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
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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。
This dissertation includes two parts:(1) Based on atomic force microscopy (AFM). combined with laser scanning confocal microscopy, flow cytometry and inverted microscope, the effects of umbilical cord mesenchymal stem cells on proliferation and activation of allogeneic lymphocytes were being preliminarily investigated. (2)Using the high-resolution and force spectrum of the AFM, detect the changes of the morphology, adhesion force and Young's modulus properties of the different T lymphocytes by stimulation. the cell surface antigen receptor molecules recognition was performed by laser scanning confocal microscopy.
In the first part, atomic force microscopy, laser scanning confocal microscopy combinationed with quantum dots, flow cytometry and inverted microscope were used to investigate the effects of umbilical cord mesenchymal stem cells on proliferation and activation of allogeneic lymphocytes. The morphology and biophysical properties of the resting, PHA activated, coculture with hUC-MSCs were analyzed and compared. Lymphocyte cells intercontact with hUC-MSCs, and adhesion on them. HUC-MSCs had a dose dependent inhibitory effect on lymphocyte proliferation induced by PHA was found by CCK-8. FCM showed hUC-MSCs could inhibit CD69 expression on PHA activated T lymphocyte cells from 52.5±4.7%to 37.9±3.4%. Laser scanning confocal microscopy further illustrated that T lymphocytes adhesion on hUC-MSCs.
The second part of this paper, on the base of lymphocytes, AFM, flow cytometry, laser scanning confocal microscopy, quantum dots mark, fluorescence microscope were used to study the biophysical properties(the parameters including morphology, membrane nanostructure, membrane pore, adhesion, distribution of antigen receptor molecules and so on) of T lymphocytes stimulated different time and different stimulation, the main results are the following(1):Studyed the cellular surface ultrastructures and nanomechanical properties of acute T lymphoblastic leukemic cell (Jurkat cell) which was treated with staphylococcal enterotoxin A (SEA) at different time and compared the adhesive force of Jurkat cell at different states. The morphosructure and cell membrane of Jurkat cell exposed to SEA changed significantly and the ultrastructures became more complex with the time prolonging. The adhesion force values of Jurkat cells treated with SEA at 24 and 48 h were five times than those of Jurkat cell treated with SEA at 6h. The changes of the ultrastructures and membrane structure of Jurkat cell have caused the change of mechanical function. (2)BCL 11B were inserted into PIRES-EGFP eukaryotic expression vector to construct recombinant PIRES-EGFP-BCL 11B plasmid, which was transfected into human Naive T cell by electroporation. the morphology, ultrastructure, Young's modulus and stiffness of different four Naive T cell changed greatly after transfection, and BCL 11B promote the proliferation of Naive T cell of human.(3)The differences of morphology and surface antigen receptor molecules of resting, PHA or SEA activated human lymphocyte were compared. PBMCs formed colony after stimulation of PHA, and scattered after stimulation of PHA.Volume of activated lymphocytes in both groups were greater than resting group, and the activation polarization occurs during lymphocyte migration to form a membrane protrusion. the mean relative of CD69 expression after stimulation of PHA (39.5±8.7%) were higher than stimulation of SEA(8.3±1.8%), also illustrate that CD3 and CD69 receptors inhomogenously distributing on cell membrane, and the surface receptors CD3 and CD69 of T-lymphocytes activated by SEA forming micro-domain in space.
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。
This dissertation includes two parts:(1) Based on atomic force microscopy (AFM). combined with laser scanning confocal microscopy, flow cytometry and inverted microscope, the effects of umbilical cord mesenchymal stem cells on proliferation and activation of allogeneic lymphocytes were being preliminarily investigated. (2)Using the high-resolution and force spectrum of the AFM, detect the changes of the morphology, adhesion force and Young's modulus properties of the different T lymphocytes by stimulation. the cell surface antigen receptor molecules recognition was performed by laser scanning confocal microscopy.
In the first part, atomic force microscopy, laser scanning confocal microscopy combinationed with quantum dots, flow cytometry and inverted microscope were used to investigate the effects of umbilical cord mesenchymal stem cells on proliferation and activation of allogeneic lymphocytes. The morphology and biophysical properties of the resting, PHA activated, coculture with hUC-MSCs were analyzed and compared. Lymphocyte cells intercontact with hUC-MSCs, and adhesion on them. HUC-MSCs had a dose dependent inhibitory effect on lymphocyte proliferation induced by PHA was found by CCK-8. FCM showed hUC-MSCs could inhibit CD69 expression on PHA activated T lymphocyte cells from 52.5±4.7%to 37.9±3.4%. Laser scanning confocal microscopy further illustrated that T lymphocytes adhesion on hUC-MSCs.
The second part of this paper, on the base of lymphocytes, AFM, flow cytometry, laser scanning confocal microscopy, quantum dots mark, fluorescence microscope were used to study the biophysical properties(the parameters including morphology, membrane nanostructure, membrane pore, adhesion, distribution of antigen receptor molecules and so on) of T lymphocytes stimulated different time and different stimulation, the main results are the following(1):Studyed the cellular surface ultrastructures and nanomechanical properties of acute T lymphoblastic leukemic cell (Jurkat cell) which was treated with staphylococcal enterotoxin A (SEA) at different time and compared the adhesive force of Jurkat cell at different states. The morphosructure and cell membrane of Jurkat cell exposed to SEA changed significantly and the ultrastructures became more complex with the time prolonging. The adhesion force values of Jurkat cells treated with SEA at 24 and 48 h were five times than those of Jurkat cell treated with SEA at 6h. The changes of the ultrastructures and membrane structure of Jurkat cell have caused the change of mechanical function. (2)BCL 11B were inserted into PIRES-EGFP eukaryotic expression vector to construct recombinant PIRES-EGFP-BCL 11B plasmid, which was transfected into human Naive T cell by electroporation. the morphology, ultrastructure, Young's modulus and stiffness of different four Naive T cell changed greatly after transfection, and BCL 11B promote the proliferation of Naive T cell of human.(3)The differences of morphology and surface antigen receptor molecules of resting, PHA or SEA activated human lymphocyte were compared. PBMCs formed colony after stimulation of PHA, and scattered after stimulation of PHA.Volume of activated lymphocytes in both groups were greater than resting group, and the activation polarization occurs during lymphocyte migration to form a membrane protrusion. the mean relative of CD69 expression after stimulation of PHA (39.5±8.7%) were higher than stimulation of SEA(8.3±1.8%), also illustrate that CD3 and CD69 receptors inhomogenously distributing on cell membrane, and the surface receptors CD3 and CD69 of T-lymphocytes activated by SEA forming micro-domain in space.
引文
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