【作者】 李臣鸿；

【导师】 徐涛；

【作者基本信息】 华中科技大学 ， 生物物理， 2005， 博士

【摘要】 细胞物质转运实质上是细胞内的一个庞大而复杂的物流系统,这一系统的异常将导致相应各种疾病的发生。囊泡是这一物流系统中的一种可调控的重要的运输工具,脂筏是膜脂双层内含有特殊脂质及蛋白质的微区,在这一物流系统中具有重要的作用。本课题引入单分子研究技术和全内反射荧光成像技术,实时动态地研究转运囊泡的动力学过程,和两类脂筏与囊泡之间的动态空间定位关系。这一研究有助于我们进一步揭示囊泡转运机制和糖尿病的发生机制,而且对于进一步认识脂筏功能及开发以GLUT4为靶的调节血糖的药物都具有重要的理论及实际指导意义。机体内葡萄糖的平衡对生命过程至关重要。胰岛素是调节血糖平衡的重要激素,它促进葡萄糖进入细胞内,从而降低血中葡萄糖。胰岛素的这一作用是通过细胞内的葡萄糖转运体(Glucose Transporter,GLUT)家族之一葡萄糖转运子4(GLUT4)将葡萄糖从细胞外转运到细胞内的。研究表明II型糖尿病的发生与细胞内GLUT4的转运障碍有关。而GLUT4的转运被认为是受多种因素控制的,这些因素目前较公认的有GLUT4在细胞器中的保持机制、动态分选、囊泡的转运、锚定和与质膜的融合以及质膜的脂质结构。GLUT4在细胞内的转运至少有两个循环,一个是细胞表面和内涵体间的循环,一个是在trans-Golgi network (TGN) 和内涵体间的循环。在这两个循环中GLUT4转位有几个严格的控制点。这两个循环似乎告诉我们GLUT4在胞内可能有两种运动形式。为了回答这个问题,我们研究了GLUT4在胞内的动力学过程。在研究过程中使用Confocal显微镜观察了GLUT4在胞内的整体分布。使用全内反射荧光显微镜时序成像获得了GLUT4的动态连续的图像,用于三维动力学分析。分析中发展了单颗粒追踪方法(SPT),使之能跟踪亚象素的位移,发现GLUT4在胞内虽然有两个循环路径,但是在无刺激的情况下,它的运动呈现限制性运动,其分散系数呈现一种连续的平滑的分布,即其运动并未因循环有两个路径而分成几种形式。此外,我们初步研究了囊泡转运与细胞膜脂筏的关系。首先构建了含荧光标记更多还原

【Abstract】 The vesicles transport is a complicated system in cell in which materials are transfered any abnormity of the system would results in corresponding deseases. Vesicles function as transport carries and can be regulated. Lipid-Raft is a microdomain containing special lipids and proteins between the two layers of cell membranes and plays an important role in vesicles transport. In this thesis, single melecular technique and total internal reflex fluorecence microscope imaging technique were used to investigate the location and mobility characteristics of Glucose Transporter 4 in the real-time dynamic and space location relation between the large dense core vesicles (LDCVs) and two kinds of lipid raft.The study will be very interesting in illustrations of the mechenisms of vesicles transport as well as of diabetes and helpneses in developing the drugs targeting at GLUT4 and benifical in further sighting into the functions of lipid-raft. It is an importance of homeostasis body’s glucose. It is known that insulin is important hormone to regulate balance of blood glucose. Insulin helps to transport glucose cross plasma membrane into cells and decrease glucose level in blood. In fact, the effect of insulin is dependent on the effect of GLUT4 in some degree. Therefore, the GLUT4 becomes a key factor of regulation in balance of blood glucose. Evidences show the type2 diabetes is related with the abnormity of transportation of GLUT4. The transport of GLUT4 is regulated by many factors, which include its sequestration mechanism in cells, dynamic sorting, assemble secretory vesicles and structure of membrane lipid. GLUT4 transportation in cells has two recycling pathways. The cycle 1 occurs between the cell surface and endosomes and cycle 2 between the trans-Golgi network (TGN) and endosomes. It seems likely that various recycling loops must be coordinated in regulating the trafficking of intracellular GLUT4. Although different GLUT4 trafficking pathways have been proposed, it is still unclear whether GLUT4 is classified into distinct patterns according to their mobility. It would be clearly helpful to investigate the mobility of a single GLUT4 in living cells. Here we have labeled the GLUT4 by constructing a fusion protein linking EGFP to the C-terminus of mouse GLUT4. The GLUT4-EGFP fusion protein was expressed in 3T3-L1 cells by transient transfection. By using total internal reflection fluorescence microscopy (TIRFM), the corresponding fluorescence images of GLUT4-EGFP fusion protein were recorded in charge coupled device (CCD) at high time resolution. In order to obtain the sub-pixel displacement of GLUT4, we employed a Gaussian-based single particle tracking (SPT) method to resolve the kinetics of vesicle motion. We found that GLUT4 moves in a constrained fashion as if it is tethered by some intracellular structure. The distribution of the three-dimensional diffusion coefficients (D(3)) was a smooth continuum. Our results demonstrate that even the existence of different recycling loops for GLUT4, they still are organized in a continuous range of mobility rather than into separated classes in intact 3T3-L1, but they are organized in two kinds of mobility pools after stimulation by insulin. In addition, we have studied the relationships between the vesicles transport and lipid rafts. Lipid rafts are specific membrane microdomains that are rich in cholesterol, glycosphingolipids and glycosylphosphatidylinositol linked molecules. They play role in the system of protein trafficking. Here we have labeled the Caveolin and flotillin, which are two kinds of marker proteins of rafts by constructing a fusion protein that links EGFP, and labeled the NPY and SNAP25 by constructing a fusion protein that links DsRed. The fusion proteins were expressed in PC12 cells by transient transfection. By using total internal reflection fluorescence microscopy (TIRFM), the corresponding fluorescence images were recorded with charge coupled device (CCD) at high time resolution. We found that Caveolin is not in colocation with NPY during free-stimulation and stimulation with high concentration K+, and neither colocation with SNAP25. Flotillin does not distinctly coexisted with NPY. The results show that rafts may not take part in the transport procedure of LDCVs with NPY. The results are not consistent with that from biochemical approaches. The difference may be interpreted by two ways: on the one hand,it is possibility that rafts take part in small vesicles transport but no do in LDCVs, because the two kinds of vesicles have actually different mechanism of transport; on the other hand different results attribute to different experimental ways. It is very necessary that we must develop new methods to study rafts.更多还原