S100B在胶质瘤生长中的作用及其机制的研究

发布时间：2018-04-01 14:00

本文选题：S100B　切入点：CCL2　出处：《山东大学》2014年博士论文

【摘要】：胶质瘤是神经外科中最常见的恶性肿瘤,它是神经上皮性肿瘤,常由间质细胞组成,其生长特点为浸润性生长,常见的临床症状为颅内压增高及相应生长部位的局灶性症状,该肿瘤的恶性程度高,与周围组织界限不清,生长迅速且具有很强的侵袭性,目前为止仍无法手术完全切除,术后容易复发,且由于血脑屏障的存在致使化疗的效果不佳。目前可用的治疗方法有手术治疗、放疗、化疗、免疫治疗、基因治疗、微波治疗等综合治疗方法。胶质瘤患者的预后非常差,中位生存期为18个月。 S100B是钙结合蛋白多基因家族中的一员,它在细胞的代谢、增殖和移动中发挥了一定作用。研究表明神经系统中的星形细胞释放出毫摩尔级别浓度的S100B蛋白至细胞外间隙,从而促进轴突的生长并能保护神经元。在肿瘤、炎症等病理条件下,S100B蛋白的浓度上升至微摩尔浓度级别,从而刺激小神经胶质细胞和星形细胞,并能升高前炎性细胞因子的表达。S100B蛋白在大部分恶性胶质瘤中过度表达。目前常认为肿瘤的发生是因为肿瘤抑制蛋白p53的功能受到抑制以及通过刺激有丝分裂激活酶Ndr和Akt来调节细胞增殖及分化。S100B蛋白的浓度与胶质瘤的生长及星形细胞的功能有关系。中枢神经系统中S100B蛋白可能会有利于星形细胞的分化并可能促进星形细胞的活化和运动。但是,S100B在胶质瘤发生中的所起的作用,以及S100B对该作用的机制,目前为止还没有广泛的研究,还是不清楚。目的目前关于S100B蛋白在胶质瘤发生、生长中的作用还没有进行广泛的研究。此前研究显示即使低浓度的S100B蛋白亦可激活Stat3从而调节肿瘤相关巨噬细胞(TAM)的活性。因此,我们假设：S100B蛋白的表达同样可以通过调节体内TAM的活性来促进胶质瘤的生长增殖。本实验通过使用多种试验手段观察S100B蛋白在胶质瘤生长方面的作用以及S100B在肿瘤生长及血管生成时起作用的途径,从而明确S100B在胶质瘤发生及生长中所起的作用,初步阐明S100B通过何种途径促进胶质瘤生长的机制,并探讨在胶质瘤治疗方面以S100B蛋白为靶点,使用S100B抑制剂来抑制肿瘤生长的潜在的利用价值。方法： 1.稳定转染的三种细胞系：使用细胞转染及筛选后稳定表达并传代的三种细胞系。 2.S100B对胶质瘤生长的作用： 2.1S100B在体外试验时对GL261细胞系生长的影响：使用Western blot及其蛋白定量和ELISA检测S100B在三种细胞系中的表达情况,用细胞计数法及流式细胞术检测三种细胞系细胞在体外培养的生长速度。 2.2S100B在胶质瘤动物模型中对GL261细胞生长的影响：使用荧光免疫组织化学、实际肿瘤大小的比较、三种细胞系小鼠生存试验、流式细胞术、活体动物血管成像来检测S100B在动物模型中对肿瘤生长的影响。 2.3抑制S100B对胶质瘤动物模型中GL261细胞生长的影响：使用Western blot、小鼠生存试验、流式细胞术、流式细胞术检测S100B受抑制时对胶质瘤生长的影响。 2.4S100B对炎症及巨噬细胞浸润情况的影响：使用NF-κB检测、RT-PCR及流式细胞术检测S100B对炎症及巨噬细胞浸润情况的影响。 3. RAGE在胶质瘤生长的影响：使用Western blot、荧光免疫组织化学、RT-PCR、流式细胞术检测RAGE在胶质瘤生长中的作用。 4.CLL2在胶质瘤生长中的影响：使用ELISA、Western blot、荧光免疫组织化学、RT-PCR检测CLL2在胶质瘤生长中的影响。 5.S100B与胶质瘤的联系：使用恶性肿瘤基因图谱分析来检测S100B与肿瘤亚型的关系及在各个亚型中S100B与CLL2的关联性。结果： 1.S100B可以促进体内胶质瘤的生长我们发现三种不同S100B表达水平的GL261细胞系在体外生长的增值速率相似。与此相反,这三种细胞系在体内肿瘤的生长速率及肿瘤血管生成速度是有区别的。 2.S100B促进了炎症的发展并增强了巨噬细胞对胶质瘤的渗透能力体内S100B高表达的细胞系S100Bhi-肿瘤表现出了增强的炎性反应,流式细胞术同时证明巨噬细胞(CD11bhi8hCD45hi8h)在S100Bh-和S100Bwt中的表达要高于在S100B1ow中的表达,并发现通过抑制S100B可以减低TAM的浸润并可以延长载胶质瘤动物的生存期。 3.S100B诱导的RAGE活化 S100B在肿瘤中可以诱导RAGE活化, S100B是唯一一个在肿瘤中可以刺激RAGE表达的RAGE配体,但RAGE可能不是S100B介导的TAM浸润模型中的必须品。 4.趋化因子的产生体内体外实验表明S100B表达的上调导致了GL261细胞中CCL2的诱导的发生,并且它是TAM浸润入胶质瘤组织过程中的决定因素。相反的是,这三种细胞系分泌的CXCL12却没有明显的区别。另外相关的一个细胞系也证实CCL2的表达随S100B的增加而增加,并且同时增加的还有TAM的浸润。 5.与人类胶质瘤的关系为了评价我们发现的结果与人类胶质瘤之间的联系,通过分析TCGA中开放数据库提供的数据,S100B的表达与原神经性、神经性、经典型胶质瘤有正相关关系,并发现S100B的表达在所有TCGA HTU133A array中与CCL2的表达有关系,并且在神经元型及原神经元型胶质瘤亚型中有更强的关联。以上结果肯定了S100B在一些胶质瘤亚型中可能是通过上调CCL2的表达来进行TAM浸润这一过程。结论：体内、体外实验证实：胶质瘤中S100B很可能是通过CCL2的上调表达及TAM的化学趋化性来促进了肿瘤的生长。胶质瘤可以分泌S100B,其可以通过影响趋化因子CCL2的化学趋化作用吸引骨髓源性的巨噬细胞从而达到促进胶质瘤生长。因此胶质瘤中S100B与CCL2在巨噬细胞的浸润及胶质瘤的生长中起着十分关键的作用。这为胶质瘤治疗中S100B抑制物提供了一个潜在的应用机会。
[Abstract]:Gliomas are the most common malignant tumor in neurosurgery , which is a neuroepithelial tumor , which is usually composed of stromal cells . Its growth characteristics are invasive growth . The common clinical symptoms are intracranial pressure increase and focal symptom of the corresponding growth part . The tumor has a high degree of malignancy , and has a very strong invasion ability . So far , it has not been completely cut off , and the effect of chemotherapy is not good . The treatment methods available now have comprehensive treatment methods such as surgical treatment , radiotherapy , chemotherapy , immunotherapy , gene therapy , microwave treatment , etc . The prognosis of the patients with glioma is very poor , and the median survival time is 18 months .

S100B is a member of the multi - gene family of calcium - binding proteins . It plays a role in the metabolism , proliferation and movement of the cells .

Purpose

It has been shown that S100B protein can also activate Stat3 to regulate the activity of tumor - related macrophages ( TAM ) . Therefore , it is assumed that the expression of S100B protein can also promote the growth and proliferation of glioma by regulating the activity of TAM in vivo .

The effects of S100B on the growth of glioma and the role of S100B in tumor growth and angiogenesis were observed by various test methods .

Method :

1 . Three cell lines stably transfected : three cell lines stably expressed and passaged using cell transfection and screening .

2 . Effect of S100B on growth of glioma :

2.1 S100B was used to detect the growth of GL261 cell line in vitro . Western blot and its protein quantification and ELISA were used to detect the expression of S100B in three cell lines . Cell counting and flow cytometry were used to detect the growth rate of three cell line cells in vitro .

2 . The effect of S100B on the growth of GL261 cells was investigated in the animal model of glioma by the comparison of fluorescence and immunohistochemistry , actual tumor size , survival test of three cell lines , flow cytometry , and vascular imaging of living animals .

2.3 Inhibition of S100B on GL261 cell growth in glioma animal model : Western blot , mouse survival test , flow cytometry and flow cytometry were used to detect the effect of S100B on glioma growth .

2 . Effect of 4S100B on inflammation and macrophage infiltration : Using NF - 魏B assay , RT - PCR and flow cytometry were used to detect the effects of S100B on inflammation and macrophage infiltration .

3 . Effects of RAGE on glioma growth : Western blot , fluorescence immunohistochemistry , RT - PCR and flow cytometry were used to detect the role of RAGE in the growth of glioma .

4 . Effects of CLL2 on the growth of glioma : ELISA , Western blot , fluorescence immunohistochemistry and RT - PCR were used to detect the effect of CLL2 on the growth of glioma .

5 . Association between S100B and glioma : The relationship between S100B and tumor subtype and S100B and CLL2 in each sub - subtype were detected by using gene map analysis of malignant tumor .

Results :

1.S100B can promote the growth of glioma in vivo

In contrast , the growth rate of these three cell lines in vivo and the rate of tumor angiogenesis were different .

2 . S100B promotes the development of inflammation and enhances the permeability of macrophages to glioma .

In vivo S100B high expression of the cell line , the tumor cell line , Bhi - tumor , showed an enhanced inflammatory response , and flow cytometry demonstrated that the expression of macrophages ( CD11bhi8hCD8h8 8h ) was higher in the expression of the expression of the expression of the tumor cells , and it was found that the inhibition of S100B could reduce the infiltration of TAM and prolong the survival time of the glioma animals .

3 . S100B - induced RAGE activation

S100B may induce RAGE activation in tumors , and S100B is the only RAGE ligand that can stimulate RAGE expression in tumors , but RAGE may not be a necessity in S100B - mediated TAM infiltration models .

4 . Generation of chemokine

In vitro experiments in vivo showed that the up - regulation of the expression of S100B led to the induction of CCL2 in GL261 cells , and it was a determinant of TAM infiltration into the glioma tissue . On the contrary , the CXCL12 secreted by these three cell lines was not significantly different . A further cell line also confirmed that the expression of CCL2 increased with the increase of S100B and also increased the infiltration of TAM .

5 . Relationship with human glioma

In order to evaluate the relationship between the findings and human glioma , the expression of S100B is related to the expression of CCL2 in all TCGA HTU133A arrays by analyzing the data provided by the open database in TCGA , and the expression of S100B is associated with the expression of CCL2 in all TCGA HTU133A arrays .

Conclusion :

In vivo , in vitro experiments confirm that S100B in glioma is likely to promote tumor growth through up - regulation of CCL2 and the chemotropism of TAM . S100B can be secreted by glioma , which can induce the growth of glioma . S100B and CCL2 in glioma play a key role in the infiltration of macrophages and growth of glioma . This provides a potential application opportunity for S100B inhibitor in glioma treatment .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

【共引文献】