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Study on Peripheral Blood Monocytes Transplantation for the Treatment of Liver Cirrhosis

Author: YanLi
Tutor: FanDaiMing；HanYing
School: Fourth Military Medical University
Course: Internal Medicine
Keywords: Liver cirrhosis Bone marrow derived stem cells Peripheral blood monocytes Hepatocyte-like cells Transplantation
CLC: R575.2
Type: PhD thesis
Year: 2008
Downloads: 296
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Abstract

Decompensated liver cirrhosis is one of the most common end-stage liver diseases. Conventional medical treatment can only improve the clinical symptoms for such patients but can not reverse the liver dysfunction caused by decreasing number of functional hepatocytes. Currently, liver transplantation provides the only effective therapy for such patients. However, its widely used was limited by such factors: lack of donor organs, risk of rejection, operative damage, and high costs. Therefore, it is very important to find an alternative therapy for decompensated liver cirrhosis.Recently, there has been great progress in the study of the regeneration of stem cells, which provide new insights into therapies of liver diseases. Some studies have suggested that bone marrow-derived stem cells had the potential of differentiating into multiple lineages, such as the endoderm-derived hepatocytes. In 1999, Petersen discovered that the stem cells from rat bone marrow can differentiate into hepatic oval cells, and then into hepatocytes and biliary epithelial cells, which was a landmark experiment of stem cells in liver diseases. In recent years, bone marrow-derived stem cell transplantation for the decompensated liver cirrhosis has had significant progress, which indicated that bone marrow derived stem cells have the potential to differentiate into hepatocytes. This opens up new possibilities for the treatment of acute liver failure, end-stage liver disease and genetic metabolic liver diseases.In our clinical trial, 20 patients recruited with the approval of our ethics committee (according to criteria determined by Xijing hospital of the Fourth Military Medical University) have accepted peripheral blood monocytes transplantation, and the results showed that this protocol could improve the liver function in the patients who were suffering from decompensated liver cirrhosis. At present, the mechanism of the clinical efficacy of PBMCs transplantation is still unclear, and in the present study, we will explore this problem through experiments in vivo and in vitro.【Aims】(1) To study whether the PBMCs from the patients with decompensated liver cirrhosis could differentiate into hepatocyte-like cells in vitro.(2) To explore whether the PBMCs from the patients with decompensated liver cirrhosis could locate in the liver of nude mice by observing the human hepatocyte-marker expression. (3) To establish the animal model of liver cirrhosis and study the role of PBMCs in liver cirrhosis. (4) To compare the therapeutic effect between G-CSF mobilization and G-CSF combined with PBMCs transplantation, and then explore the mechanism.【Methods】(1) Recombinant human hepatocyte growth factor and epidermal growth factor- 4 were used to induce the PBMCs from patients with liver cirrhosis in vitro, and an inverted microscope was then used to observe the induced cell morphology. (2) Immunocytochemistry, PAS staining, RT– PCR, Western Blot analysis were used to identify the hepatocyte-markers induced from the PBMCs from the decompensated liver cirrhosis. (3) ELISA analysis was used to measure the hepatocyte-markers in the supernatants of the induced hepatocyte-like cells. (4) PKH26 was used to label the PBMCs in vitro and trace the cells in vivo. (5) PRINS technique was used to trace the transplanted cells by detecting the sex-determining gene (SRY) of humans and rats. (6) Immunohistochemistry, RT-PCR and Western Blot analysis were used to identify the human hepatocyte-markers expressed in the livers of nude mice that accepted the transplantation of human PBMCs. (7) To establish a liver cirrhosis model in the SD rats by intraperitoneally injecting them with 0.6% CCl4 olive oil, with a dosage of 0.3 ml/100g every three days for 16 weeks. (8)To analyze liver function of the cirrhotic rats at different points using automatic biochemical analyzer. (9) To observe liver pathological changes of the cirrhotic rats in different groups with HE staining. (10) The continuous variables were expressed as mean and standard deviation for normal distribution and median and quartile were shown for the asymmetric variables. Differences between the synthetic functions of the human normal hepaticcell line QZG and hepatocyte-like cells were tested by the Wilcoxon test for paired data (two-sided test). The comparative liver functions of the two groups were investigated by analysis of two sample t-tests. The kinetics of live functions in each group was compared by post hoc Student–Newman–Keuls (SNK) multicomparison tests. A P value of <0.05 was considered as significant in all analyses. Statistical analysis was done using the SPSS version 13.0 software. 【Results】1 PBMCs derived from the cirrhotic patients could be induced into hepatocyte-like cells in vitro.In this study, the mobilized PBMCs from the cirrhotic patients were separated by density gradient centrifugation, and then cultured with HGF and FGF-4 in vitro. Seven days later, the PBMCs appeared like hepatocyte cells with polygon and oval shapes among others. Moreover, the results of immunocytochemistry, RT-PCR and Western Blot also confirmed that the induced cells with a hepatocyte-like shape also expressed some hepatocyte markers, including CK18、ALB and CYP3A4. These results suggested that the PBMCs from the cirrhotic patients could be induced into hepatocyte-like cells in vitro.2 PBMCs derived from the cirrhotic patients could locate into the liver of nude mice with human hepatocyte marker expressionIt is reported by Wang that albumin-expressing hepatocyte-like cells could develop in the livers of immune-deficient mice that had received transplants of highly purified human hematopoietic stem cells. In this study, the mobilized PBMCs from the cirrhotic patients were transplanted into the nude mice that underwent acute liver injury to identify their location and differentiation. The tracing techniques of the PKH26 and SRY gene showed that the transplanted PBMCs could locate in the liver of the nude mice. Moreover, human hepatocyte markers were detected in the nude mice liver, including ALB and CK18. The results suggested that the PBMCs derived from liver cirrhosis could migrate and locate in the liver of nude mice and then convert into hepatocyte-like cells in the liver microenviroment. 3 Successfully established an animal model of liver cirrhosis, and observing the role of PBMCs in the cirrhotic liverThe levels of liver function in rats treated with CCl4 on the16th week were significantly damgaged when compared with those of the normal rats, and the liver cirrhosis was indicated by pathohistology. The female liver cirrhosis rats were divided into four groups, (C1、T1、C2、T2). Rats in group T1 underwent PBMCs transplantation from male rats, while rats in group C1 underwent PBS transplantation as control; Rats in group T2 underwent G-CSF mobilization, followed by transplantation of PBMCs, while rats in group C2 underwent G-CSF mobilization as control. Compared with the control group (group C1), the levels of serum ALT, AST and TBIL of the rats in T1 group were significantly decreased on the second week and second month after transplantation, while at the same time the levels of serum albumin were significantly increased (p<0.05);There were still have significant differences in the levels of serum ALT, AST and TBIL between group T2 and group C2 (p<0.05),while the levels of serum albumin showed no significantly between groupT2 and group C2 (p>0.05).On the second week and second month after PBMCs transplantation, the PKH26 labeled cells were found scattered in the livers of the rats that underwent PBMCs transplantation, while these were not detected in the control groups at the corresponding points. Moreover, the SRY gene was detected in the cirrhotic rats that underwent the transplantation of PBMCs derived from male rats, while it was not detected in the control groups. These results indicated that PBMCs from the male cirrhotic rats could locate in the liver of the female cirrhotic rats.After 6 months of PBMCs transplantation, the rats were sacrificed and the pathological results indicated that Similar pathological liver changes were observed in group T1 and C1, T2 and C2. While the changes of fibrosis in the rats of group T1 was significantly alleviated when controlled with group C1 ,and the semi-quantitative classification showed that a significant difference was observed between the two groups (P <0.05).Compared with group C2, no significantly alleviated fibrosis were observed in group T2, and the semi-quantitative classification also didn’t show a significant difference between the two groups (P >0.05).【Conclusions】In present study, it revealed that PBMCs derived from the cirrhotic patients could be not only differentiated into hepatocyte-like cells in vitro, but also could be located in the injured liver and converted into hepatocytes under the liver micro-environment. The animal model with liver cirrhosis further showed that bone marrow-derived PBMCs could locate in the cirrhotic liver and contribute to the repair of the liver damage. In addition, it was also found that G-CSF mobilization could improve the liver function by repairing the liver damage to a certain degree.

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