造血细胞产生的活性蛋白LL-37/hCAP-18和EDAG功能和临床意义的研究

【作者】 安莉莉；

【导师】 吴克复；

【作者基本信息】 中国协和医科大学 ， 细胞生物学， 2005， 博士

【摘要】 造血系统的发生和功能调节涉及造血细胞产生的众多活性蛋白。本文研究其中的两种活性蛋白LL-37／hCAP-18和EDAG的功能和临床意义。 多肽抗生素LL-37／hCAP-18是天然防御系统的重要组成成分之一，是已发现的人类唯一的cathelicidin家族成员，主要由中性粒细胞产生。LL-37的前体是一个19.3 kD的前原肽，含信号肽，保守的cathelin结构域和37个氨基酸残基的活性肽，除去信号肽后的表观分子量为18 kD，称为hCAP-18。机体感染或受伤后在丝氨酸蛋白酶-3和其他蛋白水解酶的作用下释放出有活性的LL-37。LL-37具有广谱抗菌作用和中和内毒素作用，其功能受损或表达下降会减弱机体对病原微生物的抵抗能力，与很多疾病直接相关。但LL-37／hCAP-18在血液病中的表达情况和临床意义尚不清楚。 我们用ABC免疫组化方法检测了143例血液病患者和50例正常人外周血涂片中LL-37／hCAP-18蛋白的表达，发现LL-37／hCAP-18在AML患者外周血白细胞中的表达水平明显低于正常人白细胞中的表达，分类计数表明在AML患者中性粒细胞中的阳性率仅为26.6±12.8％，感染者阳性率更低，而正常人为93.8±3.5％。提示AML患者外周血不仅有中性粒细胞量的减少，也存在着功能的缺陷。LL-37蛋白的低表达可能是急性白血病患者容易感染的原因之一。研究中还发现白血病细胞的LL-37／hCAP-18蛋白表达与mRNA表达不一致，提示其表达调控主要在转录后水平，我们检测了人白血病细胞系Raji，NB4，HL-60，J6-1及U937中的LL-37／hCAP-18基因5’-UTR序列，结果与文献报导的正常人的5’-UTR序列相符，初步排除了这些白血病细胞系因5’-UTR突变而引起翻译阻滞的可能。 近年文献报道，LL-37／hCAP-18除了直接的抗菌作用外，还有免疫调节作用，如趋化作用和调节树突状细胞功能等。防御素也是一种多功能抗菌肽，有促进免疫反应效应，但是，cathelicidin有无类似作用尚无报道。我们利用我室已有的人-鼠嵌合肿瘤模型，构建以LL-37为分子佐剂的抗肿瘤DNA疫苗，进行实验治疗研究。以分别带有M-CSFR和LL-37cDNA片段的重组质粒为模板，扩增M-CSFR信号肽和胞外三个免疫球蛋白(Ig)样结构域的编码基因，插入更多还原

【Abstract】 The ontogeny and function of hematopoietic system concerns numerous proteins produced by hematopioetic cells. In this study, we explored the function and clinical significance of two of the proteins, LL-37/hCAP-18 and EDAG.Antimicrobial peptide LL-37/hCAP-18 that is mainly produced by neutrophils, is important in host innate-immune defense. LL-37/hCAP-18 is synthesized as a pre-pro-peptide with 19.3 kD including a signal peptide, a cathelin-Iike domain and a 37-residue antimicrobial domain. The pro-peptide is 18 kD and is designated as hCAP-18. Cleavage of hCAP-18 by proteinase 3 liberates its C-terminal, active biologic domain (LL-37) from the conserved cathelin-Iike prosequence. LL-37 exhibits potent antimicrobial activity against Gram-negative and Gram-positive bacteria. It also has the ability to bind and neutralize the biological activity of lipopolysaccharide (LPS). Its deficiency may increase the susceptibility to infection. Involvement of LL-37 in pathological processes has been reported in many diseases. However, the knowledge of the expression of LL-37/hCAP-18 in hematological diseases and its clinical relevance is limited.We detected LL-37/hCAP-18 expression in the peripheral blood smears of 50 healthy donors and 143 patients with various hematological diseases. Compared with that in the healthy donors, expression of the protein in the neutrophils was significantly lower in patients with AML(93.8±3.5% versus 26.6±12.8%, p<0.05), especially those with infection. LL-37/hCAP-18 has been shown to play a role in host defense, and its deficiency in AML may be one of the explanations for their susceptibilities to infection among these patients. However, no significant difference was detected on the mRNA level between the neutrophils of the healthy donors and the AML patients suggesting that post-transcriptional mechanisms govern the expression of LL-37/hCAP-18 protein. Since 5’-UTR contains elements which are important in the regulation of translation, we detected the 5’-UTR sequences of LL-37/hCAP-18 gene in Raji, NB4, HL-60, J6-1 and U937 cells. There was no abnormality in the 5’-UTR sequences in these leukemia cell lines.Recently, other effects of LL-37 related to immune response have been gradually unveiled. LL-37 is chemotactic for human monocytes, neutrophils and T lymphocytes and acts as a potent modifier of DC differentiation. It was reported that defensin, another multifunctional antimicrobial peptide with chemotactic responses, could enhance the immunogenicity of the tumor antigen or viral antigen when fused with these antigens in DNA vaccination. Whether LL-37 has similar effects in antitumor immunity remains unclear. Previously, our lab constructed an M-CSFR DNA vaccine, which could induce humoral and cellular immunity against M-CSFR bearing SP2/0 cells in a murine model and markedly prolong the survival of mice challenged with M-CSFR+ tumor. In this study, we also used this model with necessary modification and investigated whether LL-37 could enhance the antitumor activities of the vaccines against M-CSFR by altering the更多还原