- Email: [email protected]
Liver Progenitor Cells Activated After 30% Small-for-Size Liver Transplantation in Rats: A Preliminary Study
Liver Progenitor Cells Activated After 30% Small-for-Size Liver Transplantation in Rats: A Preliminary Study L. Mao, Y.-D. Qiu, S. Fang, Y.-F. Wu, H. Liu, and Y.-T. Ding ABSTRACT Aim. To explore whether liver progenitor cells were activated after 30% small-for-size liver transplantation in rats. Methods. 200 rats were arranged in three groups: 70% partial hepatectomy (PH), whole liver transplantation (WLT), and 30% liver transplantation (SLT) group. On days 1, 2, 3, 5, 7 after operation, 6 rats were sacrificed in each group at each time. One week survivals were analyzed; while liver injury and regeneration index were estimated by serum ALT AST, H&E staining and proliferating cell nuclear antigen index. The oval cell markers, including CD90 and OV6, were detected in liver sections by immunohistochemistry. Results. The 50% survival rate of the SLT group was significantly lower than those of the PH and the WLT groups. At each time after operation, the serum ALT and AST were much higher in the SLT group. Compared with the PH group on days 1, 2, and 3 postoperatively, the PCNA indices were lower among the SLT group. OV-6 positive and CD90 positive cells were detected in the SLT group from day 2 postoperatively. These progenitor cells were first dispersed in the liver but restricted to the periportal region over the following days. Conclusion. Liver progenitor cell activation after SLT may be related to the liver dysfunction caused by a small-for-size graft.
T
HE TECHNIQUES of partial liver transplantation, using either a cadaveric (split) or a living donor graft, expand the supply of organs and partially overcome the current shortage. But these benefits are limited in adult recipients when the graft volume is small. Present data document that a small graft negatively impacts the postoperative prognosis.1–2 As we know, the small liver graft needs to regenerate to enable normal function after undergoing serious injury after transplantation. The ability of liver to restore major tissue loss involves numerous interacting cells. Several studies have shown that bone marrow stem cells are activated and contribute to liver regeneration after partial liver transplantation.3– 4 But whether the liver progenitor cells, like oval cells are activated and where they are located are still unclear. The aim of present study was to investigate the course of liver dysfunction after small-for-size transplantation in rats and whether hepatic progenitor cells were activated during this process. MATERIALS AND METHODS Animals Inbred male Sprague-Dawley rats (220 –240 g) purchased from Model Animal Research Center of Nanjing University were housed
with free access to water and chow with constant 12-hour light-dark cycle environmental conditions. The rats were fasted 12 hours before the operation. All operations were performed under clean conditions. The studies met the Research Council’s guidelines for the care and use of laboratory animals.
Liver Transplantation Orthotopic liver transplantation was performed using the two-cuff method. Animals (150 rats) were divided into three groups: group 1 was the 70% partial hepatectomy (PH) group (30 rats); Group 2, whole orthotopic liver transplantation (WLT; 60 rats) and Group 3, 30% small liver transplantation (SLT) group (60 rats). In the SLT group, the donor underwent hepatectomy including left lateral and median lobes, followed by liver harvest. The small-size graft was then implanted orthotopically into the recipient. 6 rats were sacrificed at 1, 2, 3, 5, 7 days after operation in each group. At the time of sacrifice, blood was obtained from the inferior vena cava to From the Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital, Medical School of Nanjing University, China. Address reprint requests to Prof. Yi-Tao Ding, Affiliated Drum Tower Hospital, Medical School of Nanjing University, Department of Hepatobiliary Surgery, No. 321 Zhongshan Road, Nanjing, Jiangsu 210008, China. E-mail: [email protected]
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.03.133
Transplantation Proceedings, 40, 1635–1640 (2008)
1635
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MAO, QIU, FANG ET AL each step, the slides were washed with 10 mol/L PBS in 1% BSA. For each slide, positive and negative cells were counted in ten randomly selected fields under the light microscope using 200⫻ magnification.
Immunohistochemistry of CD90 Antigen Immunohistochemical detection was performed on frozen sections after 10 minutes fixation in acetone at room temperature. The 4-m-thick sections were then incubated in a 1:400 dilution of mouse anti-rat monoclonal CD90 antibody (BD Pharmingen,) at 4°C overnight. Thereafter, the slides were incubated in secondary antibody (DAKO) at 37°C for 30 minutes. The sections were counterstained with hematoxylin. At each step, slides were washed with 10 mol/L PBS in 1% BSA.
Immunohistoflowcytometry of OV-6 Antigen Fig 1. Survival curve of PH, WLT and SLT group. The survival of rats in SLT group was significantly lower compared with that of PH and WLT. estimate serum alanine transferase (ALT) and aspartate transferase (AST) activities. Portions of the liver fixed in 10% neutral buffered formalin for 18 to 24 hours were processed for light microscopy. The remainder of the liver was frozen in liquid nitrogen for storage at ⫺80°C.
Survival Study Ten samples in each group (50 rats total) were used for the survival study. Rat deaths within 24 hours of operation were considered to be technical failures; their data were excluded from further analysis.
Evaluation of Histologic Changes For histological analysis, paraffin-embedded 6-m-thick sections were stained with hematoxylin and eosin (H&E).
Evaluation of Proliferation Ability of Mature Hepatocytes For evaluation of hepatocyte proliferation, immunohistochemistry stains of proliferating cell nuclear antigen (PCNA) were performed with a commercial kit (Zymed, South San Francisco, Calif, USA). Briefly, paraffin sections were stained with antibodies at a concentration of 1:400. Thereafter, the slides were incubated with secondary antibody (DAKO Invasion System, Denmark) at 37°C for 30 minutes. The sections were counterstained with hematoxylin. After
Fig 2. Histology of the grafts on day 3 after operation. H&Estained (A) Liver section from PH group (B) Liver sections from WLT group. Note the normal histology of liver lobule. (C) Liver section from SLT group graft with focal and confluent small areas of hepatocytes necrosis in vicinity of central vein. (Original magnification: 100⫻.)
Immunohistoflowcytometry detection was performed on frozen sections after 10 minutes fixation in cold acetone. The 4-m-thick sections were incubated in a 10-g/mL dilution of anti-rat OV-6 antibody (R&D Systems Inc., Abingdon, UK) overnight at 4°C, followed by FITC-labeled rabbit anti-mouse IgG (1:100 dilution; DAKO) at 37°C in the dark for 30 minutes. Slides were observed with a Zeiss Axiovert fluorescence microscope (Carl Zeiss, Jena, Germany) at 490 nm.
Statistical Analysis All results were expressed as mean values ⫾ SD. Groups were compared using analysis of variance (ANOVA) plus StudentNewman-Keuls post-hoc test. Survival rates were assessed by the Kaplan-Meier method. A value of P ⬍ .05 was considered significant. Data analysis and statistical procedures were performed using SPSS software version 15.0 (SPSS Inc); all graphs were generated using Sigmaplot 10.0 (SPSS Inc).
RESULTS Survival Rates
Graft survivals are shown in Fig 1. In the SLT group, the rate of survival at 7 days was 50%. Most deaths happened during 2 to 4 postoperative days. In contrast, recipients in the WLT and PH groups survived permanently. Liver Injury
On day 3 liver sections from the PH and WLT groups showed normal morphology except for a few scattered areas
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Fig 3. Serum ALT and AST activity: (A) Serum ALT and (B) AST activities after operation (mean ⫾ SD) (n ⫽ 6). Vertical bars represent the SD from the mean. *P ⬍ .05 for SLT versus PH and WLT groups at the different time points. **P ⬍ .01 for SLT versus PH and WLT groups at the different time points.
of hepatocyte necrosis. In contrast, livers from the SLT showed loss of normal architecture with portal zone expansion, cholestasis, and leukocyte infiltration (Fig 2). Serum AST and ALT levels increased on day 1 and decreased on the following days among the SLT, PH, and WLT groups. But the values in SLT were significantly higher than those in the PH or WLT groups at various times (Fig 3). These findings suggested serious injury and liver dysfunction in the SLT group. Hepatocytes Proliferation
The PCNA indices are shown in Fig 4. Compared with the PH group, the livers in the SLT group showed decreasing proliferation rates from day 1 to day 3. The maximum PCNA index of hepatocytes was 54% for PH rats versus 41% for SLT rats (P ⬍ .05). These data suggested that
proliferation of mature hepatocytes in a small liver graft was decreased compared with PH. CD90 Positive Cells
Figure 5 shows the CD90 positive cells in the liver graft. There were no CD90 positive cells in WLT and PH groups (Figs 5A and 5B). But in the SLT group, many positive cells appeared from day 2 postoperatively (Figs 5C and 5D); this phenomenon persistent to the seventh say (Figs 5E and 5F). The difference was that the positive cells were dispersed into the liver on day 2 postoperatively, but decreased and mainly located in the periportal region on day 7. OV-6 Positive Cells
Figure 6 shows the OV-6 positive cells in the liver section of each group. There were nearly negative target cells in both the PH and WLT groups (Figs 6A and 6B), but many OV-6 positive cells in the sections of SLT from 2 day after operation (Fig 6C). On day 2 post operative, the OV-6 positive cells were dispersed with some arranged in ductlike structures at higher magnification (Fig 6D). But on day 7 post-operative, they were mainly located in the periportal region; there was nearly no target cells in other regions of the liver lobule (Figs 6E and 6F). DISCUSSION
Fig 4. Hepatocyte-proliferative activity of liver grafts after transplantation. Percentage of PCNA index is shown in each group (mean ⫾ SD). *P ⬍ .05 for SLT versus PH at different time points. **P ⬍ .01 for SLT versus PH at different time points.
Using a novel model of liver dysfunction, we showed that the 30% small liver graft underwent serious injury during the transplantation process. The graft was unable to function normally after transplantation, which was particularly serious between 2 to 3 postoperative days. During this period, the rats showed were weary and displayed ascites. The ALT and AST levels in serum were significantly higher than those of the PH and WLT group. The low survival rate at one week demonstrated the negative impact of a small graft volume. Our study on proliferative
1638
MAO, QIU, FANG ET AL
Fig 5. Immunohistoflowcytometrical staining of OV-6 positive cells. (A) PH group and (B) WLT group has no OV-6 positive cell on day 2 postoperation. But (C) SLT group shows many OV-6 positive cells dispersed into liver section on day 2. Fig (D) shows some positive cells arranged into a ductual-like structure in higher magnification. On day 7 postoperation in SLT group, Figs (E) and (F) show OV-6 positive cells were mainly located in periportal region. (Original magnification: A,B,C,E:100⫻; F:200⫻; D:400⫻).
ability showed the PCNA index in the SLT group to be decreased compared to the PH group from day 1 to day 3, demonstrating partially suppressed proliferation of mature hepatocytes. This finding is consistent with Zhizhong et al.5 We believe that the increased injury and suppressed proliferation in small-size grafts further decreases the functional liver mass and together leads to graft dysfunction. In rodents, OV-6 is a key surface marker of oval cells, which are considered to be a hepatic progenitor cell located in the canals of Hering.6 Farber7 first described oval cells as “small oval cells with scant lightly basophilic cytoplasm and pale blue-staining nuclei.” In subsequent studies,8 –12 many studies have shown that oval cells are activated when hepatocyte proliferation is impaired, although the actual mechanisms are largely unknown. In the classical oval
cell activation animal model (2-AAF combined with partial hepatectomy), when “oval cells” are activated to proliferate they give rise to duct-like structures originating from the periportal regions and spreading into the liver acinus. Similar manifestations have been discovered in human liver disease; it is termed “ductular reaction.”13 In this study, we discovered an abundant number of OV-6 positive cells in liver grafts after SLT; they were nearly negative in both PH and WLT groups. The immunofluorescence tests also demonstrated some OV-6 positive cells arranged in duct-like structures. These positive cells peaked at days 2 to 3 and decreased over the following days. They were dispersed into the liver at first and later located restrictedly to the periportal region. We believe that the mitogenic stimulus provided by the loss of tissue (small graft) and suppression of
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Fig 6. Immunohistochemistry staining of C90 positive cells. (A) PH group and (B) WLT group has no CD90 positive cell on day 2 postoperation. But (C) and (D) show many CD90 positive cells dispersed into liver section on day 2 in SLT group. On day 7 postoperation in SLT group, Figs (E) and (F) show the number of CD90 positive cells was decreased and they were mainly located in periportal region. (Original magnification: A,B,C,E:100⫻; F:200⫻; D: 400⫻.)
hepatocyte proliferation caused oval cell activation after SLT. Recent progress in bone marrow stem cell (BMMC biology) has revealed that these elements can be directed to differentiate into hepatocyte-like cells in vitro.14 –18 This manifestation has also been discovered in clinical research.19 In further research some interesting studies have demonstrated a relationship between bone marrow stem cells and hepatic progenitor cells.20 –22 Bone marrow stem cell markers, like CD90 and CD34, have been observed on oval cells, suggesting that a source of liver progenitor cells. In this study, we investigated many CD90 positive cells in the liver sections of the SLT group. Further, we observed that the mode of CD90 positive cells appearance was
similar to that of OV-6 positive cells. We believe that this is a hint that liver progenitor cells may come from bone marrow stem cells. The present study was unique in that we observed oval cells, which may also come from bone marrow stem cells to be activated after the liver dysfunction induced by a 30% small-for-size partial liver transplantation model in rats. Beside the serious liver injury and failure after SLT, we observed that the proliferative ability of mature hepatocyte decreased; oval cells responded to activation in this condition and bone marrow stem cells seemed to also participate in this process. The exact mechanisms of the suppressed hepatocyte proliferation and activation of hepatic progenitor cells after SLT represent foci for further study as well as
1640
the possibility of transplantation of stem cells to correct liver dysfunction after small-for-size liver transplantation.
REFERENCES 1. Kiuchi T, Kasahara M, Uryuhara K, et al: Impact of graft size mismatching on graft prognosis in liver transplantation from living donors. Transplantation 67:321, 1999 2. Sugawara Y, Makuuchi M, Takayama T, et al: Hashizume K. Small-for-size grafts in living-related liver transplantation. J Am Coll Surg 192:510, 2001 3. Liu F, Pan XB, Chen GD, et al: Hematopoietic stem cell mobilization after rat partial orthotopic liver transplantation. Transplant Proc 38:1603, 2006 4. Dirsch O, Chi H, Gu YL, et al: Influence of stem cell mobilization and liver regeneration on hepatic parenchymal chimerism in the rat. Transplantation 81:1695, 2006 5. Zhong Z, Schwabe RF, Kai Y, et al: Liver regeneration is suppressed in small-for-size liver grafts after transplantation: involvement of c-Jun N-terminal kinase, cyclin D1, and defective energy supply. Transplantation 82:241, 2006 6. Shafritz DA, Oertel M, Menthena A, et al: Dabeva, Liver stem cells and prospects for liver reconstitution by transplanted cells. Hepatology 43(2 Suppl 1):S89, 2006 7. Farber E: Similarities of the sequence of the early histological changes induced in the liver of the rat by ethionine, 2-acetylaminofluorene, and 3-methyl-4-dimethylaminoazobenzene. Cancer Res 16:142, 1956 8. Evarts RP, Nagy P, Marsden E, et al: A precursor-product relationship exists between oval cells and hepatocytes in rat liver. Carcinogenesis 8:1737, 1987 9. Evarts RP, Nagy P, Nakatsukasa H, et al: In vivo differentiation of rat liver oval cells into hepatocytes. Cancer Res 49:1541, 1989
MAO, QIU, FANG ET AL 10. Nagy P, Bisgaard HC, Thorgeirsson SS: Expression of hepatic transcription factors during liver development and oval cell differentiation. J Cell Biol 126:223, 1994 11. Petersen BE, Zajac VF, Michalopoulos GK: Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats. Hepatology 27:1030, 1998 12. Paku S, Schnur J, Nagy P, et al: Origin and structural evolution of the early proliferating oval cells in rat liver. Am J Pathol 158:1313, 2001 13. Roskams TA, Theise ND, Balabaud C, et al: Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers. Hepatology 39:1739, 2004 14. Theise ND, Badve S, Saxena R: Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 31:235, 2000 15. Alison MR, Poulsom R, Jeffery R, et al: Hepatocytes from non-hepatic adult stem cells. Nature 406:257, 2000 16. Lagasse E, Connors H, Al-Dhalimy M, et al: Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 6:1229, 2000 17. Fiegel HC, Lioznov MV, Cortes-Dericks L, et al: LiverSpecific gene expression in cultured human hematopoietic stem cells. Stem Cells 21:98, 2003 18. Krause DS, Theise ND, Collector MI, et al: Sharkis SJ. Multi-organ, multi-lineage engraftment by a single bone marrowderived stem cells. Cell 105:369, 2001 19. Theise ND, Nimmakayalu M, Gardner R, et al: Liver from bone marrow in humans. Hepatology 32:11, 2000 20. Petersen BE, Bowen WC, Patrene KD, et al: Bone marrow as a potential source of hepatic oval cells. Science 284:1168, 1999 21. Petersen BE, Goff JP, Greenberger JS, et al: Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in rat. Hepatology 27:433, 1998 22. Petersen B, Grossbard B, Hatch H, et al: Mouse A6 positive hepatic oval cells also express several hematopoietic stem cell markers. Hepatology 37:632, 2003
T
HE TECHNIQUES of partial liver transplantation, using either a cadaveric (split) or a living donor graft, expand the supply of organs and partially overcome the current shortage. But these benefits are limited in adult recipients when the graft volume is small. Present data document that a small graft negatively impacts the postoperative prognosis.1–2 As we know, the small liver graft needs to regenerate to enable normal function after undergoing serious injury after transplantation. The ability of liver to restore major tissue loss involves numerous interacting cells. Several studies have shown that bone marrow stem cells are activated and contribute to liver regeneration after partial liver transplantation.3– 4 But whether the liver progenitor cells, like oval cells are activated and where they are located are still unclear. The aim of present study was to investigate the course of liver dysfunction after small-for-size transplantation in rats and whether hepatic progenitor cells were activated during this process. MATERIALS AND METHODS Animals Inbred male Sprague-Dawley rats (220 –240 g) purchased from Model Animal Research Center of Nanjing University were housed
with free access to water and chow with constant 12-hour light-dark cycle environmental conditions. The rats were fasted 12 hours before the operation. All operations were performed under clean conditions. The studies met the Research Council’s guidelines for the care and use of laboratory animals.
Liver Transplantation Orthotopic liver transplantation was performed using the two-cuff method. Animals (150 rats) were divided into three groups: group 1 was the 70% partial hepatectomy (PH) group (30 rats); Group 2, whole orthotopic liver transplantation (WLT; 60 rats) and Group 3, 30% small liver transplantation (SLT) group (60 rats). In the SLT group, the donor underwent hepatectomy including left lateral and median lobes, followed by liver harvest. The small-size graft was then implanted orthotopically into the recipient. 6 rats were sacrificed at 1, 2, 3, 5, 7 days after operation in each group. At the time of sacrifice, blood was obtained from the inferior vena cava to From the Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital, Medical School of Nanjing University, China. Address reprint requests to Prof. Yi-Tao Ding, Affiliated Drum Tower Hospital, Medical School of Nanjing University, Department of Hepatobiliary Surgery, No. 321 Zhongshan Road, Nanjing, Jiangsu 210008, China. E-mail: [email protected]
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.03.133
Transplantation Proceedings, 40, 1635–1640 (2008)
1635
1636
MAO, QIU, FANG ET AL each step, the slides were washed with 10 mol/L PBS in 1% BSA. For each slide, positive and negative cells were counted in ten randomly selected fields under the light microscope using 200⫻ magnification.
Immunohistochemistry of CD90 Antigen Immunohistochemical detection was performed on frozen sections after 10 minutes fixation in acetone at room temperature. The 4-m-thick sections were then incubated in a 1:400 dilution of mouse anti-rat monoclonal CD90 antibody (BD Pharmingen,) at 4°C overnight. Thereafter, the slides were incubated in secondary antibody (DAKO) at 37°C for 30 minutes. The sections were counterstained with hematoxylin. At each step, slides were washed with 10 mol/L PBS in 1% BSA.
Immunohistoflowcytometry of OV-6 Antigen Fig 1. Survival curve of PH, WLT and SLT group. The survival of rats in SLT group was significantly lower compared with that of PH and WLT. estimate serum alanine transferase (ALT) and aspartate transferase (AST) activities. Portions of the liver fixed in 10% neutral buffered formalin for 18 to 24 hours were processed for light microscopy. The remainder of the liver was frozen in liquid nitrogen for storage at ⫺80°C.
Survival Study Ten samples in each group (50 rats total) were used for the survival study. Rat deaths within 24 hours of operation were considered to be technical failures; their data were excluded from further analysis.
Evaluation of Histologic Changes For histological analysis, paraffin-embedded 6-m-thick sections were stained with hematoxylin and eosin (H&E).
Evaluation of Proliferation Ability of Mature Hepatocytes For evaluation of hepatocyte proliferation, immunohistochemistry stains of proliferating cell nuclear antigen (PCNA) were performed with a commercial kit (Zymed, South San Francisco, Calif, USA). Briefly, paraffin sections were stained with antibodies at a concentration of 1:400. Thereafter, the slides were incubated with secondary antibody (DAKO Invasion System, Denmark) at 37°C for 30 minutes. The sections were counterstained with hematoxylin. After
Fig 2. Histology of the grafts on day 3 after operation. H&Estained (A) Liver section from PH group (B) Liver sections from WLT group. Note the normal histology of liver lobule. (C) Liver section from SLT group graft with focal and confluent small areas of hepatocytes necrosis in vicinity of central vein. (Original magnification: 100⫻.)
Immunohistoflowcytometry detection was performed on frozen sections after 10 minutes fixation in cold acetone. The 4-m-thick sections were incubated in a 10-g/mL dilution of anti-rat OV-6 antibody (R&D Systems Inc., Abingdon, UK) overnight at 4°C, followed by FITC-labeled rabbit anti-mouse IgG (1:100 dilution; DAKO) at 37°C in the dark for 30 minutes. Slides were observed with a Zeiss Axiovert fluorescence microscope (Carl Zeiss, Jena, Germany) at 490 nm.
Statistical Analysis All results were expressed as mean values ⫾ SD. Groups were compared using analysis of variance (ANOVA) plus StudentNewman-Keuls post-hoc test. Survival rates were assessed by the Kaplan-Meier method. A value of P ⬍ .05 was considered significant. Data analysis and statistical procedures were performed using SPSS software version 15.0 (SPSS Inc); all graphs were generated using Sigmaplot 10.0 (SPSS Inc).
RESULTS Survival Rates
Graft survivals are shown in Fig 1. In the SLT group, the rate of survival at 7 days was 50%. Most deaths happened during 2 to 4 postoperative days. In contrast, recipients in the WLT and PH groups survived permanently. Liver Injury
On day 3 liver sections from the PH and WLT groups showed normal morphology except for a few scattered areas
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Fig 3. Serum ALT and AST activity: (A) Serum ALT and (B) AST activities after operation (mean ⫾ SD) (n ⫽ 6). Vertical bars represent the SD from the mean. *P ⬍ .05 for SLT versus PH and WLT groups at the different time points. **P ⬍ .01 for SLT versus PH and WLT groups at the different time points.
of hepatocyte necrosis. In contrast, livers from the SLT showed loss of normal architecture with portal zone expansion, cholestasis, and leukocyte infiltration (Fig 2). Serum AST and ALT levels increased on day 1 and decreased on the following days among the SLT, PH, and WLT groups. But the values in SLT were significantly higher than those in the PH or WLT groups at various times (Fig 3). These findings suggested serious injury and liver dysfunction in the SLT group. Hepatocytes Proliferation
The PCNA indices are shown in Fig 4. Compared with the PH group, the livers in the SLT group showed decreasing proliferation rates from day 1 to day 3. The maximum PCNA index of hepatocytes was 54% for PH rats versus 41% for SLT rats (P ⬍ .05). These data suggested that
proliferation of mature hepatocytes in a small liver graft was decreased compared with PH. CD90 Positive Cells
Figure 5 shows the CD90 positive cells in the liver graft. There were no CD90 positive cells in WLT and PH groups (Figs 5A and 5B). But in the SLT group, many positive cells appeared from day 2 postoperatively (Figs 5C and 5D); this phenomenon persistent to the seventh say (Figs 5E and 5F). The difference was that the positive cells were dispersed into the liver on day 2 postoperatively, but decreased and mainly located in the periportal region on day 7. OV-6 Positive Cells
Figure 6 shows the OV-6 positive cells in the liver section of each group. There were nearly negative target cells in both the PH and WLT groups (Figs 6A and 6B), but many OV-6 positive cells in the sections of SLT from 2 day after operation (Fig 6C). On day 2 post operative, the OV-6 positive cells were dispersed with some arranged in ductlike structures at higher magnification (Fig 6D). But on day 7 post-operative, they were mainly located in the periportal region; there was nearly no target cells in other regions of the liver lobule (Figs 6E and 6F). DISCUSSION
Fig 4. Hepatocyte-proliferative activity of liver grafts after transplantation. Percentage of PCNA index is shown in each group (mean ⫾ SD). *P ⬍ .05 for SLT versus PH at different time points. **P ⬍ .01 for SLT versus PH at different time points.
Using a novel model of liver dysfunction, we showed that the 30% small liver graft underwent serious injury during the transplantation process. The graft was unable to function normally after transplantation, which was particularly serious between 2 to 3 postoperative days. During this period, the rats showed were weary and displayed ascites. The ALT and AST levels in serum were significantly higher than those of the PH and WLT group. The low survival rate at one week demonstrated the negative impact of a small graft volume. Our study on proliferative
1638
MAO, QIU, FANG ET AL
Fig 5. Immunohistoflowcytometrical staining of OV-6 positive cells. (A) PH group and (B) WLT group has no OV-6 positive cell on day 2 postoperation. But (C) SLT group shows many OV-6 positive cells dispersed into liver section on day 2. Fig (D) shows some positive cells arranged into a ductual-like structure in higher magnification. On day 7 postoperation in SLT group, Figs (E) and (F) show OV-6 positive cells were mainly located in periportal region. (Original magnification: A,B,C,E:100⫻; F:200⫻; D:400⫻).
ability showed the PCNA index in the SLT group to be decreased compared to the PH group from day 1 to day 3, demonstrating partially suppressed proliferation of mature hepatocytes. This finding is consistent with Zhizhong et al.5 We believe that the increased injury and suppressed proliferation in small-size grafts further decreases the functional liver mass and together leads to graft dysfunction. In rodents, OV-6 is a key surface marker of oval cells, which are considered to be a hepatic progenitor cell located in the canals of Hering.6 Farber7 first described oval cells as “small oval cells with scant lightly basophilic cytoplasm and pale blue-staining nuclei.” In subsequent studies,8 –12 many studies have shown that oval cells are activated when hepatocyte proliferation is impaired, although the actual mechanisms are largely unknown. In the classical oval
cell activation animal model (2-AAF combined with partial hepatectomy), when “oval cells” are activated to proliferate they give rise to duct-like structures originating from the periportal regions and spreading into the liver acinus. Similar manifestations have been discovered in human liver disease; it is termed “ductular reaction.”13 In this study, we discovered an abundant number of OV-6 positive cells in liver grafts after SLT; they were nearly negative in both PH and WLT groups. The immunofluorescence tests also demonstrated some OV-6 positive cells arranged in duct-like structures. These positive cells peaked at days 2 to 3 and decreased over the following days. They were dispersed into the liver at first and later located restrictedly to the periportal region. We believe that the mitogenic stimulus provided by the loss of tissue (small graft) and suppression of
LIVER PROGENITOR CELLS
1639
Fig 6. Immunohistochemistry staining of C90 positive cells. (A) PH group and (B) WLT group has no CD90 positive cell on day 2 postoperation. But (C) and (D) show many CD90 positive cells dispersed into liver section on day 2 in SLT group. On day 7 postoperation in SLT group, Figs (E) and (F) show the number of CD90 positive cells was decreased and they were mainly located in periportal region. (Original magnification: A,B,C,E:100⫻; F:200⫻; D: 400⫻.)
hepatocyte proliferation caused oval cell activation after SLT. Recent progress in bone marrow stem cell (BMMC biology) has revealed that these elements can be directed to differentiate into hepatocyte-like cells in vitro.14 –18 This manifestation has also been discovered in clinical research.19 In further research some interesting studies have demonstrated a relationship between bone marrow stem cells and hepatic progenitor cells.20 –22 Bone marrow stem cell markers, like CD90 and CD34, have been observed on oval cells, suggesting that a source of liver progenitor cells. In this study, we investigated many CD90 positive cells in the liver sections of the SLT group. Further, we observed that the mode of CD90 positive cells appearance was
similar to that of OV-6 positive cells. We believe that this is a hint that liver progenitor cells may come from bone marrow stem cells. The present study was unique in that we observed oval cells, which may also come from bone marrow stem cells to be activated after the liver dysfunction induced by a 30% small-for-size partial liver transplantation model in rats. Beside the serious liver injury and failure after SLT, we observed that the proliferative ability of mature hepatocyte decreased; oval cells responded to activation in this condition and bone marrow stem cells seemed to also participate in this process. The exact mechanisms of the suppressed hepatocyte proliferation and activation of hepatic progenitor cells after SLT represent foci for further study as well as
1640
the possibility of transplantation of stem cells to correct liver dysfunction after small-for-size liver transplantation.
REFERENCES 1. Kiuchi T, Kasahara M, Uryuhara K, et al: Impact of graft size mismatching on graft prognosis in liver transplantation from living donors. Transplantation 67:321, 1999 2. Sugawara Y, Makuuchi M, Takayama T, et al: Hashizume K. Small-for-size grafts in living-related liver transplantation. J Am Coll Surg 192:510, 2001 3. Liu F, Pan XB, Chen GD, et al: Hematopoietic stem cell mobilization after rat partial orthotopic liver transplantation. Transplant Proc 38:1603, 2006 4. Dirsch O, Chi H, Gu YL, et al: Influence of stem cell mobilization and liver regeneration on hepatic parenchymal chimerism in the rat. Transplantation 81:1695, 2006 5. Zhong Z, Schwabe RF, Kai Y, et al: Liver regeneration is suppressed in small-for-size liver grafts after transplantation: involvement of c-Jun N-terminal kinase, cyclin D1, and defective energy supply. Transplantation 82:241, 2006 6. Shafritz DA, Oertel M, Menthena A, et al: Dabeva, Liver stem cells and prospects for liver reconstitution by transplanted cells. Hepatology 43(2 Suppl 1):S89, 2006 7. Farber E: Similarities of the sequence of the early histological changes induced in the liver of the rat by ethionine, 2-acetylaminofluorene, and 3-methyl-4-dimethylaminoazobenzene. Cancer Res 16:142, 1956 8. Evarts RP, Nagy P, Marsden E, et al: A precursor-product relationship exists between oval cells and hepatocytes in rat liver. Carcinogenesis 8:1737, 1987 9. Evarts RP, Nagy P, Nakatsukasa H, et al: In vivo differentiation of rat liver oval cells into hepatocytes. Cancer Res 49:1541, 1989
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