【作者】 李晏；

【导师】 梁长虹；

【作者基本信息】 南方医科大学 ， 影像医学与核医学， 2010， 硕士

【摘要】 研究背景许多急性或慢性肝病的终末期,死亡率非常高,治疗十分棘手。目前,原位肝移植已成为临床上治疗终末期肝病的主要手段,但由于供体肝的缺乏等问题,使这一治疗措施举步维艰。因此,细胞移植治疗终末期肝病是当前研究的热点,采用肝细胞移植可望提高疗效,但存在肝细胞来源缺乏的问题。近年来,干细胞的无限增殖能力及多向分化潜能使其成为产生肝细胞治疗终末期肝病的一个肝外来源。许多实验研究证明大鼠和人不同组织来源的干细胞在体内、外能诱导分化为肝样细胞。可供选择的干细胞包括胚胎干细胞(embryonic stem cells, ESCs)、骨髓间充质干细胞(bone marrow mesenchyme stem cells, BMSCs)及脂肪干细胞(adipose tissue-derived stromal cells, ADSCs)等。由于ESCs来源有限而且受伦理道德及基因方面等问题困扰,不具有广泛开展临床应用的可行性。BMSCs是临床应用性较好的成体干细胞,然而,BMSCs来源有限、抽取骨髓有创,且患者难以接受,其广泛应用也受到限制。2001年Zuk等首次报道在成体脂肪中发现干细胞以来,ADSCs逐渐成为一个重要的干细胞来源。ADSCs具有和BMSCs相似的表型和多向分化潜能,可以向脂肪细胞、肝样细胞、成骨细胞及心肌细胞等分化。脂肪组织来源充足、取材容易,并且ADSCs分离培养简便、体外增殖能力强。ADSCs还具有免疫抑制特性,这使其没有免疫排斥风险,可以用于自体移植、同种异体移植和异种移植。因此,ADSCs更适合于临床应用,可以作为组织工程潜在最大的成体干细胞库,给肝细胞移植治疗终末期肝病的患者带来了希望。干细胞治疗疗效的评价需要反映出移植的干细胞在体内移植后的分化、分布、迁徙及归巢情况。大量研究表明,MRI是无创、在体示踪细胞的最佳手段。为了使移植的细胞在体内突出显示,必须在移植前进行细胞内磁性对比剂标记。目前最常用的磁性标记物是超顺磁氧化铁(superparamagnetic iron oxide, SPIO)。然而,未经表面修饰的SPIO颗粒很难标记干细胞。许多学者对干细胞的SPIO标记方法作了深入研究。转染剂(transfect agents, TAs)的出现是干细胞SPIO标记的一个重大突破,使得干细胞的SPIO标记工作简单、易行。TAs包括脂质体、多胺类物质、树枝状物质、硅化合物、硫酸鱼精蛋白等。目前,应用较多的是左旋多聚赖氨酸(poly-1-lysine, PLL)。SPIO标记细胞移植到体内后,由于SPIO中的氧化铁纳米粒子具有超顺磁性,通过干扰周围磁场,T2WI或T2*WI呈低信号,从而使移植细胞与周围组织区别开,可根据靶器官MRI信号的改变判断移植细胞的情况,但该标记技术对干细胞的生长和分化影响存在争议。所以,本实验利用ADSCs作为研究对象,通过对ADSCs诱导分化成肝样细胞,同时检测细胞活力和观察ADSCs分化后细胞糖原储存和ALB表达,以探讨SPIO标记技术对ADSCs|的生长以及分化成肝样细胞的影响,为该技术在干细胞移植治疗示踪应用提供实验依据。研究目的探讨(1)大鼠ADSCs的原代分离、培养和鉴定；(2)转染剂PLL介导SPIO标记大鼠ADSCs的可行性及对干细胞活力的影响；(3)使用PLL介导SPIO标记方法对大鼠ADSCs向肝样细胞分化潜能的影响。材料与方法1.大鼠ADSCs的分离、培养和鉴定。无菌条件下采集SD大鼠腹股沟脂肪组织,进行原代ADSCs的分离、培养、鉴定和传代。倒置显微镜动态观察干细胞的生长过程。采用流式细胞技术检测干细胞表面标记物CD29、CD45、CD44、CD34及CD31的表达。2.采用转染剂PLL介导SPIO进行大鼠ADSCs的磁探针标记的可行性及其对标记干细胞活力的影响。使用第一部分研究中培养的第三代(P3)干细胞。采用SPIO试剂为铁羧葡氨,原始浓度为28mg/ml。无血清培养基加入SPIO (25μg/mL)和]PLL (0.75μg/mL),室温下置于摇床混匀30min (30r/min),然后加入有贴壁干细胞且铺满瓶底约90%的培养瓶内,37℃、5%CO2孵箱内培养12h,PBS洗涤标记细胞3次,除去多余SPIO待用。普鲁士蓝染色定性检测细胞内SPIO标记情况。0.25%胰蛋白酶消化细胞,台盼兰实验检测细胞活力(取细胞悬液50μl/孔+0.2%台盼兰50μl染色检测细胞活力)3.转染剂PLL介导SPIO标记大鼠ADSCs对其向肝样细胞分化潜能的影响。本试验使用第二部分的方法对大鼠ADSCs进行SPIO标记,采用台盼蓝试验检测SPIO标记细胞的活力,将标记后的细胞进行向肝样细胞诱导分化培养。诱导方法如下：P3大鼠ADSCs氐糖DMEM+10%FBS+双抗液中培养,至细胞贴壁80%-90%时消化,以4×104/ml细胞浓度接种于六孔板；铺板的细胞分四组：标记诱导组、未标记诱导组、标记未诱导组及未标记未诱导组；首先,四组全部换培养液：低糖DMDM、20ng/mlEGF、10ng/mlβ-FGF培养48h；然后,诱导组换成诱导培养液Ⅰ：10%FBS、低糖DMDM、20ng/mlHGF、10ng/mlβ-FGF培养至第14d；再换成诱导培养液Ⅱ：10%FBS、低糖DMEM、20ng/mlHGF、1μmol/l地塞米松,培养至第21d；每3天换液一次。未诱导组则以完全培养基换液,每3d换液1次。在诱导过程中,分别在诱导前及诱导后7、14及21d采用糖原染色检测分化后肝样细胞内糖原储存,免疫细胞化学染色检测肝样细胞内白蛋白(albumin,ALB)生成,以及RT-PCR鉴定肝样细胞特定基因ALB的表达。诱导后21d采用糖原染色和普鲁士蓝染色双染对各组细胞进行糖原及细胞内铁的检测。结果1.大鼠ADSCs能在体外成功分离培养。经流式细胞(Flow cytometry, FCM)检测,细胞表达CD44占100%,CD29占99.9%,CD45仅占1.4%,CD34占7.8%, CD31占13.1%,表明分离的细胞表型均一2.转染剂PLL介导SPIO能简单高效地对大鼠ADSCs进行磁探针标记。光学显微镜下普鲁士蓝染色结果示干细胞胞浆内见大量蓝色颗粒,细胞标记率接近100%。台盼兰染色结果显示,标记细胞的活力(92.98±0.588%)和未标记细胞的活力(93.50±0.469%)之间差异无统计学显著性意义(t=1.683,P=0.123)。3.标记诱导组与未标记诱导组大鼠ADSCs均能成功分化为肝样细胞,并具有肝细胞功能的特征。PAS染色发现标记诱导组与未标记诱导组干细胞在诱导后14d分别可见细胞胞浆内出现紫红色颗粒,为PAS染色阳性,诱导21d后,两组细胞PAS染色强阳性,即出现紫红色颗粒的细胞增多。诱导21d后,PAS染色+普鲁士蓝染色的双染可见标记诱导组细胞胞浆内见紫红色糖原颗粒,少数细胞内见蓝色铁颗粒。标记未诱导组少数细胞胞浆内可见蓝色铁颗粒。未标记诱导组细胞浆内见紫红色糖原颗粒。向肝样细胞诱导分化14d、21d,通过免疫细胞化学染色观察,两组细胞胞浆内存在棕色ALB颗粒。诱导分化后7d、14d、21d, RT-PCR显示标记诱导组与未标记诱导组分别在14d可见ALB基因的表达,且21d时ALB表达增强,并且分别在14d及21d,ALB mRNA表达差异无统计学显著性意义(P>0.05)。上述结果初步表明,SPIO标记对ADSCs分化为肝样细胞无明显影响。结论本研究表明体外分离培养大鼠ADSCs具有可行性、可重复性。ADSCs培养简易,增殖能力强,是一个重要的干细胞来源。采用转染剂PLL介导SPIO可以简单、高效地标记大鼠ADSCs,并且该标记对干细胞活力及其向肝样细胞分化潜能无影响。更多还原

【Abstract】 BackgroundMany acute or chronic end-stage liver diseases have a very high mortality rate, which are intractable. Hepatocyte transplantation as an alternative to whole liver transplantation for the treatment of hepatic diseases is still hampered by the limited availability of marginal donor organs to isolate human adult hepatocytes in sufficient amounts and quality for transplantation. The ready availability and unrestricted potential to propagate and differentiate make stem cells of extrahepatic origin a feasible option to generate hepatocytes for therapeutic application in liver diseases. It has been shown in a number of recent studies that murine and human stem cells from various sources may develop into hepatocyte-like cells in vitro and in vivo. Candidates for such strategies include embryonic stem cells (ESCs), bone marrow mesenchyme stem cells (BMSCs) and adipose tissue-derived stem cells(ADSCs), and so on. Although the therapeutic potential of ESCs is enormous due to their auto-reproducibility and pluripotentiality, there are still some limitations to their practical use, including cell regulations, ethical considerations and genetic manipulation. BMSCs possess adipogenic, osteogenic, chondrogenic, myogenic and neurogenic potential in vitro. Since Zuk et al reported that the stem cells in the body fat tissue was discovered at first time in 2001, the ADSCs became an important source of stem cells gradually. ADSCs with similar characteristics to BMSCs, by nature, immunocompatible, and there are no ethical issues related to their use. Adipose tissue drepresents an abundant and accessible source of adult stem cells that can differentiate along multiple lineage pathways, including adipogenic, osteogenic, chondrogenic, and hepatocyte-like cells in a lineage-specific culture medium. It broughts the hope to the patients who suffered form liver diseases of final stage.The evaluation of stem cell therapy needs to reflect the situation of differentiation, distribution, metastasization and homing of transplanted stem cells in vivo. A number of recent studies show that MRI is the best imaging technique for tracking grafted stem cell in vivo. However, the cells have to be labeled magnetically prior to transplantation to be visible on MRI. At present, superparamagnetic iron oxide (SPIO) is one of the paramagnetic materials which commonly used. However, unmodified form of SPIO particles can not be used to efficiently label stem cells. Therefore, various approaches to label stems cell have been extensively explored. Substantial development has been made by introduction of commercially available transfection agents (TAs), including lipofectamine, poly-L-lysine protamine sulfate, arginine/lysine-rich cationic peptides, dendrimers and silica and alkoxysilane coating, allowing for nonspecific magnetic intracellular labeling with SPIO agents. And poly-1-lysine (PLL) is used mostly at present. Due to their strong harassing effect on local magnetic field homogeneity, iron-based contrast agents as SPIO can be readily detected by MRI, it shows hypo-intensity on T2WI or T2*WI.So it can distinguish grafted stem cells from surrounding tissue easily, and judge the state of labled cells according to the change of MR signal change on target organ. However, the effects of this label technique on viability and differentiation of stem cells still exist disputes. Herein, we have made ADSCs an investigation object, by inducing ADSCs differentiation into hepatocyte-like cells, evaluating cell viability at the same time, detecing glucogen depositions and expression of ALB after differentiation, in order to investigate the effects of intracellular magnetic labeling of ADSCs with SPIO on the differentiation capability into hepatocyte-like cells, and to provide experiment evidence for SPIO application in the field of stem cells transplanted therapy.PurposeTo investigate:(1) separate, cultivate and identify the primitive ADSCs from SD rat; (2) the feasibility of intracellular magnetic labeling of stem cells with SPIO using transfection agent PLL, and to analyze the effect of the viability of the labeling stem cells.; and (3) the effect of this approach on the differentiation capability into hepatocyte-like cells of the SPIO labeled stem cells.Materials and Methods1. Culture and identify the rat’s ADSCs in vitro. The fat was obtained from the inguinal area of Sague-Dawley rats, then the primary stem cells were pick-up, and cultured, and identified, and subcultured. The cell surface markers such as CD29, CD45, CD44, CD34 and CD31 of rat’s ADSCs were identified with Flow Cytometer.2. To label the stem cells with SPIO and transfection agent PLL, and to analyze the effect of the viability of the labeled stem cells. The passage 3 cells of the rat ADSCs were used. The approved, commercially available SPIO, ferucarbotran (Fe 28mg/ml,Resovist; Schering, Berlin, Germany), was used as the intracellular magnetic labeling agentr. The cells were mixtured with the SPIO- PLL complex (SPIO 25μg/mL and PLL 0.75μg/mL) in the serum free media, and shake up about 30min at 30r/min in the room temperature. The cultures with the cells adherenced to bottol bottom in 90% were kept overnight at 37℃in a couveuse contained 5% CO2. Prussian blue staining was used to evaluate glycogen and intracellular iron uptake. After incubation, the ADSCs were washed with phosphate-buffered saline three times to remove excess SPIO-PLL. Cell viability of the unlabeled and labeled ADSCs was assessed with trypan blue testing.3. The effect of intracellular magnetic labeling of stem cells with SPIO on the cell differentiation capability into hepatocyte-like cells was evaluated. We use the method in part two to label the rat ADSCs with SPIO, and trypan blue testing to evaluate cell viability of the unlabeled and labeled ADSCs. SPIO-labeled and unlabled ADSCs were subjected to differentiate into hepatocyte-like cells. And the protocol as following:the stem cells (passage 4) were divided into four groups. Group 1 was labeled-induced ADSCs, group 2 was unlabeled-induced ADSCs, group 3 was labeled-uninduced ADSCs, and group 4 was unlabeld-uninduced ADSCs. The ADSCs of group 1 and 2 were subjected to hepatocyte-like cell induction. When ADSCs cultured to Passage 4 adhered each other at 80-90%, these ADSCs were used for differentiation assays. Cells were serum deprived for 2d and pre-cultured in LG-DMEM supplemented with 20 ng/mL EGF and 10 ng/mLβ-FGF (conditioning step) to stop cell proliferation, prior to induction of differentiation toward a hepatic phenotype. Then a 2-step differentiation protocol was performed, followed by a sequential addition of growth factors, cytokines and hormones. In step-1 differentiation medium, consisting of LG-DMEM supplemented with 20 ng/mL HGF,10 ng/mLβ-FGF, the cells were cultured for 14d, followed by step-2 differentiation medium, consisting of LG-DMEM supplemented with 20 ng/mL HGF,1μmol/L dexamethasone to achieve cell maturation up to 21d. For each step, the culture medium was replaced every 3 days. Then Periodic acid-schiff (PAS) staining was used to test the glycogen storage within cytoplasm, immunohisto chemistry staining was used to detect the ALB and RT-PCR to identify the specific gene ALB of hepatocyte cells. After inducing 21 days, PAS staining together with Prussian blue staining was used to evaluate glycogen and intracellular iron.Results1. The rat’s ADSCs were separated and cultured successfully in vitro. The cell surface phenotyping of ADSCs were identified by Flow Cytometer, which expressed CD 44(100%), CD 29 (99.9%), CD45(1.4%), CD34(7.8%) and CD 31(13.1%).2. It is easy and high-performance for intracellular magnetic labeling of ADSCs with PLL mediating SPIO particles. After PLL-SPIO labeling, instant Prussian blue dye staining of the Labeled ADSCs revealed that most ADSCs (in 100%) were covered with blue iron particles at the end of the labeling process. Trypan blue exclusion testing revealed a mean viability of 92.98±0.588% for the control cells, and 93.50±0.469% for unlabeled cells. No significant difference of the cell viability between the two groups (t=1.683, P=0.123) was found.3. Both of the labeled-induced group and unlabeled-induced group were differentiated into hepatocyte-like cells successfully, and possess the functional characterization of hepatocyte. PAS method indicating glycogen deposition was positive(prunosus particles within cytoplasm) on day 14 after inducing both in the labeled-induced group and unlabeled-induced group, and on day 21, the cells were strongly positive for glycogen of the two groups. After inducing 21 days, the results of PAS staining together with Prussian blue staining were as following:glycogen deposition and intracellular iron particles were found on the labeled-induced group; glycogen deposition was on the unlabeled-induced group; intracellular iron uptake was on the labeled-uninduced group; and none was found on the uninduced-unlabeled group. Undifferentiated cells were domenstrated as negative at immunocytochemistry stain for albumin, but positive staining on day 14 and 21 in the labeled-induced group and unlabeled-induced group. The two groups both express the ALB on 14d and 21d by RT-PCR, which expressed higher on 21d, and there is no significant difference between the two groups on day 14 and 21, respectively (P>0.05). These results indicated intracellular magnetic labeling with SPIO did not affect the capability of ADSCs to differentiate into hepatocyte-like cells.ConclusionOur initial results showed that SPIO could effectively label stem cells with the need for secondary TAs and it did not alter the cell viabitly and differential potential into hepatolike-cells of the labeled stem cells.更多还原