小鼠Fcα/μR组织学定位初步研究

【作者】 王睿；

【导师】 张伟；

【作者基本信息】 中国协和医科大学 ， 免疫学， 2007， 博士

【摘要】 Fcα/μR是一种近年发现的能够与IgA、IgM结合的Fc受体,基因表达谱广泛,在小鼠多种脏器组织中均检测到其mRNA的表达。小鼠Fcα/μR(mFcα/μR)可以介导B细胞对免疫复合物的内吞,但是有关Fcα/μR生物学功能的更多研究尚在进行中。pIgR也是一种IgA、IgM受体,担负着将pIgA转运至粘膜细胞表面的重要使命,是机体粘膜免疫的重要分子。由于Fcα/μR与pIgR在染色体的基因定位相邻,且二者序列蛋白序列同源性较高,加之在与IgA、IgM结合功能方面具有相似性,因此,本论文将mFcα/μR与mpIgR进行了对比分析,以期为mFcα/μR的功能研究提供更有力的证据。首先,本论文的研究从抗体的制备着手。通过原核表达的方法得到了mFcα/μR及mpIgR重要结构域蛋白,并以纯化过的表达蛋白为抗原免疫大鼠,制备了大鼠抗mFcα/μR及mpIgR单克隆、多克隆抗体。经过ELISA、Western blot、FACS及细胞免疫化学等方法的多次鉴定,挑选出了可以特异识别mFcα/μR及mpIgR的单克隆抗体,确定了多克隆抗体的抗原识别特异性。为了探讨mFcα/μR的组织定位,本研究通过使用RT-PCR及免疫组织化学的方法对mFcα/μR及mpIgR的基因及蛋白水平的表达谱进行了检测和对比分析。实验证明,mFcα/μR比mpIgR的基因表达略广,在正常小鼠肝、心、脾、肺、肾、小肠、大肠、胸腺、睾丸、子宫及卵巢均有mRNA表达。但是在蛋白水平,通过使用不同的切片方法及尝试多种抗原修复条件对正常小鼠、DSS诱导肠炎模型小鼠及BSA免疫小鼠进行组织切片的免疫组织化学染色后,均未检测到mFcα/μR的表达。而同时对mpIgR的检测结果则基本与文献报道相符。综合以上实验结果,我们推测mFcα/μR可能与mpIgR发挥相似的作用,在二者同时存在时,mpIgR发挥主要作用,而mFcα/μR则保持较低的维持量表达。这种较低的表达水平可以在基因水平检测到,却可能已经超出免疫组织化学的检验灵敏度、从而无法在蛋白水平被检测到。当mpIgR功能缺陷(如mpIgR基因敲除)或mpIgR发挥功能障碍(如J链基因敲除)时,mFcα/μR可能会表达增强,从而代替mpIgR发挥转运IgA、IgM的功能。为了证明上述假设,还需开展大量的实验研究工作。此外,我们还对大鼠Fcα/μR(rFcα/μR)的基因及蛋白表达情况进行了初步探讨。RT-PCR结果表明,rFcα/μR的基因表达谱与mFcα/μR相似,比rCD89及rpIgR表达略广,在正常大鼠肝、心、脾、肺、肾、小肠、大肠、胸腺、睾丸、子宫均有mRNA表达。使用与rFcα/μR有交叉识别的小鼠抗人Fcα/μR单抗对正常大鼠各脏器进行免疫组织化学分析,同样没有得到明显的阳性信号。由于抗体对甲醛固定后抗原的识别特异性尚不能确定,因此本部分研究结果的生物学意义还需进一步的实验验证。本论文的工作为mFcα/μR的研究奠定了实验基础,并为mFcα/μR功能研究方向的确立提供了线索。更多还原

【Abstract】 Fcα/μR is a newly found Fc receptor specifically for IgA and IgM.Its mRNA is expressed on several tissues in normal mouse.It was found that Fcα/μR was involved in the endocytosis of immune complexes by B cell.However,the biology function of Fcα/μR is still largely unknown at present,pIgR is also a receptor for IgA and IgM,which transcytose pIgA and pIgM cross epithelial cells,so it is an important protein in mucosal immunity. The gene location of Fcα/μR is near to pIgR on mouse and human chromosome 1,and they have high amino acid homology.In this work,we analyzed the difference between Fcα/μR and pIgR in gene expression and protein distribution,in order to explore functions of Fcα/μR.First we prepared rat monoclone and polyclone antibodies against mouse Fcα/μR (mFcα/μR) and mouse pIgR(mpIgR).mFcα/μR and pIgR were expressed in E.coli and used as immunogen.Using ELISA,Western blot,FACS and immunocytochemistry,some mAbs was selected.We also identified the specificity of polyclone antibodies.We detected mRNA expression of mFcα/μR and mpIgR in several tissues of normal mice. The results showed that mpIgR was not expressed as widely as mFcα/μR,which was found in liver,heart,spleen,lung,kidney,small intestine,large intestine,thymus,testicle,uterus and ovary.However,immunohistochemistry analysis showed that mFcα/μR was negative in all tissues tested.On the other hand,the mpIgR was found in many organs.So we speculate that mFcα/μR may have similar function with mpIgR,mpIgR was the main receptor when the body is under normal conditions.When mpIgR can’t function properly, for example in pIgR knockout mouse or J chain knockout mouse,mFcα/μR may be expressed more and functions as mpIgR.More experiments are needed to support the hypothesis.At the same time,we also detected mRNA expression of rat Fcα/μR.The result showed that rFcα/μR was expressed more widely than rpIgR and rCD89.However,we also got negative result when we detected the protein expression of rFcα/μR using a mouse mAb (anti-hFcα/μR).Although this is in agreement with mFcα/μR result,we still can not make a conclusion about rFcα/μR because of the uncertainty of the mAb’s specificity. Based on the above experiments and analysis,this work has provided a useful tool and some data for the study of mFcα/μR,and more work will be carried out in the future research.更多还原