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

【摘要】： 本學位論文主要分為兩大部分：(1)應用原子力顯微鏡、共聚焦顯微鏡、流式細胞儀等分析方法,初步研究了臍帶間充質干細胞對淋巴細胞活化增殖的影響；(2)應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,應用激光共聚焦顯微鏡對細胞表面抗原分子的識別進行研究。 本文第一部分應用原子力顯微鏡、激光共聚焦顯微鏡、流式細胞儀等分析方法,探測了臍帶間充質干細胞對淋巴細胞增殖活化的影響。分析比較靜息、PHA刺激、與臍帶間充質干細胞共培養(yǎng)的三種淋巴細胞的形貌和生物物理性質,原子力顯微鏡觀察到共培養(yǎng)過程中干細胞與淋巴細胞相互接觸,淋巴細胞粘附在干細胞上,CCK-8檢測提示在hUC-MSC共培養(yǎng)條件下,絲裂原刺激T淋巴細胞增殖受到抑制,且抑制的程度與hUC-MSC的劑量正相關,流式細胞儀檢測在hUC-MSC共培養(yǎng)情況下,經絲裂原刺激后,外周血淋巴細胞CD69表達與對照組(52.5±4.7%)的陽性率相比較,下降至37.9±3.4%,激光共聚焦實驗進一步驗證了黏附在干細胞上的細胞為T淋巴細胞。觀察hUC-MSCs的免疫調節(jié)作用,進一步探討hUC-MSCs的免疫調控機制,為hUC-MSCs的臨床應用提供實驗依據。 本文第二部分基于原子力顯微術,結合激光共聚焦顯微術和熒光半導體量子點(Quantum dots, QDs)標記技術、流式細胞術和倒置熒光顯微鏡技術,以淋巴細胞為研究對象,在納米尺度研究了活化前后以及不同活化階段T細胞生物物理特性的變化,即細胞結構形態(tài)、膜表面納米結構、膜孔變化、膜表面粘附特性、膜表面受體分子分布等。主要研究結果如下：(1)對處于不同活化階段的Jurkat細胞進行了細胞全貌和細胞膜表面納米結構成像和探測研究,比較不同狀態(tài)下細胞表面的粘附力變化。隨著超抗原刺激時間的延長,Jurkat細胞的體積、高度、半寬度、粗糙度等參數發(fā)生明顯的變化,活化48h、72 h時細胞與針尖間的相互作用力大約是活化6 h時的5倍,活化過程中細胞膜表面納米結構的改變引起其機械性能的變化。(2)完成了對重組質粒真核表達載體pIRES-EGFP-BCL 11B電轉染人幼稚T細胞的研究,重組質粒轉染幼稚T細胞后,細胞的體積、高度、半寬度、粗糙度、表面顆粒大小等參數發(fā)生了變化,細胞楊氏模量以及細胞硬度也呈現(xiàn)很大變化,CCK-8結果顯示,重組質粒pIRES-EGFP-BCL 11B電轉染人幼稚T細胞后影響細胞的增殖。(3)比較了靜息、絲裂原、超抗原活化淋巴細胞的形態(tài)結構、膜表面抗原分子的表達等差異性。絲裂原PHA刺激的淋巴細胞大多呈成群聚集,而超抗原SEA刺激的淋巴細胞大多數呈分散狀。兩組活化后淋巴細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胞,形成了膜凸起。絲裂原和超抗原刺激淋巴細胞12 h后,都能使淋巴細胞表達CD69抗原分子,但在量表達上存在差異性(PHA：39.5±8.7%；SAE：8.3±1.8%), CD3和CD69分子在細胞膜上呈不均勻分布狀態(tài),且SEA活化后的T淋巴細胞表面受體CD3和CD69分子在空間上形成了微結構域。
[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.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329

【參考文獻】

相關期刊論文 前10條

1 胡曉東,郭彤,胡小唐;利用AFM動態(tài)電場在Si表面實現(xiàn)納米氧化結構[J];半導體學報;2002年11期

2 王茜;楊琨;白海;吳濤;路繼紅;;人臍血間充質干細胞抑制異體T淋巴細胞反應的實驗及臨床意義[J];第四軍醫(yī)大學學報;2007年18期

3 鐘麗云;廖問陶;王小平;蔡繼業(yè);;SNOM結合量子點標記進行T淋巴細胞體外刺激活化的研究[J];電子顯微學報;2006年05期

4 國立秋,董小瑛,趙鐵強,趙清亮,張飛虎,董申;碳納米管原子力顯微鏡針尖在結構生物學研究中的應用[J];分析化學;2004年04期

5 董颯英,王洪仁,羅國安;自組裝金電極的電化學測試及其FTIR和AFM分析[J];分析科學學報;2002年05期

6 張浩力,郭云,力虎林,楊得全;AFM誘導正十八硫醇在金基底上的選擇性生長[J];高等學�；瘜W學報;1999年09期

7 鄧曉芳;曾波航;胡偉民;陳靜琦;黃慧;孫建聰;彭燕;李冰;;超抗原金黃色葡萄球菌腸毒素A對T淋巴細胞分化的作用[J];廣州醫(yī)學院學報;2006年03期

8 鄧文禮,楊大本,方曄,白春禮;在Au表面硫醇自組裝單分子層微結構的原子力顯微鏡表征[J];化學物理學報;1996年02期

9 王蒙;楊媛;楊東明;王序全;許建中;;人臍血源性MSCs的免疫調節(jié)作用[J];免疫學雜志;2007年03期

10 段志堅;高波;徐薇;周倩;熊思東;;人TRIM22基因真核表達質粒的構建和表達及TRIM22對Jurkat T細胞增殖的影響[J];現(xiàn)代免疫學;2008年04期

，

本文編號：2038610