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作者：Shuji Teraia, Tsuyoshi Ishikawaa, Kaoru Omoria, Koji Aoyamaa, Yoshio Marumotoa, Yohei Urataa, Yuichirou Yokoyamaa, Koichi Uchidaa, Takahiro Yamasakia, Yasuhiko Fujiib, Kiwamu Okitaa, Isao Sakaidaa作者单位：aDepartment of Molecular Science Applied Medicine (Gastroenterology Hepatology), Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan;bCenter of Regenerative Medicine and Cell Therapy, Yamaguchi University Hospital, Yamaguchi, Japan
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7 z" q; @. C% F, [1 \. S 【摘要】# C+ I( o& K! Z0 M6 @. K
We here report nine liver cirrhosis (LC) patients that underwent autologous bone marrow cell infusion (ABMI) from the peripheral vein. Subjects were patients with LC with total bilirubin of less than 3.0 mg/dl, platelet count of more than 5 (1010/l), and no viable hepatocellular carcinoma on diagnostic imaging. Autologous bone marrow (BM; 400 ml) was isolated from the ilium under general anesthesia. Mononuclear cells (MNCs) were separated by cell washing and were infused via the peripheral vein. MNC characteristics were confirmed by fluorescence-activated cell sorting analysis (CD34, CD45, and c-kit). After ABMI therapy, liver function was monitored by blood examination for 24 weeks. From 400 ml of BM, we obtained 7.81 ¡À 0.98 x 109 MNCs. After washing, 5.20 ¡À 0.63 x 109 MNCs were infused into patients with LC. Significant improvements in serum albumin levels and total protein were observed at 24 weeks after ABMI therapy (p < .05). Significantly improved Child-Pugh scores were seen at 4 and 24 weeks (p < .05). -Fetoprotein and proliferating cell nuclear antigen (PCNA) expression in liver biopsy tissue was significantly elevated after ABMI therapy (p < .05). No major adverse effects were noted. In conclusion, ABMI therapy should be considered as a novel treatment for patients with decompensated LC. / s7 L) F7 D0 E. L; ~" Y
【关键词】 Liver cirrhosis Regenerative medicine Liver regeneration Autologous bone marrow cell infusion Stem cell therapy Clinical trial
9 B8 z/ Z; d1 m INTRODUCTION* w4 \, |% B& C7 P& p( i5 O* j" D
G- ?3 w3 g) x$ `# @) i" D' }Liver cirrhosis (LC) is the end stage of chronic liver disease and is very difficult to treat. Currently, liver transplantation is one of the only effective therapies available to such patients. However, serious problems are associated with liver transplantation: lack of donors, surgical complications, rejection, and high cost. Regenerative therapies have the potential to provide minimally invasive procedures with few complications.
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The potential for stem cells in bone marrow (BM) to differentiate into hepatocytes and intestinal cells was recently confirmed through detection of Y chromosome-containing cells in samples from female recipients of BM cells (BMCs) from male donors . Taken together, these findings suggest that BMCs are effective sources for regenerative liver therapy.
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1 O4 D$ a' ?3 l7 T9 mTo confirm the potential of cell therapy using BMCs, we developed an in vivo mouse model (green fluorescent protein . However, there have been no reports on autologous BMC infusion (ABMI) in patients with LC. Here, we report nine patients with LC treated with ABMI therapy.
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MATERIALS AND METHODS3 g) m1 m0 w' _& W
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Eligible patients were between 18 and 75 years of age and had a clinical diagnosis of LC. The present subjects were patients with LC with total bilirubin of less than 3.0 mg/dl, a platelet count of more than 5 (1010/l), and no viable hepatocellular carcinoma on computed tomography (CT) and magnetic resonance imaging. Patients were excluded from the study if they had problems in organs other than the liver (e.g., the heart or lungs). Patients who were observed for the 24-week period are shown in Table 1. Written informed consent was obtained from all patients.
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7 G& n' b, q6 o/ ATable 1. Patient characteristics1 v* S$ \1 \; d# k- C
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Follow-Up (Laboratory Data, Ultrasonography, and CT)
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The study protocol is shown in Figure 1. After ABMI therapy, patients were followed up every week for 4 weeks, and laboratory data were analyzed monthly for 24 weeks. During the study period, medication was unchanged before and after ABMI therapy. Patients who changed medication or those who consumed alcohol were treated as dropouts. During the study, patients did not receive antiviral therapies such as interferon, lamivudine, ribavirin, or granulocyte colony-stimulating factor (G-CSF). Primary outcomes were safety and feasibility of ABMI therapy. Recruitment started in October 2003, and the first patient began treatment on November 14, 2003. A Child-Pugh score (albumin, total bilirubin, prothromibin time activity, ascites, and encephalopathy) was used to evaluate the overall condition of patients with LC ., s# b) A s7 E* g- H( k
) ~3 P: i) C7 V( p' EFigure 1. Study protocols. Abbreviations: ABMI, autologous bone marrow cell infusion; BM, bone marrow; CT, computed tomography; MNC, mononuclear cell.
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% A0 F) W' l2 h! {9 N! T0 VABMI Therapy
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BM (400 ml) was harvested from the ilium according to standard procedures under general anesthesia and was collected in plastic bags containing heparin . Cell washing and concentration were ensured using a spinning membrane connected to a filter wash bag. The final concentrated and washed cell product was made up to a final volume of 105 ml. Five milliliters of the final cell product was subjected to trypan blue dye exclusion test, endotoxin test, and fluorescence-activated cell sorting analysis. CD34-, CD45-, and c-kit-positive cells were determined by flow cytometry (EPICS XL-MCL; Beckman Coulter, Inc., Fullerton, CA, http://www.beckmancoulter.com, 92834-310.). CD34, CD45, and c-kit antibodies were obtained from Dako (Shijou douri, Sakyoku, Kyoto, Japan, http://www.dako.com). At 6¨C8 hours after BM harvest, the final MNC preparation was administered via the peripheral vein. All protocols were approved by the Ethics Committee of Yamaguchi University, and written informed consent was obtained from all patients.- n6 L! |! a T
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Immunohistochemical Analysis of Proliferating Cell Nuclear Antigen and -Fetoprotein Expression in Liver Biopsy Tissue Before and After ABMI Therapy2 y' W; Z3 `# ]% F6 s
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Liver biopsy samples before ABMI therapy and at 4 weeks after ABMI therapy were obtained by ultrasound-guided liver biopsy to evaluate liver condition. We obtained liver biopsy samples from three patients (patients 2, 8, and 9). We used an ALOKA ProSound SSD-5500 ultrasound transducer (ALOKA Co., Ltd., Tokyo, http://www2.aloka.co.jp) and a 16-guage biopsy needle (Super-Core II Biopsy Instrument; Medical Device Technologies, Inc., Gainesville, FL, http://www.interv.net). Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded liver specimens using the standard avidin-biotin-peroxidase complex (ABC) method. Specimens were small and were thoroughly infiltrated by formalin. Briefly, 3-µm tissue sections were deparaffinized in xylene and dehydrated in an alcohol series. Endogenous peroxidase was blocked with fresh 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature, followed by microwave antigen retrieval for 6 minutes at 95¡ãC in 10 mM sodium citrate buffer (pH 6.0). Normal goat serum (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) was applied for 20 minutes and removed. This method is the same as previously described . Differences in labeling index between each group were analyzed for significance by Student¡¯s t test.
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Statistical Analysis
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Changes in laboratory data from baseline (before ABMI therapy) to 4 or 24 weeks after ABMI therapy were analyzed. Values are shown as means ¡À SE. Data were analyzed by analysis of variance with Fisher¡¯s projected least significant difference test.; O1 I8 H7 p+ s
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RESULTS
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# P3 I/ M, e* aFollow-Up' {' V+ T3 J1 `" E
) e% I+ J* R" [) ^0 y; _3 g: VBetween October 2003 and March 2006, 12 patients with LC were recruited into the study, but two were subsequently excluded. The reasons for exclusion were as follows: one patient exhibited a tendency for severe bleeding, and the other patient was diagnosed with gastric cancer. One patient with LC (etiology was alcohol) dropped out of the study because he continued to consume alcohol during the follow-up period. Thus, a total of nine patients were followed up to the end in the study. The characteristics of these patients are shown in Table 1. Patient medications are also shown in Table 1. Patients were administrated branched-chain amino acid granules, Livact Granule (Ajinomoto Co., Inc., Tokyo, http://www.ajinomoto.com), or Aminoleban EN (Otsuka Pharmaceutical Co., Ltd., Tokyo, http://www.otsuka.co.jp), which contains a high branched-chain amino acid content, to improve the nutritional disorders associated with LC . Spironolactone and Furosemide were used to control ascites. Medications were not changed during the study period, and no antiviral therapies were administered.
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Isolation of BMCs and Cell Products from Patients T; W' f1 H/ I3 m ^& e
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We isolated 400 ml of autologous BM from the ilium. As shown in Table 2, we obtained 7.81 ¡À 0.98 x 109 MNCs. After washing with Cytomate, 5.20 ¡À 0.63 x 109 MNCs were administered. MNC viability was more than 90%. The characteristics of the cell product are shown in Table 2.
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/ ^0 y& G& }* R4 N: A8 X& tTable 2. Characteristics of processed mononuclear cells (MNCs)
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2 _3 S4 w2 \$ w2 C) I6 zChanges in Laboratory Data After ABMI Therapy
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Data for serum albumin and total protein, as well as Child-Pugh scores, at 8 weeks, 4 weeks, and 1 day before ABMI therapy were not significantly different. As shown in Figure 2A, average serum albumin levels and total protein had significantly improved at 24 weeks after ABMI therapy (p + y* b& }9 ]4 P
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Figure 2. Improved liver function after ABMI therapy. (A): Serum albumin, total protein, Child-Pugh score, PIIIP, and HGF during 24-week follow-up period after ABMI therapy. (B): Serum level of PIIIP and HGF after ABMI therapy. Average: average value. indicates significant difference between baseline value and that at 24 weeks after ABMI therapy. # indicates significant difference between baseline value and that at 24 weeks after ABMI therapy. * indicates significant difference between each group (before, 4 weeks after, and 24 weeks after ABMI therapy). indicates no significant differences between the data at 8 weeks, 4 weeks, and 1 day before ABMI therapy. (Normal ranges: albumin 3.7¨C4.7 g/dl, total protein 6.8¨C8.3 g/dl, PIIIP 0.3¨C0.8 U/ml, and HGF 0¨C0.39 ng/ml). Abbreviations: ABMI, autologous bone marrow cell infusion; HGF, hepatocyte growth factor; PIIIP, pro-collagen III peptide.6 b* T. P' }' B% x! B4 V& _
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Improvement of Ascites After ABMI Therapy
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As shown in Figure 3, ascites improved after ABMI therapy. In patient 1, abdominal ultrasonography and CT showed that ascites had decreased at 2 weeks after ABMI therapy (Fig. 3C, 3D). The same trend was observed in patients 4, 5, 8, and 9.
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7 s/ [( P, ^" @5 L8 A0 MFigure 3. Abdominal ultrasonography and CT for patient 1. (A): Abdominal ultrasonography before ABMI therapy. Arrows indicate severe ascites. (B): CT shows severe ascites before ABMI therapy. Arrows indicate ascites. (C): Abdominal ultrasonography showing decrease in ascites at 2 weeks after ABMI therapy. (D): CT showing improvement in ascites at 2 weeks after ABMI therapy. Abbreviations: ABMI, autologous bone marrow cell infusion; CT, computed tomography.
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AFP and PCNA Expression in Liver Biopsy Tissue After ABMI Therapy
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We analyzed AFP and PCNA expression in liver biopsy tissue before and after ABMI therapy (patients 2, 8, and 9). As shown in Figure 4A and 4B, we found that AFP and PCNA expression was significantly elevated at 4 weeks after ABMI therapy (p
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/ A6 T) W5 o4 Z3 iFigure 4. Immunohistochemical analysis of AFP and PCNA expression. (A): AFP expression before and at 4 weeks after ABMI therapy in patient 2. Arrows indicate AFP-positive cells. Labeling index includes data for three patients (patients 2, 8, and 9). An asterisk indicates significant difference between baseline value and that at 4 weeks after ABMI therapy (p , m; i ~& B/ z0 }
" n" ^" N, d5 B3 vComplications
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c; _4 v( k4 s% G* M* n0 w5 nAll nine patients exhibited fever (approximately 38¡ãC) at 1 day after ABMI therapy, but fever was not seen after that. No adverse effects were noted in any of the present patients.
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4 B- \% K* `4 c0 `3 a3 DDISCUSSION
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We demonstrated the safety of isolation of autologous BM from patients with LC and the effects of ABMI therapy. When we began this study, there were no data regarding whether sufficient numbers of BMCs could be obtained from patients with LC.
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Previous reports suggested that anesthesia is associated with an increased risk of morbidity and mortality . Thus, we selected isolation of BMC from the ilium under general anesthesia, and despite our concerns regarding adverse effects due to general anesthesia, the present nine patients exhibited no such complications. We safely isolated 400 ml of BM from the ilium, and as shown in Table 2, we obtained 7.81 ¡À 0.91 x 109 MNCs (average). After preparation, we administered 5.20 ¡À 0.63 x 109 MNCs via the peripheral vein (average). MNC viability was found to exceed 90%. The characteristics of infused MNCs are shown in Table 2. These results suggest that we obtained sufficient numbers of MNCs from the patients with LC enrolled in this study. In addition, we did not observe any major adverse effects due to isolation of BM and ABMI therapy.
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" R6 ~. L0 T5 P8 K( M3 H/ [The changes of liver function in all nine patients after ABMI therapy are shown in Figure 2. As shown in Figure 2A, mean serum albumin levels, total protein levels, and Child-Pugh scores improved significantly after ABMI therapy (p
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) l X7 _" w7 k5 P# Jam Esch et al. reported that portal administration of autologous CD133 BMCs accelerated liver regeneration and is a novel therapy to support hepatic resection . Data from human studies corresponded with our basic study. However, further study is needed to confirm the significance of growth factor.
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When we started the study, we believed that G-CSF injection would mobilize BMCs as a treatment for patients with LC. Our previous basic study using an animal model showed that mesenchymal cells would be more useful for liver regeneration , which may support our results.: _2 X& c' y( {* m* R' Z3 q
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The present report evaluated ABMI therapy in patients with LC. Our results suggest that ABMI therapy is a candidate modality in the treatment of decompensated LC. To demonstrate the full therapeutic value of this protocol, more comprehensive long-term case-control clinical studies and/or randomized control studies are required.. e% I" C: [* B
1 r$ h1 ]0 ]% i/ QDISCLOSURES
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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3 u/ _0 z6 ^4 L) L* xWe thank Dr. Yuzuru Sato, Hiroshi Nishina, and Naomi Kojima for their valuable assistance. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 163,90211, 165,90597) and for translational research from the Ministry of Health, Labor and Welfare (H14-trans-5, H17-Specai-015).- w0 V$ ^ v4 A# t) j% ?) ^2 A8 ?
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发表于 2009-3-5 00:01
发表于 2010-1-27 11:09
