大鼠前包钦格复合体P-CaMKⅡ及PBC患者肝组织LC3B和BECLIN1的表达及意义

发布时间：2018-03-14 22:36

本文选题：前包钦格复合体　切入点：Ca2+/钙调蛋白依赖性蛋白激酶II　出处：《第四军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：本研究通过应用免疫酶组织化学、免疫荧光组织化学及免疫电镜技术，，观察了大鼠前包钦格复合体磷酸化钙/钙调蛋白依赖性蛋白激酶II (phospho-Ca2+/calmodulin-dependent protein kinase II，P-CaMKII)和原发性胆汁性肝硬化(primary biliaryecirrhosis，PBC)患者肝组织中自噬相关蛋白LC3B和BECLIN1的表达，并且探讨了这些蛋白表达的生物学意义。前包钦格复合体位于延髓腹外侧，被认为是呼吸节律的产生中枢。CaMKII在神经元中广泛表达且介导神经元细胞的各种生理过程。然而，很少有研究关注在前包钦格复合体中CaMKII的作用。本研究应用神经激肽-1受体（neuroknin-1receptor，NK1R）作为前包钦格复合体标记物，采用免疫酶组织化学、免疫荧光组织化学及包埋前免疫电镜技术，观察了P-CaMKII在正常大鼠前包钦格复合体的表达。我们尤其关注P-CaMKII在NK1R免疫反应神经元突触后致密体（post-synaptic density，PSD）的表达。实验结果显示：光镜下P-CaMKII免疫反应的神经元通常为椭圆形、纺锤形或锥形，P-CaMKII免疫反应产物分布在胞体和突起，在966个NK1R免疫反应神经元中，63.7%(615/966)与P-CaMKII双标。电镜下前包钦格复合体P-CaMKII和NK1R双标神经元，免疫金颗粒代表P-CaMKII免疫反应，免疫过氧化物酶产物代表NK1R免疫反应，在胞体，有21个突触检测出P-CaMKII免疫反应，其中8(38.1%)个是对称型突触，其余为不对称型突触。然而，在树突P-CaMKII免疫反应大多数集中在不对称型突触，我们检测了87个树突，其中81(93.1%)是不对称型突触，仅有少量是对称型突触。部分突触缺乏P-CaMKII免疫反应。此外，在突触周区域和突触外区域也可观察到P-CaMKII免疫反应。免疫过氧化物酶反应产物显示NK1R免疫反应，主要集中在突触外区域，这与我们先前研究一致，在细胞胞质中也可观察到NK1R免疫反应。P-CaMKII与NK1R可在前包钦格复合体共存。以上结果提示，P-CaMKII分布在胞体、突触、突触周区域和突触外区域，在不同区域有不同功能，在树突不对称型突触中CaMKII可以感应突触后Ca2+变化、磷酸化突触后蛋白和调节兴奋性突触传递，而在胞体中CaMKII同时作用于对称和不对称型突触，调节兴奋性和抑制性突触传递，可能影响呼吸可塑性的调节，为深入研究前包钦格复合体参与呼吸可塑性调控提供了形态学依据。 PBC是一种以血清中抗线粒体抗体滴度增高，肝脏非化脓性小胆管炎症和纤维化为特点的的自身免疫性肝脏疾病。在Sasaki M等的研究发现LC3B特异性表达于损伤小胆管上皮细胞内，自噬与PBC小胆管上皮细胞线粒体功能相关，可能参与PBC胆管损伤的发病机制。以上研究只集中在胆管上皮细胞，在肝细胞未发现明确自噬。我们的观察发现在PBC患者的肝细胞中也存在明显的自噬体，因此自噬在PBC肝细胞损伤中的作用值得进一步研究。本研究进行了PBC患者的病例收集及相关指标分析，运用免疫组化、免疫荧光及电镜技术观察所收集病例肝脏组织中肝细胞和胆管上皮细胞自噬体的分布情况。结果显示50例PBC患者，碱性磷酸酶（alkalinephosphatase，ALP）、γ-谷氨酰转肽酶（γ-glutamyl transpeptidase，γ-GGT）有明显升高，自噬相关蛋白LC3B和BECLIN1在所选抗线粒体抗体（anti-mitochondriaantibody，AMA）阳性PBC患者肝细胞和胆管上皮细胞的细胞内有表达，且大多数表达的肝细胞都邻近汇管区，且LC3B和BECLIN1在部分肝细胞里有共存的现象。透射电镜下在肝细胞和胆管上皮细胞有明显的自噬体，同时伴随肝细胞和胆管上皮细胞的损伤。以上结果提示自噬可能参与PBC患者的肝细胞和胆管上皮细胞损伤，为自噬在肝细胞和胆管上皮细胞损伤中的作用提供形态学依据。
[Abstract]:This study through the application of immunohistochemistry, immunofluorescence and immunoelectron microscopy, rats were observed pre botinger complex phosphorylation of calcium / calmodulin dependent protein kinase II (phospho-Ca2+/calmodulin-dependent protein kinase II, P-CaMKII) and primary biliary cirrhosis (primary, biliaryecirrhosis, PBC) expression of autophagy in liver tissue of patients the related protein LC3B and BECLIN1, and to investigate the biological significance of these proteins.
Pre botinger complex is located in the ventrolateral medulla, is considered to be the central respiratory rhythm generation.CaMKII is widely expressed in neurons and neuronal cells mediated by various physiological processes. However, few studies have focused on the CaMKII in the pre botinger complex in vitro. This study used by God kinin -1 receptor (neuroknin-1receptor, NK1R) as pre botinger complex markers by immunohistochemistry, immunofluorescence and pre embedding immunoelectron microscopy, observe the expression of P-CaMKII in normal rat pre botinger complex. We particularly concern P-CaMKII in synaptic NK1R immunoreactive neurons after dense bodies (post-synaptic density, PSD). Expression. Experimental results show that: under the light microscope P-CaMKII immunoreactive neurons usually oval, spindle shaped or conical, P-CaMKII immunoreactivity in cell bodies and processes, in 966 NK1 R immunoreactive neurons, 63.7% (615/966) and P-CaMKII staining. Electron microscope pre botinger complex P-CaMKII and NK1R double labeled neurons, immunogold particles representing P-CaMKII immune response, immune response to NK1R immunoperoxidase product representative in the soma, 21 synapses detected P-CaMKII immune response, of which 8 (38.1%) a symmetric synapses, the rest are asymmetrical synapses. However, in the most concentrated P-CaMKII immunoreactive dendritic synapses in the asymmetric type, we tested 87 dendrites, of which 81 (93.1%) is asymmetric synapses, only a small amount of symmetric synapses. Some synapses lacking P-CaMKII immunoreactivity in the synaptic region. In addition, week and extrasynaptic area also P-CaMKII immunoreactivity was observed. Immunoperoxidase reaction product showed NK1R immunoreactivity, mainly concentrated in the synaptic, this and our previous research, in the cytoplasm Could also be observed in NK1R and NK1R in the immune response of.P-CaMKII pre botinger complex coexist. These results suggest that the P-CaMKII distribution in the soma, synapses, synaptic and extrasynaptic region around the region, there are different functions in different regions, in asymmetric synapses in dendritic CaMKII can induced changes of Ca2+ postsynaptic phosphorylation post synaptic proteins and regulate excitatory synaptic transmission in the cell bodies of CaMKII at the same time to symmetric and asymmetric synapses, regulation of excitatory and inhibitory synaptic transmission, may affect the regulation of respiratory plasticity, and provided morphological basis in respiratory plasticity for the further study of pre botinger complex.
PBC is an elevated serum anti mitochondrial antibody titer in liver, non small bile duct inflammation and fibrosis characterized by purulent autoimmune liver disease. In the study of Sasaki M found that LC3B specifically expressed in small injury bile duct epithelial cells, autophagy and PBC on mitochondrial function of small bile duct epithelial cells, pathogenesis the mechanism may be involved in the bile duct injury of PBC. The above research focused only on bile duct epithelial cells in liver cells. We found no clear autophagy observed in PBC liver cells also showed obvious autophagosomes, so the role of autophagy in PBC liver cell injury is worthy of further study. This study analyzed the cases collected PBC patients and related indexes, using immunohistochemistry, distribution of immunofluorescence and electron microscope were used to observe the collected cases of liver tissue in liver cells and bile duct epithelial cell autophagy. The results showed that the 50 PBC patients, alkaline phosphatase (alkalinephosphatase, ALP), gamma glutamyltransferase (transpeptidase gamma gamma -glutamyl, -GGT) increased significantly, autophagy related protein LC3B and BECLIN1 in the selected anti mitochondrial antibody (anti-mitochondriaantibody, AMA) PBC positive patients with liver cells and bile duct epithelial cells in the expression, and the expression of liver cells are most near the portal area, and LC3B and BECLIN1 in liver cells have coexisted. Transmission electron microscope in liver cells and bile duct epithelial cells have obvious autophagosomes, accompanied liver cells and bile duct epithelial cells injury. These results suggest that liver cells and bile duct autophagy may be involved in epithelial cell injury in patients with PBC and provide the morphological evidence for the role of autophagy in the injury of liver cells and bile duct epithelial cells.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.2

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