The Influence Of Targeting Interference Of PERK Gene On The Autophagy In Cell Mode Of Caerulein-induced Acute Pancreatitis In Endoplastic Reticulum Stress

Acute pancreatitis is a deadly disease high morbidity and mortality rate, but there is still no effective medicine or other treatment. It is generally believed that the disease began in acinar cells, but the detailed pathological mechanism is still unknown. The characteristic manifestations of pancreatitis include hyperamyla semia, the abnormal activation of digestive enzymes（eg trypsinogen into trypsin）, the accumulation of large vacuoles in acinar cells, inflammation of the pancreas, systemic inflammatory response caused by cell invasion which followed by the release of pro-inflammatory mediators（such as key transcription factor NF-κ B） and acinar cell death induced by apoptosis or necrosis. Previous study indicated that the endoplasmic reticulum stress response（ERS） occurs in acute pancreatitis. Histological examination suggested that in all of the current experimental model, ERS can be observed in the early stage of acute pancreatitis.The changes of gene expression in pancreatitis may explain the ERS mechanisms.PART ONE Establishment of a stable EGFP-LC3-expressed AR42 J cell lineObjective: To establish a stable EGFP-LC3-expressed AR42 J cell.Methods: The EGFP-LC3-overexpressed Lentivirus was constructed and transfected into pancreatic acinar cells（AR42J） of rats with Lentiviral transfection. The transfection efficiency was detected by inverted fluorescence microscope and flow cytometry.The EGFP-LC3 protein expression in the stable cell lines were analyzed by Western blot.the LC3 gene m RNA expression was detected by real-time PCR.Results: Detected by inverted fluorescence microscope and flow cytometry, the efficiency of Lentiviral-EGFP-LC3 tansfection is higer than 85%.As demonstrated by Western blot analysis, EGFP-LC3 protein expressed in the transfectedcells.Quantitative real-time PCR show that LC3 m RNA expression of EGFP and EGFP-LC3 were 1.08 ± 0.07、9.14 ± 0.32 fold as compared with that of the untreated cells. LC3 m RNA in AR42 J cells was overexpressed by EGFP-LC3 as compared with that of the untreated cells（P<0.05）.Conclusion:A pancreatic acinar cell line（AR42J） of rat stably expressing EGFP-LC3 protein was constructed successfully in the study, and it provided a new model for further researching into the relationship between acute pancreatic inflammation and autophagy.PART TWO Construction of sh RNA eukaryotic expression vector targeting PERK gene and effect of interference targeting in AR42 J cellsObjective:To construct short hairpin RNA（sh RNA） eukaryotic expression vector targeting the gene of RNA-dependent protein kinase（PKR）-like endoplasmic reticulum kinase（PERK） using interference methods of RNAi; U6-MCS-Ubiquitin-Cherry-IRES-PUR-PERK2-sh RNA was stably transfected into AR42J-LC3 cells with Lentiviral transfection to construct a AR42J-LC3 line targeting in PERK gene. And it provided a foundation for further researching the relationship between acute pancreatic inflammation, reticulum stress and autophagy.METHOD:1.we designed RNAi target sequences of PERK gene m RNA in rat based on the nucleotide sequence of PERK of rat（NM₀31599） referring to Gen Bank database and the criteria of designing small interfering RNA（si RNA）,aided byonline sequence design software of Ambion company.We applied BLAST to analyze homology of the designed sequences and choosed three si RNA to interfere PERK gene. And then we constructed eukaryotic expression vector（Lentiviral vectors） using cloning technology,further to identify its sequence. The lentiviral vector were packaged into a lentivirus with co-transfection.2.AR42J-LC3 cells were divided to five groups:control,negative control（NC）, PERK-sh RNA-1,PERK-sh RNA-2,PERK-sh RNA-3.Then we transfected these groups with Lentiviral and measured PERK m RNA and protein expression respectively by RT-PCR and Western blot.RESULTS:1. The Sequencing indicated that sh RNA eukaryotic expression vector targeting the gene of PERK（Lentiviral vectors）was constructed,which named PERK-sh RNA-1,2,3 respectively. 2.RT-PCR showedthat PERK m RNA expression in group NC and other groups were 1.07±0.07-fold、0.54±0.04-fold、0.37±0.01-fold、0.59±0.03-fold of control. The disparity between NC with Control group do not have a statistically significant（P> 0.05）, but the difference between the control group and other experimental groups was statistically significant（P< 0.05）. The inhibition of PERK-sh RNA-2 on PERK gene expression is the most obvious. Western blot suggested: the relative expression levels of the PERK protein（PERK/β-actin）in group control,NC and other groups were 0.69±0.03、0.64±0.04、0.41±0.01、0.21±0.02、0.38±0.01 respectively. The difference between NC group and the Control group was not statistically significant（P> 0.05）,but the difference between the control group and other experimental groups was statistically significant（P< 0.05）. The inhibition of PERK-sh RNA-2 on PERK gene protein expression is the most obvious,which is consistent with the result of RT-PCR.CONCLUSION: shRNA eukaryotic expression vector targeting the gene of PERK（Lentiviral vectors）was constructed successfully, and a AR42J-LC3 line targeting inPERK gene was built stably, which provides a new model for further researching the relationship between acute pancreatic inflammation, reticulum stress and autophagy.PART THREE The influence of endoplastic reticulum stress on autophagy in caerulein-induced acute pancreatitisObjective:to observe the alteration of autophagy in vitro model of acute pancreatitis afer the endoplastic reticulum stress（ERS） was effected by PERK targeted inhibitionMethod:We divided the experiments to six groups: AR42J-LC3（control A）, AR42JLC3-sh PERK（control B）, AR42J-LC3+ caerulein, AR42J-LC3-sh PERK+ caerulein,AR42J-LC3+ aerulein+ thapsigargin, AR42J-LC3-sh PERK+ caerulein+thapsigargin. We dealed the model of ERS with thapsigargin（TG） for 8 hours, and dealed groups about caerulein（CAE） with caerulein for 8 hours.The levels of autophagy related gene such as LC3,Beclin1 m RNA and their protein expression were detected by RT-PCR and Western blot.We also applied confocal microscopy to observe the c hange of autophagy.Result: RT-PCR and Western blot results indicated down-regulation of PERK can decrease the convertion of LC3-I to LC3-II and Beclin1 expression.We can observe t he relative decrease of autophagy formation in pancreatitis under the ERS of PERK down-regulated through the confocal microscopy.Conclusion: We got further explanation on the view that PERK is involved in pancreatic autophagy formation under ERS.