脐带间充质干细胞对淋巴细胞的影响及淋巴细胞活化研究

发布时间：2018-06-19 05:12

本文选题：原子力显微镜 + 激光共聚焦显微镜　；参考：《暨南大学》2010年硕士论文

【摘要】： 本学位论文主要分为两大部分：(1)应用原子力显微镜、共聚焦显微镜、流式细胞仪等分析方法,初步研究了脐带间充质干细胞对淋巴细胞活化增殖的影响；(2)应用原子力显微镜的高分辨率和力谱特性,探测不同T淋巴细胞在刺激剂作用下的形态变化以及粘附力和杨氏模量的变化,应用激光共聚焦显微镜对细胞表面抗原分子的识别进行研究。本文第一部分应用原子力显微镜、激光共聚焦显微镜、流式细胞仪等分析方法,探测了脐带间充质干细胞对淋巴细胞增殖活化的影响。分析比较静息、PHA刺激、与脐带间充质干细胞共培养的三种淋巴细胞的形貌和生物物理性质,原子力显微镜观察到共培养过程中干细胞与淋巴细胞相互接触,淋巴细胞粘附在干细胞上,CCK-8检测提示在hUC-MSC共培养条件下,丝裂原刺激T淋巴细胞增殖受到抑制,且抑制的程度与hUC-MSC的剂量正相关,流式细胞仪检测在hUC-MSC共培养情况下,经丝裂原刺激后,外周血淋巴细胞CD69表达与对照组(52.5±4.7%)的阳性率相比较,下降至37.9±3.4%,激光共聚焦实验进一步验证了黏附在干细胞上的细胞为T淋巴细胞。观察hUC-MSCs的免疫调节作用,进一步探讨hUC-MSCs的免疫调控机制,为hUC-MSCs的临床应用提供实验依据。本文第二部分基于原子力显微术,结合激光共聚焦显微术和荧光半导体量子点(Quantum dots, QDs)标记技术、流式细胞术和倒置荧光显微镜技术,以淋巴细胞为研究对象,在纳米尺度研究了活化前后以及不同活化阶段T细胞生物物理特性的变化,即细胞结构形态、膜表面纳米结构、膜孔变化、膜表面粘附特性、膜表面受体分子分布等。主要研究结果如下：(1)对处于不同活化阶段的Jurkat细胞进行了细胞全貌和细胞膜表面纳米结构成像和探测研究,比较不同状态下细胞表面的粘附力变化。随着超抗原刺激时间的延长,Jurkat细胞的体积、高度、半宽度、粗糙度等参数发生明显的变化,活化48h、72 h时细胞与针尖间的相互作用力大约是活化6 h时的5倍,活化过程中细胞膜表面纳米结构的改变引起其机械性能的变化。(2)完成了对重组质粒真核表达载体pIRES-EGFP-BCL 11B电转染人幼稚T细胞的研究,重组质粒转染幼稚T细胞后,细胞的体积、高度、半宽度、粗糙度、表面颗粒大小等参数发生了变化,细胞杨氏模量以及细胞硬度也呈现很大变化,CCK-8结果显示,重组质粒pIRES-EGFP-BCL 11B电转染人幼稚T细胞后影响细胞的增殖。(3)比较了静息、丝裂原、超抗原活化淋巴细胞的形态结构、膜表面抗原分子的表达等差异性。丝裂原PHA刺激的淋巴细胞大多呈成群聚集,而超抗原SEA刺激的淋巴细胞大多数呈分散状。两组活化后淋巴细胞体积均大于静息组,且活化过程中发生极化作用迁移淋巴细胞,形成了膜凸起。丝裂原和超抗原刺激淋巴细胞12 h后,都能使淋巴细胞表达CD69抗原分子,但在量表达上存在差异性(PHA：39.5±8.7%；SAE：8.3±1.8%), CD3和CD69分子在细胞膜上呈不均匀分布状态,且SEA活化后的T淋巴细胞表面受体CD3和CD69分子在空间上形成了微结构域。
[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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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