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1.
目的分选肝癌MHCC97H细胞中的CD133+和CD133-细胞亚群,并初步探讨CD133+细胞亚群的干细胞特性。方法用磁珠分选技术分选MHCC97H细胞株中的CD133+和CD133-细胞亚群,用流式细胞仪检测分选前后CD133的表达,用MTT法检测其增殖能力,比较其体内成瘤能力,用Western印迹法检测其干细胞相关基因蛋白的表达。结果分选前后MHCC97H细胞中CD133表达分别为(1.09±0.43)vs(86.65±6.49)%(P<0.01)。与CD133-亚群相比,CD133+亚群的体外增殖能力较强,成瘤能力高,高表达干细胞相关基因蛋白(P<0.05,P<0.01)。结论肝癌细胞株MHCC97H中的CD133+细胞亚群具有肿瘤干细胞的特性;CD133是人肝癌MHCC97H细胞株的肿瘤干细胞的表型之一。  相似文献   

2.
人肝细胞癌细胞亚群的克隆分离及异质性机制的初步研究   总被引:5,自引:3,他引:2  
目的:通过原代培养人肝癌细胞、克隆分离异质性亚群细胞,探讨肝癌异质性机制.方法:原代培养人肝细胞癌细胞,应用有限稀释法对培养的肝癌细胞进行单细胞克隆分离异质性亚群,并应用细胞计数法测定其细胞倍增时间及倍增数,流式细胞仪检测其DNA含量确定细胞周期分布,裸鼠异体移植检测其成瘤能力.结果:分离到LCSC-1及LCSC-2两个细胞亚群.LCSC-1亚群细胞呈长梭形,裸鼠异体移植不能成瘤(0/8);LCSC-2亚群细胞呈多角形、多突起,裸鼠异体移植可成瘤(8/8).LCSC-1与 LCSC-2相比,体外增殖能力强,倍增时间(18.6 ±3.2 h vs 25.9±4.7 h)和最大倍增倍数(16.1 ±1.4 vs 12.2±1.6)有显著差异(均P<0.01);而且LCSC-1处于细胞周期S(28.4%±3.3%vs 20.2%±1.9%,P<0.01)和G2M(25.0%±6.3% vs 16.6%±4.7%,P<0.05)的比例明显高于 LCSC-2.结论:原发性肝细胞癌中存在着异质性的肿瘤细胞亚群,可能来源于肿瘤干细胞的分化.  相似文献   

3.
朱殷  郑青  汪铮  王园园  萧树东 《胃肠病学》2011,16(5):281-285
背景:肝癌是常见的恶性肿瘤之一,易复发、转移,术后5年生存率较低。目前肿瘤干细胞学说已成为肿瘤研究的热点.CD133是一种肿瘤干细胞的标记物。目的:比较肝癌细胞株SMMC7721中CD133+和CD133一亚群的生物学特性差异.并初步探讨CD133+亚群的干细胞特性。方法:采用免疫磁珠法(MACS)分选SMMC7721细胞中CD133+和CD133一亚群.以流式细胞术检测CD133表达量,平板克隆形成实验检测CDl33体外增殖能力,裸鼠成瘤实验检测体内致瘤性.CCK-8法检测对5-氟尿嘧啶(5-Fu)的敏感性。结果:MACS分选并培养1周后,CD133+亚群中CD133表达量明显下降。与CD133-亚群相比.CD133+亚群的体外克隆形成率明显增高,裸鼠肿瘤的体积明显升高,对5-Fu的敏感性降低.差异均有统计学意义(P〈0.05)。结论:肝癌SMMC7721细胞中CD133+亚群较CD133-亚群更具有肿瘤干细胞的特性.  相似文献   

4.
目的:建立分离肝癌细胞系HepG2中对罗丹明123(Rho123)染料具有不同排斥能力细胞亚群的方法,并探讨其在肝癌异质性研究中的意义.方法:利用流式细胞分选术(FCM)并结合不同浓度的Rho123染料,分析和分选肝癌细胞系HepG2细胞中具有不同染料排斥能力的肝癌细胞亚群,再通过细胞生长的测定、软琼脂克隆形成以及免疫组织化学检测和裸鼠成瘤实验等方法,以比较不同肝癌细胞亚群的差异.结果:根据不同染料排斥能力可以将肝癌细胞系HepG2细胞分为Rholow和Rhohigh2个亚群,2个亚群在倍增时间(16.9 h vs 24.7 h)、最大增生倍数(15.2 vs 12.3)、克隆形成率(50%vs 20%)以及甲胎蛋白(AFP)的表达(31/32 vs 20/26)以及裸鼠成瘤实验(7/10 vs 1/10)上具有显著性差异(均P<0.01),Rholow亚群是具有干细胞特性的.结论:Rho123结合FCM可以富集具有干细胞特性的Rholow亚群,同时进一步证实肝癌的异质性.  相似文献   

5.
目的 观察甲胎蛋白在肝癌细胞株的癌干细胞表达.方法 利用肝癌细胞株HepG-2,采用细胞克隆法分离肝癌干细胞,倒置显微镜下采用细胞克隆形态鉴别肿瘤干细胞和普通肿瘤细胞.应用免疫细胞化学技术对所用的细胞克隆进行AFP的检测,同时以宫颈癌细胞株HeLa作为阴性对照.结果 HepG-2细胞形成三种克隆形态,即Holoclones型、Meroclones型、Paraclones型,其中Holoclones型为少数,是肿瘤干细胞.免疫细胞化学技术显示,HepG-2细胞三种克隆AFP染色均为阳性,HeLa细胞AFP染色为阴性.结论 肝癌细胞株HepG-2分离的癌干细胞表达AFP,为以AFP为靶点的肝癌肿瘤干细胞治疗提供了实验基础和理论依据.  相似文献   

6.
目的 研究人前列腺癌PC3肿瘤细胞系中是否存在具有干细胞特征的细胞亚群,并对其形态及克隆生长特征进行分析,同时探讨染料Rho123着色强度是否可用于区分不同的肿瘤细胞亚群.方法 采用Rho123染色后流式细胞仪分析;Rho123与DAPI复染后荧光显微镜下形态学观察及细胞克隆生长特征分析等方法.结果 PC3细胞中存在对Rho123着色显著差异的两类细胞亚群,其形态结构、增殖能力及克隆生长方式均有明显差别.结论 人前列腺癌PC3细胞系中存在具有干细胞特性的细胞亚群,对Rho123拒染的特性可用于区分肿瘤干细胞与其他肿瘤细胞.  相似文献   

7.
目的 从人胃癌细胞株MGC-803中分离侧群(SP)细胞,研究其生物学特征.方法 利用流式细胞分选术(FACS)从MGC-803细胞株中分选出SP细胞和非SP细胞亚群进行培养,采用克隆形成实验比较两组亚群细胞的体外增殖能力,NOD/SCID鼠成瘤实验检测两组亚群细胞体内成瘤能力.结果 FACS结果显示,细胞株MGC-803中SP细胞亚群占总细胞的0.3% ~ 1.2%;SP细胞体外克隆形成率为(0.862±0.050)%,非SP细胞体外克隆形成率为(0.325 ±0.207)%,两者比较P<0.05;SP细胞最低成瘤数量是1×103/只,非SP细胞为5×103/只.结论 人胃癌细胞株MGC-803中存在SP细胞,其生物学特性与干细胞基本符合.  相似文献   

8.
胃肠道干细胞标记物的筛选   总被引:4,自引:1,他引:4  
目的研究几种常用干细胞标记物在人胃肠道黏膜干细胞中的表达。方法采用免疫组织化学SP法对5例正常胃黏膜、5例正常小肠黏膜及5例正常降结肠黏膜中Musashi—1、端粒酶逆转录酶(TERT)、Oct-4和Nestin的表达情况进行研究。结果胃黏膜中Musashi—1和TERT阳性细胞主要位于腺体峡部,Oct-4阳性细胞多位于腺体基底部,Nestin阳性细胞主要分布在胃黏膜间质以及腺体颈部。小肠黏膜中Musashi—1和TERT阳性细胞主要在距隐窝底部4~7个细胞的位置,Oct-4阳性细胞散在分布于腺体内,Nestin阳性细胞均分布在小肠黏膜间质,无腺体内着色。降结肠黏膜中Musashi—1和TERT阳性细胞分别主要在距隐窝底部1.4个和1-5个细胞的位置,Oct-4阳性细胞散在分布在腺体内和黏膜间质,Nestin阳性细胞分布黏膜间质内,腺体内无表达。结论Musashi—1和TERT的表达与胃肠道干细胞的位置较吻合,可认为是胃肠道相对特异的干细胞标记物。而Oct-4与Nestin做为胚胎干细胞和神经干细胞的标记物被广泛应用,但不能用于胃肠道干细胞的标记。  相似文献   

9.
肿瘤干细胞(cancer stem cells)是存在于肿瘤组织中具有干细胞样能力(stem—cell—like ability)的肿瘤细胞亚群,一个肿瘤干细胞能够产生整个肿瘤组织的(处于不同分化阶段的)所有肿瘤细胞。迄今,在白血病、脑肿瘤、乳腺癌、前列腺癌、结肠癌、胃癌、头颈部肿瘤、肺癌、卵巢癌、黑色素瘤、胰腺癌和肝癌等恶性肿瘤中均成功分离出肿瘤干细胞。  相似文献   

10.
目的:基于癌干细胞的耐药特性联合单细胞培养方法识别肝癌干细胞所在的克隆亚群.方法:以BEL-7404细胞系为研究对象,裸鼠体内移植瘤形成实验,并予阿霉素(adriamycin,ADM)8 mg/kg干预,待肿瘤直径为1.5 cm时取移植瘤细胞做原代培养,并在裸鼠体内连续传代4代,并将第4代移植瘤细胞命名为"BEL-7404-ADM-P4",根据成瘤时间检测肝癌干细胞富集情况.联合单细胞培养,取BEL-7404及BEL-7404-ADM-P4所形成的克隆根据克隆形态分为全克隆、部分克隆、旁克隆,检测BEL-7404及BEL-7404-ADM-P4单克隆中全克隆、部分克隆、旁克隆的癌干特性,即增殖能力、克隆形成率及悬浮球形成率;取其全克隆、部分克隆、旁克隆做hoechst33342染色,并在共聚焦显微镜下观察其染色情况;根据BEL-7404及BEL-7404-ADM-P4单克隆中全克隆、部分克隆、旁克隆的增殖能力、克隆形成率及悬浮球形成率以及hoechst33342染色情况识别癌干细胞所在的克隆亚群.结果:裸鼠体内低剂量阿霉素干预,在体内连续成瘤传代过程中,裸鼠皮下移植瘤成瘤率均为100%,且成瘤时间从第一代到第四代均有所缩短,以此达到了肝癌干细胞的初步富集;增殖能力:全克隆增殖速度最快,细胞量最大,部分克隆次之,旁克隆增殖速度最慢,并于第10天开始出现细胞皱缩死亡;克隆形成率:BEL-7404-ADM-P4-H高于BEL-7404及BEL-7404-ADM-P4-M,差异有统计学意义(P<0.05);悬浮球形成率:仅BEL-7404-H及BEL-7404-ADM-P4-H可形成悬浮球,其余细胞系不形成悬浮球,而BEL-7404-H及BEL-7404-ADM-P4-H悬浮球形成率比较,无统计学意义(P>0.05);BEL-7404及BEL-7404-ADM-P4单克隆中全克隆、部分克隆、旁克隆hoechst33342染色情况:BEL-7404及BEL-7404-A D M-P4单克隆中全克隆都有极少数不染和低染的细胞存在,但部分克隆、旁克隆全染,且BEL-7404单克隆中全克隆、部分克隆、旁克隆hoechst33342荧光强度均强于BEL-7404-ADM-P4单克隆中全克隆、部分克隆、旁克隆.结论:基于癌干细胞的耐药特性联合单细胞培养方法所形成的克隆亚群中,全克隆含有更高比例的肝癌干细胞.  相似文献   

11.
AIM: To explore the method of isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma cell line PANC-1. METHODS: The PANC-1 cells were cultured in Dulbecco modified eagle medium F12 (1:1 volume) (DMEM-F12) supplemented with 20% fetal bovine serum (FBS). Subpopulation cells with properties of tumor stem cells were isolated from pancreatic adenocarcinoma cell line PANC-1 according to the cell surface markers CD44 and CD24 by flow cytometry. The proliferative capability of these cells in vitro were estimated by 3-[4,5-dimehyl-2-thiazolyl]-2, 5-diphenyl- 2H-tetrazolium bromide (MTT) method. And the tumor growth of different subpopulation cells which were injected into the hypodermisof right and left armpit of nude mice was studied, and expression of CD44 and CD24 of the CD44^+CD24^+ cell-formed nodules and PANC-1 cells were detected by avidin-biotin-peroxidase complex (ABC) immunohistochemical staining. RESULTS: The 5.1%-17.5% of sorted PANC-1 cells expressed the cell surface marker CD44, 57.8% -70.1% expressed CD24, only 2.1%-3.5% of cells were CD44^+ CD24^+. Compared with CD44-CD24- cells, CD44^+CD24^+ cells had a lower growth rate in vitro. Implantation of 104 CD44 CD24 cells in nude mice showed no evidenttumor growth at wk 12. In contrast, large tumors were found in nude mice implanted with 103 CD44^+CD24^+ cells at wk 4 (2/8), a 20-fold increase in tumorigenic potential (P 〈 0.05 or P 〈 0.01). There was no obvious histological difference between the cells of the CD44^+CD24^+ cell-formed nodules and PANC-1 cells. CONCLUSION: CD44 and CD24 may be used as the cell surface markers for isolation of pancreatic cancer stem cells from pancreatic adenocarcinoma cell line PANC-1. Subpopulation cells CD44^+CD24^+ have properties of tumor stem cells. Because cancer stem cells are thought to be responsible for tumor initiation and its recurrence after an initial response to chemotherapy, it may be a very promising target for new dr  相似文献   

12.
Background and Aim:  Recent investigations demonstrate that adult stem cells may be targets for malignant transformation and that the stem-like cells in diseased livers possess the capacity of tumorigenicity in animal models. The aim of this study is to examine expression patterns of stem-cell markers in hepatitis B virus–associated cirrhotic livers and hepatocellular carcinomas (HCC), and to investigate the stem-like cell capacity of tumorigenicity in these tissues.
Methods:  Twenty surgically resected HCCs and corresponding adjacent tissues as well as 10 cirrhotic liver tissues were collected and immunohistochemical staining were performed to detect the expression of CD34, Thy-1, CD133, and c-kit. Then the non-cancerous tissues were transplanted into immunodeficient mice and the characteristics of the cells from primary tissue cultures were explored in vitro .
Results:  Immunohistochemical analysis characterized different expression patterns of stem-cell markers among these tissues. First, CD34 and Thy-1 expression was identified in proliferating bile ductules and it represented hepatic progenitor cells; CD133 and c-kit-positive cells were observed in the parenchyma of these tissues, and some of them were characterized as intermediate hepatocytes morphologically and spatially. Second, in two groups including three mice transplanted with tissues adjacent to HCC-initiated tumors, CD133 and c-kit expression was detected. Finally, our study also indicated that stem-like cells from tissue could express hepatic-lineage markers and possessed great capacities to proliferate, self-renew, and form clones in vitro .
Conclusion:  Our results suggest that the stem-like cells in cirrhotic livers possess the capacity of tumorigenicity and may contain candidates for HCC cancer stem cells.  相似文献   

13.
AIM: To investigate the different effects of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) on hepatic differentiation. METHODS: MSCs from rat bone marrow were isolated and cultured by standard methods. HSCs from rat bone marrow were isolated and purified by magnetic activated cell sorting. Both cell subsets were induced. Morphology, RT-PCR and immunocytochemistry were used to identify the hepatic differentiation grade. RESULTS: MSCs exhibited round in shape after differentiation, instead of fibroblast-like morphology before differentiation. Albumin mRNA and protein were expressed positively in MSCs, without detection of alpha-fetoprotein (AFP). HSCs were polygonal in shape after differentiation. The expression of albumin signal decreased and AFP signal increased. The expression of CK18 was continuous in MSCs and HSCs both before and after induction. CONCLUSION: Both MSCs and HSCs have hepatic differentiation capabilities. However, their capabilities are not the same. MSCs can differentiate into mature hepatocyte-like cells, never expressing early hepatic specific genes, while Thy-1.1(+) cells are inclined to differentiate into hepatic stem cell-like cells, with an increasing AFP expression and a decreasing albumin signal. CK18 mRNA is positive in Thy-1.1(+) cells and MSCs, negative in Thy-1.1(-) cells. It seems that CK18 has some relationship with Thy-1.1 antigen, and CK18 may be a predictive marker of hepatic differentiation capability.  相似文献   

14.
Human livers contain two pluripotent progenitors: hepatic stem cells and hepatoblasts. The hepatic stem cells uniquely express the combination of epithelial cell adhesion molecule (EpCAM), neural cell adhesion molecule (NCAM), cytokeratin (CK) 19, albumin +/-, and are negative for alpha-fetoprotein (AFP). They are precursors to hepatoblasts, which differ from hepatic stem cells in size, morphology, and in expressing the combination of EpCAM, intercellular cell adhesion molecule (ICAM-1), CK19, albumin++, and AFP++. The hepatic stem cells are located in vivo in stem cell niches: the ductal plates in fetal and neonatal livers and canals of Hering in pediatric and adult livers. The hepatoblasts are contiguous to the niches, decline in numbers with age, wax and wane in numbers with injury responses, and are proposed to be the liver's transit-amplifying cells. In adult livers, intermediates between hepatic stem cells and hepatoblasts and between hepatoblasts and adult parenchyma are observed. Amplification of one or both pluripotent cell subpopulations can occur in diseases; for example, hepatic stem cell amplification occurs in mild forms of liver failure, and hepatoblast amplification occurs in forms of cirrhosis. Liver is, therefore, similar to other tissues in that regenerative processes in postnatal tissues parallel those occurring in development and involve populations of stem cells and progenitor cells that can be identified by anatomic, antigenic, and biochemical profiles.  相似文献   

15.
16.
人胚胎肝干细胞的形态特点   总被引:5,自引:0,他引:5  
目的:观察胚胎发育早期肝干细胞的形态特征、时空分布及分化,以探讨肝干细胞的生物学特征.方法:运用发育第3-12 wk人胚标本47例(其中 3.5 wk各8例;6-8 wk各5例,9-12 wk各2例),石蜡切片,连续切片,免疫组化染色,光镜下观察人胚肝及肝干细胞的发育及其AFP、c-Met和 CK19的时空表达.结果:发育第3 wk,肝芽形成,第4 wk形成肝索,第5 wk出现原始肝血窦.第3-5 wk人胚肝芽和肝索细胞排列紧密,较小,形态不规则,核圆形或卵圆形,核质比例大,核深染,胞质颜色较淡,偏蓝色,显示出幼稚细胞的形态学特征,并呈甲胎蛋白(α-Fetoprotein,AFP)、c-Met 阳性反应.第6 wk,肝索内出现了体积大、核大、淡染的细胞,呈AFP、c-Met阴性反应.随胚龄增加,这类细胞数量增加.10-12 wk. AFP、c-Met阳性细胞主要分布于汇管区周围. CK19阳性反应在7 wk时开始出现于一些与 AFP、c-Met阳性反应的细胞形态类似的肝索细胞中.10-11 wk时,CK19阳性反应主要位于汇管区附近的肝索细胞、胆管板细胞及胆管上皮细胞,12 wk时,CK19阳性信号仅见于胆管板和胆管上皮细胞.此时所有的胆管板细胞及胆管上皮细胞均呈AFP、c-Met和CK19阳性.结论:人胚发育3-5 wk肝实质由肝干细胞组成,其表型为AFP /c-Met .6 wk,肝干细胞开始向肝细胞系分化,7 wk向胆管系分化,10-12 wk,肝干细胞主要局限于汇管区周围的肝索, 与成年肝中卵圆细胞(成年肝干细胞)的分布一致.AFP /c-Met/ CK19 细胞可能为胆管祖细胞.  相似文献   

17.
大鼠部分肝移植后自体骨髓干细胞动员的研究   总被引:1,自引:0,他引:1  
目的 探讨大鼠部分肝移植后骨髓干细胞动员的情况。方法 建立大鼠性别交叉部分肝移植模型,分为部分肝移植组、全肝移植组和假手术组,分别与术后1、3、5、7d取标本。用流式细胞法检测骨髓中干细胞标记的细胞群体的数量变化,荧光原位杂交检测移植肝脏内Y染色体特异的Sry基因的表达,免疫组织化学法检测移植肝脏内干细胞标志CD34,c—kit和Thy-1.1的表达。结果 与全肝移植组相比,部分肝移植组术后第1天,骨髓细胞中,β2微球蛋白(β2m)/Thy-1.1^+,CD45^+/CD34^+有不同程度的升高,然后呈递减趋势。免疫组织化学检测汇管区和炎性灶周围可见CD34、c—kit和Thy-1.1单个核细胞表达,并于第5至7天后减少。同时免疫组织化学双染可检出CD34^+/CD45^+细胞。全肝移植组汇管区CD34、c—kit和Thy-1.1表达较少,假手术组CD34、c-kit和Thy-1.1偶见表达。在部分肝移植物可检出Sry^+细胞,但Srg^+/CD34^+,Sry^+/Thy-1.1^+细胞较少。全肝移植组Sry^+偶见。结论 部分肝移植中,骨髓源性的干细胞发生动员;同时肝内有干细胞被激活,其中外源性干细胞较少。  相似文献   

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目的:体外诱导小鼠骨髓间充质干细胞分化(BMSCs)为肝样细胞,提高分化效率.方法:将第一代BMSCs随机分为诱导组和对照组.诱导组加肝细胞生长因子、成纤维生长因子、表皮生长因子、抑瘤素M等进行诱导培养,观察细胞形态,检测甲胎蛋白(AFP)、白蛋白(A1b)、细胞角蛋白18(CK18)、酪氨酸氨基转移酶(TAT)和细胞色素P450 2b9(CYP2b9)的mRNA表达,A1b合成以及A1b和CK18蛋白标记细胞阳性率.结果:诱导组第3天出现多边形细胞,5—7d上皮样细胞呈岛状分布,14 d呈铺路石状.对照组细胞为长梭形.第7,14,21天,诱导组细胞AFP,A1b,CK18 mRNA和A1b蛋白检测阳性;第14,21天,细胞表达TAT和CYP2b9 mRNA.对照组除AFP mRNA呈弱阳性外,其余均为阴性.第7,14,21天,诱导组CK18阳性率分别为71.4%.75.9%,80.6%:A1b阳性率分别为75.0%,79.7%.81.1%.而对照组第7天CK18和A1b阳性率仅2.3%,1.7%,与诱导组相比有显著差异(P_(CK18)=1.97×10~(-5),P_(A11b)=3.08×10~(-6)).结论:BMSCs在体外可以被诱导分化为肝样细胞,诱导率最高可达80%以上.  相似文献   

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Recent experiments show that hematopoietic progenitor cell populations contain endothelial precursor cells. We have isolated a population of CD34(+) cells that expresses fibroblast growth factor receptor-1 (FGFR-1) and that differentiates into endothelial cells in vitro. We find that 4.5% +/- 2.1% of CD34(+) cells isolated from bone marrow, cord blood, and mobilized peripheral blood express FGFR-1 and that viable CD34(+)FGFR(+) cells are small, with little granularity, and express both primitive hematopoietic and endothelial markers on their surface. The primitive hematopoietic markers AC133, c-kit, and Thy-1 are coexpressed by 75%, 85%, and 64% of CD34(+)FGFR(+) cells, respectively. Most of the CD34(+)FGFR(+) cells also express antigens found on endothelial cells, such as CD31, vascular endothelial growth factor receptor-2, and the endothelial-specific cell surface marker, vascular endothelial cadherin (VE-cadherin), whereas 56% to 60% of the cells express Tie, Tek, and the endothelial-specific marker, P1H12. The CD34(+)FGFR(+) population is enriched in cells expressing endothelial-specific antigens compared with the CD34(+) population. Isolated CD34(+)FGFR(+) cells grow slowly in culture, are stimulated by fibroblast growth factor-2 and vascular endothelial growth factor, and give rise to cells that express von Willebrand factor and VE-cadherin and that incorporate acetylated low-density lipoprotein. These experiments show that FGFR-1 is expressed by a subpopulation of CD34(+) cells that give rise to endothelial cells in vitro, indicating that this population contains endothelial stem/progenitor cells.  相似文献   

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