本文選題：前包欽格復合體　切入點：Ca2+/鈣調蛋白依賴性蛋白激酶II　出處：《第四軍醫(yī)大學》2014年碩士論文　論文類型：學位論文

【摘要】：本研究通過應用免疫酶組織化學、免疫熒光組織化學及免疫電鏡技術，，觀察了大鼠前包欽格復合體磷酸化鈣/鈣調蛋白依賴性蛋白激酶II (phospho-Ca2+/calmodulin-dependent protein kinase II，P-CaMKII)和原發(fā)性膽汁性肝硬化(primary biliaryecirrhosis，PBC)患者肝組織中自噬相關蛋白LC3B和BECLIN1的表達，并且探討了這些蛋白表達的生物學意義。 前包欽格復合體位于延髓腹外側，被認為是呼吸節(jié)律的產生中樞。CaMKII在神經元中廣泛表達且介導神經元細胞的各種生理過程。然而，很少有研究關注在前包欽格復合體中CaMKII的作用。本研究應用神經激肽-1受體（neuroknin-1receptor，NK1R）作為前包欽格復合體標記物，采用免疫酶組織化學、免疫熒光組織化學及包埋前免疫電鏡技術，觀察了P-CaMKII在正常大鼠前包欽格復合體的表達。我們尤其關注P-CaMKII在NK1R免疫反應神經元突觸后致密體（post-synaptic density，PSD）的表達。實驗結果顯示：光鏡下P-CaMKII免疫反應的神經元通常為橢圓形、紡錘形或錐形，P-CaMKII免疫反應產物分布在胞體和突起，在966個NK1R免疫反應神經元中，63.7%(615/966)與P-CaMKII雙標。電鏡下前包欽格復合體P-CaMKII和NK1R雙標神經元，免疫金顆粒代表P-CaMKII免疫反應，免疫過氧化物酶產物代表NK1R免疫反應，在胞體，有21個突觸檢測出P-CaMKII免疫反應，其中8(38.1%)個是對稱型突觸，其余為不對稱型突觸。然而，在樹突P-CaMKII免疫反應大多數(shù)集中在不對稱型突觸，我們檢測了87個樹突，其中81(93.1%)是不對稱型突觸，僅有少量是對稱型突觸。部分突觸缺乏P-CaMKII免疫反應。此外，在突觸周區(qū)域和突觸外區(qū)域也可觀察到P-CaMKII免疫反應。免疫過氧化物酶反應產物顯示NK1R免疫反應，主要集中在突觸外區(qū)域，這與我們先前研究一致，在細胞胞質中也可觀察到NK1R免疫反應。P-CaMKII與NK1R可在前包欽格復合體共存。以上結果提示，P-CaMKII分布在胞體、突觸、突觸周區(qū)域和突觸外區(qū)域，在不同區(qū)域有不同功能，在樹突不對稱型突觸中CaMKII可以感應突觸后Ca2+變化、磷酸化突觸后蛋白和調節(jié)興奮性突觸傳遞，而在胞體中CaMKII同時作用于對稱和不對稱型突觸，調節(jié)興奮性和抑制性突觸傳遞，可能影響呼吸可塑性的調節(jié)，為深入研究前包欽格復合體參與呼吸可塑性調控提供了形態(tài)學依據(jù)。 PBC是一種以血清中抗線粒體抗體滴度增高，肝臟非化膿性小膽管炎癥和纖維化為特點的的自身免疫性肝臟疾病。在Sasaki M等的研究發(fā)現(xiàn)LC3B特異性表達于損傷小膽管上皮細胞內，自噬與PBC小膽管上皮細胞線粒體功能相關，可能參與PBC膽管損傷的發(fā)病機制。以上研究只集中在膽管上皮細胞，在肝細胞未發(fā)現(xiàn)明確自噬。我們的觀察發(fā)現(xiàn)在PBC患者的肝細胞中也存在明顯的自噬體，因此自噬在PBC肝細胞損傷中的作用值得進一步研究。本研究進行了PBC患者的病例收集及相關指標分析，運用免疫組化、免疫熒光及電鏡技術觀察所收集病例肝臟組織中肝細胞和膽管上皮細胞自噬體的分布情況。結果顯示50例PBC患者，堿性磷酸酶（alkalinephosphatase，ALP）、γ-谷氨酰轉肽酶（γ-glutamyl transpeptidase，γ-GGT）有明顯升高，自噬相關蛋白LC3B和BECLIN1在所選抗線粒體抗體（anti-mitochondriaantibody，AMA）陽性PBC患者肝細胞和膽管上皮細胞的細胞內有表達，且大多數(shù)表達的肝細胞都鄰近匯管區(qū)，且LC3B和BECLIN1在部分肝細胞里有共存的現(xiàn)象。透射電鏡下在肝細胞和膽管上皮細胞有明顯的自噬體，同時伴隨肝細胞和膽管上皮細胞的損傷。以上結果提示自噬可能參與PBC患者的肝細胞和膽管上皮細胞損傷，為自噬在肝細胞和膽管上皮細胞損傷中的作用提供形態(tài)學依據(jù)。
[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.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R575.2

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