骨髓间充质干细胞移植在大鼠急性肝损伤组织中的定植能力

背景:目前对于移植后的骨髓间充质干细胞在肝组织中的分化途径以及能够在多大程度上修复损伤的肝细胞等问题,尚未达成统一共识.目的:观察经尾静脉移植的骨髓间充质干细胞在急性肝损伤大鼠肝组织中的定植分化情况.设计、时间及地点:细胞学体内对照观察,于2007-12/2008-06在兰州大学中心实验室地点完成.材料:清洁级6～8周龄雄性SD大鼠47只,取5只用于制各骨髓间充质干细胞,剩余42只随机分为3组:肝正常细胞移植组15只、肝损伤细胞移植组14只、肝损伤盐水对照组13只.方法:肝损伤细胞移植组、肝损伤盐水对照组大鼠通过腹腔注射D-氨基半乳糖建立急性肝损伤模型.造模后24 h,肝损伤细胞移植组、肝正常细胞移植组经尾静脉注入BrdU标记的第3代大鼠骨髓间充质干细胞悬液1 mL,含(1.5～2.0)×106个细胞:肝损伤盐水对照组大鼠同法注入等量生理盐水.主要观察指标:移植后肝功能恢复情况,肝脏免疫组织化学检测结果.结果:与造模后24 h比较,移植后2,3周肝正常细胞移植组、肝损伤盐水对照组血清谷丙转氨酶活性无明显变化(P>0.05);肝损伤细胞移植组血清谷丙转氨酶活性明显降低(P＜0.05),肝功能恢复较好.与肝正常细胞移植组比较,肝损伤细胞移植组Brdu+细胞数明显增多(P＜0.01),多分布在汇管区及中央静脉周围,但随着时间延长BrdU+细胞数逐渐减少.结论:经尾静脉移植的骨髓间充质干细胞可促进急性肝损伤大鼠的肝功能恢复,并能够在受损肝脏及正常肝脏中定植分化,且定植与分化的程度可能与肝脏损伤程度有关.     

人胚胎骨髓间充质干细胞大鼠脾脏移植后向肝脏的迁移与分化

目的:研究人胚胎骨髓间充质干细胞在大鼠体内移植后向肝脏迁移和在肝内的分化.方法:将人胚胎骨髓间充质干细胞移植到肝损伤合并自身肝细胞再生抑制大鼠的脾脏,术后分时段取肝脏进行免疫组化染色,了解人白蛋白(ALB)、人角蛋白8(CK8)、CK18、CK19的表达情况.结果:人胚胎骨髓间充质干细胞移植后10 d大鼠肝脏可见人CK8、CK18、CK19阳性细胞,移植后15 d大鼠肝脏组织内可见人ALB阳性细胞.移植后90 d仍有阳性细胞检出.结论:人胚胎骨髓间充质干细胞大鼠脾脏移植后能够向肝脏迁移并分化为人肝细胞.     

珍珠梅提取物对四氯化碳所致大鼠急性肝损伤的保护作用

目的：研究珍珠梅乙酸乙醇提取物对CCl4所致大鼠急性肝损伤的保护作用。方法：给动物ig给药，用CCl4造大鼠急性肝损伤模型，用比色分析法测定大鼠血清天冬氨酸转氨酶(aspartate rtansaminase,ALT)、丙氨酸转氨酶(alanine transaminase,AST)的活性；在大鼠血清、肝匀浆、肝线粒体测定超氧化歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione perioxidase,GSH)。结果：珍珠梅能显著降低因CCl4所致急性肝损伤大鼠血清ALT、AST的升高；明显回升CCl4所致肝损伤大鼠肝线粒体SDH活性的降低；对急性肝损伤大鼠血清、肝匀桨、肝线粒体SOD、GSH—PX的活性有明显的升高作用及降低MDA的含量。结论：珍珠梅乙酸乙酯提取物对CCl4所致大鼠急性肝损伤有保护作用。     

人羊膜间充质干细胞在大鼠受损肝组织中分化为肝细胞

背景：羊膜间充质干细胞在体外适当的诱导条件下可分化为肝细胞样细胞,而在受损肝脏原位是否可分化为肝细胞值得探讨。目的：观察人羊膜间充质干细胞在大鼠肝损伤原位植活及分化情况。方法：采用胰酶-胶原酶二酶消化法从羊膜组织中分离人羊膜间充质干细胞。采用腹腔注射D-氨基半乳糖建立大鼠肝损伤模型,随机分为2组,人羊膜间充质干细胞移植组造模后注射L-DMEM悬浮的人羊膜间充质干细胞悬液,对照组注射等量L-DMEM。结果与结论：①FCM分析结果显示,所分离的人羊膜间充质干细胞表达CD29、CD44和CD166;免疫荧光染色显示人羊膜间充质干细胞表达波形蛋白,不表达CK19。②与对照组比较,人羊膜间充质干细胞移植后肝病理无明显差异,均呈急性肝坏死病理改变。③免疫荧光双染色结果显示,人羊膜间充质干细胞移植后1周主要定植于肝小叶且表达CK19,至2周表达CK18,至3周表达Alb。结果表明,人羊膜间充质干细胞在大鼠受损肝组织中能被植活,且可分化为肝细胞,提示人羊膜间充质干细胞移植在临床肝病的治疗方面可能具有潜在应用价值。     

人羊膜间充质干细胞在大鼠受损肝组织中分化为肝细胞

背景:羊膜间充质干细胞在体外适当的诱导条件下可分化为肝细胞样细胞,而在受损肝脏原位是否可分化为肝细胞值得探讨。目的:观察人羊膜间充质干细胞在大鼠肝损伤原位植活及分化情况。方法:采用胰酶-胶原酶二酶消化法从羊膜组织中分离人羊膜间充质干细胞。采用腹腔注射D-氨基半乳糖建立大鼠肝损伤模型,随机分为2组,人羊膜间充质干细胞移植组造模后注射L-DMEM悬浮的人羊膜间充质干细胞悬液,对照组注射等量L-DMEM。结果与结论:①FCM分析结果显示,所分离的人羊膜间充质干细胞表达CD29、CD44和CD166;免疫荧光染色显示人羊膜间充质干细胞表达波形蛋白,不表达CK19。②与对照组比较,人羊膜间充质干细胞移植后肝病理无明显差异,均呈急性肝坏死病理改变。③免疫荧光双染色结果显示,人羊膜间充质干细胞移植后1周主要定植于肝小叶且表达CK19,至2周表达CK18,至3周表达Alb。结果表明,人羊膜间充质干细胞在大鼠受损肝组织中能被植活,且可分化为肝细胞,提示人羊膜间充质干细胞移植在临床肝病的治疗方面可能具有潜在应用价值。     

原代肝细胞的分离和移植

背景:肝细胞移植可以作为一种桥梁作用,帮助肝功能衰竭患者渡过肝衰期,并提高患者生存率和预后。目的:采用Seglen改良的原位灌注探讨SD大鼠原代肝细胞分离的影响因素和方法的改进,同时分析原代肝细胞治疗急性肝功能衰竭大鼠的疗效。方法:两步法分离大鼠肝细胞。D-氨基半乳糖诱导大鼠急性肝衰竭,24h后分2组:细胞移植组经脾脏移植约2×107个肝细胞;对照组经脾脏注射0.4mLHank’s液。观察不同时间点大鼠的生存率和血清中转氨酶、总胆红素变化情况、脾内白蛋白分泌作用及脾内肝细胞分布情况。结果与结论:大鼠肝细胞分离存活率达85%～95%。细胞移植组14d存活率(75%)显著高于对照组组(30%)(P=0.01),且肝功能改善情况明显优于对照组。移植30d后,脾内有肝细胞白蛋白绿色荧光信号;移植15d后,可以看到肝细胞在脾脏红髓中簇集在一起并定植。结果说明胶原酶、pH值、灌注液、灌注方法等均是影响肝细胞分离存活率的因素;经脾脏移植的肝细胞能提高急性肝衰竭大鼠的生存率和改善肝功能。     

核桃仁对大鼠CCl_4肝损伤的保护作用

目的:研究核桃仁的保肝作用。方法:给动物喂食给药,用CCl4制作大鼠急性肝损伤模型,用比色分析法测定大鼠血清天冬氨酸转氨酶(ALT)、丙氨酸转氨酶(AST)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性及丙二醛(MDA)的含量。结果:核桃仁显著降低因CCl4所致急性肝损伤大鼠血清ALT、AST的升高,对急性肝损伤大鼠血清SOD、GSH-Px的活性有明显的升高作用,并降低MDA的含量。结论:核桃仁对CCl4所致大鼠急性肝损伤有保护作用。     

核桃仁对大鼠CCl4肝损伤的保护作用

目的：研究核桃仁的保肝作用。方法：给动物喂食给药，用CCl4制作大鼠急性肝损伤模型，用比色分析法测定大鼠血清天冬氨酸转氨酶（ALT）、丙氨酸转氨酶（AST）、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）的活性及丙二醛（MDA）的含量。结果：核桃仁显著降低因CCl4所致急性肝损伤大鼠血清ALT、AST的升高，对急性肝损伤大鼠血清SOD、GSH-Px的活性有明显的升高作用，并降低MDA的含量。结论：核桃仁对CCl4所致大鼠急性肝损伤有保护作用。     

肝细胞生长因子和丹参对大鼠急性肝损伤保护作用的实验研究

作者观察了肝细胞生长因子（HGF）、丹参对DDl4所致大鼠急性肝损伤的影响，结果发现，HGF组、丹参组以及西药联用组对急性肝损伤大鼠的血清ALT、α-肿瘤坏死因子（TNF-α）、丙二醛（MDA）以及肝组织匀浆MDA、肝组织钙含量的升高等均有不同程度的降低作用，且能使CCl4所致降低的血清及肝组织匀浆SOD活力明显升高。光镜及电镜下观察，用药组肝细胞病变软CCl4组有明显改善。本文结果表明，HGF、丹参对CCl4所致大鼠急性肝损伤有良好的保护作用，且HGF、丹参联用效果优于单用．     

人羊膜上皮细胞移植急性肝损伤小鼠的定量效果分析

背景：目前已有较多关于人羊膜上皮细胞移植入动物体内的存活、迁徙及相关特性的初步研究,但其对移植效果的定量分析尚未见报道。目的：对脾内移植传代的人羊膜上皮细胞小鼠血清肝生化功能及人血白蛋白的定量分析。方法：40只裸小鼠随机分为4组,每组各10只。肝叶切除＋细胞移植2周组、肝叶切除＋细胞移植4周组、肝叶切除＋盐水组,行半肝叶切除,肝叶切除＋细胞移植组自脾下极移植密度为5×106传代的人羊膜上皮细胞约0.2mL,分别于移植后2周和4周采血;肝叶切除＋盐水组自脾下极注射生理盐水0.2mL;单纯细胞移植组：不行肝叶切除,自脾下极移植密度为5×106传代的人羊膜上皮细胞约0.2mL。检测其各组肝脾组织学、形态学的改变及各组血清谷丙转氨酶、谷草转氨酶、人血白蛋白的变化和人血白蛋白表达定量分析。结果与结论：人羊膜上皮细胞移植急性肝损伤小鼠4周后肝脾形态未见明显改变,组织学可检测到特异性细胞,血清谷丙转氨酶、谷草转氨酶、人血白蛋白有明显改善,血清中能检测到人血白蛋白且移植后4周较移植后2周有明显升高。因此,人羊膜上皮细胞移植入肝受损小鼠体内能存活超过4周且仍表达肝细胞样细胞的部分特性及功能,改善小鼠的肝功能,治疗小鼠急性肝损伤。     

Intrasplenic hepatocyte transplantation: Evaluation of DNA synthesis and proliferation in auxiliary transplanted cells

The value of isolated hepatocyte transplantation as a temporary support in acute hepatic failure remains controversial regarding the functional capacities of freshly isolated and transplanted hepatocytes. To evaluate the survival rate of intrasplenically transplanted liver cells and their response to a proliferation stimulus like partial hepatectomy, 3H-thymidine incorporation into hepatic and auxiliary transplanted hepatocyte DNA was determined. The survival rate of intrasplenically transplanted hepatocytes was evaluated by analyses of m-albumin-RNA within the splenic tissue and compared to the morphological findings. The histological results show a marked decrease (greater than x 100) of intrasplenically transplanted hepatocytes within 1 week after injection. The amount of surviving cells then remained constant for 3 months without any signs of proliferation. After partial hepatectomy a stimulation of hepatic regeneration was observed in the remaining liver tissue but not in auxiliary transplanted hepatocytes. M-albumin-RNA determination of auxiliary transplanted hepatocytes revealed a decrease of m-albumin-RNA concentration of greater than 100 times within 24 h after transplantation indicating early cell necrosis of the transplanted cells. Since intrasplenically transplanted hepatocytes underwent an early cell necrosis without any evidence for a directly postoperatively inducible cell proliferation, it is concluded that a sufficient metabolic support in acute hepatic failure cannot be taken into consideration.     

Transplanted fibroblast cell sheets promote migration of hepatic progenitor cells in the incised host liver in allogeneic rat model

Cell sheet engineering has been noted as a new and valuable approach in the tissue‐engineering field. The objective of this study was to explore a procedure to induce hepatic progenitor cells and biliary duct structures in the liver. Sprague–Dawley rat dermal fibroblast (DF) sheets were transplanted into the incised surface of the liver of F344 nude rats. In the control group, an incision was made without transplantation of the DF sheets. Bile duct (BD)‐like structures and immature hepatocyte‐like cells were observed in the DF sheet transplant sites. These BD‐like structures were cytokeratin‐8‐positive, while the hepatocyte‐like cells were both OV‐6‐positive and α‐fetoprotein‐positive as well. The proliferation and differentiation of liver progenitor cells were not influenced by hepatectomy. We also transplanted DF sheets transfected with a plasmid encoding the enhanced yellow fluorescent protein target to mitochondria (pEYFP–Mito) by electroporation, and found that the new structures were pEYFP–Mito‐negative. We observed new BD‐like structures and immature hepatocytes after transplantation of DF sheets onto incised liver surfaces, and clarified that the origin of these BD‐like structures and hepatocyte‐like cells was the recipient liver. The present study described an aspect of the hepatic differentiation process induced at the site of liver injury. Copyright © 2013 John Wiley & Sons, Ltd.     

Hepatic stellate cells contribute to progenitor cells and liver regeneration

Retinoid-storing hepatic stellate cells (HSCs) have recently been described as a liver-resident mesenchymal stem cell (MSC) population; however, it is not clear whether these cells contribute to liver regeneration or serve as a progenitor cell population with hepatobiliary characteristics. Here, we purified HSCs with retinoid-dependent fluorescence-activated cell sorting from eGFP-expressing rats and transplanted these GFP⁺ HSCs into wild-type (WT) rats that had undergone partial hepatectomy in the presence of 2-acetylaminofluorene (2AAF) or retrorsine, both of which are injury models that favor stem cell–based liver repair. Transplanted HSCs contributed to liver regeneration in host animals by forming mesenchymal tissue, progenitor cells, hepatocytes, and cholangiocytes and elevated direct bilirubin levels in blood sera of GUNN rats, indicating recovery from the hepatic bilirubin–handling defect in these animals. Transplanted HSCs engrafted within the bone marrow (BM) of host animals, and HSC-derived cells were isolated from BM and successfully retransplanted into new hosts with injured liver. Cultured HSCs transiently adopted an expression profile similar to that of progenitor cells during differentiation into bile acid–synthesizing and –transporting hepatocytes, suggesting that stellate cells represent a source of liver progenitor cells. This concept connects seemingly contradictory studies that favor either progenitor cells or MSCs as important players in stem cell–based liver regeneration.     

人脐血造血干/祖细胞能在鼠肝中生长

肝脏干／祖细胞的分化、再生以及它的生物特性是一个非常活跃的研究领域。本研究报告当人脐血细胞经静脉输注给肝脏受损伤的及免疫功能缺陷的小鼠后，采用流式细胞术和组织配型(HLA)的方法，结果发现，在输注9周后鼠肝脏细胞内还存在有人脐血细胞，即使在灌洗后肝脏细胞的贴壁细胞培养及细胞集落(CFU—GEMM)中，也可以发现人脐血细胞的存在。结论：证实了脐血造血干／祖细胞能长期在肝脏中生长。     

体外大鼠肝卵圆细胞的长期培养方法

背景：肝卵圆细胞是目前公认的成体肝干/祖细胞,但其体外长期培养时会不可避免地丢失干细胞的活性。目的：探索大鼠肝卵圆细胞体外长期培养的方法。方法：构建2-乙酰胺基芴/部分肝切除肝再生大鼠模型,通过酶消化和Percoll梯度离心分离纯化大鼠异质卵圆细胞并进行免疫染色鉴定,用含表皮生长因子、白血病抑制因子的培养基体外长期培养,后撤去表皮生长因子、白血病抑制因子,通过形态学的观察和分子标志物的检测判断其能否保持干/祖细胞活性。结果与结论：采用含表皮生长因子、白血病抑制因子的培养基体外培养卵圆细胞4个月后,大鼠异质卵圆细胞仍能表达肝细胞标志物ALB、胆管上皮细胞标志物CK-19,经不含表皮生长因子、白血病抑制因子的培养液继续培养后,卵圆细胞胎肝标志AFP表达量迅速下降。该研究结果表明大鼠肝卵圆细胞在表皮生长因子、白血病抑制因子等培养条件下可长期增殖并保持干细胞活性。     

CD166pos Subpopulation From Differentiated Human ES and iPS Cells Support Repair of Acute Lung Injury

Previous efforts to derive lung progenitor cells from human embryonic stem (hES) cells using embryoid body formation or stromal feeder cocultures had been limited by low efficiencies. Here, we report a step-wise differentiation method to drive both hES and induced pluripotent stem (iPS) cells toward the lung lineage. Our data demonstrated a 30% efficiency in generating lung epithelial cells (LECs) that expresses various distal lung markers. Further enrichment of lung progenitor cells using a stem cell marker, CD166 before transplantation into bleomycin-injured NOD/SCID mice resulted in enhanced survivability of mice and improved lung pulmonary functions. Immunohistochemistry of lung sections from surviving mice further confirmed the specific engraftment of transplanted cells in the damaged lung. These cells were shown to express surfactant protein C, a specific marker for distal lung progenitor in the alveoli. Our study has therefore demonstrated the proof-of-concept of using iPS cells for the repair of acute lung injury, demonstrating the potential usefulness of using patient''s own iPS cells to prevent immune rejection which arise from allogenic transplantation.     

内皮祖细胞移植对肺损伤炎症状态的影响

背景：内皮祖细胞在维持内皮系统功能及血管损伤后的修复中起重要作用,已广泛应用到心血管、下肢缺血、血管修复等多种疾病,但在炎症疾病及肺损伤中的研究较少。目的：观察内皮祖细胞移植对急性肺损伤/急性呼吸窘迫综合征肿瘤坏死因子α及白细胞介素10的影响,探讨细胞移植能否改善急性肺损伤/急性呼吸窘迫综合征的炎症状态。方法：同遗传背景SD大鼠30只随机均分为正常对照组、肺损伤组、细胞移植组。采用密度梯度离心法分离、培养SD大鼠的骨髓内皮祖细胞。肺损伤组、细胞移植组大鼠经尾静脉注射脂多糖建立急性肺损伤模型;正常对照组仅给予等量的磷酸盐缓冲溶液。造模半小时后,正常对照组、细胞移植组大鼠经尾静脉注入内皮祖细胞悬液;肺损伤组大鼠同法注入等量的磷酸盐缓冲溶液。结果与结论：与肺损伤组相比,细胞移植组中白细胞介素10的水平显著增加（P〈0.001）,肿瘤坏死因子α的表达下调,但差异无显著性意义（P〉0.05）。细胞移植促进白细胞介素10的表达,下调肿瘤坏死因子α的表达,能改善损伤肺组织的炎症状态。     

胚胎干细胞源性肝干细胞肝内移植对宿主肝功能的影响及安全性评估

背景：体外诱导胚胎干细胞分化为肝细胞已有不少成功的报道，但其体内移植后能否有效整合入宿主肝板、在肝内能否进一步生长分化并表达肝细胞功能以及成瘤的风险等情况目前还不清楚。 目的：应用治疗性肝再生模型进行胚胎干细胞源性肝干细胞肝内移植．观察其在肝组织替代、体内的生长分化及成瘤性情况。 设计：随机对照动物实验。 单位：中山大学附属第二医院小儿外科。 材料：选用BALB／c小鼠24只为受体，鼠龄6～8周，体质量20～352，雌雄不拘购自广州市实验动物中心。实验所用胚胎干细胞源性肝干细胞由作者所在课题组诱导胚胎干细胞分化而成。小鼠胚胎干细胞株E14由本院干细胞中心提供。 方法：实验于2006-07／2007-06在中山大学附属第二医院干细胞研究中心完成。将24只小鼠随机分为2组：肝再生模型＋干细胞移植组和肝切除＋干细胞移植组，每组12只。前组分两次按50mg／kg剂量腹腔内注射倒千里光碱（retrorsine），间隔2周，第2次注射4周后行70％肝部分切除制造肝损伤；然后经门静脉分别移植1×10^5羟基荧光素乙酰乙酸（CFDA-SE）荧光标记的细胞入小鼠肝内进行胚胎干细胞源性肝干细胞移植。后组在行70％肝部分切除制造肝损伤模型后进行胚胎干细胞源性肝干细胞移植。 主要观察指标：荧光显微镜下观察移植细胞组受体鼠肝脏内分布、整合与体内生长分化情况。2周后行白蛋白荧光免疫组化（双荧光染色）、血清白蛋白水平检测其功能状况。将胚胎干细胞源性肝干细胞注入治疗性肝再生小鼠肝内，将未分化的胚胎干细胞移植入小鼠腋区皮下作为对照，观察胚胎干细胞源性肝干细胞体内成瘤情况。 结果：①肝干细胞在受体鼠肝内生长情况：CFDASE标记的胚胎干细胞源性肝干细胞肝内移植1周，受体小鼠肝实质内可见散     

Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration

Recent evidence has contradicted the prevailing view that homeostasis and regeneration of the adult liver are mediated by self duplication of lineage-restricted hepatocytes and biliary epithelial cells. These new data suggest that liver progenitor cells do not function solely as a backup system in chronic liver injury; rather, they also produce hepatocytes after acute injury and are in fact the main source of new hepatocytes during normal hepatocyte turnover. In addition, other evidence suggests that hepatocytes are capable of lineage conversion, acting as precursors of biliary epithelial cells during biliary injury. To test these concepts, we generated a hepatocyte fate-tracing model based on timed and specific Cre recombinase expression and marker gene activation in all hepatocytes of adult Rosa26 reporter mice with an adenoassociated viral vector. We found that newly formed hepatocytes derived from preexisting hepatocytes in the normal liver and that liver progenitor cells contributed minimally to acute hepatocyte regeneration. Further, we found no evidence that biliary injury induced conversion of hepatocytes into biliary epithelial cells. These results therefore restore the previously prevailing paradigms of liver homeostasis and regeneration. In addition, our new vector system will be a valuable tool for timed, efficient, and specific loop out of floxed sequences in hepatocytes.