of low expression of ABCG2 protein and expression of.2. in abcg2-sirna and ABCG2 model to CD133 positive U251 cells in table As plasmid after transfection, to observe ABCG2 protein low expression and high expression, respectively, using MTT, the cell proliferation was measured by transell assay and cell scratch assay, invasion and migration of.3. in abcg2-sirna and ABCG2 to change the CD133 + U251 cells expression plasmid transfected, ABCG2 protein low expression and high expression, the expression of MMP9 protein and mRNA was determined by ELISA method and PCR method respectively, using the MMP9 spectrum method for determination of gelatinase activity changes. Results: 1. in CD133 positive U251 cells after abcg2-sirna, the expression of ABCG2 and mRNA decreased significantly. Given CD133 positive U251 cells ABCG2 expression plasmid transfected, the expression of ABCG2 and mRNA can be found obviously increased.2. in CD133 positive U251 cells with ABCG2-siRNA, ABCG2 protein expression decreased, U251 cell proliferation, invasion and migration was inhibited. Given CD133 positive U251 cells ABCG2 With the increased expression of ABCG2 expression plasmid after transfection, U251 cell proliferation, invasion and migration of.3. to ABCG2-siRNA and ABCG2 significantly enhanced CD133 positive U251 cells overexpressing plasmid transfection, although able to change the expression of ABCG2 protein, but does not affect the synthesis of MMP9 protein and mRNA in CD133 positive U251 cells. After ABCG2-siRNA, with ABCG2 protein the reduced expression of U251 stem cells, MMP9 activity was significantly decreased. In CD133 + U251 cells expression plasmid, with increased expression of ABCG2, U251 stem cells MMP9 activity was significantly enhanced. Conclusion: the expression of U251 ABCG2 protein 1. stem cells, can effectively inhibit the proliferation of U251 stem cells, invasion and migration of.2.ABCG2 gene expression regulation the molecular mechanism of U251 stem cell invasion may be due to its effect on MMP9 activity.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.41

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