| Objective Our study aims to explore a safe and effective method of mobilization of peripheral blood stem cells (PBSCs) without worsening liver damage on observing the changes of CD34+ cells expressed in the liver and peripheral blood, and the influence of liver biochemistry while recombinant human granulocyte-colony stimulating factor (rhG-CSF) was administrated to mobilize the CCl4-induced cirrhotic rats. We try to find a viable cell-tracker to label PBSCs effectively, and to investigate the efficient pathway of autologously transplanted peripheral blood stem cells developed in the cirrhotic rats' livers. Accordingly, we attempt to provide an evidence of the possibility on utilizing autologous peripheral blood stem cells transplantation to treat chronic liver disease. Methods Firstly, we established the cirrhotic rat model with 50% CCl4 solution, and confirmed it by serum biochemistry and histopathology. Then rhG-CSF was administrated subcutaneously for 5 days, whereas the control cirrhotic rats recept injection with natrium saline. Then we collected the peripheral blood to examine the quantity of CD34+ cells contained in mononuclear cells (MNC) by flow cytometry(FCM), and we studied the cells expressing CD34 antigen in rats' livers by immunofluorescence. At the same time, we examined liver function including alanine aminotransferase(ALT),aspartate aminotranferase (AST),total bilirubin(TBIL) and albumin(ALB) by serum biochemistry. Then we used PKH26 fluorescent dye to label peripheral blood mononuclear cells, and the labeling efficiency was analyzed by FCM. Meanwhile, we used phase-contrast fluorescent microscope to observe the configuration of the labelled cells. Finally we autologously transplanted 1×106 PKH26-labelled peripheral blood mononuclear cells to the cirrhotic livers through portal vein, lower pole of spleen and intrahepatic parenchyma injection. After 8 and 12 weeks, we gathered the right lobes of transplanted cirrhotic livers, and used fluorescent microscope to observe the location of the PKH26- labelled cells. Results When administrated with 50% CCl4 for 70 days, the rats developed liver cirrhosis confirmed by HE and Masson staining and serum biochemistry with ALT( 323.50±103.72 vs 36.50±5.24IU/L,P<0.01), AST( 702.83±190.54 vs 83.17±11.70 IU/L,P<0.01),ALB(21.35±1.20 vs 29.58±1.84g/L,P<0.01), and TBIL(12.87±2.01 vs2.78±0.43μmol/L,P<0.01). After mobilization of rhG-CSF at dose of 10μg/kg/day for 5days, the quantity of CD34 contained in mononuclear cells increased apparently, which was 9.5 folds than that of the control rats administered with natrium saline (4.08±1.38% vs 0.43±0.21%). In the livers of cirrhotic rats, whether mobilized or not, only vascular endothelial cells expressed CD34 antigen. When compared with the control mobilized cirrhotic rats, the serum liver function tests indicated that the administration of rhG-CSF did not worsen liver damage, including ALT (392.13±26.04 vs 378.63±24.42IU/L, P>0.05), AST (678.50±115.15 vs 691.25±135.59IU/L, P>0.05), ALB (20.69±0.94 vs 21.44±1.47g/L, P>0.05), TBIL (11.99±1.28 vs 11.90±1.72μmol/L, P>0.05). The labeling efficiency of peripheral blood mononuclear cells used PKH26 dye was 93.70±0.44% examined by FCM and the labelled cells presented red fluorescence observed by phase-contrast fluorescent microscope. Few cells presenting red fluorescence existed in the cirrhotic liver 8 and 12 weeks after transplantation of the PKH26-labelled peripheral blood mononuclear cells through portal vein, lower pole of spleen and intrahepatic parenchyma injection. Conclusions RhG-CSF mobilization can't increase the proliferation of liver-derived hepatic stem cells in the cirrhotic livers, but it can mobilize more bone marrow/hematopoietic progenitor cells-derived hepatic stem cells to peripheral blood and do not worsen the liver damage of cirrhotic rats. So it provides a possible cell biological method of using mobilized hepatic stem cells in peripheral blood to treat chronic liver disease. PKH26 dye is an efficient and convenient cell-track...
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