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。     

Origin of endothelial progenitors in human postnatal bone marrow

This study demonstrates that a CD34(-), vascular endothelial cadherin(-) (VE-cadherin(-)), AC133(+), and fetal liver kinase(+) (Flk1(+)) multipotent adult progenitor cell (MAPC) that copurifies with mesenchymal stem cells from postnatal human bone marrow (BM) is a progenitor for angioblasts. In vitro, MAPCs cultured with VEGF differentiate into CD34(+), VE-cadherin(+), Flk1(+) cells - a phenotype that would be expected for angioblasts. They subsequently differentiate into cells that express endothelial markers, function in vitro as mature endothelial cells, and contribute to neoangiogenesis in vivo during tumor angiogenesis and wound healing. This in vitro model of preangioblast-to-endothelium differentiation should prove very useful in studying commitment to the angioblast and beyond. In vivo, MAPCs can differentiate in response to local cues into endothelial cells that contribute to neoangiogenesis in tumors. Because MAPCs can be expanded in culture without obvious senescence for more than 80 population doublings, they may be an important source of endothelial cells for cellular pro- or anti-angiogenic therapies.     

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。     

Repopulation of athymic mouse liver by cryopreserved early human fetal hepatoblasts

Transplantation of hepatocytes is a promising alternative to liver transplantation for the treatment of severe liver diseases. However, this approach is hampered by the shortage of donor organs and intrinsic limitations of adult hepatocytes. To investigate whether most of the hurdles faced with adult hepatocytes could be surmounted by the use of human fetal hepatoblasts, we have developed a method to isolate, transduce, and cryopreserve hepatoblasts from human livers at an early stage of development (11-13 weeks of gestation). Cells were characterized in vitro for expression of specific markers, and in vivo for their proliferation and differentiation potential after transplantation into athymic mice. Most of the cells (80-90%) harbored a bipotent phenotype, expressing cytokeratins 8/18, albumin, and CK19. They proliferated spontaneously in culture and were efficiently transduced by a beta-galactosidase-expressing retrovirus (90%). After transplantation, cryopreserved cells engrafted into the liver of athymic mice and proliferated, resulting in up to 10% repopulation. Engrafted cells expressed markers of differentiated adult hepatocytes including albumin, alpha1-antitrypsin, cytochrome P450 3A4, and alpha-glutathione-S-transferase. When retrovirally transduced before transplantation they expressed the transgene in vivo. In summary, early human fetal hepatoblasts engraft, proliferate, and mature in athymic mouse liver, without conditioning the donor.     

人脐带间充质干细胞与肝细胞共培养可分化为肝样细胞

背景：文献报道,从骨髓与脐带中分离获得的间充质干细胞可在体外连续传代培养,仍保持干细胞的特性,并在多种细胞因子的＂鸡尾酒式＂诱导下分化为肝细胞样细胞。目的：进一步验证人脐带间充质干细胞在体外正常人肝细胞共培养体系下是否可分化为肝细胞并探讨其分化方法。方法：采用贴壁法,从脐带中分离培养间充质干细胞,流式细胞仪检测脐带间充质干细胞表面标志。人肝细胞LO2细胞与人脐带间充质干细胞建立共培养体系,不添加外源诱导因子,分别于第7,14,21天,通过RT-PCR法检测肝细胞特异标志物甲胎蛋白、白蛋白、人细胞角蛋白19mRNA的表达,糖原染色进行功能鉴定。结果与结论：从人脐带中可分离得到贴壁生长的间充质干细胞,其中CD29＋细胞比例为96.02%,CD105＋细胞比例为96.6%,CD34-细胞比例为99.65%,CD105＋CD29＋双阳性细胞比例为94.84%。与LO2细胞共培养后第7天仅有甲胎蛋白阳性表达;第14天表达白蛋白、人细胞角蛋白19,第21天时,LO2与人脐带间充质干细胞共培养组未出现甲胎蛋白表达;人细胞角蛋白19和白蛋白的表达比第14天略有增强。共培养21d后,糖原染色呈阳性。结果证实,无需额外添加外源诱导因子,脐带间充质干细胞可在人正常肝细胞共培养的微环境中,向正常肝细胞分化。     

异种肝前体细胞移植治疗急性肝损伤大鼠

背景：成体肝前体细胞可在受体肝脏内定植并分化为肝细胞。不过,异种肝前体细胞移植能否促进急性肝损伤的恢复,脾脏微环境能否促进移植物的存活和向肝细胞分化,尚没有研究。目的：评价异种肝前体细胞移植治疗急性肝损伤的作用;监测移植肝前体细胞在大鼠脾脏实质内的定植及向肝细胞的分化。方法：体外培养雄性小鼠来源的肝前体细胞系肝上皮样前体细胞。通过CCl4腹腔注射联合2/3肝切除构建急性肝损伤大鼠模型,进行肝上皮样前体细胞脾脏移植。在肝切除后1,5,14和21 d,苏木精-伊红染色观察肝脏病理改变,全自动生化分析仪监测血清转氨酶变化,PCR反应检测脾脏组织Y染色体特异性序列Sry,脾脏CK-19和Alb免疫组织化学追踪移植肝上皮样前体细胞的植入和肝细胞分化。结果与结论：肝上皮样前体细胞可在体外长期培养,保持增殖能力和双向分化潜能。肝上皮样前体细胞脾脏移植后,肝损伤大鼠肝细胞肿胀明显减轻,丙氨酸转氨酶和天门冬氨酸转氨酶下降更明显。移植后1,5,14和21 d,脾脏DNA中均能检测到Sry序列。在整个实验期间CK-19阳性细胞在大鼠脾脏实质内始终存在。Alb阳性细胞在移植后5 d在脾脏实质中出现,随后阳性细胞数逐渐增多。实验表明,移植肝前体细胞能在大鼠脾脏实质中植入,并分化为肝细胞,能有效促进CCl4腹腔注射联合2/3肝切除诱导的大鼠急性肝损伤的修复过程。     

脐带血单个核细胞体外培养分化为肝细胞的实验观察

目的探讨脐带血单个核细胞在体外定向分化为肝细胞的条件及能力,为建立脐血干细胞移植治疗慢性肝衰竭提供实验依据。方法采集脐带血分离单个核细胞后,在实验组加入成纤维细胞生长因子(FGF)及促肝细胞生长因子(HGF)、或FGF加胎肝上清液培养,对照组只加FGF培养,观察细胞生长分化状况,免疫组化检测两组人甲胎蛋白(AFP)和白蛋白(Alb)。结果实验组可见类圆形及多边形细胞,呈类肝细胞形态,对照组大多为梭形细胞,未见多边形细胞;实验组细胞AFP、Alb免疫组化染色为阳性,对照组未见AFP,Alb阳性细胞。HGF的诱导分化效果优于胎肝上清液。结论脐带血单个细胞在HGF或胎肝上清液的诱导下可以定向分化为能分泌Alb及AFP的类肝细胞。     

Rapid clearance of syngeneic transplanted hepatocytes following transduction with E-1-deleted adenovirus indicates early host immune responses and offers novel ways for studying viral vector, target cell and host interactions.

To distinguish between transduced cell clearance and transgene regulation following adenoviral gene transfer, we infected F344 rat hepatocytes with an E-1-deleted adenovirus (Ad beta gal) and studied cell survival in the liver of dipeptidyl peptidase IV-deficient (DPPIV-) F344 rats. Transplanted cells were localized with histochemical staining for DPPIV and transgene expression localized with staining for beta-galactosidase (lacZ). The transgene was expressed in 90-100% hepatocytes without impairment in cell viability in vitro, although transplanted cells were cleared mostly within 1 day by infiltrates containing activated macrophages, CD4+ or CD8+ lymphocytes, and phagocytes. When Ad beta gal-transduced hepatocytes were transplanted repeatedly at 14-day intervals, transplanted cells were cleared rapidly each time. LacZ expression following Ad beta gal administration to intact animals was associated with apoptosis and unscheduled DNA synthesis in the liver. To determine whether adenoviral antigen expression activated consequential MHC-restricted liver injury, we transplanted Ad beta gal-hepatocytes followed subsequently by transplantation of nontransduced hepatocytes. Transplanted cells expressing Ad beta gal were rapidly cleared as before, whereas nontransduced hepatocytes engrafted with progressive liver repopulation. The findings indicated that adenovirally transduced cells are cleared early in the host liver. Use of ex vivo strategies will facilitate analysis of modified adenoviral vectors in the context of immunoregulatory, cellular and viral mechanisms.     

干细胞诱导分化为肝细胞的鉴定方法

背景：目前关于干细胞体外诱导分化为肝细胞的各种方法较多,需要总结一套具有说服力的鉴定方法去验证。目的：总结干细胞经诱导分化为肝细胞鉴定方法的研究进展,使之更安全的应用。方法：应用计算机检索西方生物医学期刊及PubMed数据库1985-01/2010-06有关干细胞分离、培养及诱导分化为肝细胞的文章,检索词为＂stemcells,induced,differentiation,hepatocytes,identification＂等;同时检索中国期刊全文数据库2000-01/2010-06有关干细胞分离、培养诱导分化为肝细胞的文章,检索词为＂干细胞,诱导,分化,肝细胞,鉴定＂。共检索到文献43篇。结果与结论：干细胞具有可塑性特性,通过体外刺激因子、体内利用特定的微环境均可诱导干细胞分化为肝前体细胞和成熟肝细胞,理论上可提供丰富的肝细胞来源,为人们提供种子细胞,可以进行实验研究、临床应用。但是经诱导后的细胞是否为所预期的种子细胞需要一套科学的、系统的检测方法,根据肝细胞的诸多特性,如表面标志、合成、代谢功能可以证明。     

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～ 35 g,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50 mg/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1×105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:CFDA SE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在绿色荧光分布.2周后,肝实质内绿色荧光分布区域明显扩大,且可见类似肝索样结构排列.②肝功能:共焦白蛋白荧光免疫组化(双荧光染色)结果表明,受体小鼠肝组织内可见标记细胞表达白蛋白阳性信号(呈黄色荧光),肝再生模型 干细胞移植组和肝切除 干细胞移植组血清白蛋白水平则无明显差异(P > 0.05).③肝干细胞移植安全性:6周内未见畸胎瘤形成,而将未分化的胚胎干细胞移植入小鼠腋区皮下6周后则可见畸胎瘤形成.结论:胚胎干细胞源性肝干细胞移植入治疗性肝再生模型小鼠肝内后可有效在肝内能进一步生长分化并部分表达肝细胞功能;且此移植安全性较好.     

骨髓间充质干细胞及其向肝样细胞分化的潜能

背景：研究表明,在一定条件下骨髓间充质干细胞可诱导分化为肝样细胞,为治疗急性肝衰竭等终末期肝病提供了新的思路。目的：对骨髓间充质干细胞的发现、分离培养、诱导分化及应用前景等方面做一综述。方法：计算机检索中国期刊网全文数据库以及PubMed数据库1999至2014年期间有关骨髓间充质干细胞及其向肝样细胞分化的文章。检索词分别为“肝样细胞,骨髓间充质干细胞,细胞分化”和“hepatocyte-like cel s, bone marrow mesenchymal stem cel s,differentiation”。最后选择52篇文章纳入结果分析。结果与结论：目前,肝组织工程的首要问题是寻找性状稳定具有肝特异性功能的种子细胞,成熟肝细胞获取难度较大,并具有产生免疫排斥反应、体外培养困难等缺陷,严重限制了肝移植的开展。骨髓间充质干细胞具有多向分化、自我更新快、易于扩增及培养等优点,被认为是最有前途的细胞来源。已有研究表明骨髓间充质干细胞在体内外均可分化为肝细胞,并具有肝细胞的合成和分泌功能。但如何大量扩增此类细胞的同时又能保持其良好的分化潜能、体外培养的最佳条件、诱导分化机制和临床应用的安全性等尚需进一步研究和探讨。     

胚胎干细胞体外分化为功能性肝细胞过程中肝细胞生长因子诱导系统中瘀胆血清的促进作用

背景:目前各种诱导胚胎干细胞(ESC)分化为肝细胞的方法中,大多忽略了对分化细胞功能的诱导与鉴定。是否表达肝细胞功能应作为ESC向肝细胞分化的鉴定指标之一。目的:观察在肝细胞生长因子(HGF)体外诱导小鼠胚胎干细胞向肝细胞分化的体系中,瘀胆血清病理环境对分化细胞表达肝细胞功能的作用。设计:观察对比,体外细胞学实验。单位:中山大学附属第二医院肝胆外科。材料:实验于2004-10/2007-02在中山大学附属第二医院医学研究中心完成。小鼠E14ESC系由中山大学干细胞与组织工程中心提供;SD大鼠20只,鼠龄2周,购自中山大学动物实验中心。实验过程中对动物的处置符合动物伦理学要求。方法:对SD大鼠施以胆总管结扎切断手术,制作瘀胆模型,饲养10d后取全血制备瘀胆血清。用悬滴培养ESC发育5～7d的拟胚体,将其离散细胞种植于不同的分化体系,分别进行自主分化、20μg/L肝细胞生长因子、5%瘀胆血清 20μg/L肝细胞生长因子诱导分化。主要观察指标:①倒置显微镜下动态观察细胞形态变化。②分化4周时进行白蛋白、甲胎蛋白、CK18/19、糖原及吲哚氰绿和荧光二乙酯染色。③采用相应试剂盒每3天检测细胞合成白蛋白、三酰甘油及尿素氮功能。结果:①ESC自主分化难以控制,分化为3个胚层的细胞。肝细胞生长因子促进ESC向内脏内胚层和中胚层(心肌)分化,但两者仅能表达低水平的肝细胞特异性功能。②引入瘀胆血清的肝细胞生长因子诱导体系中ESC能分化为较为形态均一的多角形细胞,其糖原、吲哚氰绿和荧光二乙酯染色均为阳性;白蛋白、三酰甘油和尿素氮合成能力显著高于自发分化和肝细胞生长因子诱导结果(P<0.05～0.01)。结论:采用瘀胆血清体外模拟病理性微环境可促进HGF诱导的ESC源性肝细胞表达高水平的肝特异性代谢功能。     

人外周血单个核细胞向肝样细胞转化的体外诱导培养方法

背景：外周血单个核细胞在体外条件下可向肝样细胞转化。目的：探索体外诱导外周血单个核细胞向肝样细胞分化的培养方法。方法：采用密度梯度离心法联合贴壁法纯化肝硬化患者外周血单个核细胞,以含巨噬细胞集落刺激因子、白细胞介素3及β-巯基乙醇培养基培养6d后,诱导组用含肝细胞生长因子,成纤维细胞生长因子4的培养基培养14d;以未诱导培养的单个核细胞及L02肝脏细胞系为对照。结果与结论：外周血单个核细胞呈透明圆形、类圆形,用巨噬细胞集落刺激因子、白细胞介素3及β-巯基乙醇培养基培养6d后,部分细胞向成纤维样细胞生长,诱导后部分细胞呈多边形,多角形,14d后细胞形态逐渐接近肝细胞,并表达白蛋白、甲胎蛋白、角蛋白18。说明在一定的诱导条件下,外周血单个核细胞在体外可以向肝样细胞分化。     

诱导大鼠骨髓Thy-1+ β2 M-细胞分化为肝脏细胞

目的探讨定向诱导大鼠骨髓来源的Thy-1^ β1M^-细胞(bone marrow—derived Thy—1^ β2M^-cell,BDTC)分化为成熟的、有功能的肝脏细胞及其可能的机制：方法分别采用Transwell培养板联合培养BDTC和丙烯醇(allyl alcohol,AA)损伤的肝脏细胞,并用含有肝细胞生长因子和碱性成纤维细胞生长因子的条件诱导液单独培养BDTC,通过光镜和电镜观察BDTC的形态变化,免疫细胞化学和RT—PCR检测BDTC诱导前后肝细胞特异性基因的表达;用靛青绿(indocyanine green,ICG)摄取、排泌实验及白蛋白、尿素分泌检测肝细胞的相关功能。结果联合培养及单独以条件诱导液培养7d,部分BDTC体积明显增大,出现大而圆的单个或多个细胞核,胞浆富含线粒体、内质网和糖原颗粒等;这些细胞表达幼稚或成熟肝细胞特异性的AFP、OV-6、CK18等基因,并具有靛青绿摄取、排泌和分泌白蛋白、氨基代谢生成尿素等功能：结论大鼠BDTC在AA所致肝损伤环境中或在特定诱导体系内均可以向成熟的、有生理功能的肝脏细胞分化,而且该过程无需通过细胞融合而实现?     

Diphtheria toxin-murine granulocyte-macrophage colony-stimulating factor-induced hepatotoxicity is mediated by Kupffer cells

DT388GMCSF, a fusion toxin composed of the NH2-terminal region of diphtheria toxin (DT) fused to human granulocyte-macrophage colony-stimulating factor (GMCSF) has shown efficacy in the treatment of acute myeloid leukemia. However, the primary dose-limiting side effect is liver toxicity. We have reproduced liver toxicity in rats using the rodent cell-tropic DT-murine GMCSF (DT390mGMCSF). Serum aspartate aminotransferase and alanine aminotransferase were elevated 15- and 4-fold, respectively, in DT390mGMCSF-treated rats relative to controls. Histologic analysis revealed hepatocyte swelling; however, this did not lead to hepatic necrosis or overt histopathologic changes in the liver. Immunohistochemical staining showed apoptotic cells in the sinusoids, and depletion of cells expressing the monocyte/macrophage markers, ED1 and ED2, indicating that Kupffer cells (KC) are targets of DT390mGMCSF. In contrast, sinusoidal endothelial cells seemed intact. In vitro, DT390mGMCSF was directly cytotoxic to primary KC but not hepatocytes. Two related fusion toxins, DT388GMCSF, which targets the human GMCSF receptor, and DT390mIL-3, which targets the rodent IL-3 receptor, induced a less than 2-fold elevation in serum transaminases and did not deplete KC in vivo. In addition, DTU2mGMCSF, a modified form of DT390mGMCSF with enhanced tumor cell specificity, was not hepatotoxic and was significantly less toxic to KC in vivo and in vitro. These results show that DT390mGMCSF causes liver toxicity by targeting KC, and establish a model for studying how this leads to hepatocyte injury. Furthermore, alternative fusion toxins with potentially reduced hepatotoxicity are presented.     

The perturbation of apoptosis and mitosis by drugs and xenobiotics.

Drugs such as the barbiturate phenobarbitone and fibrate hypolipidaemic agents, in addition to a range of chemicals of environmental and industrial significance, are able to perturb rodent tissue homeostasis, leading to tissue enlargement. Many of these xenobiotics are rodent nongenotoxic carcinogens since they do not damage DNA, yet cause tumours in the rat and mouse. These nongenotoxic carcinogens display both species and tissue specificity; for example, rat and mouse hepatocytes display S-phase induction and a suppression of apoptosis in response to drugs such as phenobarbitone or the hypolipidaemic peroxisome proliferators (PPs). In contrast, human hepatocytes or other types of rodent cells are refractory to these effects. However, in the absence of a discrete mechanism of action, the clear species differences preclude extrapolation of rodent data to provide an accurate human risk assessment. Recent data have demonstrated that PPs activate the PP-activated receptor alpha in rodent liver, leading to enzyme induction, stimulation of S-phase, and a suppression of apoptosis. How these acute effects may lead to hepatocarcinogenesis and the relevance of this for humans will be discussed.     

骨髓间充质干细胞体内分化为肾小管上皮细胞的实验研究

目的探讨骨髓间充质干细胞(mesenchymal stem cells,MSCs)能否在体内分化为肾小管上皮细胞。方法取SD雄性大鼠胫、股骨骨髓,密度梯度离心法分离MSCs,采用4,6-联脒-2-苯基吲哚(4,6-diamidino-2-phenylindole,DAPI)进行标记。32只雌性SD大鼠复制缺血再灌注肾损伤模型后随机分为移植组和对照组,移植组于缺血45min后经下腔静脉注入用DAPI标记的MSCs,对照组注入等量的生理盐水。分别于术后1d、2d、3d、4d处死大鼠,留取肾组织,荧光显微镜观察移植的MSCs在肾组织中的分布,采用能与肾小管内腔壁特异结合的蓖麻血凝素(Ricinus communis agglurinin,RCA)对迁移入肾脏的MSCs分化状况进行鉴定。结果移植组术后第三天肾组织内可见DAPI标记细胞,第四天DAPI标记细胞明显增多(P<0.05),且多数DAPI标记细胞能结合RCA。对照组没有发现DAPI标记细胞。结论移植的外源性MSCs能够迁移、定居于肾组织中并分化为肾小管上皮细胞。     

鼠骨髓间充质干细胞体外诱导分化肝干细胞的研究

目的:探讨体外诱导大鼠骨髓间充质干细胞(rat bone marrow stromal cells,rBMSCs)定向分化为肝干细胞的可行性。方法:采用全骨髓培养法体外培养rBMSCs,设实验组及对照组,应用肝细胞生长因子(HGF)诱导分化,采用倒置相差显微镜观察细胞形态变化,免疫组化检测CK18、CK19及波形蛋白Vimentin;ELASA等方法检测甲胎蛋白,白蛋白及碱性磷酸酶。结果:rBMSCs经体外培养诱导后呈多角形、卵圆形改变,白蛋白,AFP,CK18、CK19表达阳性,碱性磷酸酶出现明显改变。结论:rBMSCs体外经HGF诱导下,具有向肝干细胞分化的能力,可成为肝组织工程主要种子细胞来源。