【作者】 开心；

【导师】 白增亮； Shiv Pillai；

【作者基本信息】 山东大学 ， 发育生物学， 2010， 博士

【摘要】 慢性B淋巴细胞白血病(B cell Chronic lymphocytic leukemia, B-CLL)作为最普遍的白血病类型之一,主要发生在老年人群中。其主要特征是具有成熟细胞表型但其功能缺失的CD5+, CD23+ B淋巴细胞在外周血、骨髓、淋巴节、脾和肝等免疫器官中的增殖与累积。B-CLL的病因及发病机制在发育免疫学研究中迄今尚不清楚,但已发现在B-CLL患者体内,白血病细胞经历了从正常B细胞转变为具有B-CLL临床表征克隆的演化,在此过程受到作为决定B细胞命运的B细胞受体(B cell receptor, BCR)编辑及其信号通路的持续影响。最近的一系列研究进展,加深了对于B-CLL细胞中BCR及其信号通路的了解。例如表面BCR的低表达,IgHV基因的突变状态以及ZAP70和CD38在不同亚型B-CLL中的差异表达。这些发现清楚地显示了在B-CLL细胞中存在BCR信号通路的异常调控,对此核心问题的深入研究,将从解读B-CLL的演变过程的分子机制中对此疾病发生环节的靶向治疗提供理论依据。本论文以BCR相关分子及其介导的信号通路作为研究对象,利用western blotting, real-time RT-PCR,免疫共沉淀,流式细胞术,RNA干扰等技术比较系统地研究了在B-CLL细胞与正常人外周血B细胞,以及不同亚型外周血B细胞之间的重要基因和蛋白的差异表达及其互作关系,并对B细胞癌变及其与发育相关的分子机制进行了理论概括。BCR抑制受体CD22相关因子及其介导的信号通路在B-CLL细胞中的表达：本研究首先采集了63个B-CLL患者和60个正常人外周血临床样本,利用多抗体混合物交联多余细胞增加细胞密度后,Ficoll缓冲液密度梯度离心收集到纯度为83%±8%的B细胞,提取其总蛋白和nRNA作为主要研究材料。利用Western blotting技术及N-糖苷酶F(PNase F)消化糖蛋白N’端寡糖单元的方法检测了BCR抑制受体CD22的表达,确定其同工型CD22a和CD22(3同时存在于B-CLL细胞中,且蛋白表达量出现了适当的下调。通过比较B-CLL细胞和正常B细胞的CD22胞质尾区ITIMs基序磷酸化,发现了CD22起抑制作用的激活状态在B-CLL细胞中显著降低。利用免疫共沉淀技术观察到CD22对其下游信号分子SHP-1的更新作用明显减少,显示出B-CLL细胞中CD22介导的BCR抑制信号通路的缺失。通过磷酸化CD22发现抑制BCR功能的Lyn蛋白在B-CLL细胞中高表达,real-time RT-PCR结果表明该现象不依赖于mRNA转录调控,而其活性和无活性磷酸化形式均存在不同程度的增强,但相对于过表达的Lyn总蛋白无一状态占明显优势。依赖Lyn活化的酪氨酸激酶Syk,是介导BCR激活信号通路的重要分子,其蛋白在B-CLL细胞内表达正常,但其激活磷酸化形式过表达,显示B-CLL细胞中Syk下游信号通路的持续激活。E3泛素蛋白连接酶c-Cbl在B-CLL细胞中的表达及功能研究：为深入了解B-CLL细胞内对BCR信号通路相关蛋白起降解作用的蛋白泛素化-蛋白酶体降解系统,我们检测了该系统中的核心因子E3泛素蛋白连接酶c-Cbl的表达。Western blotting结果显示c-Cbl蛋白在B-CLL细胞中显著高表达,而依据real-time RT-PCR结果分析该现象为转录后现象。利用RNA干扰技术对BJAB细胞系进行c-Cbl siRNA基因转染,流式细胞术检测基因沉默前后膜表面CD22表达,结果显示CD22表面表达明显下调,提示B-CLL细胞内增强的c-Cbl蛋白可能导致其表面CD22内在化和降解。免疫共沉淀c-Cbl及其相关的磷酸化酪氨酸激酶,确定PI3-kinase P85与c-Cbl在B-CLL细胞内具有明显的交互作用,且此作用随B-CLL重要预后判断因子ZAP70的表达升高而增强。此结果表明c-Cbl对P85的磷酸化更新可能是ZAP70阳性B-CLL患者的重要调控事件。BCR相关基因和蛋白在外周血B细胞亚型及B-CLL细胞中的差异表达：为阐明BCR相关分子在B细胞发育过程中所发挥的作用,并推测B-CLL细胞的起源,我们利用CD19、CD27、和IgM作为荧光标记表型将外周血B细胞通过流式细胞术成功分选为naive B-cells、IgM memory B-cells(MZ B-cells)和switch memory B-cells三个亚型,分离提纯度达到90%。以3个正常人外周血样本分选的B细胞亚型及B-CLL临床样本的mRNA为模板,对SIAE、c-Cbl、ST6Gal1、ZAP70、Syk和Lyn 6个基因的real-time RT-PCR结果分析确定在记忆性B细胞中SIAE和c-Cbl基因上调,而S T6Gall基因下调,而中SIAE是一种乙酰基酯酶,可特异性去除位于α2-6,端连接的唾液酸9-OH上的乙酰基部分,而睡液酸9-0-乙酰化状态可以调节CD22的功能。ST6Gall则是位于高尔基体上的唾液酸转移酶,可将CD22上包含的唾液酸配基O-乙酰基化。这两种蛋白酶对CD22的联合调控可能是记忆性B细胞的一种特性。B-CLL细胞过表达的Lyn和c-Cbl货白在不同类型B细胞中表达观察,揭示了Lyn蛋白的上调可能是由于B-CLL患者机体衰老引发的记忆性B细胞集聚所导致,而c-Cbl蛋白的表达不受基因调控，可能是B-CLL发让过程序中的关键事件。综上的述,能过对BCR抑制和激活通路以及c-Cbl与相关蛋白交互作用在B-CLL细胞中的研究结果,我们构建了B-CLL细胞内BCR相关因子及其信号通路表达与交互关系的动态平衡模型(见下图I)。在B-CLL细胞中,细胞表而表达BCR减少,但Lyn蛋白表达上调,过表达的Lyn包括有活性和无活性的Lyn两种状态。其中激活的Lyn磷酸化酷氨酸激酶Syk并造成BCR激活信号增强,而未活化的Lyn阻碍了CD22上ITIMs的磷酸化,导致发挥抑制作用的CD22显著降低,无法对SHP-1进行更新,并最终造成B-CLL细胞中BCR抑制信号通路的缺失。E3连接酶c-Cbl在B-CLL细胞明显高表达,并作用于表面的CD22,标记其发生细胞内吞作用,所造成的CD22内在化成为B-CLL的重要表型之一。C-Cbl还可磷酸化PI3-Kinase的功能调节部分P85,该现象受ZAP70表达的调控,可能潜在增强AKT和PKCδ信号通路的激活,为B-CLL细胞的异常集聚提供条件。此外通过对不同亚型B细胞BCR相关基因和蛋白表达的研究,阐述了SIAE、c-Cbl基因上调和S T6Gall基因下调具有成为记忆性B细胞标记的潜在的临床应用价值；而B-CLL细胞内,Lyn蛋白表达增强可能是机体衰老的表现,c-Cbl蛋白表达升高可能是肿瘤发生中的关键事件。更多还原

【Abstract】 B cell Chronic lymphocytic leukaemia (B-CLL) is the commonest form of leukaemia, which develops in the ageing population. B-CLL is characterized by the progressive accumulation of functionally incompetent, mature looking, monoclonal CD5+, CD23+ B lymphocytes in blood, bone marrow, lymph nodes and spleen/liver. The etiopathogenesis of B-CLL in developmental immunology research remains unclear. But it has already found that the leukemic cells perform the evolution from a normal B cell to a clinically manifested B-CLL clone in vivo of B-CLL patient. And these processes influence by B cell receptror (BCR) and its signaling pathway which is critical in determining B cell fate.Recently, there are several advances have enhenced the understanding of BCR and BCR signaling pathway in B-CLL. Such as low level of surface BCR, mutational status of IGHV genes, and differential expression of ZAP70 and in differnet kinds of B-CLL. These advanves shed light on the abnormal regulation of BCR signaling pathway in B-CLL cells. Further studies to this project will offer clues for improving the design and targeting of therapeutic strategies.In this paper, we studied BCR related moleculars and signaling pathway. And we compared important differential expressions and interactions of several genes and proteins between B-CLL cells and normal peripheral B cells as well as in different B cells subgroup using western blotting, real-time RT-PCR, immunoprecipitation, flow cytometry and RNA interfere techniques, which will imporve the understanding of mechanisms for B-cell tumor pathogenesis and B-cell development.Expression of BCR inhibitory coreceptor CD22 related factors and signaling pathway via CD22 in B-CLL cells:Peripheral blood samples were collected from 63 typical B-CLL patients and 60 healthy donors. After increased the density of the unwanted cells by crosslinking antibody cocktail, we purified 83%±8% B cells by centrifugation over Ficoll buoyant density medium and isolated total protein and mRNA as research samples. We detected the protein expression of BCR inhibitory receptor CD22 by western blotting and N-Glycosidase F (PNGase F) which cleaves oligosaccharides of N-linked glycans on glycoproteins. Both CD22a and CD22βisoform are determined in CLL B cells, but the CD22 protein expression is less. We analyzed the phosphorylation of ITIMs motif in the cytoplasmic tails of CD22 in B-CLL cells compared with normal B cells, and found the significant decrease of active CD22 was dramatically. We determined that the recruitement of SHP-1 by CD22 was significant decreased using an co-immunoprecipitation approach, and further suggested BCR inhibitory pathway via CD22 is defective in B-CLL cells.Lyn proteins are overexpressed in B-CLL cells and block BCR funciton by phophrylated CD22. The results of real-time RT-PCR showed it does not depend on mRNA transcription. Both active Lyn and inactive Lyn are moderated up-regulation, but neither of them is as the same level as overexpressed total Lyn protein. Tyrosine kinase Syk is a key molecule in BCR active pathway, which requires phosphorylated by Lyn during activation. In B-CLL cells, Syk protein level is normal but activated phosphorylated Syk is increased, which means the constitutive activation of Syk downstream signaling pathway.Expression and function of c-Cbl E3 ubiquitin-ligase in B-CLL cells:In order to further understand the ubiquitination-proteasome system which targets BCR related proteins for degradation in B-CLL cells, we detected protein expressions of the system key factor c-Cbl E3 ubiquitin-ligase. The results of western blotting show the enhanced levels of c-Cbl protein, which is a post-transcriptional phenomena based on real-time RT-PCR results.We used an RNA interfere approach to transfect c-Cbl siRNA into in BJAB B cells, and detected surface CD22 expressions during gene knocking down process by flow cytometry. The results show that CD22 surface levels are upregulated. Enhanced c-Cbl expression in CLL B cells may therefor result in CD22 internalization and degradation. We co-immunoprecipated c-Cbl with phosphorylated tyrosine kinase, and determined a strong association between PI3-kinase P85 and c-Cbl in B-CLL This interaction is enhanced by increasing protein levels of B-CLL prognostic factor ZAP70, which suggest that p85 recuitment by c-Cbl may be an important regulation event in ZAP70 positive B-CLL. Differential expression of BCR related genes and proteins in peripheral blood B cells subgroups and B-CLL cells:To investigate the effect of BCR associated factors in B cell development and hypothesize the origins of CLL B cells, we separated naive B-cells, IgM memory B-cells (MZ B-cells) and switch memory B-cells by flow cytometry sorting using CD 19, CD27 and IgM as fluorescent marked phenotype. The purification rate is about 90%.B cell subgroups sorting was performed in 3 healthy donors and B-CLL patients samples, and 6 genes SIAE, c-Cbl, ST6Gall, ZAP70, Syk and Lyn were analysised by real-time RT-PCR using isolated mRNA as template. SIAE, c-Cbl is upregulated but ST6Gall is down-regulated in memory B cells. SIAE is an acetyl esterase that specifically removes acetyl moieties from 9-OH position of a2-6 liked sialic acid moieties to make ligands accessible to CD22. ST6GalI is a glycosyltransferase in the Golgi that can O-acetylated sialic acid-contaning ligands for CD22. The CD22 regulation by these two enzyme may be an characteristic of memory B cells. We also investigated protein levels of B-CLL overexpressed Lyn and c-Cbl in different kinds of B cells. The data indicates the up-regulation of Lyn protein may results from memory B cells accumulation following B-CLL patients aging. But c-Cbl protein overexperssion which is not regulated by gene may be an key event in B-CLL tumorigenesis.In conclusion, we constructed a hypothetical model about BCR associated signaling pathway based on research of BCR inhibitory and activation pathway and related proteins interaction in CLL B cells. In CLL B cells, following the low levels of surface BCR expression, CD22 is down-regulated but CD22 alpha and CD22 beta can be expressed equally well. At the same time, protein tyrosine Lyn is upregulated in CLL which included more active Lyn and inactive Lyn. More active Lyn contribute to phosphorylate protein tyrosine Syk that propagates the BCR signaling, while more inactive Lyn block phosphorylation of ITIMs in CD22, and results in abnormal recruit SHP-1, which results in defective BCR inhibitory signaling in B-CLL. E3 ligase c-Cbl is significant overexpressed in B-CLL, and affects the surface CD22 by leading it degradation. CD22 internalization in a c-Cbl dependent fashion becomes one of the most important phenotype of B-CLL. C-Cbl can phosphorylat regulatory subunit of PI3-kinase P85 which is associated with ZAP70 expression. This interaction may enhance activation of AKT and PKCδsignaling, and result in B-CLL cells abnormal accumulation. According to BCR related genes and proteins expression in B cell subgroups, SIAE and c-Cbl gene up-regulation and ST6Gall gene down-regulation could be potential molecular markers in memory B cells. However, Lyn protein levels enhanced may results from B-CLL patient aging, and c-Cbl protein overexpression may be an key event in tumorigenesis.更多还原