外周T细胞淋巴瘤中转录因子GATA3的预后分析和体外研究
发布时间：2018-10-25 06:30
【摘要】：背景和目的外周T细胞淋巴瘤(PTCL)是一组异质性强、侵袭性高的非霍奇金淋巴瘤,缺乏规范统一的治疗方案,预后较其他类型淋巴瘤差。寻找新的分子标记物对于深入探索其成瘤机制、研发新药都有重要意义。近几年国外基于高通量基因组层面的研究首次揭示TH2细胞分化指标转录因子GATA3可能在PTCL发生发展中起到作用。本研究拟进行GATA3的预后研究,并在体外条件下探索GATA3对肿瘤增殖、浸润等恶性行为的影响,为淋巴瘤发病机理研究提供新的实验证据。方法回顾性收集PTCL患者的福尔马林固定石蜡包埋的病理标本及临床资料,所有病例均为2007年至2013年间由北京协和医院病理科诊断的初治患者。对GATA3、TBX21、CD68进行免疫组化染色,分析其表达与患者临床特征及预后的关系。在体外条件下通过慢病毒转染法建立GATA3稳定敲低的Hut78淋巴瘤细胞系,应用流式细胞检测技术研究敲低细胞系的凋亡变化,应用实时定量PCR技术检测敲低细胞系的细胞因子表达,通过条件培养方法研究敲低细胞系对M2巨噬细胞分化的影响。结果本研究共纳入109例初治PTCL患者,其中PTCL非非特指为60例。在PTCL组织的肿瘤细胞中54例GATA3表达阳性(49.5%),47例TBX21表达阳性(43.1%),57例CD68表达阳性(52.3%)。GATA3和CD68与PTCL的不良预后相关。K-M曲线法发现,GATA3阳性组的中位总生存期(OS)为120天,GATA3阴性组的中位OS为511天(Log-rank, P=0.000); CD68阳性组的中位OS为180天,CD68阴性组的中位OS为450天(Log-rank, P=0.037)。多因素分析提示ECOG评分≥2分、GATA3阳性为PTCL预后不良的独立危险因子。相关性分析提示GATA3高表达与出现B症状(Pearson P=0.006)、CD68高表达相关(Pearson P0.01)。体外研究筛选出Hut78为GATA3高表达的T细胞淋巴瘤细胞系。应用慢病毒转染shGATA3序列成功后,与对照组相比,Hut78-shGATA3细胞的GATA3 mRNA和蛋白表达量均明显下调,早期凋亡阶段细胞百分比明显增高。在细胞因子方面,Hut78-shGATA3转录IL-4、IL-5、IL-13及VEGF的mRNA水平均明显下降。应用淋巴瘤细胞上清诱导U937向巨噬细胞分化,研究发现与对照组相比,Hut78-shGATA3上清诱导巨噬细胞向M2分化的比例显著下降。结论在PTCL中,GATA3阳性患者较GATA3阴性患者OS明显缩短,且与ECOG评分≥2分一并为预后不良的独立影响因素,提示GATA3可以作为PTCL患者评估预后的因素。GATA3表达量与CD68表达量呈正相关,提示(GATA3可能促进了巨噬细胞在PTCL肿瘤组织中浸润。从绝对例数来看,GATA3高表达与嗜血综合征的发生相关。体外实验证实,T淋巴瘤细胞系Hut78的GATA3表达量显著增高。GATA3敲低后,T细胞淋巴瘤细胞的早期凋亡比例增加,TH2相关细胞因子及VEGF表达水平下降,且诱导巨噬细胞向M2型分化的能力降低。GATA3可能通过这些途径对PTCL患者预后产生影响。
[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，
本文编号：2292902
[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，
本文编号：2292902
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