| Practice and studies have shown that the pathogenesis of acute pancreatitis (AP) was a pathophysiological process involved in complex and multi-factor. And its pathogenesis has not been fully elucidated. The theories of "Inflammatory mediators or cytokines" and "calcium overload" are the focus of attention in the recent years. What mechanism will play an important role in these theories? Our previous animal experiments showed that expression of ghrelin was increased in the rats’pancreatic tissue of AP, and its expression levels were increased with the severity of AP. Previous studies have verified that exogenous of ghrelin could reduce the release of inflammatory mediators and inflammatory factors in AP, furthermore, it could also increased calcium in pancreatic acinar cells. Because ghrelin is widely distributed and has a variety of biological functions, we hypothesize that endogenous ghrelin may play an important role in the development of AP, and whether its mechanism is similar to exogenous ghrelin? This article intends to study it. Part OnePancreatic Acinar Cell Model Building of Acute Pancreatitis in Vitro and Expression of Ghrelin in This Cell ModelObjective:Rats’pancreatic acinar cells AR42J were induced by caerulin (CAE) or lipopolysaccharide (LPS) in order to build the pancreatic acinar cell model of AP in vitro. Study the expression of ghrelin in AR42J cells as well as builted in AR42J cells induced by caerulin.Methods:AR42J cells were induced by different concentration of CAE (0,10-8,10-7,10-6mol/L)or LPS(0,1,10,100mg/L)for24hrs.Amylase(AMS) content in cell supernatant was measured by bottom objects enzymatic. Expression of NF-kB p65, phosphorylation IkB-α, Cavl.2, Cav1.3, Cav2.1, Cav2.2Cav3.1Cav3.2and ghrelin mRNA was determined by RT-PCR. Proteins expression of NF-kB p65, phosphorylation IkB-α, Cav1.2, Cav1.3and ghrelin were determined by Western Blot. Proteins expression of IL-1β, IL-6, TNF-α in cell culture supernatant were determined by Elisa.[Ca2+]i was determined by calcium fluorescence probe Fluo-4-am loading combined with confocal laser scanning microscope (CLSM).Results:1.There was no statistically significant difference with AMS between control group cells and cells induced by CAE or LPS(P>0.05).2. Compared to control group, expression of NF-kB p65, IkB-α, Cav1.2, Cav1.3, Cav2.1,Cav3.1,Cav3.2mRNA(F=9.259,15.370,19.189,14.735,42.459,23.000,16.63, P<0.05) and NF-kB p65, IkB-α, Cav1.2, Cav1.3proteins (F=7.325,11.341,7.862,42.616, P<0.05) were increased significantly. Comparison in each group, some factors were dose-dependently increased (NF-kB p65, Cav1.3, Cav2.1, Cav3.2mRNA and NF-kB p65, IkB-α, Cavl.2, Cavl.3proteins, P<0.05). There was no significant difference (F=2.121, P=0.138>0.05) in Cav2.2mRNA. There was no significant difference in mRNA expression of aboved genes between control group cells and cells induced by LPS(F=0.552,0.174,0.491,0.182,0.185,0.509,0.125,0.350, P>0.05).3. Proteins expression of IL-1β and IL-6were increased significantly in AR42J cells induced by CAE, which was a dose-dependent manner (F=102.185,349.787, P<0.05). However, there was no significant difference in proteins expression of IL-1β and IL-6in AR42J cells induced by LPS (F=1.720,0.514, P=0.203,0.679>0.05). Because of OD value of TNF-a was too low, the corresponding protein concentration can not be calculated.4. Compared to control group,[Ca2+]i was increased significantly in AR42J cells induced by CAE, which was a dose-dependent manner (F=21.939, P＜0.05).5. Expressions of ghrelin mRNA and protein were increased in AR42J cells induced by CAE, which was a dose-dependent manner (F=45.128,67.2358, P<0.05).Conclusions:1. There is no significant change of AMS in cell culture supernatant between normal AR42J cells and AR42J cells induced by CAE or LPS.2. CAE can enhance mRNA and protein levels of a variety of inflammatory cytokines and calcium channel, as well as [Ca2+]i in AR42J cells. It may indicate pancreatic acinar cell model of acute pancreatitis to be built successfully.3. LPS can not affect mRNA and protein levels of a variety of inflammatory cytokines and calcium channel in AR42J cells.4. High expression of ghrelin in AR42J cells. Compared with control group, ghrelin mRNA and protein expression levels are elevated in AR42J cells induced by CAE. Part TwoBuilding and Screen of Lentiviral Interference Vector of Ghrelin miRNA and Its Targeted Interference Effect in AR42J CellsObjective:To screen the effective sequence of ghrelin miRNA of rat in order to build lentiviral vector of ghrelin miRNA. After AR42J cells were transfected with the lentiviral vector of ghrelin miRNA, we observed interference effect of ghrelin gene in cells.Methods:l.Four interference plasmids were constructed: pcDNATM6.2-X207-1-3,pcDNATM6.2-X207-2-1,pcDNATM6.2-X207-3-1,pcDN ATM6.2-X207-4-1. Expression vector pCDNA3.1(+) was constructed; After HEK293cells were co-transfected with the interference vector and expression vector, expression of target gene was determined by qPCR so as to screen out the best interference vector X207-1-3. Target fragment was restructured from miRNA vector to the lentiviral vector pLenti6.3/v5DEST through BP recombination system and LR recombination system, and positive clones were screened out and sequenced. After293T cells were cotransfected with lentiviral vector pLenti6.3-X207-1and packaging plasmid, packing virus, collecting virus stoste, ultracentrifugation to concentrate, and determining the titer.2. AR42J cells were divided into5groups:blank control group, negative control group (MOI=100), ghrelin-miRNA group A (MOI=50), ghrelin-miRNA group B (MOI=80), ghrelin-miRNA C group (MOI=100). Expression of ghrelin mRNA and protein in each group was detected by RT-PCR and Western Blot.Results:1.The four miRNA sequences to ghrelin gene silencing effection were detected by qPCR and the result showed that pcDNA TM6.2-X207-1-3was the most significant silencing effected sequence (86%).2. To pack lentivirus with the most obvious silencing effected sequence (86%) and the viral titer was1×108TU/ml.3. Ghrelin miRNA could be transfected efficiently into AR42J cells. And the transfection efficiency was up to80%. The results of RT-PCR and Western Blot showed that the most significant interference effect of ghrelin mRNA and protein expression in AR42J cells transfected by ghrelin-miRNA C group (MOI=100)(P<0.05).Conclusions:We successfully built lentiviral vector of ghrelin-miRNA and packed high titers of virus, so as to lay the foundation for the subsequent experiments. The lentiviral vector of ghrelin-miRNA can effectively silence ghrelin gene in AR42J cells. Part ThreeEffects of Ghrelin Gene Silence on Inflammation Pathway and Calcium Channel in Pancreatic Acinar Cells of Acute PancreatitisObjective:Study the changes of the inflammation pathway and calcium channel by ghrelin gene targeting suppression in pancreatic acinar cell model of acute pancreatitis induced by caerulin.Method:1. AR42J cells were divided into four groups:blank control group, CAE group, negative control (NG)+CAE group and miRNA+CAE group.2. Expression of NF-κB p65, phosphorylation IκB-α, Cav1.2, Cav1.3, Cav2.1mRNA and proteins was determined by RT-PCR and Western Blot. Proteins expression of IL-1β, IL-6and TNF-αwere determined by Elisa.3.[Ca2+]i in AR42J cells of each group was determined by Calcium Crimson TM, AM calcium fluorescent probe loading combined with confocal laser scanning microscope.Results:Compared with CAE group and NC+CAE group, the following results of miRNA+CAE group showed that:expression of NF-KBp65and IkB-α mRNA (F=51.255,9.266, P<0.05) and proteins (F=7.533,18.876, P<0.05) was elevated; proteins expression of IL-1β, IL-6in cell culture supernatant were elevated (F=334.554,336.462, P<0.05); expression of Cav1.2, Cav1.3, Cav2.1mRNA (F=37.208,55.651,42.640, P<0.05) and Cav1.2, Cavl.3proteins (F=6.894,64.239, P<0.05) were significantly decreased;[Ca2+]i in cells was significantly decreased (F=61.358, P<0.05).Conclusions:Targeting suppression of ghrelin gene in pancreatic acinar cells of acute pancreatitis can up-regulate the expression of inflammatory cytokines, as well as can reduce calcium overload in cells. Part FourAnalysing the expression profiles characteristic of ghrelin gene silencing gene in pancreatic acinar cells of AP with whole genome microarrayObjective:Screen out differentially expressed genes of targeting suppression of ghrelin gene in pancreatic acinar cells of acute pancreatitis. And further analysis of the relevant signal pathway was employed to explore the mechanism of molecular biology. Method:1. AR42J cells were divided into two groups:miRNA+CAE group and NC+CAE group.2. AR42J cells were collected to extract RNA after intervention of lentivirus and CAE. RNA quality was assessed. Synthesize total RNA into ds-DNA and mark it which was hybridized with NimbleGen rat whole genome microarray. Then, we scanned the microarray by Axon GenePix4000B gene chip scanner.3. All gene-level files were input Agilent GeneSpring GX11.5.1software to clustering classify, and further GO analysis and pathway analysis were worked.Results:1. There are2938genes of differentially expression including more than tow times of up-regulation (1435) and down-regulation (1503).2. The results of GO analysis showed that the differentially expressed genes which were associated with various metabolic processes appeared most frequently in the kind of biological process, followed by biological regulation, evolutionary process, regulation of cell process. In the kind of cellular component, the differentially expressed genes of the most significant change mainly located in the cells, nucleus, membrane type organelles and other cellular components. In the kind of molecular function, the most amount of differentially expression genes was associated with combination, including protein binding, metal ion binding, cation binding, nucleotide binding and small molecules binding.3. The results of pathway analysis showed that31signaling pathways involved in up-regulated genes and30signaling pathways involved in down-regulated genes.4. There are60differentially expressed genes involved in CAMP/Ca2+signal pathway and199differentially expressed genes involved in inflammation pathway.Conclusions:Through the results of gene chip, pathway and GO analysis, we preliminarily speculate that the mechanism of ghrelin on pancreatic acinar cells of acute pancreatitis may be related with metabolic processes in cells and binding of ion, protein, etc. And the related pathways of this mechanism may include MAPK signal pathway, cell adhesion molecules signal pathway, focal adhesion, Wnt signal pathway and p53signal pathway, etc. |
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