本文選題：原子力顯微鏡 + 激光共聚焦顯微鏡��； 參考：《暨南大學(xué)》2010年碩士論文

【摘要】： 本學(xué)位論文主要分為兩大部分：(1)應(yīng)用原子力顯微鏡、共聚焦顯微鏡、流式細(xì)胞儀等分析方法,初步研究了臍帶間充質(zhì)干細(xì)胞對淋巴細(xì)胞活化增殖的影響；(2)應(yīng)用原子力顯微鏡的高分辨率和力譜特性,探測不同T淋巴細(xì)胞在刺激劑作用下的形態(tài)變化以及粘附力和楊氏模量的變化,應(yīng)用激光共聚焦顯微鏡對細(xì)胞表面抗原分子的識別進(jìn)行研究。 本文第一部分應(yīng)用原子力顯微鏡、激光共聚焦顯微鏡、流式細(xì)胞儀等分析方法,探測了臍帶間充質(zhì)干細(xì)胞對淋巴細(xì)胞增殖活化的影響。分析比較靜息、PHA刺激、與臍帶間充質(zhì)干細(xì)胞共培養(yǎng)的三種淋巴細(xì)胞的形貌和生物物理性質(zhì),原子力顯微鏡觀察到共培養(yǎng)過程中干細(xì)胞與淋巴細(xì)胞相互接觸,淋巴細(xì)胞粘附在干細(xì)胞上,CCK-8檢測提示在hUC-MSC共培養(yǎng)條件下,絲裂原刺激T淋巴細(xì)胞增殖受到抑制,且抑制的程度與hUC-MSC的劑量正相關(guān),流式細(xì)胞儀檢測在hUC-MSC共培養(yǎng)情況下,經(jīng)絲裂原刺激后,外周血淋巴細(xì)胞CD69表達(dá)與對照組(52.5±4.7%)的陽性率相比較,下降至37.9±3.4%,激光共聚焦實(shí)驗(yàn)進(jìn)一步驗(yàn)證了黏附在干細(xì)胞上的細(xì)胞為T淋巴細(xì)胞。觀察hUC-MSCs的免疫調(diào)節(jié)作用,進(jìn)一步探討hUC-MSCs的免疫調(diào)控機(jī)制,為hUC-MSCs的臨床應(yīng)用提供實(shí)驗(yàn)依據(jù)。 本文第二部分基于原子力顯微術(shù),結(jié)合激光共聚焦顯微術(shù)和熒光半導(dǎo)體量子點(diǎn)(Quantum dots, QDs)標(biāo)記技術(shù)、流式細(xì)胞術(shù)和倒置熒光顯微鏡技術(shù),以淋巴細(xì)胞為研究對象,在納米尺度研究了活化前后以及不同活化階段T細(xì)胞生物物理特性的變化,即細(xì)胞結(jié)構(gòu)形態(tài)、膜表面納米結(jié)構(gòu)、膜孔變化、膜表面粘附特性、膜表面受體分子分布等。主要研究結(jié)果如下：(1)對處于不同活化階段的Jurkat細(xì)胞進(jìn)行了細(xì)胞全貌和細(xì)胞膜表面納米結(jié)構(gòu)成像和探測研究,比較不同狀態(tài)下細(xì)胞表面的粘附力變化。隨著超抗原刺激時間的延長,Jurkat細(xì)胞的體積、高度、半寬度、粗糙度等參數(shù)發(fā)生明顯的變化,活化48h、72 h時細(xì)胞與針尖間的相互作用力大約是活化6 h時的5倍,活化過程中細(xì)胞膜表面納米結(jié)構(gòu)的改變引起其機(jī)械性能的變化。(2)完成了對重組質(zhì)粒真核表達(dá)載體pIRES-EGFP-BCL 11B電轉(zhuǎn)染人幼稚T細(xì)胞的研究,重組質(zhì)粒轉(zhuǎn)染幼稚T細(xì)胞后,細(xì)胞的體積、高度、半寬度、粗糙度、表面顆粒大小等參數(shù)發(fā)生了變化,細(xì)胞楊氏模量以及細(xì)胞硬度也呈現(xiàn)很大變化,CCK-8結(jié)果顯示,重組質(zhì)粒pIRES-EGFP-BCL 11B電轉(zhuǎn)染人幼稚T細(xì)胞后影響細(xì)胞的增殖。(3)比較了靜息、絲裂原、超抗原活化淋巴細(xì)胞的形態(tài)結(jié)構(gòu)、膜表面抗原分子的表達(dá)等差異性。絲裂原PHA刺激的淋巴細(xì)胞大多呈成群聚集,而超抗原SEA刺激的淋巴細(xì)胞大多數(shù)呈分散狀。兩組活化后淋巴細(xì)胞體積均大于靜息組,且活化過程中發(fā)生極化作用遷移淋巴細(xì)胞,形成了膜凸起。絲裂原和超抗原刺激淋巴細(xì)胞12 h后,都能使淋巴細(xì)胞表達(dá)CD69抗原分子,但在量表達(dá)上存在差異性(PHA：39.5±8.7%；SAE：8.3±1.8%), CD3和CD69分子在細(xì)胞膜上呈不均勻分布狀態(tài),且SEA活化后的T淋巴細(xì)胞表面受體CD3和CD69分子在空間上形成了微結(jié)構(gòu)域。
[Abstract]:This dissertation is divided into two main parts: (1) the effect of umbilical cord mesenchymal stem cells on lymphocyte activation and proliferation was preliminarily studied by atomic force microscopy, confocal microscopy and flow cytometry. (2) the effects of different T lymphocytes on stimulants were detected by the high resolution and force spectrum characteristics of atomic force microscopy. The changes of morphology, adhesion force and Young's modulus were studied. Confocal laser scanning microscopy was used to identify cell surface antigen molecules.
In the first part of this paper, the effects of umbilical cord mesenchymal stem cells on lymphocyte proliferation and activation were detected by atomic force microscopy, confocal laser scanning microscopy and flow cytometry. The morphologies and biophysical properties of three kinds of lymphocytes co cultured with umbilical cord mesenchymal stem cells were analyzed and compared. Microscope observed the interaction of stem cells with lymphocytes and lymphocytes adhered to stem cells during co culture. CCK-8 detection suggested that mitogen stimulated T lymphocyte proliferation under hUC-MSC co culture conditions, and the degree of inhibition was positively related to the dose of hUC-MSC. Flow cytometry was used to detect hUC-MSC in the case of co culture. After mitogen stimulation, the expression of CD69 in peripheral blood lymphocytes was decreased to 37.9 + 3.4% compared with the positive rate of the control group (52.5 + 4.7%). The laser confocal experiment further verified that the cells attached to the stem cells were T lymphocytes. The immunoregulation effect of hUC-MSCs was observed and the immunoregulation mechanism of hUC-MSCs was further explored, which was hUC-MSCs The clinical application provides the experimental basis.
The second part of this paper, based on atomic force microscopy, combined with laser confocal microscopy and fluorescent semiconductor quantum dots (Quantum dots, QDs) labeling, flow cytometry and inverted fluorescence microscopy, studied the biological and physical properties of T cells before and after activation and at different activation stages in nanoscale. Changes in cell structure morphology, membrane surface nanostructure, membrane pore change, membrane surface adhesion properties, membrane surface receptor molecular distribution, etc. the main results are as follows: (1) Jurkat cells at different activation stages were examined and studied on cell surface and membrane surface nanostructures, and the surface of cells in different states were compared. With the prolongation of the time of superantigen stimulation, the volume, height, half width and roughness of Jurkat cells changed obviously. The interaction between the cell and the needle tip was about 5 times that of the activation of 6 h at 72 h. The change of the surface surface nano structure of cell membrane in the process of activation caused the change of mechanical properties. (2) After the transfection of recombinant plasmid pIRES-EGFP-BCL 11B to human immature T cells, the volume, height, half width, roughness, surface particle size and other parameters of the recombinant plasmid were changed, and the young's modulus and cell hardness of the cells changed greatly. The CCK-8 results showed that the recombinant plasmid was reorganized. Plasmids pIRES-EGFP-BCL 11B transfected human immature T cells after transfection. (3) the morphologic structure of resting, mitogen, superantigen activated lymphocytes and the expression of membrane surface antigen molecules were compared. Most of the lymphocytes stimulated by mitogen PHA were clustered, and most of the lymphocytes stimulated by superantigen SEA were dispersed. The volume of lymphocyte in the two groups was greater than that in the resting group, and the migration of the lymphocyte in the process of activation had a membrane protruding. After the mitogen and superantigen stimulated the lymphocyte 12 h, the lymphocyte expressed the CD69 antigen, but there was a difference in the quantity expression (PHA:39.5 + 8.7%; SAE:8.3 + 1.8%), CD3 and C D69 molecules are not evenly distributed on the cell membrane, and SEA activated T lymphocytes surface receptors CD3 and CD69 molecules form a micro domain in space.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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