| Objective:Autophagy is a highly conservative degration process of the materials in cells.Intracellular materials are transferred into lysosome to recycle by autophagosome,w hich is a two-layer membrane structure formed by cell membrane.The role of auto phagy in acute pancreatitis pathology is one of the most popular things recently.A utophagy related gene Beclin1 is an important molecule regulating the beginning of autophagy,Our research firstly aimed to knockdown the expression of Beclin1 i n AR42 J by shRNA technology.We knew the differences of proliferation,apoptosis,autophagy and the expression of chymotrypsin among them by contrasting the pri mitive and knockdown cells.Furthermore,we explored the function of Beclin1 in caerulein induced AR42 J acute pancreatitis model in the aspect of inflammatory fa ctors,apoptosis,autophagy and ultramicrostructure.Method:1.Interference plasmids coding mRNA against Beclin1 were constructed and transfected into AR42 J of rat.The expression of Beclin1 mRNA were detected by RT-PCR.And then,we choosed the most efficient silencing plasmid and packaged them into lentiviral particles.AR42 J were transduced with the particles and screened by puromycin.AR42Jbeclin1-/-were defined throughout RT-PCR and western blot.2.We used CCK8 kit to know the influence of Beclin1 on proliferation of A R42 J by drawing their growth curves.We used flow cytometry to know the influe nce of Beclin1 on apoptosis of AR42 J.We used transmission electron microscope to know the influence of Beclin1 on autophagy of AR42 J by observing their ultra microstructure.We used immunofluorescence technique to know the influence of B eclin1 on expression of chymotrypsin in AR42 J.3.We explored the influence of Beclin1 on inflammatory factors levels in cae rulein induced AR42 J acute pancreatitis model by using ELISA kits to detect infl ammatory factors including IL-1??IL-6?TNF-? at different time points?0h,1h,4h,6h,8h,12 h,24h,36 h and 48h?.We explored the influence of Beclin1 on apoptosis,auto phagy in caerulein induced AR42 J acute pancreatitis model by using western blot to detect BCL2 and LC3 at different time points?0h,1h,4h,6h,8h,12 h,24h,36h?.We explored the influence of Beclin1 on ultramicrostructure in caerulein induced AR42 J acute pancreatitis model by using transmission electron microscope.Results:1.Interference plasmids were separated by agarose gel electrophoresis and we have sequenced them as our expectation.The Beclin1 mRNA expression sciencing rates of AR42 J transfected with p-sh-Beclin1-1,p-sh-Beclin1-2,p-sh-Beclin1-3,p-shRNA-NC were?17.8±4.0?%,?30.6±2.8?%,?45.8±7.7?%,?7.0±11.8?%,respectively.The sciencing rates of three interference plasmids were different from the primary AR42 J statistically?P<0.05?.The difference between AR42 J transfected with p-shRNA-NC and primary AR42 J was not statistical significantly?P>0.05?.We choosed p-sh-Beclin1-3 to package into lentivirals,transduce with AR42 J,and screened by puromycin.Consistent with the inhibition rate of Beclin1 protein?87.9±2.8?%,The sciencing rate of Beclin1 mRNA in AR42 J transduced with lentiviral particles was?86.1±1.2?% compared with primary AR42 J.The difference between them was significant statistically?P<0.01?.2.The difference between the primitive cell and the AR42Jbeclin1-/-wasn't significant statistically?P>0.05?.While Beclin1 gene could inhibit apoptosis of AR42 J.The number of autophagic vacuoles in AR42Jbeclin1-/-was less than that in AR42 J.While Beclin1 gene had no influence on the expression of chymotrypsin in AR42 J.3.The result of ELISA showed the level of inflammatory factors escalated in the beginning of acute pancreatitis,reached the first peak at 6h/8h,then decreased,and then increased again,reached the second peak at 36h/48 h.When Beclin1 gene was knockdown,the level of IL-1? decreased,that of IL-6 kept invariable,that of TNF-? increased at the same points.The result of western blot indicated that targeting interference Beclin1 gene made protein BCL2 decrease while LC3 increase at different points in caerulein induced AR42 J acute pancreatitis model.When caerulein worked at the concentration of 1×10-8mol/L for 4 hours,we could observe acute pancreatitis in AR42 J,while there was not inflammation in AR42Jbeclin1-/-,and less autophagic vacuole than that in AR42 J.Conclusion:we have already built the stable AR42 J line silencing Beclin1 successfully.Beclin1 gene takes part in autophagosome formation,inhibits apoptosis,has no effect on proliferation and the chymotrypsin expression of AR42 J cell.The level of inflammatory factors in caerulein induced AR42 J acute pancreatitis model fluctuate according to the time.Targeting interference Beclin1 gene has an impact on inflammation,apoptosis,autophagy and works as a protective factor in caerulein induced AR42 J acute pancreatitis model.We provide a new cell model for profound research of relationship between Beclin1 and pancreatitis. |
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