【摘要】：背景和目的外周T細(xì)胞淋巴瘤(PTCL)是一組異質(zhì)性強(qiáng)、侵襲性高的非霍奇金淋巴瘤,缺乏規(guī)范統(tǒng)一的治療方案,預(yù)后較其他類型淋巴瘤差。尋找新的分子標(biāo)記物對(duì)于深入探索其成瘤機(jī)制、研發(fā)新藥都有重要意義。近幾年國外基于高通量基因組層面的研究首次揭示TH2細(xì)胞分化指標(biāo)轉(zhuǎn)錄因子GATA3可能在PTCL發(fā)生發(fā)展中起到作用。本研究擬進(jìn)行GATA3的預(yù)后研究,并在體外條件下探索GATA3對(duì)腫瘤增殖、浸潤(rùn)等惡性行為的影響,為淋巴瘤發(fā)病機(jī)理研究提供新的實(shí)驗(yàn)證據(jù)。方法回顧性收集PTCL患者的福爾馬林固定石蠟包埋的病理標(biāo)本及臨床資料,所有病例均為2007年至2013年間由北京協(xié)和醫(yī)院病理科診斷的初治患者。對(duì)GATA3、TBX21、CD68進(jìn)行免疫組化染色,分析其表達(dá)與患者臨床特征及預(yù)后的關(guān)系。在體外條件下通過慢病毒轉(zhuǎn)染法建立GATA3穩(wěn)定敲低的Hut78淋巴瘤細(xì)胞系,應(yīng)用流式細(xì)胞檢測(cè)技術(shù)研究敲低細(xì)胞系的凋亡變化,應(yīng)用實(shí)時(shí)定量PCR技術(shù)檢測(cè)敲低細(xì)胞系的細(xì)胞因子表達(dá),通過條件培養(yǎng)方法研究敲低細(xì)胞系對(duì)M2巨噬細(xì)胞分化的影響。結(jié)果本研究共納入109例初治PTCL患者,其中PTCL非非特指為60例。在PTCL組織的腫瘤細(xì)胞中54例GATA3表達(dá)陽性(49.5%),47例TBX21表達(dá)陽性(43.1%),57例CD68表達(dá)陽性(52.3%)。GATA3和CD68與PTCL的不良預(yù)后相關(guān)。K-M曲線法發(fā)現(xiàn),GATA3陽性組的中位總生存期(OS)為120天,GATA3陰性組的中位OS為511天(Log-rank, P=0.000); CD68陽性組的中位OS為180天,CD68陰性組的中位OS為450天(Log-rank, P=0.037)。多因素分析提示ECOG評(píng)分≥2分、GATA3陽性為PTCL預(yù)后不良的獨(dú)立危險(xiǎn)因子。相關(guān)性分析提示GATA3高表達(dá)與出現(xiàn)B癥狀(Pearson P=0.006)、CD68高表達(dá)相關(guān)(Pearson P0.01)。體外研究篩選出Hut78為GATA3高表達(dá)的T細(xì)胞淋巴瘤細(xì)胞系。應(yīng)用慢病毒轉(zhuǎn)染shGATA3序列成功后,與對(duì)照組相比,Hut78-shGATA3細(xì)胞的GATA3 mRNA和蛋白表達(dá)量均明顯下調(diào),早期凋亡階段細(xì)胞百分比明顯增高。在細(xì)胞因子方面,Hut78-shGATA3轉(zhuǎn)錄IL-4、IL-5、IL-13及VEGF的mRNA水平均明顯下降。應(yīng)用淋巴瘤細(xì)胞上清誘導(dǎo)U937向巨噬細(xì)胞分化,研究發(fā)現(xiàn)與對(duì)照組相比,Hut78-shGATA3上清誘導(dǎo)巨噬細(xì)胞向M2分化的比例顯著下降。結(jié)論在PTCL中,GATA3陽性患者較GATA3陰性患者OS明顯縮短,且與ECOG評(píng)分≥2分一并為預(yù)后不良的獨(dú)立影響因素,提示GATA3可以作為PTCL患者評(píng)估預(yù)后的因素。GATA3表達(dá)量與CD68表達(dá)量呈正相關(guān),提示(GATA3可能促進(jìn)了巨噬細(xì)胞在PTCL腫瘤組織中浸潤(rùn)。從絕對(duì)例數(shù)來看,GATA3高表達(dá)與嗜血綜合征的發(fā)生相關(guān)。體外實(shí)驗(yàn)證實(shí),T淋巴瘤細(xì)胞系Hut78的GATA3表達(dá)量顯著增高。GATA3敲低后,T細(xì)胞淋巴瘤細(xì)胞的早期凋亡比例增加,TH2相關(guān)細(xì)胞因子及VEGF表達(dá)水平下降,且誘導(dǎo)巨噬細(xì)胞向M2型分化的能力降低。GATA3可能通過這些途徑對(duì)PTCL患者預(yù)后產(chǎn)生影響。
[Abstract]:Background and objective Peripheral T-cell lymphoma (PTCL) is a group of non-Hodgkin 's lymphoma with strong heterogeneity and high invasiveness. Searching for new molecular markers is of great significance for further exploring its tumorigenesis mechanism and developing new drugs. In recent years, foreign studies based on high-throughput genome level for the first time revealed that TH2 cell differentiation index transcription factor GATA3 may play a role in the development of PTCL. This study is intended to study the prognosis of GATA3 and to explore the effects of GATA3 on malignant behaviors such as tumor proliferation and invasion in vitro, providing new experimental evidence for the study of the pathogenesis of lymphoma. Methods the pathological specimens and clinical data of formalin fixed paraffin embedded PTCL patients were collected retrospectively. All the patients were newly diagnosed by the Department of Pathology of Peking Union Hospital from 2007 to 2013. Immunohistochemical staining was used to analyze the relationship between the expression of GATA3,TBX21,CD68 and clinical features and prognosis. GATA3 stable knockout Hut78 lymphoma cell lines were established by lentivirus transfection in vitro. Apoptosis of knockout cell lines was studied by flow cytometry and cytokine expression was detected by real-time quantitative PCR. The effect of knock-down cell line on M 2 macrophage differentiation was studied by conditioned culture. Results A total of 109 patients with PTCL were enrolled in this study, including 60 patients with PTCL. 54 cases (49.5%) were positive for GATA3, 47 (43.1%) for TBX21 and 57 (52.3%) for CD68 in PTCL. GATA3 and CD68 were correlated with the poor prognosis of PTCL. K-M curve showed that the median total survival time of GATA3 positive group was 120 days, and that of GATA3 negative group was 120 days. The median OS of CD68 positive group was 180 days and the median OS of CD68 negative group was 450d (Log-rank, P0. 037). Multivariate analysis showed that ECOG score 鈮，

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