体外诱导外周血单个核细胞向肝样细胞分化的研究

发布时间：2018-05-25 15:27

本文选题：外周血单个核细胞 + 肝细胞　；参考：《大连医科大学》2008年硕士论文

【摘要】： 干细胞是一类具有无限或永生的自我更新能力的细胞,能产生至少一种类型的、高度分化的子代细胞。在个体发育的不同阶段及成体不同组织中均存在着干细胞,来源于各种组织的成体干细胞在适当的微环境中能分化为其他类型的细胞,即成体干细胞具有可塑性。人外周血单个核细胞包括单核细胞、干细胞和淋巴细胞,通过密度梯度离心法,收集外周血单个核细胞,利用单核细胞可粘着于塑料培养器皿表面的特性,可去除未贴壁细胞,进一步纯化单核细胞。巨噬细胞集落刺激因子(macrophage colony stimulating factor,M-CSF)可促进单核细胞增殖分化,白细胞介素-3(interleukin-3,IL-3)可促进单核细胞分化及多能干细胞自我更新。这两种因子的联合应用对单核细胞的可塑性具有协同作用,可使外周血单核细胞产生部分未分化,具有重新分化能力并对刺激它们终末分化的因子反应下调,使这些单核细胞来源的细胞被诱导分化为肝样细胞成为可能。细胞的分化和再生受细胞因子或生长因子的控制,在大多数的研究中,肝细胞生长因子(hepatocyte growth factor,HGF)、表皮生长因子(epidermal growth factor, EGF)、转化生长因子(transforming growth factor,TGF)、成纤维细胞生长因子(fibroblast growth factor,FGF)应用最多。曾有报道,在研究多潜能人祖细胞(hMAPCs)转分化实验时,分析比较了多种细胞因子的作用,如FGF、白血病抑制因子(leukaemia inhibitory factor,LIF)、HGF、制瘤素M(oncostatin M,OSM)、EGF等,得出在HGF和FGF-4存在的情况下分化为肝样细胞的培养条件较好。目前对急性或晚期肝病引起的肝功能衰竭,最佳治疗方法是肝移植,原位肝移植由于供体器官紧缺,其应用受到限制。采用细胞移植技术来修复、治疗终末期肝病患者,帮助患者度过危险期等待肝移植或者直接修复衰竭肝脏,是一个理想的方法,因此寻找合适的肝细胞来源备受关注。外周血有收集方便、供体不需要麻醉、无骨髓穿刺痛苦、易于患者接受等优点,因此用外周血干细胞移植的方法来获得转分化后的肝样细胞,其临床应用前景非常突出。目的:探索人外周血单个核细胞在HGF及FGF-4诱导下分化为肝样细胞的可行性。方法: 1.采集正常健康志愿献血者的外周血,密度梯度离心法分离外周血单个核细胞。 2.用RPMI1640培养液对外周血单个核细胞培养2小时后,去除未贴壁细胞。 3.在含10%胎牛血清、140uM ?-巯基乙醇、5ng/ml M-CSF、0.4ng/ml IL-3的RPMI1640培养液中对贴壁细胞培养6天。 4.6天后,用含10%胎牛血清、20ng/ml HGF、10ng/ml FGF-4的RPMI1640培养液继续诱导培养20天,即对外周血单个核细胞总共培养约26天。 5.观察细胞培养过程中的形态变化,加入HGF、FGF-4诱导前及诱导后的第6、10、14、20天采用免疫荧光法检测肝细胞标志物甲胎蛋白(α-fetoprotein,AFP)、白蛋白(albumin,ALB)的表达情况。结果: 1.刚分离的单个核细胞形态上呈较为均一的圆形,台盼兰染色法测定活细胞率 95 %,培养2小时后去除未贴壁细胞,贴壁细胞形态呈毛毛糙糙的圆形。 2.在M-CSF、IL-3存在的条件下,对贴壁细胞培养6天后,可见细胞呈集落样生长,细胞趋向融合。未用M-CSF、IL-3培养或单用M-CSF培养的对照组细胞呈单个细胞生长,随培养时间延长,贴壁伸展,培养2周后大部分呈纤维样或梭形,少部分呈圆形。 3.加入HGF、FGF-4培养后4天左右,可见部分细胞体积变大,向类圆形细胞转变,随培养时间延长,类圆形细胞比例增加。培养20天后,细胞数量较未加入HGF、FGF-4培养前减少。未加入HGF、FGF-4培养的对照组细胞,随培养时间的延长,部分细胞贴壁伸展,培养20天后大部分呈梭形或纤维样,少部分呈圆形。 4.实验组加入HGF、FGF-4培养后第6、10、14、20天免疫荧光法检测AFP均为阳性表达。实验组未加入HGF、FGF-4培养前及对照组于实验组相应检测日期免疫荧光法检测AFP均为阴性表达。 5.实验组加入HGF、FGF-4培养后第10、14、20天免疫荧光法检测ALB均为阳性表达。实验组未加入HGF、FGF-4培养前及对照组于实验组相应检测日期免疫荧光法检测ALB均为阴性表达。结论:外周血单个核细胞在一定诱导条件下具有向肝样细胞分化的潜能,有可能成为干细胞移植治疗肝病的细胞来源。
[Abstract]:Stem cells are a class of cells with unlimited or permanent self - renewal ability to produce at least one type of differentiated , highly differentiated progeny . Stem cells are present in different stages of ontogeny and in different tissues of the adult body , and adult stem cells derived from various tissues can be differentiated into other types of cells in an appropriate microenvironment , i.e . adult stem cells have plasticity .

Human peripheral blood mononuclear cells include monocytes , stem cells and lymphocytes , and the peripheral blood mononuclear cells are collected by density gradient centrifugation . The monocytes can be adhered to the surface of the plastic culture vessel to remove non - adherent cells and further purify the monocytes . The macrophage colony stimulating factor ( M - CSF ) can promote the differentiation of monocytes and further purify the monocytes . The macrophage colony stimulating factor ( M - CSF ) can promote the differentiation of monocytes , and can be regulated by the factors that stimulate their terminal differentiation , so that the cells derived from these monocytes are induced to differentiate into liver - like cells .

The differentiation and regeneration of cells were controlled by cytokines or growth factors . In most studies , hepatocyte growth factor ( HGF ) , epidermal growth factor ( EGF ) , transforming growth factor ( TGF ) and fibroblast growth factor ( FGF ) were most widely used .

The present invention has the advantages of convenient collection , no need of anesthesia , no bone marrow puncture pain , easy patient acceptance and the like , and the clinical application prospect of the liver - like cell after the transformation is very prominent .

Objective : To explore the feasibility of differentiation of human peripheral blood mononuclear cells into hepatocyte - like cells induced by HGF and FGF - 4 .

Method :

1 . Peripheral blood and density gradient centrifugation of normal healthy volunteers were collected to separate mononuclear cells from peripheral blood .

2 . After 2 - hour incubation with RPMI1640 medium on peripheral blood mononuclear cells , no adherent cells were removed .

3 . The adherent cells were cultured for 6 days in RPMI1640 medium containing 10 % fetal bovine serum , 140 uM ? - mercaptoethanol , 5 ng / ml M - CSF , 0.4 ng / ml IL - 3 .

After 4 . 6 days , the culture was continued for 20 days with RPMI1640 medium containing 10 % fetal bovine serum , 20 ng / ml HGF , 10 ng / ml FGF - 4 , i.e . , for a total of about 26 days for peripheral blood mononuclear cells .

5 . Observe the morphological changes in the cell culture process , add HGF , FGF - 4 induction and the 6th , 10th , 14th , and 20th days after induction , and detect the expression of alpha - fetal protein ( AFP ) , albumin ( albumin , ALB ) by immunofluorescence .

Results :

1 . The morphology of individual nuclear cells was uniform round , trypan blue staining method was used to determine the viable cell rate of 95 % , the unadherent cells were removed after 2 hours of culture , and the morphology of adherent cells was rough .

2 . In the presence of M - CSF and IL - 3 , the cells showed colony - like growth and the cells tended to fuse .

3 . After adding HGF and FGF - 4 for 4 days , the volume of some cells became larger and the proportion of round cells increased . After 20 days of culture , the number of cells was decreased compared with that of the control group without HGF and FGF - 4 .

4 . The positive expression of AFP was detected by immunofluorescence assay at 6 , 10 , 14 and 20 days after the growth of HGF and FGF - 4 in the experimental group .

5 . The expression of ALB was detected by immunofluorescence assay at 10 , 14 and 20 days after the growth of HGF and FGF - 4 in the experimental group .

Conclusion : Peripheral blood mononuclear cells have the potential to differentiate into liver - like cells under certain induction conditions .
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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