【作者】 申芳芳；

【导师】 潘兴瑜；

【作者基本信息】 中国医科大学 ， 医学免疫学， 2010， 硕士

【摘要】 目的根据细B胞的发育来源,可将B淋巴细胞分为两个亚群,B1细胞和B2细胞。根据能否表达CD5分子,B1细胞又分为B1-a细胞(CD5+)和B1-b细胞(CD5-)。B1细胞是由早期的前体细胞发育而来,在胚胎期及出生后分布于脾脏,成年后主要定位于腹膜、胸膜大网膜及扁桃体等处,在外周血和淋巴器官中较少见,几乎不依赖于骨髓来源,是一类具有重要功能,可以自我更新的长寿细胞。研究发现,B1细胞与B2细胞在起源、分化、表面标志及其基因表达等方面存在很大差异,尤其是二者在起源和具体发育分化过程中的关系存在很大争议。目前的研究主要有以下观点：①B细胞是在祖B细胞阶段就已经分为不同的谱系,然而该研究中的祖B细胞分别来源于胚胎肝脏和成年骨髓,而二者在时间和空间上已经存在了差异,也就是说B细胞的发育过程中可能于所处的微环境有一定的关系；②B细胞在发育后期由于某种信号介导而导致向不同的方向分化,但在祖细胞阶段还是属于同一个谱系。那么给予B1细胞一定的信号或者提供相应B2细胞发育所需要的干预因素,在这种情况下B1细胞能否向B2细胞分化需要我们近一步探讨；③在一定的条件下B1细胞可以由成熟的B2细胞发育而来。然而B1细胞相对于B2细胞出现较早,有研究表明产生于妊娠8.5～9.0天的主动脉旁的脏壁层(PAS)的B细胞的表型为CD5+CD45RlowsIgMhi,这一表型即所谓的B1-a细胞,而不是B1-b细胞或者B2细胞。因此,我们认为B细胞发育的第一波峰为B1-a细胞。依照物种进化的观点,如果B2细胞分化为B1细胞属于返祖的个别现象。那么,B1细胞有没有可能在一定的条件下分化为B2细胞有待于我们进一步研究。本实验通过建立骨髓体外微环境,对采用流式细胞仪分选纯化的B1-a细胞进行体外培养,在培养第2至6天进行流式检测。从而观察B1-a细胞能否分化为B2细胞。方法各取15只C57BL／1d新生鼠和2d新生鼠,分别从脾脏中提取淋巴细胞,将提取的淋巴细胞采用抗小鼠IgM-(APC)和抗CD5-(PE-CY5)的荧光抗体标记,用流式细胞仪分选出B-1a细胞。把分选好的B-1a细胞标记CFSE,置于体外骨髓微环境中培养,将培养第2天至第6天的细胞再次标记抗IgD-(PE)的荧光抗体,并通过FACS检测,观察B1-a细胞能否分化为B2细胞。结果新生鼠／1d较新生鼠／2d,B1-a细胞分化为B2细胞的相对分化率较高,其分化率分别为52.93±1.93%(1d)和26.98±2.94%(2d),有显著统计学差异(P<0.01)。讨论B1细胞在胚胎发育期达到高峰,成年后明显下降。因此,选择新生鼠的脾脏,提取淋巴细胞,再通过流式细胞仪分选纯化B1-a细胞,进行CFSE标记后转入体外骨髓微环境进行培养,对培养第2天至第6天的细胞再次标记抗IgD-(PE)的荧光抗体,并通过FACS检测,结果显示标记CFSE的B1-a细胞在第2天大部分分化为B2细胞。通过体外培养B1-a细胞可以分化为B2细胞,这可能是B细胞亚群分化的一个途径,B1-a细胞可能为B2细胞分化的过渡阶段。在此基础之上,应当继续完善有关体外或者体内实验体系,以便清楚准确地阐明B细胞的分化途径。结论1、通过体外培养新生鼠脾脏B1-a细胞可以分化为B2细胞,二者在分化和发育上有着很大的相关性。2、新生鼠／1d较新生鼠／2d,B1-a细胞分化为B2细胞的分化率明显升高,有显著的统计学差异(P<0.01)。更多还原

【Abstract】 ObjectiveAccording to the development of B cells, B lymphocytes can be divided into two groups:B1 cells and B-2 cells. According to whether CD5 molecules can be express, B1 cells can be divided into two subsets:Bl-a cell(CD5+) and B1-b cell(CD5-). B1 cells,which developed from the early precursors, distributed in the spleen at Embryos and after birth, and mainly engaged in adulthood peritoneal, pleural omental and tonsil, etc.while B1 cells are rare in peripheral blood and lymph organs in adult. Almost not dependenting on bone marrow sources, B1 cells,which have important functions and can updated independently,is a kind of long-lived cells.The relevant research suggested that there is a big difference between B1 cells and B2 cells in origin, differentiation, surface mark and gene expression.Especially in the development and differentiation, there has been an controversion. The current research shows that: (DB cells has been divided into different lineages in the develpoment of progenitors. Thece cells were taken separately from embryonic liver and adult bone marrow.which has already been an difference both in time and space.As a result, the development of B cells may has a certain relationship with their environment. (2)B cells may developed into different directions in the late differentiation due to a mediated signal, while these cells is belong to the same lineage at the progenitor stage. If we provide certain environment or interference factors, B1 cells can be transformed to B2 cells?③B1 cells appeared relatively earlier than B2 cells. Studies show that the B cells produced in pregnancy 8.5-9.0 days near the aorta (PAS) dirty walls were CD5+CD45RlowsIgMhi phenotype, this phenotype was so called B1-a cells, which is not B1-b or B2 cells. Therefore, we think the first developmen wave of B cell was B1-a cells.However, B2 cells can be transformed to B1 cells in certain conditions. This transformation belongs to atavism. Then B1 cells may be transformed to B2 cells under certain conditions,which shoud be in our further study. We provide environment via establishing bone marrow in vitro, in which B1 cells purified by FACS(facial action coding systerm) can be cultured and developed. At the first to sixth day, we will make a detection by FACS to observe whether B1-a cells could be transformed to B2 cells.MethodsTaking 15 C57BL newborn mouse and extracting lymphocytes from splenic, which would be markered by APC-anti-mouseIgM, PE-CY5-anti-mouseCD5, PE-anti-mouse IgD fluorescent antibodys. B1-a cells purified by FACS,would be marked by CFSE and then transfered into a space micro-environment via establishing bone marrow form C57BL/6 weeks mice,further cultuerd in vitro. At the first to sixth day, we will use PE-anti-mouse IgD fluorescent antibody markers and make a detection by FACS to observe whether B1-a cells could be transformed to B2 cells.ResultAt the second to sixth day, we will make a detection by FACS.We can detect B 2 cells with CFSE maked. B1-a cells could be transformed to B2 cells. The conversion percentage is respectively,52.93±1.93%(1d),26.98±2.94%(2d). The conversion percentage of the former(1d) was more than the latter(2d). The results was proved had statistical difference (P<0.01).DiscussionThe development peak of B1 cells was in period of embryonic,and decreased obviously in adult. Therefore,extracting splenic lymphocytes from new born 1d mice, from which we sorting B1-a cells and then marked by CFSE.These cells were cultured in a space bone marrow micro-environment in vitro.At the first to sixth day, we will make a detection by FACS. The results prove that B1-a cells could be transformed into B 2 cells in vitro.Both in differentiation and development,they has great relevance. But B1-a cells purified by FACS,may contained some B 2 cells.Therefore,the results may be false positive,and we should make more progress in the research to make the rults more validity. Conclusion1.The results prove that B1-a cells purified by FACS could be transformed into B2 cells in vitro.2. The conversion percentage is respectively,52.93±1.93%(1d),26.98±2.94%(2d). The conversion percentage of the former(1d) was more than the latter(2d). The results was proved had statistical difference (P<0.01).更多还原