Study Of Bone Marrow Progenitor Cells Of Liver Sinusoidal Endothelial Promote The Fast Liver Regeneration After The First Step Of Associated Liver Partition And Portal Vein Ligation For Staged Hepectomy

objective : Associated Liver Partition and Portal vein ligation for Staged Hepectomy(ALPPS)could stimulate liver regeneration greatly,however,the mechanism is still unclear.Meanwhile,bone marrow derived liver sinusoidal endothelial progenitor cells(BM SPCs)could promote liver proliferation by secreting hepatic growth factor,which is regulate by vascular endothelial growth factor(VEGF).Therefore,we assume that BM SPCs would be involved in liver regeneration after the first step of ALPPS.In our study,we first build a stable model of ALPPS in rat and using liver remnant to body weight(FLR/BW),liver biochemical determinations and histopathology to test our model.Secondly,immunofluorescence,PCR test and WB test were used to figure out the recruitment of BM SPCs in liver and the function of BM SPCs in the liver regeneration after ALPPS.The first step: establishment of ALPPS model in ratMethod: 90 SD rats were randomly distributed to ALPPS group(n =30),portal vein ligation(PVL)group(n = 30)and Sham group(n=30);rats were scarified at day 1,day 2 day 4 and day 7.ALPPS group: 80%portal vein ligation and liver partition;PVL group: 80% portal vein ligation;Sham group: only finding portal vein and then close abdomen.All rats in every groups were scarified at each same time point.Blood and liver tissues were collected to run tests as follow:(1)the rate of right middle lobe weight to body weight;(2)hematoxylin-eosin staining to find out the extent of liver damage;(3)immunohistochemically staining to find out the extent of liver proliferation by the Ki-67 positive cells;(4)the level of ALT,AST,TBil.Result:(1)ALPPS group showed a higher growth rate comparing with PVL group;(2)ALPPS group showed a more severe liver damage and more Ki-67 positive cells;(3)The level ALS,AST and TBIL increased dramatically at day 1(p<0.05),and backed to normal level at day 3.The second step: finding out the function of BM SPCs at liver regeneration after ALPPSObjective: finding out the function of BM SPCs at liver regeneration after ALPPSMethod: using immunofluorescence to test surface antigen of BM SPCs(CD31,CD45,CD133);using PCR test to figure out the level of mRNA of HGF,VEGF in liver tissue;WB test to determine the level of HGF and VEGF.The above results are analyzed by SPSS17.0 statistical software.Result:(1)the test of immunofluorescence tells us that there were much more CD133,CD31 and CD45 positive cells,which are the surface antigen of BM SPCs,in the ALPPS group than in the PVL group and in the Sham group the CD133,CD31 and CD45 remain negative.Besides that,there were more CD133,CD31 and CD45 positive cells in the ligated side of ALPPS group than they in the remnant side of ALPPS at any time point after the operation;(2)Western Blot test shows that in the ligated side of ALPPS group,the protein level of HGF continuously increased after the surgeon while in the other side the protein level of HGF decreases regularly after the operation,p<0.05;As for the level of VEGF protein,in the remnant side of ALPPS,it decreases at the first two days after the operation and started to increase at day 3.In the day 7 the level of VEGF was even higher than it in the day 1.While in the ligated side,the level of VEGF continuously increases at the first two days after the operation,and it reaches the top at day 2.Then the level of VEGF starts to reduce.The extent of protein of VEGF in the ligated side of ALPPS is always higher than it in the remnant side of ALPPS except day1 and day 7,p<0.05.(3)PCR test shows that in the remnant side of ALPPS,the level of mRNA of persistently reduces after the operation ofALPPS,while in the ligated side,the mRNA level of VEGF increases immediately after the first step of ALPPS and it reached the crest at day 2,then the level of mRNA of VEGF in the ligated side start to decrease after the second day of the two steps surgeon,p<0.05.As for the mRNA of HGF,in the remnant side,it immediately reduces after the operation,while in the ligated side,the level of mRNA of HGF persistently increases after the first step of ALPPS,p<0.05.(4)Comparing with the PVL group,the WB test shows the level of protein of HGF and VEGF are obviously much higher in the ALPPS group.And there are no striking changes in the level of protein of HGF and VEGF in the Sham group,p<0.05.Conclusion: 1: establish rat ALPPS model successfully;2: BM SPCs could be recruited to liver and then secreting high level HGF after ALPPS;3: VEGF could regulate the process of recruitment of BM SPCs to be involved in liver regeneration.