The Regulatory Mechanism Of LAMP2 In The Location Of MRP2 On The Hepatocyte Canalicular Membrane

Cholestatic liver disease is caused by excessive depositing of bile in liver,which may involve both intrahepatic and extrahepatic bile ducts.And if a person whose ALP is beyond three-fold of the normal limit,and GTT is half as much again as its range of normal value,he or she can be diagnosed with the disease.And it can be diagnosed by two main indicators in clinic,one of them is ALP,and another is GGT.At present,the therapeutic methods of Cholestatic liver disease are all based on removing the pathogeny.Unfortunately,there are no effective drugs to cure these diseases which have unclear pathogenesis,such as PBC and PSC.So it is significant for us to further explore the exact mechanism of cholestasis.In previous works,we prepared a monoclonal antibody(m Ab1F9)by immunizing the mice with the cell membrane which is nearby the bile duct and obtained from the PBC patients.In addition,this antibody was identified as Lysosomal membrane proteins(LAMP2).We found that LAMP2 rise obviously in the blood of PBC patients,and the change of the protein can be used to estimate if UDCA is efficient to the patient who was treated with the drug for three months.At the same time,we observed that cholestasis appeared in the LAMP2 knockout rats,and the location of MRP2 also changed.All of these results remind us of the close relationship between LAMP2 and bile excretion.In order to illustrate how dose LAMP2 influence the excretion of bile and the location of MRP2,we did the following research.In the first place,to clarify whether the change of the location of the MRP2 is caused by lacking of LAMP2 or by the complications,we examined the orientation of MRP2 and other related in primary hepatocyte of mice.In the second place,we select Hep G2 cell line to probe the relationship between LAMP2 and MRP2.And the reason why we choose this cell line is that it can form canalicular membrane.In the end,gene editing techniques Cas-9 were used to knockout LAMP2 A and LAMP2 B respectively in Hep G2 cell line,then we can know the effect of the two subtype of the protein on MRP2 and its mechanism.【Objectives】 To deeply study the way that LAMP2 influence the excretion of bile in liver;to explore the mechanism of the location changing of MRP2 which have been caused by LAMP2.【Methods】 1.The primary liver cells were separated from mice by collagenase perfusion method,and then these cells were cultured in sandwich medium.And then,immunofluorescence technique was used to observe if the location of MRP2 have changed in LAMP2 knockout mice,compared with normal mice.To explicit the transfer function of MRP2,we used direct immunofluorescence test to examine the transport of CMFDA.By comparing the fluorescence intensity both in cell and the field nearby bile duct,we can know the transport of MRP2.2.In order to get a deeper understanding the relationship between LAMP2 and MRP2,we chose Hep G2 cell line to do the subsequent experiments since it can form bile duct in vitro.Firstly,Hep G2 cell line was transfected with sh-LAMP2 and then builted a stable transfected cell lines.QRT-PCR and Western blot were used to confirm the efficacy of transfection.Secondly,the shape and quantity of bile duct as well as the location of polar molecular were observed by immunofluorescence.Moreover,CD59 transcytosis can represent the transport pathway.Finally,to better understand the abnormal positioning of MRP2,we examined the co-colalization of MRP2 and EEA1 in Hep G2 by immunofluorescence method.3.Craspre gene editing techniques were used to knockout LAMP2 A and LAMP2 B respectively,and the efficacy of which were test by q RT-PCR and Western blot.In the end,we would know cause the abnormal position of MRP2.【Results】 1.The primary liver cells were cultivated in sandwich medium,and in which small bile duct began to take on gradually.The number and the stability of bile duct increased as the time went by,five days later we compared the amount and the shape of the duct which were obtained from normal group liver and knockout group liver separately.Although there was no obvious difference between the two groups,the location of MRP2 have changed evidently.Almost all MRP2 located at the membrane of bile duct in normal group,but they not only lie in the membrane of bile duct,but also diffused in cytoplasm in knockout group.We also found that CMFDA can not be expelled from liver cells lacking of LAMP2,which means that LAMP2 is very important for MRP2 to perform its transport function.2.Hep G2 was transfected with lentivirus to build a LAMP2 knockdown cell line.The cellular morphology in knocking down cell line was different from the negative control group,however the number of bile duct were same in two group.Similar to primary cell,MRP2 were mainly lies in cytoplasm in Hep G2 lacking of LAMP2.In addition,being short of LAMP2 can also change the location of MDR1 and BSEP,both of them are polar molecules and positioning in the membrane of bile duct by direct pathway which is similar to MRP2.Then we examined the translocation path of CD59,transported by indirect method,whose transportational process was interrupted by knocking down LAMP2.All these results illustrate that LAMP2 could influence the translocation of MRP2 by changing the transportation of some molecular.A large number of abnormally located MRP2 assembled in the early endosome,and this remind us that LAMP2 may have some effect on the integration of endosome which caused by the change of some molecular’s transportation.3.Western blot,q RT-PCR and PCR were used to ensure LAMP2 A and LAMP2 B were knockout in Hep G2,then after a series of experiments we found that both of the two subtype of LAMP2 could intrigue MRP2 location altered.【Conclusion】 1.LAMP2 can regulate the correct location and transport function of MRP2 in primary liver cells,and as a result of which the transportation of other molecular may be changed.2.The alteration of LAPM2 in cancer cell line Hep G2 can also cause the abnormal location of MRP2.And this phenomenon may be related to the endosome fusion in liver cell.3.We built two new Hep G2 cell lines,and they were knocked down LAMP2 A and LAMP2 B respectively.Both of them occurred the positioning alteration of MRP2 compared with normal cell lines.