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  本文選題：瘦素　切入點：單核巨噬細胞　出處：《南方醫(yī)科大學》2011年碩士論文

【摘要】：瘦素是一種由167個氨基酸組成的激素樣細胞因子,主要由脂肪細胞分泌。人編碼瘦素的肥胖基因定位于人類第7號染色體,而鼠的編碼基因則位于第6號染色體。人們對瘦素最初的研究表明,瘦素主要是通過作用于下丘腦的神經(jīng)核團,調(diào)節(jié)食物攝取與能量消耗之間的平衡。隨著研究的不斷深入,人們發(fā)現(xiàn),瘦素受體不僅表達于神經(jīng)系統(tǒng),也表達于許多外周組織,如肝、肺、睪丸。因此,近年來,瘦素對免疫應答和自身免疫性疾病的影響受到越來越多的關注。 人類的單核巨噬細胞是一類非常重要的免疫細胞,無論在獲得性免疫的初始階段還是在天然免疫中都發(fā)揮著舉足輕重的功能。在天然免疫中,單核巨噬細胞可以依靠表面的模式識別受體迅速識別入侵的病原體,并由此觸發(fā)后續(xù)的炎癥反應和殺菌效應。它還能依靠表面的甘露糖受體(MR)、Fc受體和C3b受體等介導對病原體的吞噬,同時可以分泌趨化因子募集炎癥細胞,控制感染。在獲得性免疫的初始階段,單核巨噬細胞作為重要的抗原遞呈細胞可以遞呈抗原給淋巴細胞,從而促使淋巴細胞激活或分化。 我們首先以人THP-1細胞作為研究對象,加入PMA誘導其分化成巨噬細胞,并在此基礎上評價了瘦素對其吞噬功能、增殖活性、殺菌效應、趨化能力以及共刺激分子表達的影響,同時對造成這些變化的可能機制做了初步的探討。殺菌效應是單核巨噬細胞的一個重要功能,我們利用金黃色葡萄球菌為對象研究了不同劑量瘦素處理的THP-1及其來源的巨噬細胞對它們的殺傷作用。結果表明,足夠濃度的瘦素能明顯增強THP-1及其來源的巨噬細胞對細菌的殺傷作用,但1ng/ml的瘦素無此作用。這種殺菌作用的誘導可能有賴于腫瘤壞死因子(TNF-α)和活性氧(ROS)的產(chǎn)生增加而與一氧化氮(NO)的生成之間關系不明顯。 對吞噬功能的評價,我們采用了流式細胞術。我們用熒光素FITC對白色念珠菌進行了標記,然后用流式細胞術評價了瘦素處理對THP-1及其來源的巨噬細胞對細菌的吞噬作用。結果顯示,足夠濃度的瘦素能明顯增強THP-1及其來源的巨噬細胞對白色念珠菌的吞噬能力。雖然MR是與吞噬作用密切相關的受體,但我們發(fā)現(xiàn)瘦素的這種作用并不是通過上調(diào)MR表達來實現(xiàn)的。此外我們還發(fā)現(xiàn),瘦素可以刺激THP-1及其來源的巨噬細胞的增殖。 趨化功能是單核巨噬細胞的另外一種重要功能,我們用Boyden小室測定了瘦素對THP-1及其來源的巨噬細胞的趨化功能的影響。結果表明,足夠濃度的瘦素顯著增強了對HL-60粒細胞系和自身的趨化作用,這種作用可能和IL-8以及MCP-1分泌的增強有關。 我們還對THP-1及其來源的巨噬細胞和人外周血單核細胞表面的共刺激分子的表達進行了評價。結果顯示,瘦素可以上調(diào)這些細胞表面共刺激分子的表達,但結果同時也發(fā)現(xiàn),對于不同類型的細胞,瘦素所需的有效刺激濃度存在差異。 第一章瘦素對THP-1細胞及其來源的巨噬細胞殺菌活性的影響及其機制的研究 單核巨噬細胞有很強的殺傷能力,可非特異性殺傷多種病原微生物,是機體非特異性免疫防御中的重要細胞。單核巨噬細胞的這種殺傷作用可被補體的免疫黏附作用、特異性免疫中抗體的調(diào)理作用及細胞因子加強。單核巨噬細胞也能利用此功能清除體內(nèi)衰老損傷細胞,參與免疫自穩(wěn)作用。 我們首先參照相關文獻用PMA對人THP-1細胞進行了誘導使其成功分化成巨噬細胞樣細胞。結果表明,PMA誘導24h后可見細胞表面伸出偽足,細胞體積增大,胞漿內(nèi)出現(xiàn)空泡,說明細胞向巨噬細胞轉化。我們利用金黃色葡萄球菌為對象研究了不同劑量瘦素處理的THP-1及其來源的巨噬細胞對它們的殺傷作用。結果表明,10ng/ml以上組瘦素能明顯增強THP-1及其來源的巨噬細胞對細菌的殺傷作用(F=73.79,P=0.000；F=25.01,P=0.000),但1ng/ml的瘦素無此作用。由于單核巨噬細胞激活后主要通過活性氧簇(ROS)、NO的生成來實現(xiàn)其對細菌的殺傷。因此,我們分別測定了瘦素處理后,單核巨噬細胞分泌這些生物活性分子的能力。結果發(fā)現(xiàn),10、50、100ng/ml的瘦素均可以顯著刺激ROS的產(chǎn)生(F=168.95,P=0.000；F=113.00,P=0.000),其量可以達到對照的數(shù)倍甚至數(shù)十倍。然而,各個劑量組卻對NO的生成無明顯的刺激作用(F=1.179,P=0.377;F=1.978,P=0.174)。而且1ng/ml組對這幾種活性物質(zhì)均無誘導作用。TNF-α是單核巨噬細胞分泌的促炎癥因子,在感染的控制中也具有重要作用。我們通過ELISA的方法測定了瘦素處理的THP-1細胞及其來源的巨噬細胞產(chǎn)生這種細胞因子的能力。結果顯示,lOng/ml以上組瘦素可以刺激細胞產(chǎn)生TNF-α(F=502.48,P=0.000;F=2979.01,P=0.000)。這些結果提示,足夠濃度的瘦素可以顯著刺激THP-1細胞及其來源的巨噬細胞殺菌活性物質(zhì)的分泌并顯著增強其殺菌活性。 本部分實驗主要評價了瘦素對THP-1細胞及其來源的巨噬細胞的殺菌功能的影響。實驗中首先通過殺菌試驗和平板計數(shù)的方法檢測了瘦素誘導的殺菌效應。瘦素誘導了TNF-α和ROS的產(chǎn)生,但對NO的合成影響不顯著,這在一定程度上說明了瘦素誘導的殺菌機制。 第二章瘦素對THP-1細胞及其來源的巨噬細胞吞噬功能的影響及其機制的研究 吞噬功能是單核巨噬細胞的一個重要功能,我們希望通過本部分實驗評價瘦素對這種功能的作用。我們利用流式細胞術檢測了瘦素刺激對THP-1細胞及其來源的巨噬細胞吞噬功能的影響。流式結果表明,THP-1細胞及其來源的巨噬細胞經(jīng)過10ng/ml以上濃度的瘦素處理后,其對白色念珠菌的吞噬活性可以達到30%以上,說明其吞噬功能有所增強。50ng/ml以上組的瘦素處理后的THP-1細胞增殖活性顯著增高(F=64.39,P=0.000)。但和其他實驗結果不同,10ng/ml以下組增殖不明顯。對于THP-1來源的巨噬細胞也有類似作用(F=93.95,P=0.000)。MR是單核巨噬細胞表面的一種受體,與其吞噬功能密切相關。不同劑量的瘦素刺激THP-1細胞及其來源的巨噬細胞后,我們用real-time PCR的方法檢測了MR的表達。結果顯示,不同劑量瘦素處理后,MR表達無顯著改變(F=0.343,P=0.843；F=0.644,P=0.644)。 本部分實驗主要評價了瘦素對THP-1細胞及其來源的巨噬細胞的增殖和吞噬功能的影響。結果顯示足夠劑量的瘦素可以誘導THP-1細胞及其來源的巨噬細胞的增殖,并增強其吞噬功能。但這種作用并不依賴MR的表達變化,這說明瘦素誘導的吞噬功能的變化可能依賴其他受體的作用。 第三章瘦素對THP-1細胞及其來源的巨噬細胞趨化功能的影響及其機制的研究 趨化功能在控制炎癥感染中具有重要意義。單核巨噬細胞可以依靠這種功能募集炎癥細胞向炎癥感染的部位聚集,從而有效控制感染。我們希望通過本部分實驗觀察瘦素對單核巨噬細胞趨化能力的影響。我們采用了兩種細胞系一—-THP-1和HL-60來分別代表單核細胞和粒細胞。我們的研究結果表明,和未刺激對照相比,lOng/ml以上組瘦素可以將THP-1細胞及其來源的巨噬細胞的趨化指數(shù)提高數(shù)倍(F=72.59,P=0.000；F=220.78,P=0.000),說明其趨化功能顯著增強。IL-8和MCP-1是單核巨噬細胞分泌的兩種主要的趨化因子,ELISA結果顯示,瘦素刺激的細胞能夠分泌更多的IL-8和MCP-1。 本部分實驗主要評價了瘦素對THP-1細胞及其來源的巨噬細胞的趨化功能的影響。結果顯示瘦素可以增強THP-1細胞及其來源的巨噬細胞的趨化功能,這種作用可能是通過誘導IL-8和MCP-1的產(chǎn)生來實現(xiàn)的,這在一定程度上說明了瘦素誘導的趨化機制。 第四章瘦素對人單核巨噬細胞共刺激分子表達的影響 單核巨噬細胞是最重要的一類抗原呈遞細胞。外來抗原經(jīng)單核吞噬細胞處理后呈遞給T細胞,這是誘發(fā)免疫應答的先決條件。此外,在抗原呈遞過程中單核巨噬細胞產(chǎn)生的IL-1也是TH活化不可缺少的刺激信號。這種抗原呈遞功能與其表面共刺激分子表達的強弱密切相關。因此我們通過流式細胞術對它們表面的CD86、HLA-DR分子進行了檢測。我們發(fā)現(xiàn),和未刺激對照相比,lOng/ml以上組的瘦素可以明顯上調(diào)及THP-1細胞表面這些共刺激分子的表達(F=449.32,P=0.000；F=436.55 P=0.000)。而對于人外周血單核細胞而言,這種有效濃度則需要達到100ng/ml。這說明對于不同的細胞類型,瘦素的作用強度存在著差異。 本部分實驗主要評價了瘦素對人單核巨噬細胞共刺激分子表達的影響。通過流式檢測,我們發(fā)現(xiàn),瘦素可以明顯上調(diào)CD86和HLA-DR的表達。這提示瘦素可能具有增強單核巨噬細胞抗原呈遞的能力。
[Abstract]:Leptin is a hormone - like cytokine composed of 167 amino acids , mainly secreted by fat cells . Human - coded leptin is located on chromosome 7 , while the mouse ' s coding gene is located on chromosome 6 . As the study progresses , leptin receptors are found not only in the nervous system , but also in many peripheral tissues , such as liver , lung , and testis . Therefore , leptin has been more and more concerned with immune responses and autoimmune diseases in recent years .

Human mononuclear phagocytes are very important immune cells , play an important role in the initial stage of acquired immunity or in innate immunity . In natural immunity , mononuclear phagocytes can quickly identify invading pathogens by means of pattern recognition receptors on the surface , and trigger subsequent inflammatory responses and bactericidal effects . It can also secrete chemokine to recruit inflammatory cells and control infection . In the initial stage of acquired immunity , mononuclear phagocytes can be presented to lymphocytes as important antigen presenting cells , thus promoting the activation or differentiation of lymphocytes .

The effects of leptin on the phagocytic function , proliferation activity , bactericidal effect , chemoattractant ability and co - stimulatory molecule expression of THP - 1 and its origin were evaluated . The results showed that leptin could significantly enhance the killing effect of THP - 1 and its derived macrophages on bacteria by using Staphylococcus aureus as the subject .

Flow cytometry was used to evaluate the phagocytic function of THP - 1 and its origin . The results showed that leptin could significantly enhance the phagocytosis of THP - 1 and its derived macrophages against Candida albicans . The results showed that leptin could significantly enhance the phagocytosis of THP - 1 and its derived macrophages against Candida albicans .

Chemotaxis function is another important function of mononuclear phagocytes . We measured the effects of leptin on THP - 1 and its origin by Boyden chamber . The results showed that leptin significantly enhanced the effect of leptin on HL - 60 granulocyte and its own , which could be related to the enhancement of IL - 8 and MCP - 1 secretion .

We also evaluated the expression of co - stimulatory molecules on the surface of macrophages and human peripheral blood mononuclear cells from THP - 1 and its origin . The results showed that leptin could up - regulate the expression of co - stimulatory molecules on these cell surfaces , but also found that there was a difference in the effective stimulation concentrations required for leptin for different types of cells .

The effect and mechanism of leptin on the bactericidal activity of THP - 1 cell and its origin

Monocyte macrophages have a strong killing ability , and can kill various pathogenic microorganisms in a non - specific manner , which is an important cell in nonspecific immune defense of the organism . The killing effect of mononuclear phagocytes can be enhanced by the immune adherence of the complement , the regulating action of the antibody in specific immunity and the enhancement of the cytokines .

The effects of THP - 1 and their origin on the killing of THP - 1 and their derived macrophages were studied by PMA . The results showed that leptin could significantly enhance the killing effect of THP - 1 and its derived macrophages on bacteria ( F = 73.79 , P = 0.000 ) .
The results showed that 10 , 50 , 100 ng / ml leptin could significantly stimulate the production of ROS ( F = 168.95 , P = 0.000 ) .
There was no significant irritation to NO production ( F = 1.179 , P = 0.377 , F = 1.978 , P = 0.174 ) . TNF - 偽 is the pro - inflammatory factor secreted by mononuclear phagocytes and plays an important role in the control of infection . The results showed that leptin in leptin treated by leptin could stimulate the production of TNF - 偽 ( F = 502.48 , P = 0.000 ; F = 2979.01 , P = 0.000 ) . These results suggest that leptin with a sufficient concentration can significantly stimulate the secretion of THP - 1 cells and their derived macrophages bactericidal active substances and significantly enhance their bactericidal activity .

In this experiment , the effects of leptin on the bactericidal function of THP - 1 cells and their origin were evaluated . The bactericidal effect of leptin on THP - 1 cells and their origin was first determined by bactericidal test and plate counting . Leptin induced TNF - 偽 and ROS production , but the effect of leptin on NO synthesis was not significant , which indicated a certain extent the mechanism of leptin - induced bactericidal action .

Study on the Effect of the second chapter on the phagocytic function of THP - 1 cell and its origin and its mechanism

The phagocytic function of THP - 1 cells and their origin was evaluated by flow cytometry . The results showed that the phagocytic activity of THP - 1 cells and their origin was 30 % or more , and the proliferation activity of THP - 1 cells increased significantly ( F = 64.39 , P = 0.000 ) . However , unlike other experimental results , the proliferation of the following groups was not significant at 10 ng / ml . Similar effects were observed for macrophages from THP - 1 sources ( F = 93.95 , P = 0.000 ) . MR was one of the receptors on the surface of mononuclear phagocytes and was closely related to its phagocytic function . After stimulation of THP - 1 cells with different doses of leptin and macrophages from their origin , the expression of MR was detected by real - time PCR . The results showed that MR expression did not change significantly after leptin treatment ( F = 0.343 , P = 0.843 ;
F=0.644,P=0.644).


The results showed that leptin could induce the proliferation of THP - 1 cells and their derived macrophages and enhance their phagocytic function . However , this effect did not depend on the expression of leptin , suggesting that leptin - induced changes in the phagocytic function might depend on the role of other receptors .

Study on the Effect and Mechanism of Leptin on the Chemotactic Function of THP - 1 Cells and Their Origin ;

We hope to observe the effect of leptin on the chemoattractant capacity of mononuclear phagocytes by means of this experiment . We hope to observe the effect of leptin on the chemoattractant capacity of mononuclear phagocytes by means of this experiment . We have used two cell lines one - THP - 1 and HL - 60 to respectively represent monocytes and leukocytes . Our results show that the leptin in the above group can increase the chemoattractant index of macrophages from THP - 1 cells and their origin by multiple times ( F = 72.59 , P = 0.000 ) .
The results showed that leptin - stimulated cells could secrete more IL - 8 and MCP - 1 .

In this part , the effects of leptin on THP - 1 cells and their derived macrophages were evaluated . The results showed that leptin could enhance the chemoattractant function of THP - 1 cells and their derived macrophages , which could be achieved by inducing the production of IL - 8 and MCP - 1 .

Effects of leptin on the expression of costimulatory molecules in human mononuclear phagocytes

In addition , IL - 1 produced by mononuclear phagocytes in the process of antigen presentation is a prerequisite for TH activation . In addition , IL - 1 produced by mononuclear phagocytes in the process of antigen presentation is a prerequisite for TH activation .
F=436.55 P=0.000). For human peripheral blood mononuclear cells , this effective concentration needs to reach 100 ng / ml . This suggests that there is a difference in the intensity of action of leptin for different cell types .

In this part , the effects of leptin on the expression of costimulatory molecules of human mononuclear phagocytes were evaluated . By means of flow cytometry , we found that leptin could significantly increase the expression of HLA - DR . This suggests that leptin may have the ability to enhance the antigen presentation of monocytes .

【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363

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2 陳少宗;李艷梅;;辰時、申時電針對成年女性單純性肥患者瘦素影響的研究[A];2011年全國時間生物醫(yī)學學術會議論文集[C];2011年

3 周鵬;朱大龍;陳南衡;;瘦素和胰島素抵抗及糖代謝異常的關系[A];中華醫(yī)學會第六次全國內(nèi)分泌學術會議論文匯編[C];2001年

4 蔣卓勤;張彩霞;陳月嫦;;單純性肥胖兒童瘦素、胰島素及血糖關系的研究[A];中國營養(yǎng)學會第五屆婦幼營養(yǎng)學術會議論文摘要匯編[C];2002年

5 陳忠倫;王大模;李作孝;;多發(fā)性硬化患者血清瘦素水平的變化及其臨床意義[A];第九次全國神經(jīng)病學學術大會論文匯編[C];2006年

6 孫長顥;于春媛;王舒然;韓錫民;孫艷潔;劉慶敏;;肥胖和正常兒童性成熟時間與血中瘦素和性激素水平的關系[A];中國營養(yǎng)學會第八次全國營養(yǎng)學術會議論文摘要匯編[C];2000年

7 張燕軍;張靜;羅慶鋒;;瘦素防止甘氨鵝去氧膽酸誘發(fā)肝竇內(nèi)皮細胞凋亡[A];科技、工程與經(jīng)濟社會協(xié)調(diào)發(fā)展——中國科協(xié)第五屆青年學術年會論文集[C];2004年

8 史秀琴;袁志剛;程瑤;徐濱華;劉殿新;;瘦素與代謝綜合征的關系[A];全國首屆代謝綜合征的基礎與臨床專題學術會議論文匯編[C];2004年

9 李真;蔣麗艷;林桂蘭;成婭;;瘦素協(xié)同纖連蛋白對早孕絨毛細胞滋養(yǎng)細胞浸潤特性的影響[A];第八次全國婦產(chǎn)科學學術會議論文匯編[C];2004年

10 柴嬋娟;楊志明;康玉明;肖傳實;;核因子-κB信號途徑介導血管緊張素Ⅱ誘導THP-1巨噬細胞基質(zhì)金屬蛋白酶-9的表達[A];中華醫(yī)學會第11次心血管病學術會議論文摘要集[C];2009年

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1 蘭克;以嘗試用巨噬細胞治癱瘓[N];科技日報;2000年

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3 張驍；束梅英;瘦素研究進展[N];中國醫(yī)藥報;2004年

4 暨南大學醫(yī)藥生物技術研究開發(fā)中心 姚成燦 李校坤;根本減肥始自瘦素[N];醫(yī)藥經(jīng)濟報;2001年

5 曉曹;瘦素香料帶來內(nèi)衣革命[N];市場報;2003年

6 暨南大學醫(yī)藥生物技術研究開發(fā)中心 姚成燦 李校坤;“減肥” 突破口在哪？[N];醫(yī)藥經(jīng)濟報;2001年

7 ;抗肥胖研究獲突破“瘦素”能抑制食欲[N];新華每日電訊;2004年

8 ;綠原菌素與紅亮瘦素源對斷奶仔豬生產(chǎn)性能的影響[N];中國畜牧報;2004年

9 ;紅亮瘦素源[N];中國畜牧報;2004年

10 唐穎 倪兵 陳代杰;巨噬細胞泡沫化抑制劑研究快步進行[N];中國醫(yī)藥報;2006年

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4 馬翠卿;GAS下調(diào)巨噬細胞炎癥因子的作用及機制研究[D];河北醫(yī)科大學;2011年

5 鞠瑞;羧胺三唑的抗癌新機制抑制腫瘤相關巨噬細胞中促炎細胞因子的釋放[D];北京協(xié)和醫(yī)學院;2011年

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7 尚Z腪，

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