鼠疫耶尔森氏菌在小鼠巨噬细胞上的受体—Murine SIGN-R1

发布时间：2018-05-20 13:57

本文选题：鼠疫耶尔森氏菌 + DC-SIGN　；参考：《大连医科大学》2008年硕士论文

【摘要】： 目的:鼠疫耶尔森氏菌是一类引起人与动物鼠疫的革兰氏阴性菌,其自然宿主是啮齿类动物。它能够利用抗原呈递细胞(抗原presenting cells, APCs)的捕获,转移到淋巴结,引起淋巴结鼠疫。但是APCs最初如何捕捉鼠疫菌仍然未知。由于近来发现的C型凝集素受体与某些革兰氏阴性菌的核心脂多糖(核心LPS)有作用。因此,我们推测鼠疫菌也是通过某种C型凝集素进入到APCs的,例如巨噬细胞。本次实验将对这个机制加以验证,并找到相应的配体和受体。方法:1通过转染,建立稳定表达小鼠C型凝集素受体的细胞株。含有5种C型凝集素受体cDNA保存在表达载体pcDNA3.1中,转染至CHO细胞株,得到CHO-mDC-SIGN、CHO-mSIGN-R1、CHO-mSIGN-R3、CHO-mDEC-205 (CD205)和CHO-mLangerin (CD207)转染株,经过长时间G418筛选,找到能稳定表达的单克隆株,并用流式细胞仪检测。 2构建鼠疫菌的光滑型和深度粗糙型。野生鼠疫菌是粗糙型,核心脂多糖暴露在最外面。用自杀质粒同源重组的方法缺失编码核心脂多糖起始位的糖基转移酶,将阻断核心脂多糖的表达,得到鼠疫菌深度粗糙型。在核心脂多糖外侧表达O-抗原将得到光滑型。 3粘附和侵袭实验细胞悬浮于含2%FBS的RPMI,以4 x105/ml铺在96或24孔平板中。按照1:50体积加入浓度为1 x 107CFU/ml的细菌。在37℃,5% CO2下孵育2.5小时。细胞经过洗涤后用0.5%的saponin裂解,将裂解液稀释后涂在平板上,得到粘附和侵袭细菌的总量。用庆大霉素处理细菌和细胞混合液,能够杀死细胞外的细菌而不透过宿主细胞膜,得到的是侵入细菌的数量。100 ug/ml的庆大霉素作用1小时后洗去抗生素,裂解细胞进行涂菌计数。细菌侵袭的程度以细胞裂解液中细菌的CFU来估算。所有的实验重复3次。 4流式细胞仪检测细菌的侵袭能力。细菌在荧光试剂CFDA-SE中染色,洗去多余的染色液后,把标定好的细菌加入到细胞中培养2小时。细胞液经过洗涤除去未结合的细菌后,用2%的多聚甲醛固定。在做流式细胞仪检测前,以1:10加入Trypan blue (0.4%)混匀,室温10分钟以淬灭细胞外细菌的荧光。Trypan blue能阻止荧光但不能透过细胞膜。被摄取细菌的比例以细胞发出的荧光强度来判定,强度越高,细菌被吞噬的程度越高。 5外源物质抑制实验加入外源物质包括抗体,甘露糖,低聚糖,肽等来封阻细胞的吞噬作用,验证细菌和细胞相互作用的特异性。 6体内吞噬实验向活体小鼠腹腔注射细菌悬液,1.5小时后处死小鼠,提纯腹腔巨噬细胞后按照体外侵袭实验方法进行。结果:鼠疫菌野生型较光滑型和深度粗糙型在体内外具有更高的侵袭巨噬细胞和CHO-SIGN-R1的能力。在五种转染的CHO-C型凝集素细胞中,只有CHO-SIGN-R1获得了吞噬鼠疫菌的能力。这种侵袭现象能够被SIGN-R1的抗体所抑制结论: Murine SIGN-R1是鼠疫耶尔森氏菌在小鼠巨噬细胞上的受体。其识别机理是野生型鼠疫菌天然暴露核心脂多糖部分,能够被小鼠巨噬细胞上的SIGN-R1识别。阻断这种识别作用为我们在预防和治疗鼠疫菌上提供新的思路。
[Abstract]:The purpose of this study is to investigate the role of mouse plague bacteria in human and animal plague . The natural host is rodent . It is able to use antigen presenting cells to transfer to lymph nodes and cause the plague of lymph nodes . However , it is speculated that the C - type lectin receptors are still unknown to certain gram - negative bacteria , such as macrophages . This experiment will validate this mechanism and find the corresponding ligands and receptors .

Methods : 1 . The expression vector pcDNA3.1 ( CHO - mDC - SIGN , CHO - mSIGN - R1 , CHO - mSIGN - R3 , CHO - mDEC - 205 ( CD205 ) and CHO - mLangerin ( CD207 ) were transfected into CHO cell line .

2 . To construct the smooth and deep rough type of plague bacteria . The wild plague bacteria are rough type , and the core lipopolysaccharide is exposed to the outside . The deletion of glycosyltransferase of the starting position of core lipopolysaccharides by means of homologous recombination of suicide plasmid will block the expression of core lipopolysaccharide , so as to obtain the deep coarse type of plague bacteria .

3 adherent and invasive test cells were suspended in RPMI containing 2 % FBS at 4 & # xd7 ; 105 / ml in 96 or 24 well plates . The cells were incubated at 37.degree . C.for 2.5 hours at 37.degree . C. , 5 % CO2 . After washing , the cells were diluted with 0.5 % lysate , diluted with lysis solution and coated on a plate to obtain the total amount of adhesion and invasive bacteria .

The bacterial and cell mixture was treated with gentamicin , which was able to kill bacteria outside of the cell without passing through the host cell membrane , resulting in the number of invading bacteria . 100 ug / ml of gentamicin acted for 1 hour to wash away antibiotics and lysis cells were counted . The extent of bacterial invasion was estimated by the CFU of bacteria in the cell lysate . All experiments were repeated three times .

4 Flow cytometry was used to detect the invasion ability of bacteria . The bacteria were stained in the fluorescent reagent CFDA - SE . After washing the excess staining solution , the labeled bacteria were added to the cells for 2 hours . After washing and removing unbound bacteria , the cells were fixed with 2 % polyformaldehyde . Trypan blue inhibited the fluorescence but was unable to pass through the cell membrane . The proportion of the ingested bacteria was determined by the fluorescence intensity of the cells . The higher the intensity , the higher the extent of the bacteria being phagocytosed .

5 Exogenous material inhibition experiments include antibody , mannose , oligosaccharide , peptide and so on to seal the phagocytosis of cells , verify the specificity of the interaction of bacteria and cells .

Six mice were injected with bacterial suspension intraperitoneally . After 1.5 hours , mice were sacrificed and the peritoneal macrophages were purified .

Results : The wild type of plague bacteria has higher ability to invade macrophages and CHO - SIGN - R1 in vitro . In five transfected CHO - C - type lectin cells , only CHO - SIGN - R1 has the ability to phagocytose plague bacteria . This invasion can be inhibited by the antibody of SIGN - R1 .

Conclusion : Sign - R1 is a receptor on mouse peritoneal macrophages . Its recognition mechanism is that the natural exposed core lipopolysaccharide fraction of wild - type plague bacteria can be recognized by SIGN - R1 in mouse macrophages . Blocking this recognition can provide a new idea for preventing and treating plague bacteria .
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【共引文献】