本文選題：S100B　切入點(diǎn)：CCL2　出處：《山東大學(xué)》2014年博士論文

【摘要】：膠質(zhì)瘤是神經(jīng)外科中最常見的惡性腫瘤,它是神經(jīng)上皮性腫瘤,常由間質(zhì)細(xì)胞組成,其生長特點(diǎn)為浸潤性生長,常見的臨床癥狀為顱內(nèi)壓增高及相應(yīng)生長部位的局灶性癥狀,該腫瘤的惡性程度高,與周圍組織界限不清,生長迅速且具有很強(qiáng)的侵襲性,目前為止仍無法手術(shù)完全切除,術(shù)后容易復(fù)發(fā),且由于血腦屏障的存在致使化療的效果不佳。目前可用的治療方法有手術(shù)治療、放療、化療、免疫治療、基因治療、微波治療等綜合治療方法。膠質(zhì)瘤患者的預(yù)后非常差,中位生存期為18個(gè)月。 S100B是鈣結(jié)合蛋白多基因家族中的一員,它在細(xì)胞的代謝、增殖和移動(dòng)中發(fā)揮了一定作用。研究表明神經(jīng)系統(tǒng)中的星形細(xì)胞釋放出毫摩爾級別濃度的S100B蛋白至細(xì)胞外間隙,從而促進(jìn)軸突的生長并能保護(hù)神經(jīng)元。在腫瘤、炎癥等病理?xiàng)l件下,S100B蛋白的濃度上升至微摩爾濃度級別,從而刺激小神經(jīng)膠質(zhì)細(xì)胞和星形細(xì)胞,并能升高前炎性細(xì)胞因子的表達(dá)。S100B蛋白在大部分惡性膠質(zhì)瘤中過度表達(dá)。目前常認(rèn)為腫瘤的發(fā)生是因?yàn)槟[瘤抑制蛋白p53的功能受到抑制以及通過刺激有絲分裂激活酶Ndr和Akt來調(diào)節(jié)細(xì)胞增殖及分化。S100B蛋白的濃度與膠質(zhì)瘤的生長及星形細(xì)胞的功能有關(guān)系。中樞神經(jīng)系統(tǒng)中S100B蛋白可能會有利于星形細(xì)胞的分化并可能促進(jìn)星形細(xì)胞的活化和運(yùn)動(dòng)。但是,S100B在膠質(zhì)瘤發(fā)生中的所起的作用,以及S100B對該作用的機(jī)制,目前為止還沒有廣泛的研究,還是不清楚。 目的 目前關(guān)于S100B蛋白在膠質(zhì)瘤發(fā)生、生長中的作用還沒有進(jìn)行廣泛的研究。此前研究顯示即使低濃度的S100B蛋白亦可激活Stat3從而調(diào)節(jié)腫瘤相關(guān)巨噬細(xì)胞(TAM)的活性。因此,我們假設(shè)：S100B蛋白的表達(dá)同樣可以通過調(diào)節(jié)體內(nèi)TAM的活性來促進(jìn)膠質(zhì)瘤的生長增殖。 本實(shí)驗(yàn)通過使用多種試驗(yàn)手段觀察S100B蛋白在膠質(zhì)瘤生長方面的作用以及S100B在腫瘤生長及血管生成時(shí)起作用的途徑,從而明確S100B在膠質(zhì)瘤發(fā)生及生長中所起的作用,初步闡明S100B通過何種途徑促進(jìn)膠質(zhì)瘤生長的機(jī)制,并探討在膠質(zhì)瘤治療方面以S100B蛋白為靶點(diǎn),使用S100B抑制劑來抑制腫瘤生長的潛在的利用價(jià)值。 方法： 1.穩(wěn)定轉(zhuǎn)染的三種細(xì)胞系：使用細(xì)胞轉(zhuǎn)染及篩選后穩(wěn)定表達(dá)并傳代的三種細(xì)胞系。 2.S100B對膠質(zhì)瘤生長的作用： 2.1S100B在體外試驗(yàn)時(shí)對GL261細(xì)胞系生長的影響：使用Western blot及其蛋白定量和ELISA檢測S100B在三種細(xì)胞系中的表達(dá)情況,用細(xì)胞計(jì)數(shù)法及流式細(xì)胞術(shù)檢測三種細(xì)胞系細(xì)胞在體外培養(yǎng)的生長速度。 2.2S100B在膠質(zhì)瘤動(dòng)物模型中對GL261細(xì)胞生長的影響：使用熒光免疫組織化學(xué)、實(shí)際腫瘤大小的比較、三種細(xì)胞系小鼠生存試驗(yàn)、流式細(xì)胞術(shù)、活體動(dòng)物血管成像來檢測S100B在動(dòng)物模型中對腫瘤生長的影響。 2.3抑制S100B對膠質(zhì)瘤動(dòng)物模型中GL261細(xì)胞生長的影響：使用Western blot、小鼠生存試驗(yàn)、流式細(xì)胞術(shù)、流式細(xì)胞術(shù)檢測S100B受抑制時(shí)對膠質(zhì)瘤生長的影響。 2.4S100B對炎癥及巨噬細(xì)胞浸潤情況的影響：使用NF-κB檢測、RT-PCR及流式細(xì)胞術(shù)檢測S100B對炎癥及巨噬細(xì)胞浸潤情況的影響。 3. RAGE在膠質(zhì)瘤生長的影響：使用Western blot、熒光免疫組織化學(xué)、RT-PCR、流式細(xì)胞術(shù)檢測RAGE在膠質(zhì)瘤生長中的作用。 4.CLL2在膠質(zhì)瘤生長中的影響：使用ELISA、Western blot、熒光免疫組織化學(xué)、RT-PCR檢測CLL2在膠質(zhì)瘤生長中的影響。 5.S100B與膠質(zhì)瘤的聯(lián)系：使用惡性腫瘤基因圖譜分析來檢測S100B與腫瘤亞型的關(guān)系及在各個(gè)亞型中S100B與CLL2的關(guān)聯(lián)性。 結(jié)果： 1.S100B可以促進(jìn)體內(nèi)膠質(zhì)瘤的生長 我們發(fā)現(xiàn)三種不同S100B表達(dá)水平的GL261細(xì)胞系在體外生長的增值速率相似。與此相反,這三種細(xì)胞系在體內(nèi)腫瘤的生長速率及腫瘤血管生成速度是有區(qū)別的。 2.S100B促進(jìn)了炎癥的發(fā)展并增強(qiáng)了巨噬細(xì)胞對膠質(zhì)瘤的滲透能力 體內(nèi)S100B高表達(dá)的細(xì)胞系S100Bhi-腫瘤表現(xiàn)出了增強(qiáng)的炎性反應(yīng),流式細(xì)胞術(shù)同時(shí)證明巨噬細(xì)胞(CD11bhi8hCD45hi8h)在S100Bh-和S100Bwt中的表達(dá)要高于在S100B1ow中的表達(dá),并發(fā)現(xiàn)通過抑制S100B可以減低TAM的浸潤并可以延長載膠質(zhì)瘤動(dòng)物的生存期。 3.S100B誘導(dǎo)的RAGE活化 S100B在腫瘤中可以誘導(dǎo)RAGE活化, S100B是唯一一個(gè)在腫瘤中可以刺激RAGE表達(dá)的RAGE配體,但RAGE可能不是S100B介導(dǎo)的TAM浸潤模型中的必須品。 4.趨化因子的產(chǎn)生 體內(nèi)體外實(shí)驗(yàn)表明S100B表達(dá)的上調(diào)導(dǎo)致了GL261細(xì)胞中CCL2的誘導(dǎo)的發(fā)生,并且它是TAM浸潤入膠質(zhì)瘤組織過程中的決定因素。相反的是,這三種細(xì)胞系分泌的CXCL12卻沒有明顯的區(qū)別。另外相關(guān)的一個(gè)細(xì)胞系也證實(shí)CCL2的表達(dá)隨S100B的增加而增加,并且同時(shí)增加的還有TAM的浸潤。 5.與人類膠質(zhì)瘤的關(guān)系 為了評價(jià)我們發(fā)現(xiàn)的結(jié)果與人類膠質(zhì)瘤之間的聯(lián)系,通過分析TCGA中開放數(shù)據(jù)庫提供的數(shù)據(jù),S100B的表達(dá)與原神經(jīng)性、神經(jīng)性、經(jīng)典型膠質(zhì)瘤有正相關(guān)關(guān)系,并發(fā)現(xiàn)S100B的表達(dá)在所有TCGA HTU133A array中與CCL2的表達(dá)有關(guān)系,并且在神經(jīng)元型及原神經(jīng)元型膠質(zhì)瘤亞型中有更強(qiáng)的關(guān)聯(lián)。以上結(jié)果肯定了S100B在一些膠質(zhì)瘤亞型中可能是通過上調(diào)CCL2的表達(dá)來進(jìn)行TAM浸潤這一過程。 結(jié)論： 體內(nèi)、體外實(shí)驗(yàn)證實(shí)：膠質(zhì)瘤中S100B很可能是通過CCL2的上調(diào)表達(dá)及TAM的化學(xué)趨化性來促進(jìn)了腫瘤的生長。膠質(zhì)瘤可以分泌S100B,其可以通過影響趨化因子CCL2的化學(xué)趨化作用吸引骨髓源性的巨噬細(xì)胞從而達(dá)到促進(jìn)膠質(zhì)瘤生長。因此膠質(zhì)瘤中S100B與CCL2在巨噬細(xì)胞的浸潤及膠質(zhì)瘤的生長中起著十分關(guān)鍵的作用。這為膠質(zhì)瘤治療中S100B抑制物提供了一個(gè)潛在的應(yīng)用機(jī)會。
[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 .

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.41

【共引文獻(xiàn)】

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