The Effect Of Qingyi Decoction On Protection Of Structure And Expression Of Proteome In Ancreatic Acinar Cell Of SAP Rat

Object:To research the pathological mechanisms of severe acute pancreatitis(SAP) from proteomics, and to discuss Qingyitang protective role in the treatment process of the ERP rat pancreatic acinar cell structure and function, and the impact of Proteome of early pancreatic acinar cells in SAP rat. Meanwhile, in the course of experimental study, to perfect and optimize the separation of rat pancreatic acinar cells in vivo and in vitro, purification and training methods.Method:Selected SPF level 69 adult male Wistar rats, weighing 250~300g, according to computer-generated random number table were randomly divided into sham group(9), SAP model group(30), and drug treatment group(30). SAP model group and drug treatment group rats were treated with retrograde injection of 5% taurocholate sodium to make SAP rat models. Sham-operated rats injected in the same manner and dose as 5% saline. After animals anesthesia, drug treatment group fed every 12 h Qingyi decoction 5ml(equivalent to 30 g crude drug/kg). SAP model group and the sham group fed every 12 h isodose normal saline. After each group were fed at first Qingyitang and saline 24 h, 48 h, 72 h three time points, by cell separation medium being retrograde cholangiopancreatography injected and in vitro digestion, respectively, pancreatic acinar cells were isolated, purified, as well as made determination of cell viability and purification. Comparing three groups of pancreatic acinar cells and a variety of cell ultrastructural differences in the transmission electron microscope three time points. Take 48 h time point every rat pancreatic acinar cells in vitro to extract and purificate within each rat pancreatic acinar cell protein. Separately for every group of pancreatic acinar cell protein extracted were made 2-DE electrophoresis, by PDQuest analysis of the electrophoresis patterns. Then select meaningful differences significantly better resolution and stable expression of proteins with trypsin to hydrolyz. The peptides were hydrolyzed to make MALDI-TOF-MS mass spectrometry to obtain the corresponding peptide mass fingerprinting(PMF). Finally, according to the MALDI-TOF-MS data with Mascot online data retrieval tool to search through the Internet for free in the Protein Data Bank. Identified differences in protein expression stable results and to compare the final results of SAP model and Qingyitang drug treatment groups.Result:(1)Under TEM, in SAP model group, pancreatic acinar cells and intracellular organelles, including the nucleus, mitochondria, rough endoplasmic reticulum, Golgi apparatus, zymogen granules and others appear obvious structural abnormalities and marrow appear scaly body in the cytoplasm, pathological structure vacuoles, lysosomes, secondary lysosomes. with time, acinar cell ultrastructure damage has been increasing. Drug treatment group pancreatic acinar cell ultrastructure pathologic changes significantly lighter than the SAP group. In 72 h time point observed in pancreatic acinar cell structure is more complete, and normal cells physiological function gradually recovery.(2)Compared with the sham group, in SAP model group the expression of "30 differential protein spots" in pancreatic acinar cells are up-regulated 17 times, the expression of "38 differential protein spots" in pancreatic acinar cells of SAP group increases 22 times. Compared with SAP group, the expression of "30 different protein spots" of drug treatment group pancreatic acinar cells down about 1/3, and in the drug treatment group, "38 different protein spots" of pancreatic acinar cells in an amount from about down-regulation 1/2. Meanwhile experimentally determined difference of the two proteins are chymotrypsin-like protein and heat shock protein.Conclusion:(1)Qingyitang by maintaining SAP early pancreatic acinar cell structures intact, reducing the intracellular mitochondria, rough endoplasmic reticulum, zymogen granules and other organelles injury, the degree of functional disorders structure, play a therapeutic SAP purpose;(2)The abnormal expression of precursor small protein molecule(chymotrypsin-like proteins, etc.) leads to normal pancreatic acinar cell dysfunction, leading to development of disease progression;(3)Qingyitang abnormal expression of rat pancreatic acinar cells within the SAP small protein(heat shock proteins, etc.) has a regulatory role. By interfering with the expression and modification of these specific proteins, Qingyitang achieves the purpose of the treatment of SAP.