本文選題：鼠疫耶爾森氏菌 + DC-SIGN��； 參考：《大連醫(yī)科大學》2008年碩士論文

【摘要】： 目的:鼠疫耶爾森氏菌是一類引起人與動物鼠疫的革蘭氏陰性菌,其自然宿主是嚙齒類動物。它能夠利用抗原呈遞細胞(抗原presenting cells, APCs)的捕獲,轉移到淋巴結,引起淋巴結鼠疫。但是APCs最初如何捕捉鼠疫菌仍然未知。由于近來發(fā)現(xiàn)的C型凝集素受體與某些革蘭氏陰性菌的核心脂多糖(核心LPS)有作用。因此,我們推測鼠疫菌也是通過某種C型凝集素進入到APCs的,例如巨噬細胞。本次實驗將對這個機制加以驗證,并找到相應的配體和受體。 方法:1通過轉染,建立穩(wěn)定表達小鼠C型凝集素受體的細胞株。含有5種C型凝集素受體cDNA保存在表達載體pcDNA3.1中,轉染至CHO細胞株,得到CHO-mDC-SIGN、CHO-mSIGN-R1、CHO-mSIGN-R3、CHO-mDEC-205 (CD205)和CHO-mLangerin (CD207)轉染株,經過長時間G418篩選,找到能穩(wěn)定表達的單克隆株,并用流式細胞儀檢測。 2構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。用自殺質粒同源重組的方法缺失編碼核心脂多糖起始位的糖基轉移酶,將阻斷核心脂多糖的表達,得到鼠疫菌深度粗糙型。在核心脂多糖外側表達O-抗原將得到光滑型。 3粘附和侵襲實驗細胞懸浮于含2%FBS的RPMI,以4 x105/ml鋪在96或24孔平板中。按照1:50體積加入濃度為1 x 107CFU/ml的細菌。在37℃,5% CO2下孵育2.5小時。細胞經過洗滌后用0.5%的saponin裂解,將裂解液稀釋后涂在平板上,得到粘附和侵襲細菌的總量。 用慶大霉素處理細菌和細胞混合液,能夠殺死細胞外的細菌而不透過宿主細胞膜,得到的是侵入細菌的數(shù)量。100 ug/ml的慶大霉素作用1小時后洗去抗生素,裂解細胞進行涂菌計數(shù)。細菌侵襲的程度以細胞裂解液中細菌的CFU來估算。所有的實驗重復3次。 4流式細胞儀檢測細菌的侵襲能力。細菌在熒光試劑CFDA-SE中染色,洗去多余的染色液后,把標定好的細菌加入到細胞中培養(yǎng)2小時。細胞液經過洗滌除去未結合的細菌后,用2%的多聚甲醛固定。在做流式細胞儀檢測前,以1:10加入Trypan blue (0.4%)混勻,室溫10分鐘以淬滅細胞外細菌的熒光。Trypan blue能阻止熒光但不能透過細胞膜。被攝取細菌的比例以細胞發(fā)出的熒光強度來判定,強度越高,細菌被吞噬的程度越高。 5外源物質抑制實驗加入外源物質包括抗體,甘露糖,低聚糖,肽等來封阻細胞的吞噬作用,驗證細菌和細胞相互作用的特異性。 6體內吞噬實驗向活體小鼠腹腔注射細菌懸液,1.5小時后處死小鼠,提純腹腔巨噬細胞后按照體外侵襲實驗方法進行。 結果:鼠疫菌野生型較光滑型和深度粗糙型在體內外具有更高的侵襲巨噬細胞和CHO-SIGN-R1的能力。在五種轉染的CHO-C型凝集素細胞中,只有CHO-SIGN-R1獲得了吞噬鼠疫菌的能力。這種侵襲現(xiàn)象能夠被SIGN-R1的抗體所抑制 結論: Murine SIGN-R1是鼠疫耶爾森氏菌在小鼠巨噬細胞上的受體。其識別機理是野生型鼠疫菌天然暴露核心脂多糖部分,能夠被小鼠巨噬細胞上的SIGN-R1識別。阻斷這種識別作用為我們在預防和治療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 .
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

【共引文獻】

相關期刊論文 前10條

1 程金波;王加啟;李珊珊;甄云鵬;劉光磊;卜登攀;劉開朗;;不同熱處理方式對牛奶中IgG和乳鐵蛋白的影響[J];華北農學報;2010年S1期

2 李文明;王逸群;趙仁貴;周志華;;動物的乳鐵蛋白[J];西華師范大學學報(自然科學版);2007年03期

3 閆威;李和平;鐘凱;�；�;謝佳喜;李鵬;王月影;;不同泌乳期小鼠乳腺乳鐵蛋白基因的表達[J];江西農業(yè)學報;2009年10期

4 程金波;王加啟;卜登攀;劉光磊;張春剛;董曉麗;魏宏陽;周凌云;劉開朗;;免疫小鼠乳腺乳鐵蛋白基因表達變化研究[J];中國農業(yè)大學學報;2009年01期

5 紀長謹;荊強;劉儀;張源淑;;初乳中乳鐵蛋白的分離純化及鑒定[J];畜牧與獸醫(yī);2010年02期

6 許長娣;賁曉明;;乳鐵蛋白防治圍產期新生兒病毒感染的研究進展[J];中國新生兒科雜志;2007年02期

7 占今舜;張彬;胡金杰;;乳鐵蛋白的生物學功能及在仔豬生產上的應用[J];養(yǎng)豬;2011年05期

8 李翠玲;;乳鐵蛋白的研究現(xiàn)狀[J];中國奶牛;2011年14期

9 申艷麗;張華林;王美華;孫麗萍;陳世林;楊利國;;牛乳鐵蛋白的研究與應用[J];中國奶牛;2012年06期

10 裴杰;閻萍;馮瑞林;程勝利;梁春年;郭憲;曾玉峰;包鵬甲;朱新書;褚敏;;大通牦牛LF基因的克隆與分子特征[J];中國獸醫(yī)科學;2009年09期

相關博士學位論文 前1條

1 白雪晶;牛乳鐵蛋白衍生抗菌片段分子設計與構效關系[D];中國農業(yè)科學院;2010年

相關碩士學位論文 前6條

1 徐文洲;載乳鐵蛋白殼聚糖微球促進nHA/Co復合材料修復骨缺損的實驗研究[D];吉林大學;2011年

2 李美君;乳鐵蛋白對早期斷奶仔豬腸道結構和免疫功能的影響研究[D];湖南農業(yè)大學;2011年

3 程金波;牛奶中乳鐵蛋白含量變化及其影響因素的研究[D];中國農業(yè)科學院;2008年

4 崔麗華;益氣活血運脾方治療小兒反復呼吸道感染的臨床研究[D];北京中醫(yī)藥大學;2009年

5 王嘉榕;小鼠乳鐵蛋白cDNA克隆及其大腸桿菌和酵母表達[D];蘭州大學;2009年

6 詹黎明;飼糧蛋白來源對早期斷奶仔豬生產性能和免疫功能的影響[D];四川農業(yè)大學;2010年

，

本文編號：1914885