人胎儿间充质干细胞体外分离培养及诱导分化为类肝细胞的实验研究
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摘要
背景
近年来,干细胞因其作为生物医学组织工程技术和治疗性克隆技术的种子细胞,为实现人类健康长寿的梦想带来了新的希望,而成为生物医学领域研究的热点。根据干细胞的分化阶段,分为胚胎干细胞(embryo stem cells,ESCs)和成体干细胞(adult stem cells,ASCs)。目前异体来源的ESCs在临床应用上还需要攻克细胞分化机制、免疫排斥等问题,而治疗性克隆获得的ESCs虽可以解决免疫排斥问题,但其技术复杂、离分化完全的功能细胞距离较远。ASCs存在于成体组织中,具有来源丰富、可塑性强、离成熟细胞近和多向分化潜能等特点,而且可以实现个体化治疗,无免疫排斥问题,因此是最具有临床应用优势的种子细胞。目前已在成人骨髓、软骨、牙髓、血液、神经、肌肉、脂肪、皮肤、角膜缘、肝脏、胰岛等多种成体组织中发现了干细胞。现已证实成体干细胞有多向分化潜能,还可以横向分化为不同类型的特化细胞甚至发生逆分化为更原始的干细胞。
骨髓间充质干细胞(marrow mesenchymal stem cells,MMSCs)作为ASCs中最易获得的细胞,具有向多种中胚层和神经外胚层甚至内胚层来源的组织细胞分化的能力,这些细胞包括肌细胞、成骨细胞、软骨细胞、成纤维细胞、皮肤细胞、神经细胞、脂肪细胞等。大量研究显示MMSCs或者造血干细胞(hematopoietic stem cells,HSCs)可在人为诱发肝脏病变后实验鼠体内分化为肝细胞,重建肝脏,使病鼠肝脏功能恢复正常。同时多家研究者在体外利用小鼠骨髓细胞或来自骨髓的成体多潜能干细胞在不同条件下分化为类肝细胞。MSCs具有可反复多次采集和自体移植或应用而不出现免疫排斥反应的优势。如果能够利用易于获得的MSCs在体外诱导分化为成熟肝细胞,将为肝细胞移植及生物人工肝提供大量的细胞来源。
中国肝病患者众多,对肝硬化、肝坏死或遗传性肝脏疾病的病变部位实行细胞替代或基因转移治疗是新发展起来并极有前景的肝脏病学治疗策略。适合肝脏疾病细胞治疗的供体细胞应具有以下特点:①易于获得、培养、增殖,或建立细胞系;②能定向分化
国家高技术研究发展计划(863)资助项目(人肝细胞扩增分化及人源化血细胞研究,No.2001AA216161)
为所需的肝细胞或胆管上皮细胞;③移植入体内能长期存活;④不诱导免疫排斥反应,对宿主无损害;⑤如果用于基因治疗,目的基因可高效转染供体细胞。MMSCs具备上述大部分条件,因此引起肝病研究者的高度关注。如果利用人自身骨髓间充质干细胞横向分化为肝细胞则为肝脏疾病的细胞治疗提供了另一个选择。
虽然ASCs的研究已经取得较大进展,但在临床上应用还有许多问题,目前的多数实验结果来自动物体内实验,而且许多是群体细胞研究,并未在克隆细胞水平得到证实。人ASCs的特性是否与动物一致,体外结果是否与体内一致还不知道。更重要的是ASCs的分化潜能到底有多大及分化的分子机制还不十分清楚。ASCs在临床上应用还存在体外分离、扩增、诱导及体内移植后增殖调控等技术问题。MSCs体内移植尚不能达到大规模增殖,体外向肝细胞转化尚未见报道。ASCs的应用研究刚刚起步,但现代生物技术在ASCs相关研究中的应用必将加快其在临床移植修复治疗中的应用进程。
因此我们尝试利用人胎儿MMSCs在体外向类肝细胞诱导,观察人胎儿MMSCs的特性,期望在成体干细胞横向分化中作一些基础研究工作。尝试为临床肝病治疗中肝细胞来源提供新的可行性途径。
目的
1. 建立人胎儿骨髓、脐带血和肝脏间充质干细胞(mesenchymal stem cells,MSCs)的分离培养方法,观察细胞生物学特性和细胞表型,获得大量的人源性MSCs。
2. 人胎儿MMSCs体外向类肝细胞诱导分化的条件建立及优化。
3. 人胎儿MMSCs来源类肝细胞的分子生物学鉴定。
4. 人胎儿MMSCs来源类肝细胞的功能和超微结构评估。
方法
1. 共收集12个人胎儿,平均孕周为18.5±4.6周,引产后4℃低温保存。收集15份脐带血。采用体外细胞培养技术,分离培养人胎儿骨髓、脐带血和肝非实质细中MSCs,并研究其增殖及生长特征。
2. 采用体外分离培养的MMSCs为研究对象,设计不同的诱导分化条件进行体外诱导,观察细胞诱导分化过程中形态结构变化及细胞生物学特性,应用免疫组化检测肝细胞标志ALB和CK18的表达,建立人胎儿MMSCs向类肝细胞分化的优化条件。
3. 收集诱导培养第4d、7d、14d、21d、28d时的细胞爬片,SABC免疫组化法DAB显色检测肝细胞早期标志AFP、CK19及早期转录因子GATA4,成熟肝细胞标志ALB、CK18、GST-π、肝细胞转录因子HNF1α;提取诱导分化第10d和第28d细胞RNA及蛋白质,设计AFP、CK19、ALB、CK18、CYP1B1、CYP2B6引物,进行RT-PCR,观
察在mRNA水平肝细胞标志的表达,采用Western-Blot检测CK18、AFP、ALB的表达量。
4. 以未诱导的MMSCs作对照,对诱导4d、7d、14d、21d、28d MMSCs采用ELISA法检测培养上清中人白蛋白水平,脲酶法检测合成尿素的能力,过碘酸希夫试验进行糖原染色,电镜观察细胞超微结构。
结果
1. 0.5～2h内尽快分离骨髓,24h换液后可获得约300±80个贴壁细胞,5个细胞以上的集落为15±6个;培养细胞在种植后1～3d为生长滞留期,第4d达到对数生长期,以后进入到平台期,细胞分裂指数曲线的趋势与生长曲线基本类似,在达到对数生长期后,分裂相?
Background
In .the last few years, as seeds cells of biological medicine tissue engineering technique and therapeutic cloning technique, stem cell brought the new hope to dream about human health and longevial, became the hot spot of biological medicine research. Stem cells were divided into embryo stem cells (ESCs) and adult stem cells (ASCs) according to the stage differentiation. Up to now, problems of the cell differentiation mechanism and immunological rejection of xenogenic resource ESCs meeted during clinical application were still not resolved. Although the ESCs obtained from therapeutic cloning technique could resolve the immunological rejection, but the technique was very complicated, and the functions of the ESCs were much different from the function cell that differentiated completely. ASCs resided in adult tissue, had characteristics of abundance resource, powerful plasticity, recent to mature cell and multiple differentiation potentiality, could conduct individualize treatment, had not immunological rejection problem, so that it was the seeds cells for clinical application prevalently. Stem cells had been discovered in many adult tissues such as adult marrow, cartilage, tooth pulp, blood, nerve, muscle, fat, skin, limbus of cornea, liver, islands of pancreas. The multiple differentiation potentiality of ASCs had been confirmed. ASCs could trans-differentiate different type special cells, even retro-differentiate furthermore initial stem cells.
Marrow mesenchymal stem cells (MMSCs) was a kind of stem cell that obtained easily, had the potential capability to differentiate to mesoblastema and neuroepidermal, even endoblastic layer tissue cells, such as muscle cell (myocyte), skeletogenous cell, cartilage cell, fibroblast, skin cell, nerve cell and lipocyte. A great quantity researches showed that MMSCs or hematopoietic stem cells (HSCs) could differentiate to hepatocyte of experimental rat that
This work was supported by the 863 program of China (Research of human hepatocyte amplification differentiation and humanized blood cell No.2001AA216161)
was induced with liver pathological changes, reconstruct liver and restore the liver function of disease rat to normal in vivo. At the same time, many researchers differentiated rat bone marrow cell or adult multi-potentiality stem cells (MPSCs) of rat bone marrow to hepatocyte-like cell under different condition in vitro. MSCs had advantages of collection repeatedly and auto-transplantation or application but did not appeared immunological rejection. When MSCs could be induced differentiation into mature hepatocytes in vitro, it would be provided a great deal of cells for liver cell transplantation and bioartificial liver.
There were many patients with liver disease in China. Cell replacement or gene transfer therapy was a new and prospective hepatology therapeutical strategy for the pathological position of liver cirrhosis, hepatic necrosis and heritage liver diseases. The donor cell of cell therapy for liver diseases should have such as characteristics: obtained, cultured, proliferated or established easily; could differentiate directional special hepatocytes or biliary tract epithelial cells; survived for long time after transplanting in vivo; could not induce immunological rejection and had not harmful to host; objective gene could transfection the donor cell high performance if using for gene therapy. MMSCs had many of above characteristics, so that attracted the height attention of liver diseases researchers. Using human autogenic MMSCs trans-differentiated to hepatocyte would provide alterative for the cell therapy of liver diseases.
Although comparatively large advancement had been gained in ASCs research, but there were many problems needed to be resolved in clinical application. Many experimental results obtained from the experimental animal in vivo, many were side population research, and these were not confirmed on clonal cell level. It was unclear that whether coincidence of the characteristics of human and animal ASCs or the results
近年来,干细胞因其作为生物医学组织工程技术和治疗性克隆技术的种子细胞,为实现人类健康长寿的梦想带来了新的希望,而成为生物医学领域研究的热点。根据干细胞的分化阶段,分为胚胎干细胞(embryo stem cells,ESCs)和成体干细胞(adult stem cells,ASCs)。目前异体来源的ESCs在临床应用上还需要攻克细胞分化机制、免疫排斥等问题,而治疗性克隆获得的ESCs虽可以解决免疫排斥问题,但其技术复杂、离分化完全的功能细胞距离较远。ASCs存在于成体组织中,具有来源丰富、可塑性强、离成熟细胞近和多向分化潜能等特点,而且可以实现个体化治疗,无免疫排斥问题,因此是最具有临床应用优势的种子细胞。目前已在成人骨髓、软骨、牙髓、血液、神经、肌肉、脂肪、皮肤、角膜缘、肝脏、胰岛等多种成体组织中发现了干细胞。现已证实成体干细胞有多向分化潜能,还可以横向分化为不同类型的特化细胞甚至发生逆分化为更原始的干细胞。
骨髓间充质干细胞(marrow mesenchymal stem cells,MMSCs)作为ASCs中最易获得的细胞,具有向多种中胚层和神经外胚层甚至内胚层来源的组织细胞分化的能力,这些细胞包括肌细胞、成骨细胞、软骨细胞、成纤维细胞、皮肤细胞、神经细胞、脂肪细胞等。大量研究显示MMSCs或者造血干细胞(hematopoietic stem cells,HSCs)可在人为诱发肝脏病变后实验鼠体内分化为肝细胞,重建肝脏,使病鼠肝脏功能恢复正常。同时多家研究者在体外利用小鼠骨髓细胞或来自骨髓的成体多潜能干细胞在不同条件下分化为类肝细胞。MSCs具有可反复多次采集和自体移植或应用而不出现免疫排斥反应的优势。如果能够利用易于获得的MSCs在体外诱导分化为成熟肝细胞,将为肝细胞移植及生物人工肝提供大量的细胞来源。
中国肝病患者众多,对肝硬化、肝坏死或遗传性肝脏疾病的病变部位实行细胞替代或基因转移治疗是新发展起来并极有前景的肝脏病学治疗策略。适合肝脏疾病细胞治疗的供体细胞应具有以下特点:①易于获得、培养、增殖,或建立细胞系;②能定向分化
国家高技术研究发展计划(863)资助项目(人肝细胞扩增分化及人源化血细胞研究,No.2001AA216161)
为所需的肝细胞或胆管上皮细胞;③移植入体内能长期存活;④不诱导免疫排斥反应,对宿主无损害;⑤如果用于基因治疗,目的基因可高效转染供体细胞。MMSCs具备上述大部分条件,因此引起肝病研究者的高度关注。如果利用人自身骨髓间充质干细胞横向分化为肝细胞则为肝脏疾病的细胞治疗提供了另一个选择。
虽然ASCs的研究已经取得较大进展,但在临床上应用还有许多问题,目前的多数实验结果来自动物体内实验,而且许多是群体细胞研究,并未在克隆细胞水平得到证实。人ASCs的特性是否与动物一致,体外结果是否与体内一致还不知道。更重要的是ASCs的分化潜能到底有多大及分化的分子机制还不十分清楚。ASCs在临床上应用还存在体外分离、扩增、诱导及体内移植后增殖调控等技术问题。MSCs体内移植尚不能达到大规模增殖,体外向肝细胞转化尚未见报道。ASCs的应用研究刚刚起步,但现代生物技术在ASCs相关研究中的应用必将加快其在临床移植修复治疗中的应用进程。
因此我们尝试利用人胎儿MMSCs在体外向类肝细胞诱导,观察人胎儿MMSCs的特性,期望在成体干细胞横向分化中作一些基础研究工作。尝试为临床肝病治疗中肝细胞来源提供新的可行性途径。
目的
1. 建立人胎儿骨髓、脐带血和肝脏间充质干细胞(mesenchymal stem cells,MSCs)的分离培养方法,观察细胞生物学特性和细胞表型,获得大量的人源性MSCs。
2. 人胎儿MMSCs体外向类肝细胞诱导分化的条件建立及优化。
3. 人胎儿MMSCs来源类肝细胞的分子生物学鉴定。
4. 人胎儿MMSCs来源类肝细胞的功能和超微结构评估。
方法
1. 共收集12个人胎儿,平均孕周为18.5±4.6周,引产后4℃低温保存。收集15份脐带血。采用体外细胞培养技术,分离培养人胎儿骨髓、脐带血和肝非实质细中MSCs,并研究其增殖及生长特征。
2. 采用体外分离培养的MMSCs为研究对象,设计不同的诱导分化条件进行体外诱导,观察细胞诱导分化过程中形态结构变化及细胞生物学特性,应用免疫组化检测肝细胞标志ALB和CK18的表达,建立人胎儿MMSCs向类肝细胞分化的优化条件。
3. 收集诱导培养第4d、7d、14d、21d、28d时的细胞爬片,SABC免疫组化法DAB显色检测肝细胞早期标志AFP、CK19及早期转录因子GATA4,成熟肝细胞标志ALB、CK18、GST-π、肝细胞转录因子HNF1α;提取诱导分化第10d和第28d细胞RNA及蛋白质,设计AFP、CK19、ALB、CK18、CYP1B1、CYP2B6引物,进行RT-PCR,观
察在mRNA水平肝细胞标志的表达,采用Western-Blot检测CK18、AFP、ALB的表达量。
4. 以未诱导的MMSCs作对照,对诱导4d、7d、14d、21d、28d MMSCs采用ELISA法检测培养上清中人白蛋白水平,脲酶法检测合成尿素的能力,过碘酸希夫试验进行糖原染色,电镜观察细胞超微结构。
结果
1. 0.5～2h内尽快分离骨髓,24h换液后可获得约300±80个贴壁细胞,5个细胞以上的集落为15±6个;培养细胞在种植后1～3d为生长滞留期,第4d达到对数生长期,以后进入到平台期,细胞分裂指数曲线的趋势与生长曲线基本类似,在达到对数生长期后,分裂相?
Background
In .the last few years, as seeds cells of biological medicine tissue engineering technique and therapeutic cloning technique, stem cell brought the new hope to dream about human health and longevial, became the hot spot of biological medicine research. Stem cells were divided into embryo stem cells (ESCs) and adult stem cells (ASCs) according to the stage differentiation. Up to now, problems of the cell differentiation mechanism and immunological rejection of xenogenic resource ESCs meeted during clinical application were still not resolved. Although the ESCs obtained from therapeutic cloning technique could resolve the immunological rejection, but the technique was very complicated, and the functions of the ESCs were much different from the function cell that differentiated completely. ASCs resided in adult tissue, had characteristics of abundance resource, powerful plasticity, recent to mature cell and multiple differentiation potentiality, could conduct individualize treatment, had not immunological rejection problem, so that it was the seeds cells for clinical application prevalently. Stem cells had been discovered in many adult tissues such as adult marrow, cartilage, tooth pulp, blood, nerve, muscle, fat, skin, limbus of cornea, liver, islands of pancreas. The multiple differentiation potentiality of ASCs had been confirmed. ASCs could trans-differentiate different type special cells, even retro-differentiate furthermore initial stem cells.
Marrow mesenchymal stem cells (MMSCs) was a kind of stem cell that obtained easily, had the potential capability to differentiate to mesoblastema and neuroepidermal, even endoblastic layer tissue cells, such as muscle cell (myocyte), skeletogenous cell, cartilage cell, fibroblast, skin cell, nerve cell and lipocyte. A great quantity researches showed that MMSCs or hematopoietic stem cells (HSCs) could differentiate to hepatocyte of experimental rat that
This work was supported by the 863 program of China (Research of human hepatocyte amplification differentiation and humanized blood cell No.2001AA216161)
was induced with liver pathological changes, reconstruct liver and restore the liver function of disease rat to normal in vivo. At the same time, many researchers differentiated rat bone marrow cell or adult multi-potentiality stem cells (MPSCs) of rat bone marrow to hepatocyte-like cell under different condition in vitro. MSCs had advantages of collection repeatedly and auto-transplantation or application but did not appeared immunological rejection. When MSCs could be induced differentiation into mature hepatocytes in vitro, it would be provided a great deal of cells for liver cell transplantation and bioartificial liver.
There were many patients with liver disease in China. Cell replacement or gene transfer therapy was a new and prospective hepatology therapeutical strategy for the pathological position of liver cirrhosis, hepatic necrosis and heritage liver diseases. The donor cell of cell therapy for liver diseases should have such as characteristics: obtained, cultured, proliferated or established easily; could differentiate directional special hepatocytes or biliary tract epithelial cells; survived for long time after transplanting in vivo; could not induce immunological rejection and had not harmful to host; objective gene could transfection the donor cell high performance if using for gene therapy. MMSCs had many of above characteristics, so that attracted the height attention of liver diseases researchers. Using human autogenic MMSCs trans-differentiated to hepatocyte would provide alterative for the cell therapy of liver diseases.
Although comparatively large advancement had been gained in ASCs research, but there were many problems needed to be resolved in clinical application. Many experimental results obtained from the experimental animal in vivo, many were side population research, and these were not confirmed on clonal cell level. It was unclear that whether coincidence of the characteristics of human and animal ASCs or the results
引文
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