经肝动脉自体骨髓干细胞移植治疗急、慢性肝病的实验研究
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摘要
前言
肝硬化是各种慢性肝病的终末期表现,目前尚无特别有效的治疗方法,对其治疗方法的研究一直处于不断的探索之中。肝移植因其能够较好地改善肝功能、延长肝硬化患者的生存期,已成为较理想的治疗选择,然而由于其治疗费用昂贵、供肝数量有限以及伦理道德等方面的问题,又限制了其在临床上的广泛应用,因此,探索和寻找一种经济易行、又无供求限制的新方法,已成为目前研究的热点。近年来,随着对干细胞研究的不断深入,特别是骨髓干细胞具有的多向分化潜能及超强的可塑性逐渐受到重视,部分领域在临床已得到初步应用,如心肌缺血、糖尿病,肝病等。基础研究表明骨髓干细胞在体外诱导培养时可转化为功能成熟的肝细胞,在活体实验中骨髓干细胞可在肝损伤和肝纤维化的环境内存留、生长和转化,改善肝功能。在目前肝病的干细胞治疗实验研究中大多采用大鼠四氯化碳(CCL4)诱导的肝硬化模型,其采用的异体干细胞移植与局部注射移植方法离临床实用有很大差别,而大动物的肝硬化模型尚不成熟。目前骨髓干细胞移植在肝脏疾病治疗的实验研究中,已经证明骨髓干细胞对肝损伤和重症肝病较好的治疗效果,但对肝硬化似乎不很理想,因此探索增强干细胞移植对肝硬化治疗效果的方法非常必要。本研究从中等动物兔肝硬化模型和肝损伤模型中,利用自体骨髓干细胞作为种子细胞,采用临床最方便、实用的经肝动脉途径的移植方法进行实验研究,通过对肝功能的跟踪检测和动物肝脏组织病理对照观察,来评价骨髓干细胞移植对肝损伤和肝硬化治疗的安全性及有效性。并用促肝细胞生长素作为干扰因素探索提高骨髓干细胞对肝硬化治疗效果的方法。
实验目的
探索应用四氯化碳制作兔急性肝损伤及肝硬化模型的方法,评价该方法的可行性和制模成功率。
材料与方法
雄性新西兰大白兔70只,分成3组:正常对照组(10只)、急性肝损伤模型组(15只)、肝硬化组(45只),急性肝损伤组每只每日皮下注射50%CCL4橄榄油溶液0.8ml/kg,连续注射4天,第7天检测肝功能及肝脏病理。肝硬化组给予皮下注射注射50%CCL4橄榄油溶液0.2ml/kg,每周2次,12周后检测肝功能及肝脏病理。对照组分两组分别按相同剂量和时间给予皮下注射橄榄油溶液。
实验结果
1.兔急性肝损伤模型组:在四氯化碳注射4天后,化验肝功能明显改变,肝酶活性显著升高,谷丙转氨酶由35.83±8.04u/l上升至331.00±29.15u/l,总胆红素由6.41±0.52umol/l上升至11.64±0.58 umol/l。病理可见大面积肝细胞肿胀、坏死、并可见脂肪变性、肝组织结构紊乱,炎性细胞浸润明显。模型制备成功率为100%。
2.肝硬化模型组:在四氯化碳注射12周后,肝功能检查部分指标变化明显,白蛋白由40.28±2.30g/l减少至27.46±1.76g/l。凝血酶原活动度由64.32%降低至28.28%。HE病理染色及Masson纤维染色显示肝组织出现明显的纤维增生和较典型的假小叶,肝细胞可见浊肿,并见部分炎性细胞浸润。模型制备成功率为55%。
实验结论
应用皮下注射CCL4的方法可成功地建立兔急性肝损伤和肝硬化动物模型。但肝硬化模型制作时间长,动物死亡率高。
实验目的
评价自体骨髓干细胞经肝动脉移植对兔急性肝损伤模型的治疗作用,并观察促肝细胞生长素在干细胞移植治疗中的作用。
材料和方法
新西兰雄性大白兔15只,每日皮下注射50%CCL4橄榄油溶液0.8ml/kg连续4天制作急性肝损伤模型。模型形成后将动物随机分为2个实验组和对照组(各5只),3天后实验组经兔胫骨抽取骨髓约5ml,采用密度梯度离心法分离骨髓单个核细胞(干细胞),制成5ml细胞悬液。动物麻醉后在透视下股动脉穿刺插管将分离的骨髓干细胞经肝动脉注入肝脏。实验2组在干细胞移植前后隔日静脉注射促肝细胞生长素(pHGF)2.0mg/kg,共持续20天。干细胞移植后2周、4周、8周分别抽血进行肝功能检测,8周后取兔肝脏组织标本进行病理学观察。
实验结果
连续注射CCL44天后,血清肝酶及总胆红素明显上升,病理显示肝组织结构紊乱,大量肝细胞坏死、水肿和脂肪变性,汇管区及周围有大量炎性细胞浸润。骨髓干细胞肝动脉移植后肝功能逐渐改善,至移植后8周,血清AST、ALT活性和TBIL含量显著低于对照组(P＜0.05),而TP、ALB含量明显高于对照组(P＜0.05),而且实验2组上述指标改变更明显,与对照组和实验1组之间均有明显差异。
病理组织学观察实验组肝脏组织结构清楚,细胞排列规则,二组之间无明显差别,而对照组有纤维组织增生和肝细胞变性。免疫组化染色发现肝组织内散在分布有不同数量的CD34+肝样细胞,并且这些肝细胞在各组数量差异较大,对照组每高倍镜视野约1～2个,干细胞移植组3～5个,移植+pHGF组6～8个。
实验结论
1.自体骨髓干细胞经肝动脉移植对兔急性肝损伤模型有明显治疗作用,可改善急性肝损伤动物的肝功能,并促进肝组织结构重建。
2.促肝细胞生长素(pHGF)能保护肝细胞,促进干细胞在肝脏内的归巢和分化,加速肝组织的修复和肝功能的改善。
实验目的
评价自体骨髓干细胞经肝动脉移植对兔肝硬化模型的治疗作用,并比较促肝细胞生长素在干细胞移植治疗中的协同作用,为干细胞移植的临床应用提供实验研究基础。
实验方法
用皮下注射四氯化碳(CCl4)橄榄油溶液的方法复制兔肝硬化模型,模型动物分为实验1组、实验2组和对照组。在无菌条件下从兔胫骨上端抽取骨髓4～5ml,采用密度梯度离心法分离纯化骨髓干细胞,然后在透视下将分离的骨髓干细胞经肝动脉移植于肝脏。术后实验2组隔日静脉内注射pHGF2mg/kg共20天,然后在移植后4、8、12周分别检测肝功能,12周行肝脏病理学检查。实验数据资料采用SPSS11.5统计软件处理,P＜0.05为差异有显著性。
实验结果
在干细胞移植12周后,动物谷丙转氨酶(ALT)、谷草转氨酶(AST)逐渐降低,;总蛋白(TP)、白蛋白(ALB)逐渐升高,部分凝血酶原时间(APTT)逐渐上升,但这些改变与对照组相比无差异显著性(P＞0.05)。而移植+pHGF组肝功能指标改变较移植组迅速,与对照组相比有明显差异(P＜0.05)。
病理学检查显示干细胞移植组肝细胞形态较规整,无明显水肿和变性,假小叶结构仍存在,但肝组织纤维化程度较对照组明显,形态上有较明显的差异。免疫组化染色发现肝组织内散在分布有CD34+肝样细胞,并且这些肝细胞在各组数量差异较大,对照组每高倍镜视野约2～3个,干细胞移植组3～6个,移植+pHGF组6～9个。
实验结论
1、自体骨髓干细胞移植对肝硬化有一定的治疗作用。
2、促肝细胞生长素可促进干细胞在肝脏的归巢与分化,与骨髓干细胞移植有协同治疗作用。
Preface
Cirrhosis is a final stage of various kinds of liver disases,which treatment is always explored for many years.Liver transplantation is a better method to cirrhosis,but owning to the problem of its expansive cost,limited donator and ethics,its extensive application is also limited.thus,to look for a simple new way to cirrhosis is a aim of study. Recently,as research of stem cell progress,application of marrow stem cells in clinic is gradually payed close attention.Basis studys founded marrow stem cells may differentiate into function liver cells in vitro,and it can grow and differentiate within surroundings of hepatic injury or fibrosis in vivo.meantime it can improve alnormal liver function.Many cirrhosis models were induced by injection carbon tetrachloride in rats,which has great difference to clinical application,but big animal cirrhosis model is immaturity.Now bone marrow stem cells have been applicated in animal experiment and clinic,such as myocardial ischemia,diabetes and liver diease et al.Study founded bone marrow stem cells transplantion has a better effect to hepatic injury and severe liver dieases,but it seems not satisfactory to cirrhosis.It is very necessary to search for a new method enhancing its effect.Our project is that autologous bone marrow stem cells were transplanted through the hepatic artery in rubbit's hepatic injury and cirrhosis model.and the safety and efficacy of autologous bone marrow stem cells transplantion were evaluated by detecting liver function and pathohistology,Besides,the affect of hepatocyte growth-promoting factor to stem cell transplantation to cirrhosis was observed.
Part 1
The Development of Carbon Tetrachloride-Induced Hepatic injury and Cirrhotic Model in Rabbits
Objective
To search for a method of carbon tetrachloride-induced Hepatic injury and cirrhotic model in rabbit,and evaluate the feasibility and achievement ratio of the models through this method.
Methods
70 Male New Zealand white rabbits were divided into 3 group.The rabbits were subcutaneously injected with carbon tetrachloride once per day for 4 days in acute Hepatic injury model group,and liver pathology and function ware examined.Meanwhile,The rabbits were subcutane- ously injected twice weekly in cirrhotic model group,and liver pathology and function ware examined 12 weeks after the injection.
Results
In acute Hepatic injury model group,after carbon tetrachloride administration,the enzyme activity was steped up.The number of ALT goes up from 35.83±8.04u/l to 331.00±29.15u/l,and TBIL goes from 6.41±0.52 umol/1 up to 11.64±0.58 umol/l。and the serious cellular necrosis was founded,and hepatic structure was alnormal.
However,in cirrhotic model group,the albumn(ALB) and thrombozyme activity(PTA) decreased evidently compared to normal control group.The number of ALB decreased from 40.28±2.30g/l to 27.46±1.76g/l.and PTA decrease from 6.41±0.52 umol/l up to 11.64±0.58 umol/l。In pathohistology,we founded that the fiber hyperplasy was evident and typical pseudo-lobule can be seen in the rabbit liver,
Conclusion
A successive method to induce rabbit Hepatic injury and liver cirrhosis was developed.But the mortality is high in cirrhotic model group.
Part 2
Experimental Study of Autologous Bone Marrow Stem Cells for the Tr eatment of Acute Hepatic Injury in Rabbit.
Objective
A rubbit model was used to evalute the feasibility of treating acute hepatic injury through transplanting autologous bone marrow stem cells via the hepatic artery.
Methods
Fifteen rubbits were induced to develop acute hepatic injury model by daily subcutaneous injection with 50%CCL4 0.8ml/kg for 4 days.Then the animals were random- ly divided into two experimental and control groups. In the experimental group,autologous bone marrow stem cells(1×10~8) were disassociated and transplanted into liver via the hepatic artery.Besides,in the second experimental group,the hepatocyte growth- promoting factor(pHGF) was given via vein injection with 2.0mg/kg every other day for 20 days.The change of liver functions had been monitored by biochemical methods in 2,4,8weeks and histopatho-logic examination at 8 weeks after transplantation.
Results
After CCL4 injection,hepatocyte necrosis was noted.An increase in AST,ALT and TBIL activity(P＜0.05) was also observed obviously.But the levels of TP,and ALB were not obviously changed(P＞0.05) in plasma. 8 weeks after stem cells transplantation,the activity of AST and ALT,and the levels of TBIL were lower than the control group(P＜0.05).and the levels of TP and ALB were higher than the control group(P＜0.05).Moreove,the above changes in the second experimental group were more obvious than that of the first experimental group(P＜0.05).
In pathohistology,we founded that liver tissue structure in the experi-mental group was more close to normal than that of the control group,and fiber hyperplasy and hepatic cell degeneration was founded in the control group.Besides,the number of hepatic CD34+ cells of liver tissue in the experimental group was much than that of the control group.The number of the CD34+ cells can be seen per high power field of vision is 1～2 cells in the control group.3～5 cells in the first experimental group,and 6～8cells in the second experimental group.
Conclusion
It is feasible that autologous bone marrow stem cells transplanted through the hepatic artery in the rubbit liver necrosis model,and the transplanted stem cells can regenerate in the acute injurey liver.
The hepatocyte growth-promoting factor can help to improve hepatic function.
Part 3
Treatment of chronic hepatic cirrhosis with autologous bone marrow stem cells transplantation
Objective
To evalute the feasibility of treatment for rubbit model with hepatic cirrhosis by transplantation of autologous bone marrow-derived stem cells via the hepatic artery.and the effect of hepatocyte growth factor for treatment of stem cells transplantation to liver cirrhosis.
Methods
Fourty-five rubbits were induced to develop acute hepatic injury model by daily subcutaneous injection with 50%CCL4 0.8ml/kg for 4 days.Then twenty-five animals were randomly divided into two exper- imental and control groups.In the experimental group,autologous bone marrow stem cells(1×10~8) were disassociated and transplanted into liver via the hepatic artery.Besides,in the second experimental group,the hepatocyte growth-promoting factor(pHGF) was given via vein injection with 2.0mg/kg every other day for 20 days.The change of liver functions had been monitored by biochemical methods in 4,8,12weeks and histopathologic examination at 12 weeks after transplantation..
Results
After transplantation of bone marrow stem cells,the liver function of patients improved.Eight weeks after transpl-anttation,the activity of AST and ALT,and the levels of TBIL were lower than the control group,and the levels of TPA and ALB were higher than the control group,but these changes have not obvious difference.However,the above changes in the second experimental group were more obvious than that of the first experimental group.And the difference between the second experimental group and control group was very significant(P＜0.05).
In pathohistology,we founded that hepatic cells in the experimental group was more close to normal,but cell degeneration was founded in the control group,the fiber hyperplasy and hepatic pseudo-lobule was lessevident than that of the control group.Besides,the number of hepatic CD34+ cells of liver tissue in the experimental group was much than that of the control group.The number of the CD34+ cells can be seen per high power field of vision is 2～3 cells in the control group.3～6 cells in the first experimental group,and 6～9cells in the second experimental group.
Conclusion
It is effective that autologous bone marrow stem cells transplanted via the hepatic artery for the rubbit liver injury model,and the transpl-anted stem cells can regenerate in the acute injurey liver.
The hepatocyte growth-promoting factor can help to improve hepatic function.
肝硬化是各种慢性肝病的终末期表现,目前尚无特别有效的治疗方法,对其治疗方法的研究一直处于不断的探索之中。肝移植因其能够较好地改善肝功能、延长肝硬化患者的生存期,已成为较理想的治疗选择,然而由于其治疗费用昂贵、供肝数量有限以及伦理道德等方面的问题,又限制了其在临床上的广泛应用,因此,探索和寻找一种经济易行、又无供求限制的新方法,已成为目前研究的热点。近年来,随着对干细胞研究的不断深入,特别是骨髓干细胞具有的多向分化潜能及超强的可塑性逐渐受到重视,部分领域在临床已得到初步应用,如心肌缺血、糖尿病,肝病等。基础研究表明骨髓干细胞在体外诱导培养时可转化为功能成熟的肝细胞,在活体实验中骨髓干细胞可在肝损伤和肝纤维化的环境内存留、生长和转化,改善肝功能。在目前肝病的干细胞治疗实验研究中大多采用大鼠四氯化碳(CCL4)诱导的肝硬化模型,其采用的异体干细胞移植与局部注射移植方法离临床实用有很大差别,而大动物的肝硬化模型尚不成熟。目前骨髓干细胞移植在肝脏疾病治疗的实验研究中,已经证明骨髓干细胞对肝损伤和重症肝病较好的治疗效果,但对肝硬化似乎不很理想,因此探索增强干细胞移植对肝硬化治疗效果的方法非常必要。本研究从中等动物兔肝硬化模型和肝损伤模型中,利用自体骨髓干细胞作为种子细胞,采用临床最方便、实用的经肝动脉途径的移植方法进行实验研究,通过对肝功能的跟踪检测和动物肝脏组织病理对照观察,来评价骨髓干细胞移植对肝损伤和肝硬化治疗的安全性及有效性。并用促肝细胞生长素作为干扰因素探索提高骨髓干细胞对肝硬化治疗效果的方法。
实验目的
探索应用四氯化碳制作兔急性肝损伤及肝硬化模型的方法,评价该方法的可行性和制模成功率。
材料与方法
雄性新西兰大白兔70只,分成3组:正常对照组(10只)、急性肝损伤模型组(15只)、肝硬化组(45只),急性肝损伤组每只每日皮下注射50%CCL4橄榄油溶液0.8ml/kg,连续注射4天,第7天检测肝功能及肝脏病理。肝硬化组给予皮下注射注射50%CCL4橄榄油溶液0.2ml/kg,每周2次,12周后检测肝功能及肝脏病理。对照组分两组分别按相同剂量和时间给予皮下注射橄榄油溶液。
实验结果
1.兔急性肝损伤模型组:在四氯化碳注射4天后,化验肝功能明显改变,肝酶活性显著升高,谷丙转氨酶由35.83±8.04u/l上升至331.00±29.15u/l,总胆红素由6.41±0.52umol/l上升至11.64±0.58 umol/l。病理可见大面积肝细胞肿胀、坏死、并可见脂肪变性、肝组织结构紊乱,炎性细胞浸润明显。模型制备成功率为100%。
2.肝硬化模型组:在四氯化碳注射12周后,肝功能检查部分指标变化明显,白蛋白由40.28±2.30g/l减少至27.46±1.76g/l。凝血酶原活动度由64.32%降低至28.28%。HE病理染色及Masson纤维染色显示肝组织出现明显的纤维增生和较典型的假小叶,肝细胞可见浊肿,并见部分炎性细胞浸润。模型制备成功率为55%。
实验结论
应用皮下注射CCL4的方法可成功地建立兔急性肝损伤和肝硬化动物模型。但肝硬化模型制作时间长,动物死亡率高。
实验目的
评价自体骨髓干细胞经肝动脉移植对兔急性肝损伤模型的治疗作用,并观察促肝细胞生长素在干细胞移植治疗中的作用。
材料和方法
新西兰雄性大白兔15只,每日皮下注射50%CCL4橄榄油溶液0.8ml/kg连续4天制作急性肝损伤模型。模型形成后将动物随机分为2个实验组和对照组(各5只),3天后实验组经兔胫骨抽取骨髓约5ml,采用密度梯度离心法分离骨髓单个核细胞(干细胞),制成5ml细胞悬液。动物麻醉后在透视下股动脉穿刺插管将分离的骨髓干细胞经肝动脉注入肝脏。实验2组在干细胞移植前后隔日静脉注射促肝细胞生长素(pHGF)2.0mg/kg,共持续20天。干细胞移植后2周、4周、8周分别抽血进行肝功能检测,8周后取兔肝脏组织标本进行病理学观察。
实验结果
连续注射CCL44天后,血清肝酶及总胆红素明显上升,病理显示肝组织结构紊乱,大量肝细胞坏死、水肿和脂肪变性,汇管区及周围有大量炎性细胞浸润。骨髓干细胞肝动脉移植后肝功能逐渐改善,至移植后8周,血清AST、ALT活性和TBIL含量显著低于对照组(P＜0.05),而TP、ALB含量明显高于对照组(P＜0.05),而且实验2组上述指标改变更明显,与对照组和实验1组之间均有明显差异。
病理组织学观察实验组肝脏组织结构清楚,细胞排列规则,二组之间无明显差别,而对照组有纤维组织增生和肝细胞变性。免疫组化染色发现肝组织内散在分布有不同数量的CD34+肝样细胞,并且这些肝细胞在各组数量差异较大,对照组每高倍镜视野约1～2个,干细胞移植组3～5个,移植+pHGF组6～8个。
实验结论
1.自体骨髓干细胞经肝动脉移植对兔急性肝损伤模型有明显治疗作用,可改善急性肝损伤动物的肝功能,并促进肝组织结构重建。
2.促肝细胞生长素(pHGF)能保护肝细胞,促进干细胞在肝脏内的归巢和分化,加速肝组织的修复和肝功能的改善。
实验目的
评价自体骨髓干细胞经肝动脉移植对兔肝硬化模型的治疗作用,并比较促肝细胞生长素在干细胞移植治疗中的协同作用,为干细胞移植的临床应用提供实验研究基础。
实验方法
用皮下注射四氯化碳(CCl4)橄榄油溶液的方法复制兔肝硬化模型,模型动物分为实验1组、实验2组和对照组。在无菌条件下从兔胫骨上端抽取骨髓4～5ml,采用密度梯度离心法分离纯化骨髓干细胞,然后在透视下将分离的骨髓干细胞经肝动脉移植于肝脏。术后实验2组隔日静脉内注射pHGF2mg/kg共20天,然后在移植后4、8、12周分别检测肝功能,12周行肝脏病理学检查。实验数据资料采用SPSS11.5统计软件处理,P＜0.05为差异有显著性。
实验结果
在干细胞移植12周后,动物谷丙转氨酶(ALT)、谷草转氨酶(AST)逐渐降低,;总蛋白(TP)、白蛋白(ALB)逐渐升高,部分凝血酶原时间(APTT)逐渐上升,但这些改变与对照组相比无差异显著性(P＞0.05)。而移植+pHGF组肝功能指标改变较移植组迅速,与对照组相比有明显差异(P＜0.05)。
病理学检查显示干细胞移植组肝细胞形态较规整,无明显水肿和变性,假小叶结构仍存在,但肝组织纤维化程度较对照组明显,形态上有较明显的差异。免疫组化染色发现肝组织内散在分布有CD34+肝样细胞,并且这些肝细胞在各组数量差异较大,对照组每高倍镜视野约2～3个,干细胞移植组3～6个,移植+pHGF组6～9个。
实验结论
1、自体骨髓干细胞移植对肝硬化有一定的治疗作用。
2、促肝细胞生长素可促进干细胞在肝脏的归巢与分化,与骨髓干细胞移植有协同治疗作用。
Preface
Cirrhosis is a final stage of various kinds of liver disases,which treatment is always explored for many years.Liver transplantation is a better method to cirrhosis,but owning to the problem of its expansive cost,limited donator and ethics,its extensive application is also limited.thus,to look for a simple new way to cirrhosis is a aim of study. Recently,as research of stem cell progress,application of marrow stem cells in clinic is gradually payed close attention.Basis studys founded marrow stem cells may differentiate into function liver cells in vitro,and it can grow and differentiate within surroundings of hepatic injury or fibrosis in vivo.meantime it can improve alnormal liver function.Many cirrhosis models were induced by injection carbon tetrachloride in rats,which has great difference to clinical application,but big animal cirrhosis model is immaturity.Now bone marrow stem cells have been applicated in animal experiment and clinic,such as myocardial ischemia,diabetes and liver diease et al.Study founded bone marrow stem cells transplantion has a better effect to hepatic injury and severe liver dieases,but it seems not satisfactory to cirrhosis.It is very necessary to search for a new method enhancing its effect.Our project is that autologous bone marrow stem cells were transplanted through the hepatic artery in rubbit's hepatic injury and cirrhosis model.and the safety and efficacy of autologous bone marrow stem cells transplantion were evaluated by detecting liver function and pathohistology,Besides,the affect of hepatocyte growth-promoting factor to stem cell transplantation to cirrhosis was observed.
Part 1
The Development of Carbon Tetrachloride-Induced Hepatic injury and Cirrhotic Model in Rabbits
Objective
To search for a method of carbon tetrachloride-induced Hepatic injury and cirrhotic model in rabbit,and evaluate the feasibility and achievement ratio of the models through this method.
Methods
70 Male New Zealand white rabbits were divided into 3 group.The rabbits were subcutaneously injected with carbon tetrachloride once per day for 4 days in acute Hepatic injury model group,and liver pathology and function ware examined.Meanwhile,The rabbits were subcutane- ously injected twice weekly in cirrhotic model group,and liver pathology and function ware examined 12 weeks after the injection.
Results
In acute Hepatic injury model group,after carbon tetrachloride administration,the enzyme activity was steped up.The number of ALT goes up from 35.83±8.04u/l to 331.00±29.15u/l,and TBIL goes from 6.41±0.52 umol/1 up to 11.64±0.58 umol/l。and the serious cellular necrosis was founded,and hepatic structure was alnormal.
However,in cirrhotic model group,the albumn(ALB) and thrombozyme activity(PTA) decreased evidently compared to normal control group.The number of ALB decreased from 40.28±2.30g/l to 27.46±1.76g/l.and PTA decrease from 6.41±0.52 umol/l up to 11.64±0.58 umol/l。In pathohistology,we founded that the fiber hyperplasy was evident and typical pseudo-lobule can be seen in the rabbit liver,
Conclusion
A successive method to induce rabbit Hepatic injury and liver cirrhosis was developed.But the mortality is high in cirrhotic model group.
Part 2
Experimental Study of Autologous Bone Marrow Stem Cells for the Tr eatment of Acute Hepatic Injury in Rabbit.
Objective
A rubbit model was used to evalute the feasibility of treating acute hepatic injury through transplanting autologous bone marrow stem cells via the hepatic artery.
Methods
Fifteen rubbits were induced to develop acute hepatic injury model by daily subcutaneous injection with 50%CCL4 0.8ml/kg for 4 days.Then the animals were random- ly divided into two experimental and control groups. In the experimental group,autologous bone marrow stem cells(1×10~8) were disassociated and transplanted into liver via the hepatic artery.Besides,in the second experimental group,the hepatocyte growth- promoting factor(pHGF) was given via vein injection with 2.0mg/kg every other day for 20 days.The change of liver functions had been monitored by biochemical methods in 2,4,8weeks and histopatho-logic examination at 8 weeks after transplantation.
Results
After CCL4 injection,hepatocyte necrosis was noted.An increase in AST,ALT and TBIL activity(P＜0.05) was also observed obviously.But the levels of TP,and ALB were not obviously changed(P＞0.05) in plasma. 8 weeks after stem cells transplantation,the activity of AST and ALT,and the levels of TBIL were lower than the control group(P＜0.05).and the levels of TP and ALB were higher than the control group(P＜0.05).Moreove,the above changes in the second experimental group were more obvious than that of the first experimental group(P＜0.05).
In pathohistology,we founded that liver tissue structure in the experi-mental group was more close to normal than that of the control group,and fiber hyperplasy and hepatic cell degeneration was founded in the control group.Besides,the number of hepatic CD34+ cells of liver tissue in the experimental group was much than that of the control group.The number of the CD34+ cells can be seen per high power field of vision is 1～2 cells in the control group.3～5 cells in the first experimental group,and 6～8cells in the second experimental group.
Conclusion
It is feasible that autologous bone marrow stem cells transplanted through the hepatic artery in the rubbit liver necrosis model,and the transplanted stem cells can regenerate in the acute injurey liver.
The hepatocyte growth-promoting factor can help to improve hepatic function.
Part 3
Treatment of chronic hepatic cirrhosis with autologous bone marrow stem cells transplantation
Objective
To evalute the feasibility of treatment for rubbit model with hepatic cirrhosis by transplantation of autologous bone marrow-derived stem cells via the hepatic artery.and the effect of hepatocyte growth factor for treatment of stem cells transplantation to liver cirrhosis.
Methods
Fourty-five rubbits were induced to develop acute hepatic injury model by daily subcutaneous injection with 50%CCL4 0.8ml/kg for 4 days.Then twenty-five animals were randomly divided into two exper- imental and control groups.In the experimental group,autologous bone marrow stem cells(1×10~8) were disassociated and transplanted into liver via the hepatic artery.Besides,in the second experimental group,the hepatocyte growth-promoting factor(pHGF) was given via vein injection with 2.0mg/kg every other day for 20 days.The change of liver functions had been monitored by biochemical methods in 4,8,12weeks and histopathologic examination at 12 weeks after transplantation..
Results
After transplantation of bone marrow stem cells,the liver function of patients improved.Eight weeks after transpl-anttation,the activity of AST and ALT,and the levels of TBIL were lower than the control group,and the levels of TPA and ALB were higher than the control group,but these changes have not obvious difference.However,the above changes in the second experimental group were more obvious than that of the first experimental group.And the difference between the second experimental group and control group was very significant(P＜0.05).
In pathohistology,we founded that hepatic cells in the experimental group was more close to normal,but cell degeneration was founded in the control group,the fiber hyperplasy and hepatic pseudo-lobule was lessevident than that of the control group.Besides,the number of hepatic CD34+ cells of liver tissue in the experimental group was much than that of the control group.The number of the CD34+ cells can be seen per high power field of vision is 2～3 cells in the control group.3～6 cells in the first experimental group,and 6～9cells in the second experimental group.
Conclusion
It is effective that autologous bone marrow stem cells transplanted via the hepatic artery for the rubbit liver injury model,and the transpl-anted stem cells can regenerate in the acute injurey liver.
The hepatocyte growth-promoting factor can help to improve hepatic function.
引文
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10 McKinney Freeman SL,Jackson KA,Camargo FD,et al.Muscle derived hematopoietic stem cells are hematopoietic in origin[J].Proc Natl Acad Sci USA,2002,99:1341-1346.
11 Petersen B E,Bowen WC,Patrene KD,et al.Bone marrow as a potential source of hepatic oval cells[J].Science,1999,284(5417):1168-1170
12 Alison M R,Poulsom R,Jeffery R,et al.Hepatocytes from nonhepatic adult stem cells[J].Nature,2000,406:257
13 Theise ND,Nimmakayalu M,Gardner R,et al.Liver from bone marrow in humans [J].Hepatology,2000,32(1):11-16
14 Theise ND,Badve S,Saxena R,et al.Derivation of hepatocytes from bone marrow cells in mice after radiation induced myelablation.Hepatology,2000,31(1):235-240.
15 Kang XQ,Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes.World Journal Of Gastroenterology:2005,11(47),7461-5;
16李文晰,段芳龄人骨髓单个核细胞向肝细胞诱导分化的体外研究。胃肠病学和肝脏病学杂志,2004,12(2):144-147
17万荣,聂广 骨髓干细胞:肝细胞移植的新来源 中国中西医结合消化杂志 2005,13(5):345-347
18吴理茂,李连达,刘红,等.自体骨髓干细胞移植与归元方联用治疗急慢性肝损伤的实验研究[J].中国工程科学,2004,6(7):34-42
19 Kollet O,Shivtiel S,Chen Y Q,et al.HGF,SDF-1,and MMP-9are involved in stress2induced human CD34 + stem cell recruitment to the liver[J].J Clin Invest,2003,112(1):160-169
20 Lagasse E,Connors H,A12Dhalimy M,et al.Purified hematopoietic stem cells can differentiate into hepatocytes in vivo[J].Nat Med,2000,6(11):1229-1234
21潘兴华,庞荣清,陈系古等 猕猴自体骨髓干细胞移植治疗急性肝损伤的实验研究 热带医学杂志2006,6(3):247-250
22王豪勋 马 军段芳龄等 骨髓间充质干细胞治疗肝硬化的动物实验研究 胃肠病学和肝病学杂志2006,15(4):365-368
23姚鹏,胡大,王帅等,自体骨髓干细胞移植治疗慢性重症肝病60例实用医学杂志2005,21(19):2143-2145
14张强:李京雨:徐力扬:经肝动脉骨髓干细胞移植治疗肝硬化的初步临床应用 中国介入影像与治疗学,2005,2(4):261-263
25朱晓玲:邢煜:时冰:李艳:自体骨髓干细胞肝移植1例世界华人消化杂志,2006,2:176-178
27 Zhao DC, Lei JX, Chen R et al. Bone marrow-derived mesenchymal stem cells protect against experimental liver fibrosis in rats. World J Gastroenterol. 2005 11(22):3431-40.
28 Abdel Aziz MT, Atta HM, Mahfouz S et al . Therapeutic potential of bone marrow-derived mesenchymal stem cells on experimental liver fibrosis. World J Gastroenterol. 2005 11(22):3425-30.
29 Isao Sakaida, Shuji Terai, Naoki Yamamoto , et al. Transplantation of bone marrow cells reduces CC14-induced liver fibrosis in mice. Stem cell 2005 14(32) :3842-49
30 Sakaida I, Terai S, Nishina H, et al. Development of cell therapy using autologous bone marrow cells for liver cirrhosis. Hepatology 2006, 24(23):3254-62
31 Mohamadnejad M, Alimoghaddam K, Mohyeddin-Bonab M et al. Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis. Hepatology 2005,23(21):3124-32
32 Terai S, Ishikawa T, Omori K, et al. Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Gastroenterology 2007,25 (14): 3324-30
33 Mohamadnejad M, Alimoghaddam K, Mohyeddin-Bonab M et al. Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis. Hepatology 2005,22(28):3862-71
34 Stuart J Forbes .Stem cell therapy for chronic liver disease—choosing the right tools for the job Gastroenterology 2007,24 (18): 3621—29
35 Zhao DC, Lei JX, Chen R, et al. Bone marrow-derived mesenchymal stem cells protect against experimental liver fibrosis in rats. World J Gastroenterol. 2005 11(22): 3445-52.
36 Jan Schulte am Esch, IIa, Wolfram Trudo Knoefela, et al. Portal Application of Autologous CD133+ Bone Marrow Cells to the Liver: A Novel Concept to Support Hepatic Regeneration Gastroenterology 2006,23 (11): 2321—28
37 Gunter Furst, MD, Jan Schulte am Esch, MD, et al. Portal Vein Embolization and Autologous CD133+ Bone Marrow Stem Cells for Liver Regeneration: Initial Experience. Hepatology 2005,22(23):3712-18
38 Stefania Lorenzini, Pietro Andreone . Stem Cell Therapy for Human Liver Cirrhosis: A Cautious Analysis of the Resu Its . Hepatology 2005, 22(23):3768-78
39 Tian-Xue Fana, Hiroko Hishaa, b, Tie-Nan Jina, et al. Successful Allogeneic Bone Marrow Transplantation (BMT) by Injection of Bone Marrow Cells via Portal Vein:Stromal Cells as BMT-Facilitating Cells. World J Gastroenterol. 2005 10(12):2334-42.
40 Li-Mao Wu, Lian-Da Li, Hong Liu, etal. Effects of Guiyuanfang and autologous transplantation of bone marrow stem cells on rats with liver fibrosis. World J Gastroenterol 2005 February 28;11(8):1155-1160.
41 Mehdi Mohamadnejad, Mehrnaz Namiri, Mohamad Bagheri, et al. Phase 1 human trial of autologous bone marrow-hematopoietic stem cell transplant- tation in patients with decompensated cirrhosis . World J Gastroenterol 2006,11(10):1253-61.
42 Dong-Chang Zhao, Jun-Xia Lei, Rui Chen, et al. Bone marrow-derived mesenchymal stem cells protect against experimental liver fibrosis in rats. World J Gastroenterol 2006,13(12):1355-64.
43 Shuji Teraia, Tsuyoshi Ishikawaa, Kaoru Omoria, et al. Improved Liver Function in Patients with Liver Cirrhosis After Autologous Bone Marrow Cell Infusion Therapy.Stem cell 2005 14(32):3866-77
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5李连达,吴理茂,刘红 自体骨髓干细胞移植研究进展中国工程科学2004年8月
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17 Petersen B E,Bowen W C,Patrene K D,et al.Bone marrow as a potential source of hepatic oval cells[J].Science,1999,284(5417):1168-1170
18 Alison M R,Poulsom R,Jeffery R,et al.Hepatocytes from nonhepatic adult stem cells[J].Nature,2000,406:257
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25 Kang XQ,Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes.World Journal Of Gastroenterology:2005,11(47),7461-5;
26李文晰,段芳龄 人骨髓单个核细胞向肝细胞诱导分化的体外研究。胃肠病学和肝脏病学杂 志,2004,12(2):144-147
27万荣,聂广 骨髓干细胞:肝细胞移植的新来源 中国中西医结合消化杂志2005,13(5):345-347
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29吴理茂,李连达,刘红,等.自体骨髓干细胞移植与归元方联用治疗急慢性肝损伤的实验研究[J].中国工程科学,2004,6(7):34-42
30潘兴华1,2,庞荣清2,陈系古等猕猴自体骨髓干细胞移植治疗急性肝损伤的实验研究 热带医学杂志2006,6(3):247-250
31王豪勋马军段芳龄骨髓间充质干细胞治疗肝硬化的动物实验研究胃肠病学和肝病学杂志2006,15(4):365-368
32姚鹏,胡大,王帅等,自体骨髓干细胞移植治疗慢性重症肝病60例实用医学杂志2005,21(19):2143-2145
33朱晓玲;邢煜;时冰;李艳:自体骨髓干细胞肝移植1例世界华人消化杂志,2006,2:176-178
34张强;李京雨;徐力扬;经肝动脉骨髓干细胞移植治疗肝硬化的初步临床应用中国介入影像与治疗学,2005,2(4):261-263
35 Zhao DC,Lei JX,Chen R et al.Bone marrow-derived mesenchymal stem cells protect against experimental liver fibrosis in rats.World J Gastroenterol.200511(22):3431-40.
36 Abdel Aziz MT,Atta HM,Mahfouz S et al.Therapeutic potential of bone marrow-derived mesenchymal stem cells on experimental liver fibrosis.World J Gastroenterol.2005 11(22):3425-30.
37 Isao Sakaida,Shuji Terai,Naoki Yamamoto,et al.Transplantation of bone marrow cells reduces CC14-induced liver fibrosis in mice.Stem cell 2005 14(32):3842-49
38 Sakaida I,Terai S,Nishina H,et al.Development of cell therapy using autologous bone marrow cells for liver cirrhosis.Hepatology 2006,24(23):3254-62
39 Mohamadnejad M,Alimoghaddam K,Mohyeddin-Bonab M et al.Phase 1 trial of autologous bone marrow mesenchyraal stem cell transplantation in patients with decompensated liver cirrhosis. Hepatology 2005, 23(21):3124-32
40 Terai S, Ishikawa T, Oraori K, et al. Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Gastroenterology 2007,25 (14): 3324-30
41 Mohamadnejad M, Alimoghaddam K, Mohyeddin-Bonab M et al. Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis. Hepatology 2005,22(28):3862-71
42 Stuart J Forbes .Stem cell therapy for chronic liver disease—choosing the right tools for the job Gastroenterology 2007,24 (18): 3621—29
43 Zhao DC, Lei JX, Chen R, et al. Bone marrow-derived mesenchymal stem cells protect against experimental liver fibrosis in rats. World J Gastroenterol. 2005 11(22):3445-52.
44 Jan Schulte am Esch, IIa, Wolfram Trudo Knoefela, et al. Portal Application of Autologous CD133+ Bone Marrow Cells to the Liver: A Novel Concept to Support Hepatic Regeneration Gastroenterology 2006,23 (11): 2321-28
45 Gunter Furst, MD, Jan Schulte am Esch, MD,et al. Portal Vein Embolization and Autologous CD133+ Bone Marrow Stem Cells for Liver Regeneration: Initial Experience. Hepatology 2005,22(23):3712-18
46 Stefania Lorenzini, Pietro Andreone .Stem Cell Therapy for Human Liver Cirrhosis:A Cautious Analysis of the Resu Its . Hepatology 2005, 22(23):3768-78
47 Tian-Xue Fana, Hiroko Hishaa, b, Tie-Nan Jina, et al. Successful Allogeneic Bone Marrow Transplantation (BMT) by Injection of Bone Marrow Cells via Portal Vein:Stromal Cells as BMT-Facilitating Cells. World J Gastroenterol. 2005 10(12):2334-42.
48 Li-Mao Wu, Lian-Da Li, Hong Liu, etal. Effects of Guiyuanfang and autologous transplantation of bone marrow stem cells on rats with liver fibrosis. World J Gastroenterol 2005 February 28;11(8):1155-1160.
49 Mehdi Mohamadnejad, Mehrnaz Namiri, Mohamad Bagheri, et al. Phase 1 human trial of autologous bone marrow-hematopoietic stem cell transplant- tation in patients with decompensated cirrhosis.World J Gastroenterol 2006,11(10):1253-61.
50 Dong-Chang Zhao,Jun-Xia Lei,Rui Chen,et al.Bone marrow-derived mesenchymal stem cells protect against experimental liver fibrosis in rats.World J Gastroenterol 2006,13(12):1355-64.
51 Shuji Teraia,Tsuyoshi Ishikawaa,Kaoru Omoria,et al.Improved Liver Function in Patients with Liver Cirrhosis After Autologous Bone Marrow Cell Infusion Therapy.Stem cell 2005 14(32):3866-77
52姚鹏,王帅,胡大荣等 肝动脉自体骨髓干细胞移植治疗失代偿期肝硬化30例。世界华人消化杂志2005,13(13):1639-1640
53穆丽雅,韩明子 骨髓干细胞分化为肝细胞的多种移植途径 世界华人消化杂志,2006,14(6):607-610
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56彭小东,王波,王天才等,四氯化碳皮下注射构建大鼠肝纤维化模型的研究,江西医学院学报,2005,45(2):5-6,10
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1 Avital I,Feraresso C,Aoki T,et al.Bone marrow2derived liver stem cell and mature hepatocyte engraftment in livers undergoing rejection.Surgery,2002,132(2):384-390.
2 Malhi H,Gupta S.Hepatocyte t ransplantation:new horizon and challenges.J Hepatobiliary Pancreat Surg,2001,8(1):40-50.
3 Li L,Teng GJ.Study on t he isolation,culture,cryopreservation,and thawing of rat hepatocyte in hepatocyte t ransplantation experimental research.Chin J Radiol (Chinese),2002,36(4):369-372.
4 Jiang Y,J ahagirdar BN,Reinhardt RL,et al.Pluripotency of mesenchymal stem cells derived f rom adult marrow.Nature,2002,418(6893):41-49.
5李连达,吴理茂,刘红 自体骨髓干细胞移植研究进展中国工程科学2004年8月
6孙明晖综述 自体骨髓干细胞治疗缺血性心脏病的研究进展 中国心血管杂志2005年4月第10卷第2期147-149
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8 Issarachai S,Priestley GV,Nakamoto B,et al.Cells with hemopoietic potential residing in muscleare it inerrant bone marrow derived cells[J].Exp Hemato,2002,30:366-373.
9 Kawada H,Ogawa M.Bone marrow origin of hematopoietic progenitors and stem cell sinmurine muscle[J].Blood,2001,98:2008-2013.
10 McKinney Freeman SL,Jackson KA,Camargo FD,et al.Muscle derived hematopoietic stem cells are hematopoietic in origin[J].Proc Natl Acad Sci USA,2002,99:1341-1346.
11 Petersen B E,Bowen WC,Patrene KD,et al.Bone marrow as a potential source of hepatic oval cells[J].Science,1999,284(5417):1168-1170
12 Alison M R,Poulsom R,Jeffery R,et al.Hepatocytes from nonhepatic adult stem cells[J].Nature,2000,406:257
13 Theise ND,Nimmakayalu M,Gardner R,et al.Liver from bone marrow in humans [J].Hepatology,2000,32(1):11-16
14 Theise ND,Badve S,Saxena R,et al.Derivation of hepatocytes from bone marrow cells in mice after radiation induced myelablation.Hepatology,2000,31(1):235-240.
15 Kang XQ,Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes.World Journal Of Gastroenterology:2005,11(47),7461-5;
16李文晰,段芳龄人骨髓单个核细胞向肝细胞诱导分化的体外研究。胃肠病学和肝脏病学杂志,2004,12(2):144-147
17万荣,聂广 骨髓干细胞:肝细胞移植的新来源 中国中西医结合消化杂志 2005,13(5):345-347
18吴理茂,李连达,刘红,等.自体骨髓干细胞移植与归元方联用治疗急慢性肝损伤的实验研究[J].中国工程科学,2004,6(7):34-42
19 Kollet O,Shivtiel S,Chen Y Q,et al.HGF,SDF-1,and MMP-9are involved in stress2induced human CD34 + stem cell recruitment to the liver[J].J Clin Invest,2003,112(1):160-169
20 Lagasse E,Connors H,A12Dhalimy M,et al.Purified hematopoietic stem cells can differentiate into hepatocytes in vivo[J].Nat Med,2000,6(11):1229-1234
21潘兴华,庞荣清,陈系古等 猕猴自体骨髓干细胞移植治疗急性肝损伤的实验研究 热带医学杂志2006,6(3):247-250
22王豪勋 马 军段芳龄等 骨髓间充质干细胞治疗肝硬化的动物实验研究 胃肠病学和肝病学杂志2006,15(4):365-368
23姚鹏,胡大,王帅等,自体骨髓干细胞移植治疗慢性重症肝病60例实用医学杂志2005,21(19):2143-2145
14张强:李京雨:徐力扬:经肝动脉骨髓干细胞移植治疗肝硬化的初步临床应用 中国介入影像与治疗学,2005,2(4):261-263
25朱晓玲:邢煜:时冰:李艳:自体骨髓干细胞肝移植1例世界华人消化杂志,2006,2:176-178
27 Zhao DC, Lei JX, Chen R et al. Bone marrow-derived mesenchymal stem cells protect against experimental liver fibrosis in rats. World J Gastroenterol. 2005 11(22):3431-40.
28 Abdel Aziz MT, Atta HM, Mahfouz S et al . Therapeutic potential of bone marrow-derived mesenchymal stem cells on experimental liver fibrosis. World J Gastroenterol. 2005 11(22):3425-30.
29 Isao Sakaida, Shuji Terai, Naoki Yamamoto , et al. Transplantation of bone marrow cells reduces CC14-induced liver fibrosis in mice. Stem cell 2005 14(32) :3842-49
30 Sakaida I, Terai S, Nishina H, et al. Development of cell therapy using autologous bone marrow cells for liver cirrhosis. Hepatology 2006, 24(23):3254-62
31 Mohamadnejad M, Alimoghaddam K, Mohyeddin-Bonab M et al. Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis. Hepatology 2005,23(21):3124-32
32 Terai S, Ishikawa T, Omori K, et al. Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Gastroenterology 2007,25 (14): 3324-30
33 Mohamadnejad M, Alimoghaddam K, Mohyeddin-Bonab M et al. Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis. Hepatology 2005,22(28):3862-71
34 Stuart J Forbes .Stem cell therapy for chronic liver disease—choosing the right tools for the job Gastroenterology 2007,24 (18): 3621—29
35 Zhao DC, Lei JX, Chen R, et al. Bone marrow-derived mesenchymal stem cells protect against experimental liver fibrosis in rats. World J Gastroenterol. 2005 11(22): 3445-52.
36 Jan Schulte am Esch, IIa, Wolfram Trudo Knoefela, et al. Portal Application of Autologous CD133+ Bone Marrow Cells to the Liver: A Novel Concept to Support Hepatic Regeneration Gastroenterology 2006,23 (11): 2321—28
37 Gunter Furst, MD, Jan Schulte am Esch, MD, et al. Portal Vein Embolization and Autologous CD133+ Bone Marrow Stem Cells for Liver Regeneration: Initial Experience. Hepatology 2005,22(23):3712-18
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