The Expression Of Recombinant Adeno-associated Virus IκBαM In Mesenchymal Stem Cells And It’s Regulation Effect Of Pancreatic Stellate Cells In Vitro

Background and ObjectiveChronic pancreatitis（CP） is caused by different kinds of reasons to the essence and duct ofthe pancreatic irreversible chronic inflammation, its slow development of final can causepancreatic lesions necrosis, fibrosis, gland bubble and islet cell atrophy disappear, lead to astructure destroy the pancreas. CP typical symptoms for pancreas fibrosis, acinar cellsshrinking and the pancreas fibrosis is one of the major features of large fiber cell hyperplasiaand extracellular matrix precipitation. In pancreatic fibrosis, pancreatic stellate cells（PSC） inpancreatic fibrosis is the main effect cells. PSC still and activation is divided into twophenotype.Not damage in pancreatic tissue, PSC in a quiescent state, pancreatic damage, in all kinds ofstimulating factor that under the effect of PSC activation. Therefore, inhibit the activation andinduce the PSC apoptosis of pancreatic fibrosis to become possible.Mesenchymal stem cells（MSCs） from the bone marrow support structure is a group of adultstem cells, it has the update, multiplex differentiation skills and strong ability of proliferation,is the important seed cells for cell therapy and gene engineering. MSCs can secrete a varietyof cell factors, such as interleukin（IL）, stem cell factors, transformation growth factor, tumornecrosis factor（TNF）, liver cell growth factors and nerve growth factor etc. Not only caninhibit the pancreas inflammation, still can prevent pancreas produce fibrosis, reduce theoccurrence of fibrosis. IκBα the intimacy competency scale mutation forms IκBαM is able toand the NF-κB happen not reversible combined with three together to form the body withoutactive of complex forms exist in the cytoplasm. Make the NF-κB impose the resting state, andso to restrain inflammatory reaction and promote cell apoptosis role. Therefore, thisexperiment through the transfection into IκBαM gene MSCs and trained PSC indirectly,observation the MSCs for PSC have inhibition and discuss the mechanism and limitinflammation factors cause whether inflammation and inhibit the NF-κB activity, thus inhibiting PSC activation and promote its apoptosis related, also in vivo tests provide theoryfoundation. This study was divided into two parts.Materials and MethodsPart Ⅰ The expression of recombinant adeno-associated virus IκBαM in mesenchymalstem cellsThe proliferation of MSCs according to choose keeping5×10⁴cell/well vaccination into theboard, eight holes, among them2hole for the cell count with.37℃,5%CO₂incubatortraining12h. Observe whether normal cells, count cells. With the DMED serum-free mediumwill cell line twice. According to the MOI value （v.g/cell） respectively for2×10⁴,1×10⁵,5×10⁵calculate necessary to join with green fluorescent protein rAAV-IκBαM virus. Willneed to join in a test tube rAAV-IκBαM virus with and without the serum DMEM mediummix. The rAAV-IκBαM virus with and without the serum DMEM mixture to join each holecells separately, each hole with200μl cells.37℃,5%CO₂incubator training5⁶h, add in1ml/hole containing10%of bovine serum DMEM medium tyres. Continue to37℃,5%CO₂incubator training. In48hours after, the fluorescence microscope whether MSCs a greenfluorescent and Western blot method to detect IκBαM expression.PartⅡ In vitro regulation effect of mesenchymal stem cells on pancreatic stellate cellsThe purified rAAV-MSCs and PSC were set up in TranswelI co-culture system. Theincubation density2×10⁴cells/well. MSCs was set up instead of rAAV-MSCs as negativecontrol. Pancreatic stellate cells cultured alone served as blank control group. The culture wasperformed for96hours. ELISA test in medium, IL-1, IL-6, TNF-α and PDGF concentrationand cells the NF-κBp65concentration. Apoptosis of PSC was determined by flow cytometry.Polymerase chain reaction（PCR） were used to assay the expression of apoptosis genes BNIP3in PSC.ResultsPart Ⅰ The expression of recombinant adeno-associated virus IκBαM in mesenchymalstem cells1rAAV-IκBαM infection MSCs cells after48h, in fluorescence microscope to GFPexpression. 2Western blot tests showed that rAAV-IκBαM transfection group have IκBαM strongpositive strip, rAAV-GFP transfection group and not transfection group for weak positivestrip. rAAV-IκBαM transfection group IκBαM expression was significantly higher than therAAV-GFP transfection group and control group.PartⅡ In vitro regulation effect of mesenchymal stem cells on pancreatic stellate cells1In the three groups of medium, compared with the group C, group A and B IL-1, IL-6,TNF-α, PDGF concentration decreased, the difference was statistically significant （P﹤0.05）;Group A and B of differences between was statistically insignificant （P>0.05）.2In the three groups of medium, compared with the group B and C, group A NF-κBp65concentration decreased, the difference was statistically significant （P﹤0.05）; Group B and Cof differences between was statistically insignificant （P>0.05）.Conclusion1rAAV-IκBαM could be amplified in293cell and effectively infect to target cells MSCs,and could be expressed stably in cells, and the growth of MSCs to insignificant effect.2rAAV-IκBαM-MSCs can reduce the in vitro PSC cultures solution inflammation factorsIL-1, IL-6, TNF-α and PDGF level, and can restrain the NF-κB transcription activity. As aninflammatory reaction and promote cell apoptosis of the dual role.