【摘要】：[目的]膠質(zhì)母細(xì)胞瘤(GBM)是中樞神經(jīng)系統(tǒng)常見的惡性腫瘤,目前綜合治療效果不佳,免疫逃逸可能在其惡性進(jìn)展中發(fā)揮了重要作用。調(diào)節(jié)性T細(xì)胞(Treg)可以誘導(dǎo)機(jī)體的免疫抑制,在膠質(zhì)瘤組織中也有浸潤,已有研究試圖探究Treg對GBM病人的預(yù)后作用,但是結(jié)論不一,此外,Treg在膠質(zhì)瘤中的分化機(jī)制亦罕有報道。本課題希望首先明確Treg在GBM中的聚集情況,然后深入分析這類免疫抑制細(xì)胞對臨床轉(zhuǎn)歸的預(yù)后價值,最后揭示naive CD4+T細(xì)胞向Treg分化的可能機(jī)制。[方法]在第一部分中,首先采用Western Blot檢測了不同級別膠質(zhì)瘤組織、腦外傷組織中Foxp3蛋白的含量,然后對62例GBM石蠟切片進(jìn)行了Foxp3、CD8、 p53、MGMT、Ki-67免疫組化染色,鏡下計數(shù)各指標(biāo)的表達(dá)情況,并對Foxp3和CD8實施了線性相關(guān)分析,根據(jù)臨床病理資料對病人分組,比較不同組間Foxp3CD8等指標(biāo)的差異。在第二部分中,以Foxp3的中位表達(dá)密度為分界點將上述62例病人等分為兩組,結(jié)合預(yù)后資料,針對無進(jìn)展生存期(PFS)和總生存期(0S)進(jìn)行了Kaplan-Meier生存分析,采用同樣的方法對CD8、Foxp3+/CD8+的預(yù)后作用進(jìn)行了分析,此外,還根據(jù)p53、MGMT、Ki-67等病理指標(biāo)對病人分組,進(jìn)一步研究了Foxp3、 CD8、Foxp3+/CD8+在各個亞組內(nèi)的預(yù)后價值,最后,納入所有臨床、病理、免疫指標(biāo),進(jìn)行了單因素和多因素分析。在第三部分中,首先,收集了不同級別膠質(zhì)瘤病人的外周血、腦脊液、腫瘤組織勻漿液,采用FlowCytomix及ELISA的方法檢測其中IL-6、IL-10的含量并做比較,再以不同濃度的IL-6、IL-10分別孵育從健康人外周血提取的naiveCD4+T細(xì)胞72小時,用流式細(xì)胞術(shù)檢測CD4+細(xì)胞中CD25+Foxp3+田胞的比例；其次,從新鮮膠質(zhì)母細(xì)胞瘤標(biāo)本中分選得到膠質(zhì)瘤干細(xì)胞(GSC)及CD133-細(xì)胞,再分別用GSC及上清、CD133-細(xì)胞及上清孵育naive CD4+T細(xì)胞,檢測Treg的比例；最后,通過慢病毒轉(zhuǎn)染健康人巨噬細(xì)胞而獲得B7-H4陽性巨噬細(xì)胞,再分別用這類細(xì)胞及未轉(zhuǎn)染B7-H4的對照細(xì)胞孵育naive CD4+T細(xì)胞,同上檢測Treg的比例。[結(jié)果]膠質(zhì)瘤級別越高,腫瘤組織中Foxp3蛋白含量越高,而正常腦組織中幾乎無Foxp3表達(dá)。Foxp3主要位于細(xì)胞核,Foxp3+淋巴細(xì)胞在腫瘤標(biāo)本中呈散在分布,平均密度為8.6個/mm2,CD8+淋巴細(xì)胞平均密度為49.6個/mm2,顯著高于前者(P0.001),而且這兩者之間呈正相關(guān)(rs=0.375、P=0.003)。此外,Foxp3表達(dá)密度和臨床病理特征無關(guān)。Foxp3高表達(dá)組的中位PFS為9個月,顯著低于Foxp3低表達(dá)組的中位PFS12個月(P=0.001),兩組的中位0S分別為16個月和20個月,Foxp3高表達(dá)組也顯著短于Foxp3低表達(dá)組(P=0.003)。CD8高、低表達(dá)的兩組間的PFS和OS均無顯著性差異。高Foxp3+/CD8+組的中位PFS為9個月,而低Foxp3+/CD8+組為12個月,兩者間的差異有統(tǒng)計學(xué)意義(P=0.031),兩者間的OS的差異尚不夠顯著。不同病理分型下Foxp3的預(yù)后意義也不同,比如,在Ki-67低表達(dá)組,Foxp3能夠提示不良預(yù)后,而在高表達(dá)組,卻無陽性發(fā)現(xiàn)。針對PFS和OS的多因素分析均顯示,Foxp3是GBM病人的一個獨(dú)立預(yù)后因素。IL-6在不同級別膠質(zhì)瘤病人的體液中無明顯差異,但在高級別膠質(zhì)瘤的組織勻漿液中卻顯著富集,1 ng/ml IL-6處理后的CD4+細(xì)胞中Treg的比例就顯著高于未經(jīng)處理的細(xì)胞(P 0.05),5 ng/ml IL-6處理后的細(xì)胞中Treg的比例顯著高于1ng/ml IL-6處理后的細(xì)胞(P0.01),而5 ng/ml和10 ng/ml IL-6處理的兩組間無顯著差異。IL-10在不同級別膠質(zhì)瘤病人的體液中無明顯差異,在高級別膠質(zhì)瘤的組織勻漿液中呈增多趨勢,但無統(tǒng)計學(xué)意義,用不同濃度IL-10處理的naive CD4+T細(xì)胞中Treg比例無明顯差異。GSC及其上清處理組的Treg比例顯著高于其余各組,而這兩組之間無顯著性差異,CD133-腫瘤細(xì)胞及其上清處理組與對照組間均無顯著性差異。經(jīng)LV-B7-H4轉(zhuǎn)染的巨噬細(xì)胞處理的CD4+T細(xì)胞中,Treg比例顯著高于由正常培養(yǎng)基、正常巨噬細(xì)胞或LV-GFP轉(zhuǎn)染的巨噬細(xì)胞處理的其他各組。[結(jié)論]Treg在膠質(zhì)瘤中的浸潤程度隨腫瘤級別升高而上調(diào),GBM腫瘤組織中Treg和CD8+細(xì)胞的數(shù)目呈正相關(guān)。Treg是GBM病人的一個獨(dú)立預(yù)后因素,腫瘤微環(huán)境中Treg越多,病人生存期越短。IL-6、GSC、B7-H4陽性巨噬細(xì)胞均可以促進(jìn)naive CD4+T細(xì)胞向Treg的分化。
[Abstract]:[Objective] glioblastoma (GBM) is a common malignant tumor in the central nervous system. The effect of integrated treatment is not good at present. Immune escape may play an important role in its malignant progress. Regulatory T cells (Treg) can induce immune suppression in the body and also have infiltration in glioma tissue. Research has been made to explore Treg to GBM patients. In addition, the differentiation mechanism of Treg in glioma is rarely reported. In this subject, we hope to clarify the aggregation of Treg in GBM, and then analyze the prognostic value of this kind of immunosuppressive cells for clinical outcome, and finally reveal the possible mechanism of naive CD4+T cell to Treg differentiation. [method] in the first part First, Western Blot was used to detect the content of Foxp3 protein in different grades of glioma tissue and brain trauma. Then 62 cases of GBM paraffin section were treated with Foxp3, CD8, p53, MGMT, Ki-67 immunohistochemical staining, and the expression of each index was counted under the microscope. The linear correlation analysis was carried out on Foxp3 and CD8, and the disease was based on the clinicopathological data. In the second part, the 62 patients were divided into two groups with the median expression density of Foxp3 in the second part, and the Kaplan-Meier survival analysis was carried out for the progression free survival (PFS) and the total survival time (0S), and the same method was used for CD8, Foxp3+/CD8+. The post effect was analyzed. In addition, the patients were grouped according to p53, MGMT, Ki-67 and other pathological indexes, and the prognostic value of Foxp3, CD8, Foxp3+/CD8+ in each subgroup was further studied. Finally, all clinical, pathological and immune indexes were included in the analysis of single factor and multiple factors. In the third part, first, the different grade gliomas were collected. The patients' peripheral blood, cerebrospinal fluid and tumor tissue homogenate were used to detect the content of IL-6 and IL-10 by FlowCytomix and ELISA, and then the naiveCD4+T cells extracted from the peripheral blood of healthy people were incubated with IL-6 and IL-10 at different concentrations for 72 hours, and the proportion of CD25+Foxp3+ in CD4+ cells was detected by flow cytometry; secondly, the proportion of CD25+Foxp3+ in the CD4+ cells was detected; secondly, the proportion of CD25+Foxp3+ in the CD4+ cells was detected by flow cytometry. From the fresh glioblastoma specimens, the glioma stem cells (GSC) and CD133- cells were obtained, and the naive CD4+T cells were incubated with GSC and supernatant, CD133- cell and supernatant respectively, and the proportion of Treg was detected. Finally, B7-H4 positive macrophages were obtained by transfection of the healthy human macrophage by lentivirus, and then the cells and the untransfected B were respectively used. The control cells of 7-H4 incubated the naive CD4+T cells, and the ratio of Treg was detected. [results] the higher the level of the glioma, the higher the Foxp3 protein content in the tumor tissue, while the Foxp3 expression of.Foxp3 in the normal brain was mainly located in the nucleus, and the Foxp3+ lymphocytes were scattered in the tumor specimens, with an average density of 8.6 /mm2 and CD8+ lymph thin. The average density of the cell was 49.6 /mm2, significantly higher than the former (P0.001), and there was a positive correlation between the two (rs=0.375, P=0.003). In addition, the Foxp3 expression density and the clinicopathological features were not related to the middle PFS of the high expression group of.Foxp3, which was significantly lower than the median PFS12 month (P=0.001) of the low expression group of Foxp3, and the median 0S of the two groups was 16 months and 20, respectively. The Foxp3 high expression group was also significantly shorter than the Foxp3 low expression group (P=0.003).CD8, and there was no significant difference in PFS and OS between the two groups of low expression. The median PFS of the high Foxp3+/CD8+ group was 9 months, and the low Foxp3+/CD8+ group was 12 months. The difference between the two groups was statistically significant (P= 0.031). The difference between the two groups was not significant. The prognostic significance of Foxp3 is different, for example, in the Ki-67 low expression group, Foxp3 can indicate poor prognosis, but in the high expression group, there is no positive discovery. The multivariate analysis of PFS and OS shows that Foxp3 is an independent prognostic factor of GBM patients,.IL-6 is not significantly different in the body fluids of patients with different levels of glioma, but in the high level gel The proportion of Treg in the CD4+ cells after 1 ng/ml IL-6 treatment was significantly higher than that of untreated cells (P 0.05). The proportion of Treg in the cells treated with 5 ng/ml IL-6 was significantly higher than that of the 1ng/ml IL-6 treated cells (P0.01), but there was no significant difference between the 5 ng/ and 10 groups. There was no significant difference in the body fluid of the patients with glioma at different levels, but there was an increasing trend in the tissue homogenate of high grade glioma, but there was no statistical significance. There was no significant difference in the proportion of Treg in the naive CD4+T cells treated with different concentrations of IL-10. The Treg ratio of.GSC and the supernatant treatment group was significantly higher than that of the other groups, but there was no significant difference between the two groups. There was no significant difference between the CD133- tumor cell and its supernatant treatment group and the control group. In the CD4+T cells treated by LV-B7-H4 transfected macrophages, the proportion of Treg was significantly higher than that of other groups treated by normal medium, normal macrophage or LV-GFP transfected macrophages. [conclusion]Treg in glioma infiltration degree. The number of Treg and CD8+ cells in GBM tumor tissue is positively correlated with the number of CD8+ cells..Treg is an independent prognostic factor of GBM patients. The more Treg in the tumor microenvironment, the shorter the patient's survival time, the.IL-6, GSC, and B7-H4 positive macrophages can promote naive CD4+T cell to differentiate to Treg.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.41

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 徐建X,徐庚;膠質(zhì)母細(xì)胞瘤的治療進(jìn)展[J];神經(jīng)疾病與精神衛(wèi)生;2005年01期

2 張帆;;立體適形放療在治療腦膠質(zhì)母細(xì)胞瘤術(shù)后患者的應(yīng)用[J];中國冶金工業(yè)醫(yī)學(xué)雜志;2007年02期

3 劉向輝;張之營;李鴻源;申斌;;顱內(nèi)膠質(zhì)母細(xì)胞瘤的術(shù)前診斷[J];山東醫(yī)藥;2008年36期

4 周及彤;王士杰;周建;;原發(fā)性膠質(zhì)母細(xì)胞瘤1例分析[J];中國誤診學(xué)雜志;2008年10期

5 劉維生;肖新如;王碩;;膠質(zhì)母細(xì)胞瘤中腦組織特異性血管生成抑制因子1的研究進(jìn)展[J];國際神經(jīng)病學(xué)神經(jīng)外科學(xué)雜志;2008年01期

6 楊培業(yè);膠質(zhì)母細(xì)胞瘤的放射線治療[J];國外醫(yī)學(xué)參考資料.神經(jīng)病學(xué)神經(jīng)外科學(xué)分冊;1976年05期

7 吳超權(quán);膠質(zhì)母細(xì)胞瘤放射抵抗的可能解釋[J];國外醫(yī)學(xué)(臨床放射學(xué)分冊);1984年03期

8 周東,馬大程,郭京,黃輝;7例膠質(zhì)母細(xì)胞瘤[J];中國腫瘤臨床與康復(fù);1998年S1期

9 白茫茫,張劍寧,章翔;炎性肉芽腫轉(zhuǎn)變?yōu)槟z質(zhì)母細(xì)胞瘤1例[J];第四軍醫(yī)大學(xué)學(xué)報;1999年01期

10 秦德興,墨浩,歐廣飛,宋永文,康恭禮,胡郁華,谷銑之;腦膠質(zhì)母細(xì)胞瘤放化療療效觀察[J];中華腫瘤雜志;2001年02期

相關(guān)會議論文 前10條

1 黃丙倉;耿道穎;紀(jì)毅敏;李郁新;朱莉;尹波;鮑奕仿;;膠質(zhì)母細(xì)胞瘤磁共振成像的一個重要征象——“絲瓜瓤樣壞死征”[A];中華醫(yī)學(xué)會第16次全國放射學(xué)學(xué)術(shù)大會論文匯編[C];2009年

2 顧金海;李剛;蘇雨行;申杰;李蓉暉;賀峭偉;鄧林;;STAT3 Decoy ODN抑制膠質(zhì)母細(xì)胞瘤增殖實驗研究[A];中華醫(yī)學(xué)會神經(jīng)外科學(xué)分會第九次學(xué)術(shù)會議論文匯編[C];2010年

3 秦立森;梁徑山;石瓊;于如同;;膠質(zhì)母細(xì)胞瘤中強(qiáng)化與壞死比例與信號傳導(dǎo)相關(guān)性的研究[A];中華醫(yī)學(xué)會神經(jīng)外科學(xué)分會第九次學(xué)術(shù)會議論文匯編[C];2010年

4 熊劍;章翔;程光;林洪;費(fèi)舟;;Ardipusilloside Ⅰ誘導(dǎo)膠質(zhì)母細(xì)胞瘤細(xì)胞凋亡的研究[A];中國醫(yī)師協(xié)會神經(jīng)外科醫(yī)師分會第六屆全國代表大會論文匯編[C];2011年

5 張成龍;李康印;郝曉東;陳虎義;高崎煒;強(qiáng)海霞;;兒童多發(fā)膠質(zhì)母細(xì)胞瘤一例[A];2005年全國醫(yī)學(xué)影像技術(shù)學(xué)術(shù)會議西部論壇論文匯編[C];2005年

6 顏偉;張偉;游贛;王永志;劉彥偉;楊沛;江濤;;甲基鳥嘌呤甲基轉(zhuǎn)移酶蛋白表達(dá)和促進(jìn)甲基化在中國膠質(zhì)母細(xì)胞瘤患者中的臨床關(guān)系[A];2011中華醫(yī)學(xué)會神經(jīng)外科學(xué)學(xué)術(shù)會議論文匯編[C];2011年

7 江濤;陳寶師;袁芳;李桂林;李少武;邱曉光;王忠誠;;膠質(zhì)母細(xì)胞瘤臨床治療經(jīng)驗[A];中國醫(yī)師協(xié)會神經(jīng)外科醫(yī)師分會第二屆全國代表大會論文匯編[C];2007年

8 鄭秀玨;黃欣;李谷;曹飛;龔江標(biāo);;膠質(zhì)母細(xì)胞瘤術(shù)后同步放化療的臨床分析[A];2009年浙江省神經(jīng)外科學(xué)術(shù)年會論文匯編[C];2009年

9 路翠平;;四十例膠質(zhì)母細(xì)胞瘤術(shù)后護(hù)理體會[A];2011中華醫(yī)學(xué)會神經(jīng)外科學(xué)學(xué)術(shù)會議論文匯編[C];2011年

10 賀虎;李明武;牛朝詩;;膠質(zhì)母細(xì)胞瘤干細(xì)胞能夠分化為內(nèi)皮細(xì)胞以促進(jìn)腫瘤血管生成[A];2011中華醫(yī)學(xué)會神經(jīng)外科學(xué)學(xué)術(shù)會議論文匯編[C];2011年

相關(guān)重要報紙文章 前7條

1 匡遠(yuǎn)深;治療首次術(shù)后腦膠質(zhì)母細(xì)胞瘤 放化療同步優(yōu)于單純放療[N];中國醫(yī)藥報;2008年

2 吳劍鵬;南方醫(yī)院牽手多家醫(yī)院聯(lián)合開展膠質(zhì)母細(xì)胞瘤研究[N];科技日報;2014年

3 煒聞;Avastin獲準(zhǔn)用于治療膠質(zhì)母細(xì)胞瘤[N];醫(yī)藥經(jīng)濟(jì)報;2009年

4 谷文;FDA批準(zhǔn)Avastin 用于治療膠質(zhì)母細(xì)胞瘤[N];中國醫(yī)藥報;2009年

5 王小龍;一種化合物可引發(fā)癌細(xì)胞“自爆”[N];科技日報;2014年

6 孫行之邋(編譯);抵御腫瘤,疫苗將成為利器[N];第一財經(jīng)日報;2008年

7 Tracy Staton 編譯 石軍;羅氏公開數(shù)據(jù)證“有效”[N];醫(yī)藥經(jīng)濟(jì)報;2014年

相關(guān)博士學(xué)位論文 前10條

1 岳琪;調(diào)節(jié)性T細(xì)胞在膠質(zhì)母細(xì)胞瘤中的預(yù)后作用及分化機(jī)制研究[D];復(fù)旦大學(xué);2014年

2 陳鑫;CKS2在膠質(zhì)母細(xì)胞瘤中的表達(dá)及其對腫瘤生物學(xué)行為的影響和機(jī)制研究[D];山東大學(xué);2015年

3 呂磊;阿西替尼對膠質(zhì)母細(xì)胞瘤及其腫瘤干細(xì)胞治療作用的研究[D];復(fù)旦大學(xué);2013年

4 周濟(jì);NTS/NTR1信號對人膠質(zhì)母細(xì)胞瘤干細(xì)胞自我更新及侵襲能力的影響及機(jī)制[D];第三軍醫(yī)大學(xué);2015年

5 熊劍;膠質(zhì)母細(xì)胞瘤誘導(dǎo)凋亡治療及其機(jī)制研究[D];第四軍醫(yī)大學(xué);2009年

6 楊曉亮;實驗性分子靶向治療膠質(zhì)母細(xì)胞瘤[D];第四軍醫(yī)大學(xué);2011年

7 孫種夷;膠質(zhì)母細(xì)胞瘤預(yù)后評價的數(shù)學(xué)模型[D];中南大學(xué);2013年

8 丁麗娟;膠質(zhì)母細(xì)胞瘤干細(xì)胞的培養(yǎng)及其對TRAIL耐藥機(jī)制的研究[D];吉林大學(xué);2011年

9 張耀;膠質(zhì)母細(xì)胞瘤術(shù)后早期復(fù)發(fā)與假性進(jìn)展的鑒別診斷模型[D];中南大學(xué);2014年

10 李竹青;復(fù)發(fā)性膠質(zhì)母細(xì)胞瘤再手術(shù)治療的預(yù)后評價[D];中南大學(xué);2014年

相關(guān)碩士學(xué)位論文 前10條

1 邱獻(xiàn)新;Notch通路血管配體DLL4和Jagged1與原發(fā)性膠質(zhì)母細(xì)胞瘤瘤周水腫及預(yù)后的關(guān)系[D];福建醫(yī)科大學(xué);2015年

2 林國詩;pSTAT3-VEGF信號通路與新診斷幕上膠質(zhì)母細(xì)胞瘤瘤周水腫及預(yù)后的關(guān)系[D];福建醫(yī)科大學(xué);2015年

3 韓曉;miR-584對人膠質(zhì)母細(xì)胞瘤侵襲和遷移能力的影響[D];山東大學(xué);2015年

4 杜池剛;擴(kuò)散因子受體對膠質(zhì)母細(xì)胞瘤復(fù)發(fā)影響的相關(guān)研究[D];山東大學(xué);2009年

5 馬揚(yáng);膠質(zhì)母細(xì)胞瘤患者術(shù)前預(yù)后評分的研究[D];山東大學(xué);2012年

6 許豪;膠質(zhì)母細(xì)胞瘤術(shù)后放化療與單純手術(shù)治療方案的療效觀察[D];山東大學(xué);2014年

7 郭欣茹;腦膠質(zhì)母細(xì)胞瘤血漿差異蛋白質(zhì)分析[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2014年

8 王宗寶;秋水仙堿對小鼠膠質(zhì)母細(xì)胞瘤化療初步研究[D];昆明醫(yī)學(xué)院;2008年

9 師惠;人膠質(zhì)母細(xì)胞瘤中白藜蘆醇代謝產(chǎn)物及其代謝相關(guān)酶表達(dá)的分析[D];大連醫(yī)科大學(xué);2011年

10 淦作松;三氧化二砷誘導(dǎo)膠質(zhì)母細(xì)胞瘤細(xì)胞凋亡作用的研究[D];江西醫(yī)學(xué)院;2004年

，

本文編號：2172020