异体骨髓间充质干细胞移植治疗慢加急性乙肝肝衰竭可行性研究：单中心小样本随机对照试验
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摘要
【研究背景】
慢加急性肝衰竭病死率高。目前原位肝移植是唯一特效的治疗，但由于供体肝源紧缺，移植后排斥反应及移植后大量免疫抑制剂的应用带来的不良反应限制它的应用。抗病毒治疗对改善预后有一定的疗效，但其病死率仍然高达50%以上。故而迫切需要寻找一种安全、有效、方便的治疗方法。
骨髓间充质干细胞（Bone marrow mesenchymal stem cells BM-MSCs）是一类具有自我更新能力和多向分化能力的成体干细胞。研究发现BM-MSCs具有向受损肝脏归巢、向肝样细胞分化、抗肝纤维化的作用，以及在体外无成瘤性的特点。此外BM-MSCs免疫原性低，可通过分泌细胞因子以调节免疫，临床上已将其运用于器官移植后的免疫抑制治疗，以及激素抵抗的严重移植物抗宿主反应等。课题组前期研究提示自体骨髓间充质干细胞对肝功能的改善有一定的作用，但对生存率没有提高，移植细胞量不足可能是主要原因。鉴于自体BM-MSCs移植的局限性，以及BM-MSCs低免疫原性和免疫调节功能，本研究通过对慢加急性乙肝肝衰竭患者进行2种不同剂量各4次异体BM-MSCs移植，初步探讨不同剂量异体BM-MSCs移植治疗慢加急性乙肝肝衰竭的安全性及疗效。
【研究目的】
本研究通过提高移植细胞数量和移植次数，评估经外周静脉途径进行异体骨髓间充质干细胞（Bone marrow mesenchymal stem cells BM-MSCs）移植治疗慢加急性乙肝肝衰竭患者的安全性及疗效。
【研究方法】
入组25名确诊为慢加急性乙肝肝衰竭的住院患者，随机分为3组，第1组接受标准内科综合治疗（Standard Medicine Treatment， SMT组），第2、3组在标准内科综合治疗基础上，通过外周静脉按体重每周分别给予异体骨髓间充质干细胞移植1×10~5/Kg （MSC-1组）和1×10~6/Kg（MSC-2组）1次，共4次，随访24周（MSC-1组和MSC-2组合称MSC组）。观察患者治疗后不同时间点不良反应、肝肾功能、MELD评分、并发症发生率及生存情况。
【结果】
在24周的观察时间内未发现严重不良反应。入组后1-4周3组患者白细胞（WBC）、血红蛋白（Hb）、血小板（PLT）差异无统计学意义。入组后24周3组患者血肌酐（Cr）水平无统计学差异。无1例患者发展为肝脏或其它器官肿瘤。MSC组24周累计生存率较SMT组高（P=0.020）。移植后1-4周MSC组肝功能较SMT组改善，4周后两组肝功能差别无统计学意义。移植后24周内两组患者感染、肝性脑病、肝肾综合症、消化道出血、中毒性鼓肠5种并发症的发生率无统计学差异。MSC-1组与MSC-2组比较，移植后第1、2周，MSC-2组ALT水平较MSC-1组改善（P值分别为0.011,0.013），移植后第4周MSC-1组MELD评分较MSC-2组改善（P=0.048），两组患者24周生存率及并发症发生率无统计学差异。
【结论】
异体骨髓间充质干细胞移植治疗慢加急性乙肝肝衰竭是安全的，并可提高患者生存率，改善肝功能。高剂量（1×10~6/Kg）组疗效与低剂量组（1×10~5/Kg）的疗效无显著差异。
Background Acute-on-chronic liver failure (ACLF) caused by hepatitis B virus(HBV) is a severe disease with high mortality. Liver transplantation is the onlytherapy that had been proven beneficial, but the rapidity of progression and variablecourse of ACLF limit it application, and effective use of this limited resource requiresaccurate prognostication and subsequent lifelong immunosuppression. Antiviraltreatment contributed to the improved outcomes, but its mortality was still more than50%. So it is necessary to find a safe, effective and convenient method.Bone marrow mesenchymal stem cells (BM-MSCs) are characterized by theproperties of self-renewal and multipotentiality. The previous studies showed thatBM-MSCs performed the abilities of homing to the damaged liver, differentiating intohepatocytes, inhibiting the progress of liver fibrosis and having no tumorigenicity invitro. Besides, BM-MSCs possess the properties of low immunogenicity andimmunomodulatory. Clinically, BM-MSCs were used in the immunosuppressivetreatment after organ transplantation and in the steroid-resistant, severeGraft-versus-host reaction (GvHD), which were proven effectively. Our early studyrevealed that autologous bone marrow mesenchymal stem cells (BM-MSCs)transplantation for liver failure patients caused by hepatitis B improves liver functions,but without any survival benefit. The inadequate BM-MSCs dose for transfusion maybe the main cause for it. In view of the factors mentioned above, we administrate thepatients with ACLF related with hepatitis B with two different doses of allogeneicBM-MSCs via peripheral veins for4times, in order to investigate the safety andefficacy of this therapy.
Aims This study aimed to assess the safety and efficacy of allogeneic BM-MSCstransplantation via peripheral veins for the patients with ACLF caused by hepatitis B by increasing BM-MSCs dose and transplantating frequence.
Methods Twenty five inpatients with ACLF related with HBV were divided into3groups randomly. Group SMT received the standard medicine treatment. GroupMSC-1and Group MSC-2underwent transplantations of allogeneic BM-MSCs1×10~5/Kg and1×10~6/Kg by peripheral veins once a week for4weeks respectively, inaddition to the standard medicine treatment. Group MSC were defined as thecombined of Group MSC-1and MSC-2. All the groups were followed up for24weeks.
Results No serious adverse reactions were observed from1-24weeks aftertransplantations. There were no significant differences in the levels of white bloodcells (WBC), hemoglobin (Hb) and platelet (PLT) at1-4weeks after transplantationamong the3groups. And no dramatic differences were found in the levers ofcreatinine (Cr) among the3groups from1-24weeks. There was no patient developinghepatocellular carcinoma (HCC) or tumors originated from other organs. Comparingwith Group SMT, Group MSC achieved higher24-week cumulative survival rate(P=0.020), and Group MSC acquired markedly improvements in liver functions from1to4weeks after transplantations. There were no significant differences in theincidence of complications (i.e., infection, encephalopathy, hepatorenal syndrome,gastrointestinal bleeding, and toxic enteroparalysis) from1-24weeks aftertransplantations between Group SMT and Group MSC. The lever of alanineaminotransferase (ALT) of patients in Group MSC-2was markedly improved at week1and2after transplantation (P value were0.011,0.013respectively), compared withGroup MSC-1. However, Group MSC-1got dramatic improvement in levers of Modelfor End-Stage Liver Disease (MELD) score at week4(P=0.048). There were nosignificant differences in24-week cumulative survival rates and the incidence ofcomplications between Group MSC-1and Group MSC-2.
Conclusion Allogeneic BM-MSCs transplantation via peripheral veins is safe foracute-on-chronic liver failure patients caused by hepatitis B. It can improve the liverfunctions and24-week survival rate. There were no significant differences in theefficacy between Group MSC-1and Group MSC-2.
慢加急性肝衰竭病死率高。目前原位肝移植是唯一特效的治疗，但由于供体肝源紧缺，移植后排斥反应及移植后大量免疫抑制剂的应用带来的不良反应限制它的应用。抗病毒治疗对改善预后有一定的疗效，但其病死率仍然高达50%以上。故而迫切需要寻找一种安全、有效、方便的治疗方法。
骨髓间充质干细胞（Bone marrow mesenchymal stem cells BM-MSCs）是一类具有自我更新能力和多向分化能力的成体干细胞。研究发现BM-MSCs具有向受损肝脏归巢、向肝样细胞分化、抗肝纤维化的作用，以及在体外无成瘤性的特点。此外BM-MSCs免疫原性低，可通过分泌细胞因子以调节免疫，临床上已将其运用于器官移植后的免疫抑制治疗，以及激素抵抗的严重移植物抗宿主反应等。课题组前期研究提示自体骨髓间充质干细胞对肝功能的改善有一定的作用，但对生存率没有提高，移植细胞量不足可能是主要原因。鉴于自体BM-MSCs移植的局限性，以及BM-MSCs低免疫原性和免疫调节功能，本研究通过对慢加急性乙肝肝衰竭患者进行2种不同剂量各4次异体BM-MSCs移植，初步探讨不同剂量异体BM-MSCs移植治疗慢加急性乙肝肝衰竭的安全性及疗效。
【研究目的】
本研究通过提高移植细胞数量和移植次数，评估经外周静脉途径进行异体骨髓间充质干细胞（Bone marrow mesenchymal stem cells BM-MSCs）移植治疗慢加急性乙肝肝衰竭患者的安全性及疗效。
【研究方法】
入组25名确诊为慢加急性乙肝肝衰竭的住院患者，随机分为3组，第1组接受标准内科综合治疗（Standard Medicine Treatment， SMT组），第2、3组在标准内科综合治疗基础上，通过外周静脉按体重每周分别给予异体骨髓间充质干细胞移植1×10~5/Kg （MSC-1组）和1×10~6/Kg（MSC-2组）1次，共4次，随访24周（MSC-1组和MSC-2组合称MSC组）。观察患者治疗后不同时间点不良反应、肝肾功能、MELD评分、并发症发生率及生存情况。
【结果】
在24周的观察时间内未发现严重不良反应。入组后1-4周3组患者白细胞（WBC）、血红蛋白（Hb）、血小板（PLT）差异无统计学意义。入组后24周3组患者血肌酐（Cr）水平无统计学差异。无1例患者发展为肝脏或其它器官肿瘤。MSC组24周累计生存率较SMT组高（P=0.020）。移植后1-4周MSC组肝功能较SMT组改善，4周后两组肝功能差别无统计学意义。移植后24周内两组患者感染、肝性脑病、肝肾综合症、消化道出血、中毒性鼓肠5种并发症的发生率无统计学差异。MSC-1组与MSC-2组比较，移植后第1、2周，MSC-2组ALT水平较MSC-1组改善（P值分别为0.011,0.013），移植后第4周MSC-1组MELD评分较MSC-2组改善（P=0.048），两组患者24周生存率及并发症发生率无统计学差异。
【结论】
异体骨髓间充质干细胞移植治疗慢加急性乙肝肝衰竭是安全的，并可提高患者生存率，改善肝功能。高剂量（1×10~6/Kg）组疗效与低剂量组（1×10~5/Kg）的疗效无显著差异。
Background Acute-on-chronic liver failure (ACLF) caused by hepatitis B virus(HBV) is a severe disease with high mortality. Liver transplantation is the onlytherapy that had been proven beneficial, but the rapidity of progression and variablecourse of ACLF limit it application, and effective use of this limited resource requiresaccurate prognostication and subsequent lifelong immunosuppression. Antiviraltreatment contributed to the improved outcomes, but its mortality was still more than50%. So it is necessary to find a safe, effective and convenient method.Bone marrow mesenchymal stem cells (BM-MSCs) are characterized by theproperties of self-renewal and multipotentiality. The previous studies showed thatBM-MSCs performed the abilities of homing to the damaged liver, differentiating intohepatocytes, inhibiting the progress of liver fibrosis and having no tumorigenicity invitro. Besides, BM-MSCs possess the properties of low immunogenicity andimmunomodulatory. Clinically, BM-MSCs were used in the immunosuppressivetreatment after organ transplantation and in the steroid-resistant, severeGraft-versus-host reaction (GvHD), which were proven effectively. Our early studyrevealed that autologous bone marrow mesenchymal stem cells (BM-MSCs)transplantation for liver failure patients caused by hepatitis B improves liver functions,but without any survival benefit. The inadequate BM-MSCs dose for transfusion maybe the main cause for it. In view of the factors mentioned above, we administrate thepatients with ACLF related with hepatitis B with two different doses of allogeneicBM-MSCs via peripheral veins for4times, in order to investigate the safety andefficacy of this therapy.
Aims This study aimed to assess the safety and efficacy of allogeneic BM-MSCstransplantation via peripheral veins for the patients with ACLF caused by hepatitis B by increasing BM-MSCs dose and transplantating frequence.
Methods Twenty five inpatients with ACLF related with HBV were divided into3groups randomly. Group SMT received the standard medicine treatment. GroupMSC-1and Group MSC-2underwent transplantations of allogeneic BM-MSCs1×10~5/Kg and1×10~6/Kg by peripheral veins once a week for4weeks respectively, inaddition to the standard medicine treatment. Group MSC were defined as thecombined of Group MSC-1and MSC-2. All the groups were followed up for24weeks.
Results No serious adverse reactions were observed from1-24weeks aftertransplantations. There were no significant differences in the levels of white bloodcells (WBC), hemoglobin (Hb) and platelet (PLT) at1-4weeks after transplantationamong the3groups. And no dramatic differences were found in the levers ofcreatinine (Cr) among the3groups from1-24weeks. There was no patient developinghepatocellular carcinoma (HCC) or tumors originated from other organs. Comparingwith Group SMT, Group MSC achieved higher24-week cumulative survival rate(P=0.020), and Group MSC acquired markedly improvements in liver functions from1to4weeks after transplantations. There were no significant differences in theincidence of complications (i.e., infection, encephalopathy, hepatorenal syndrome,gastrointestinal bleeding, and toxic enteroparalysis) from1-24weeks aftertransplantations between Group SMT and Group MSC. The lever of alanineaminotransferase (ALT) of patients in Group MSC-2was markedly improved at week1and2after transplantation (P value were0.011,0.013respectively), compared withGroup MSC-1. However, Group MSC-1got dramatic improvement in levers of Modelfor End-Stage Liver Disease (MELD) score at week4(P=0.048). There were nosignificant differences in24-week cumulative survival rates and the incidence ofcomplications between Group MSC-1and Group MSC-2.
Conclusion Allogeneic BM-MSCs transplantation via peripheral veins is safe foracute-on-chronic liver failure patients caused by hepatitis B. It can improve the liverfunctions and24-week survival rate. There were no significant differences in theefficacy between Group MSC-1and Group MSC-2.
引文