结内滤泡性淋巴瘤的新标识—原钙粘蛋白γA3

发布时间：2018-09-08 07:32

【摘要】：滤泡性淋巴瘤(FL)是一种常见的惰性B细胞淋巴瘤,占惰性淋巴瘤的70%。在滤泡性淋巴瘤中80%-90%的病例存在14号与18号染色体异位,导致这些病例中BCL-2呈阳性表达,但其余10%-20%左右的病例由于缺乏t(14;18)(q32;q21)而不表达BCL-2。目前,对于BCL-2表达为阴性的滤泡性淋巴瘤即使通过免疫化学染色方法检测其他标志物的表达对其进行诊断仍有困难,因此找到一个在各分级结内滤泡性淋巴瘤高表达同时在BCL-2阴性样本中高表达的滤泡性淋巴瘤诊断标志物对于滤泡性淋巴瘤的临床诊断具有重要意义。本实验拟通过基因芯片找到结内滤泡性淋巴瘤与反应性滤泡增生病例相比具有差异性表达的基因,并对其功能进行研究,意在找到一个新型的FL诊断标志物并初步探索其影响FL发生、发展的可能的分子机制。具体实验方法和结果如下:方法(1)通过基因芯片分析找到结内滤泡性淋巴瘤与反应性滤泡增生相比具有差异性表达的基因,进一步筛选出差异最明显的基因,综合考虑现有抗体产品的局限性等因素,确定PCDHGA3为研究对象。(2)运用免疫组织化学染色方法检测PCDHGA3在各分级的结内滤泡性淋巴瘤及反应性滤泡增生病例中的表达情况,并进行统计学分析,探讨PCDHGA3是否可以成为结内滤泡性淋巴瘤的诊断标志物。(3)采用RT-PCR检测PCDHGA3在结内滤泡性淋巴瘤细胞系中的表达情况,应用si RNA下调PCDHGA3的表达水平,研究PCDHGA3对细胞增殖的影响,通过实时定量PCR检测NF-κB信号通路相关基因表达,以探索PCDHGA3调节细胞增殖的可能的分子机制。对基因芯片数据中差异表达基因进行分析,找到与PCDHGA3基因表达相关的共表达基因,通过免疫荧光化学染色的方法检测共表达基因在滤泡性淋巴瘤细胞中的表达情况。结果(1)基因芯片分析结果显示,原钙粘蛋白γ家族13个成员基因(原钙粘蛋白γA2,A3,A5,A7,A8,A9,A12,B1,B3,B4,B6和B7)在结内滤泡性淋巴瘤中高表达,其中上调最明显的5个基因分别为原钙粘蛋白A3,A5,B2,B3和B4。(2)免疫组织化学染色结果显示,原钙粘蛋白γA3在1级结内滤泡性淋巴瘤的表达率为93.1%(27/29),2级结内滤泡性淋巴瘤的表达率为83.3%(5/6),3级结内滤泡性淋巴瘤的表达率为55.6%(5/9)。同时在结内滤泡性淋巴瘤弥漫区域其表达率为100%(10/10),但在反应性滤泡增生的表达率仅为5.9%(1/17),并且在BCL-2呈阴性表达的结内滤泡性淋巴瘤病例中原钙粘蛋白γA3仍为高表达(86.7%,13/15)。(3)PCR结果证实,原钙粘蛋白γA3在滤泡性淋巴瘤细胞系F18和F318中均表达。运用电转染的方法将PCDHGA3 si RNA转染入FL18细胞后,实时定量PCR及Western blot结果证实结果PCDHGA3 si RNA3可使原钙粘蛋白γA3的表达下调60%(p=0.005)。细胞计数结果显示,下调原钙粘蛋白γA3的表达后,FL18细胞增殖与未转染细胞相比明显减慢(26±2.16vs 43.3±1.43,p0.001)。同时,实时定量PCR检测发现,转染PCDHGA3si RNA3的FL18细胞内与细胞增殖密切相关的NF-κB信号通路相关基因RELA、NFKB1、NFKB2表达下调,提示原钙粘蛋白γA3可能是通过NF-κB信号通路调节滤泡性淋巴瘤细胞的增殖。通过对结内滤泡性淋巴瘤及反应性滤泡增生病例中差异表达的基因数据进行分析,找到与PCDHGA3基因表达呈正相关且相关度最大的五个基因,分别为OPRL1,TNFRSF6B,ADAMTSL5,ACCSL和CHST4,其中TNFRSF6B蛋白与PCDHGA3蛋白在FL18细胞膜共表达。结论(1)结内滤泡性淋巴瘤与反应性滤泡增生病例相比,差异表达最明显的5个基因分别为原钙粘蛋白γ家族A3,A5,B2,B3和B4。(2)与反应性滤泡增生病例相比,PCDHGA3在各分级结内滤泡性淋巴瘤病例中呈现高表达,其中BCL-2阴性FL病例PCDHGA3亦呈现高表达。因此PCDHGA3是结内滤泡性淋巴瘤潜在的诊断标志物。(3)沉默PCDHGA3基因后FL18细胞增殖减慢,经典NF-κB信号通路相关基因RELA,NFKB1和NFKB2表达水平下调。说明沉默PCDHGA3基因可抑制FL细胞增殖,并且可能是通过下调经典NF-κB信号通路相关基因发挥抑制作用。(4)具有通过与Fas配体相结合抑制细胞凋亡作用的TNFRSF6B蛋白在结内滤泡性淋巴瘤中表达且其表达与PCDHGA3相关,提示PCDHGA3可能是通过TNFRSF6B调节FL18细胞增殖。
[Abstract]:Follicular lymphoma (FL) is a common inert B-cell lymphoma, accounting for 70% of inert lymphoma. 80% to 90% of follicular lymphoma cases have heterotopic chromosomes 14 and 18, resulting in positive expression of BCL-2 in these cases, but the remaining 10% to 20% of cases due to lack of t (14; 18) (q32; q21) but not expression of BCL-2. It is still difficult to diagnose follicular lymphoma with negative expression of-2 even if the expression of other markers is detected by immunochemical staining. Therefore, it is difficult to find a diagnostic marker for follicular lymphoma with high expression in all grades of follicular lymphoma and high expression in BCL-2 negative samples for follicular lymphoma. The purpose of this study is to identify the differentially expressed genes in intranodal follicular lymphoma (IFL) and reactive follicular hyperplasia (RFH) by gene chip, and to study their functions in order to find a novel FL diagnostic marker and explore the possible molecular mechanism of FL. Methods: (1) The genes differentially expressed between intranodal follicular lymphoma and reactive follicular hyperplasia were identified by gene chip analysis, and the most distinct genes were screened out. PCDHGA3 was selected as the research object by considering the limitation of antibody products. The expression of PCDHGA3 in different grades of intranodal follicular lymphoma and reactive follicular hyperplasia was detected by histochemical staining, and statistical analysis was carried out to explore whether PCDHGA3 could be used as a diagnostic marker of intranodal follicular lymphoma. (3) The expression of PCDHGA3 in intranodal follicular lymphoma cell lines was detected by RT-PCR. SiRNA was used to down-regulate the expression of PCDHGA3, and the effect of PCDHGA3 on cell proliferation was studied. The expression of genes related to NF-kappa B signaling pathway was detected by real-time quantitative PCR to explore the possible molecular mechanism of PCDHGA3 regulating cell proliferation. Results (1) Gene chip analysis showed that 13 members of the procadherin gamma family (procadherin gamma A2, A3, A5, A7, A8, A9, A12, B1, B3, B4, B6 and B7) were highly expressed in intranodal follicular lymphoma. (2) Immunohistochemical staining showed that the expression of procadherin gamma A3 in primary intranodal follicular lymphoma was 93.1% (27/29), that in grade 2 intranodal follicular lymphoma was 83.3% (5/6), and that in grade 3 intranodal follicular lymphoma was 55.6%. (5/9). At the same time, the expression rate was 100% (10/10) in the diffuse region of intranodal follicular lymphoma, but only 5.9% (1/17) in reactive follicular hyperplasia, and the expression of pro-cadherin gamma A3 was still high in BCL-2 negative intranodal follicular lymphoma (86.7%, 13/15). (3) PCR results confirmed that pro-cadherin gamma A3 was in follicular lymphoma. Both F18 and F318 were expressed in lymphoma cell lines. PCDHGA3 Si RNA was transfected into FL18 cells by electrotransfection. Real-time quantitative PCR and Western blot results showed that PCDHGA3 Si RNA 3 could down-regulate the expression of procadherin gamma A3 by 60% (p = 0.005). Cell counts showed that FL18 cells were down-regulated by down-regulating the expression of procadherin gamma A3. The proliferation of FL18 cells transfected with PCDHGA3si RNA 3 was significantly slower than that of untransfected cells (26 Proliferation of follicular lymphoma cells. By analyzing the differentially expressed gene data in intranodal follicular lymphoma and reactive follicular hyperplasia, five genes with the greatest positive correlation with the expression of PCDHGA3 gene were identified, namely OPRL1, TNFRSF6B, ADAMTSL5, ACCSL and CHST4, in which TNFRSF6B and PCDHGA3 proteins were FL. Conclusion (1) Compared with reactive follicular hyperplasia, PCDHGA3 was overexpressed in all grades of intranodal follicular lymphoma, and BCL-2 was negative. PCDHGA3 was also highly expressed in FL patients. Therefore, PCDHGA3 was a potential diagnostic marker for intranodal follicular lymphoma. (3) After PCDHGA3 gene silencing, FL18 cells proliferated slowly, and the expression levels of classical NF-kappa B signaling pathway related genes RELA, NFKB1 and NFKB2 were down-regulated. (4) The expression of TNFRSF6B protein in intranodal follicular lymphoma is related to PCDHGA3, suggesting that PCDHGA3 may regulate FL18 cell proliferation through TNFRSF6B.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.1

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