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目的 建立PKH26荧光标记肝纤维化大鼠骨髓内皮祖细胞(EPC)的方法,观察PKH26标记的EPC输注后在肝纤维化大鼠肝内的迁移和分化情况.方法 分离和培养肝纤维化大鼠骨髓源性EPC,并在体外进行PKH26荧光标记,通过荧光共聚焦显微镜观察和采用流式细胞仪检测PKH26的标记率,并观察荧光标记对细胞生长状态的影响;将PKH26荧光标记的EPC经尾静脉输注入肝纤维化大鼠体内,观察其在大鼠肝内的迁移示踪情况,并用免疫荧光法检测内皮细胞标志物CD31和血管性假血友病因子(vWF)的表达.结果 PKH26标记的细胞呈红色荧光,标记率为96.65%;PKH26标记的EPC生长状态良好,与未标记的EPC相比,生长曲线基本一致;EPC迁移入肝后主要存在于沿纤维分布的血管内皮和肝小叶内的窦内皮,并表达内皮细胞特异性抗原CD31和vWF.结论 PKH26可在体外成功标记肝纤维化大鼠骨髓源性EPC,该细胞输注入肝纤维化大鼠体内后,可迁移至肝内血管周围,并向成熟的内皮细胞分化. 相似文献
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间充质干细胞联合输注促进造血干细胞移植后的造血重建 总被引:7,自引:1,他引:7
目的探讨间充质干细胞(MSCs)与造血干细胞(HSCs)共同移植对造血重建的影响。方法将扩增的Balb/c小鼠骨髓MSCs与骨髓共同经尾静脉输入经致死剂量照射的同系小鼠体内(联合输注组),并设单输骨髓细胞的HSCs组、单输间充质干细胞的MSCs组以及输培养基的对照组,每隔5d计数白细胞,计算动物死亡率。将乙酰乙酸碳氧荧光素标记的人间充质干细胞输入SCID小鼠尾静脉,24h后取肺、心、肝、脾、肾和小肠组织做冰冻切片;取骨髓、肝脏及脾脏,制成单细胞悬液,涂片,在荧光显微镜下观察计数;用逆转录聚合酶链反应测定鼠骨髓中人特异性β2-微球蛋白。结果细胞输注后,联合移植组的白细胞恢复快,12d左右恢复正常,死亡率(3/11)低于HSCs组(5/10)、MSCs组(4/4)、输培养基的对照组(11/11)。人MSCs输入SCID小鼠后24h,其在体内的分布依次为肺、肾、脾、肝,小肠及心脏组织中几乎无阳性细胞,骨髓中有极少MSCs。结论MSCs与造血干细胞共输能促进造血恢复。 相似文献
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《中国普外基础与临床杂志》2016,(8)
目的确定一种更加稳定、有效的活体内转染干细胞的方法。方法从人的脐带提取干细胞,体外培养,并分别用PKH 26和慢病毒-GFP两种方法标记干细胞。取60只SD大鼠,随机分成PKH 26转染组和慢病毒-GFP转染组,将用PKH 26和慢病毒-GFP标记的第3代干细胞液分别注入大鼠门静脉,继续饲养。然后分别于转染后第3 d、8 d及13 d时各处死大鼠10只,取出肝脏,用流式细胞仪检测标记转染细胞百分比和凋亡细胞百分比并进行比较。结果 PKH 26和慢病毒-GFP两种方法标记的第3代人脐带间充质干细胞呈梭形,PKH 26红色染料可以均匀分布在干细胞的细胞膜内,标记清晰;慢病毒-GFP标记的干细胞在荧光显微镜下发出绿色荧光,清晰、稳定。转染后的干细胞在活体内第3 d时,两种方法转染的干细胞在镜下颜色清晰、明显,区分度大,转染效率高;但随着时间的推移,PKH 26转染的红色逐渐变淡,颜色模糊,到第13 d时,干细胞的红色逐渐消退;而慢病毒-GFP转染的干细胞,3个时间点的颜色都较清晰,在第8 d时达到高峰。通过流式细胞仪检测发现,在第8 d和第13 d时慢病毒-GFP细胞转染方法的效率明显高于PKH 26转染(P0.05),而两种转染方法的细胞凋亡率在3个时间点比较差异均无统计学意义(P0.05)。结论慢病毒-GFP转染是一种更加稳定、高效的活体内细胞示踪方法,可用于活体内细胞移植的长期观察。 相似文献
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目的探讨骨髓间充质干细胞(BMSC)分泌的外泌体调控肿瘤相关巨噬细胞(TAM)极化对胰腺癌的治疗作用。方法取4周龄C57BL/6雄性小鼠10只, 体重约20 g, 提取BMSC分泌的外泌体并用PKH26标记。取4~6周龄雌性BALB/c-nu/nu品系裸鼠30只, 无特定病原体级, 体重(18.56±0.85)g, 建立胰腺癌裸鼠模型, 并将其分为3组, 每组10只:空白对照组经鼠尾静脉注射磷酸盐缓冲液(PBS);门静脉治疗组经门静脉注射含外泌体的PBS悬液;尾静脉治疗组经尾静脉注射含外泌体的PBS悬液。8周后处死裸鼠, 检测胰腺组织中PKH26表达阳性的细胞百分比, 测定原位肿瘤体积、抑瘤率, 观察肿瘤转移情况、腹水情况、瘤重、瘤肝重。随后检测外周血M1型以及M2型巨噬细胞活化标志物的基因表达、外周血胰腺癌细胞标志物B7-H4、肿瘤糖类抗原199的表达以及胰腺肿瘤组织细胞中Survivin和基质金属蛋白酶-9(MMP-9)的表达。结果门静脉治疗组抑瘤率(72.4±21.6)%与尾静脉治疗组抑瘤率(70.1±20.7)%相比, 差异无统计学意义(t=0.24, P=0.811)。与空白... 相似文献
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目的观察磁粒子和荧光染料双标人骨髓间充质干细胞归巢至大鼠急性损伤肝组织的示踪。方法以超顺磁性氧化铁(SPIO)和氯甲基苯甲酰氨(CM-Dil)标记永生化人骨髓间充质干细胞(UE7T-13)。普鲁士蓝染色检测细胞内铁,荧光显微镜和流式细胞仪检测CM-Dil标记阳性率。建立12只急性肝损伤大鼠模型,分为实验组(n=6)和对照组(n=6),将标记细胞经尾静脉移植入实验组大鼠,未标记细胞移植入对照组大鼠。分别于移植前3h、移植后3h、3天、5天、7天应用MR T2*W、T2*map对大鼠活体成像,获得肝脏R2*值。并与肝脏组织切片普鲁士蓝染色和CM-Dil荧光表达情况对照。结果双标细胞的SPIO-PLL标记率接近100%,流式细胞仪检测CM-Dil标记阳性率达99.97%。实验组细胞移植后3天、5天肝脏R2*值均较移植前明显升高(t=7.282、7.608,P均0.01),对照组细胞移植后各时间点与移植前比较,R2*值差异均无统计学意义(F=0.99,P0.05)。普鲁士蓝阳性细胞主要分布于肝小叶中央静脉周围病变区,荧光显微镜下观察红色荧光阳性细胞与普鲁士蓝阳性细胞分布基本一致。结论 SPIO和CM-Dil可有效标记人永生化骨髓间充质干细胞(UE7T-13),不影响细胞增殖能力,临床应用型1.5T MR可对磁粒子标记的UE7T-13进行肝归巢活体示踪,CM-Dil有助于证实存活干细胞的肝内定植。 相似文献
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目的 探讨PKH26标记骨髓间充质干细胞(BMSCs)的效果及其应用于组织工程神经种子细胞体内示踪实验的可行性.方法 取Wistar大鼠股骨和胫骨的骨髓,用全骨髓贴壁的方法分离、培养BMSCs.用PKH26荧光染料进行标记,荧光显微镜下观察标记效果,流式细胞仪检测荧光标记率,MTT法测定细胞的活性,体外成脂和成骨诱导鉴定细胞的分化能力,并与未标记细胞进行比较;使用显微注射的方法将BMSCs植入去细胞神经支架内制备成组织工程神经,于体外培养3 d、5 d、7 d、14 d,行组织切片,观察BMSCs在支架内存活、迁移的情况.另外,将体外构建组织工程神经桥接Wistar大鼠坐骨神经15 mm的缺损,于术后1周、4周、6周、8周取出移植物,行组织切片观察植入的BMSCs在支架内的存活情况.结果 PKH26能有效地标记BMSCs,标记率达95%以上,标记后细胞活性及诱导成骨成脂分化能力与未标记细胞无明显差异.在体外培养的环境下,BMSCs能在去细胞神经支架里粘附生长,并沿着支架迁移;在体内外周围神经再生的微环境下,BMSCs可存活8周以上.结论 PKH26荧光染料标记法是一种有效的标记BMSCs的方法,可用于组织工程神经种子细胞体内示踪的研究. 相似文献
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目的 探讨自体骨髓单核细胞移植对血管新生及胆道缺血病变的影响.方法 将30只日本大耳白兔随机分为3组:假手术组(A组)、实验模型组(B组)和骨髓单核细胞移植组(C组),每组10只.3组均游离胆总管、肝总动脉及其间的疏松结缔组织,B组和C组阻断胆总管远端及肝总动脉2 h,C组经肝总动脉注射PKH26标记的自体骨髓单核细胞.术后监测生化指标的变化情况.术后4周开腹行胆道造影.同时,取第一肝门处汇管区肝组织制作石蜡切片,应用免疫组织化学方法观察自体骨髓单核细胞在肝内缺血环境中的分布及分化情况,并检测微血管密度.结果 术后C组生化指标恢复明显优于B组,骨髓单核细胞可分化成血管内皮细胞.术后4周,C组胆道破坏较B组轻,且胆道周围新生毛细血管多于B组.结论 自体骨髓单核细胞移植可以促进局部缺血组织毛细血管的增生,改善局部缺血胆道组织的微循环,从而减轻缺血型胆道病变的程度,甚至预防缺血型胆道病变的发生. 相似文献
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目的 探讨应用临床型1.5T磁共振活体示踪磁标记猪骨髓间充质干细胞(MSCs)自体移植肝脏的可行性.方法 获取、分离、培养、扩增猪骨髓MSCs.应用菲立磁标记细胞,普鲁士蓝染色鉴定,建立急性肝损伤模型,分为标记细胞组(n=6)和未标记细胞组(n=4)经门静脉行肝内移植,分别于移植前,移植后6 h、3 d、7 d、14 d行磁共振FFE(T_2WI)序列成像,14 d后行组织切片普鲁士蓝染色.结果 普鲁士蓝染色表明MSCs的标记率达100%,磁标记MSCs肝内移植后行磁共振T_2WI序列呈明显低信号改变,并持续至细胞移植后14 d,组织切片普鲁士蓝染色显示14 d后仍有磁标记细胞存在于肝实质及肝窦中.结论 利用菲立磁可以在体外成功标记猪骨髓间充质干细胞,磁共振成像可以对经门静脉移植到肝内的磁标记MSCs进行活体示踪. 相似文献
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H. Zhang 《Transplantation proceedings》2009,41(9):3887-3892
Small hepatocytes as hepatic stem cells or progenitors may be transplanted to treat several end-stage liver diseases. To identify the characteristics of epithelial cells enriched from fetal liver, we used immunocytochemistry and electron micrography. All cells in the colonies were immunocytochemically positive for alpha fetoprotein and cytokeratins (CK) 7, CK8, and CK18, which are markers of hepatic progenitor. Under transmission electron microscopy, we observed the cultured cells to show naive characteristics of stem cells and to be significantly distinct from mature hepatocytes. To identity whether these small hepatocytes were able to proliferate and differentiate into mature hepatocytes, we cultured them in vitro, and, through the portal vein, and transplanted elements whose membrane were stained with red fluorescence using PKH26 linker dye, into the livers of CCl4-treated rats that had been subjected to two-thirds partial hepatectomy. Significant liver regeneration was observed 30 days later in rats that did or did not receive the cells. The livers of hepatocytes recipients showed sharper edges and smoother surfaces than the control group. Diffused cells labeled with red fluorescence were observed in the portal area, with branch-like red fluorescence in regions near portal areas of some lobules, suggesting that these elements were involved in the repair of liver lobules and differentiation into mature hepatocytes. Our results revealed that small hepatocytes not only have characteristics of hepatic stem cells, but also may be a source of cellular transplantation to treat liver diseases. 相似文献
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Differentiation of hematopoietic stem cells into hepatocytes in liver fibrosis in rats 总被引:6,自引:0,他引:6
Zhan Y Wang Y Wei L Chen H Cong X Fei R Gao Y Liu F 《Transplantation proceedings》2006,38(9):3082-3085
It has been reported that hematopoietic stem cells (HSC) can differentiate into hepatocytes in the normal liver and in some pathologic environments. The aim of this study was to investigate whether HSC can differentiate into hepatocytes in cases of established liver fibrosis. Rat liver fibrosis was induced by subcutaneous injection of tetrachloride (CCl4). Thy+ CD3- CD45RA- HSC in bone marrow cells, which had been enriched by fluorescence-activated cell sorting (FACS), were labeled with PKH26-GL, and autologously transplanted into CCl4-treated rats. The expressions of albumin (Alb), cytokeratin 8 (CK8), and alpha-smooth muscle actin (SMA) were determined by immunofluorescence methods. The PKH26-GL labeled Thy+ CD3- CD45RA- HSC expressed the hepatocyte-specific markers Alb and CK8, but did not express alpha-SMA in liver fibrosis. Thy+ CD3- CD45RA- HSC differentiated into hepatocytes, but not into hepatic stellate cells. In conclusion, autologous stem cell transplantation may be helpful to treat hepatic fibrosis. 相似文献
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A novel sub-population of bone marrow-derived myocardial stem cells: potential autologous cell therapy in myocardial infarction. 总被引:3,自引:0,他引:3
Harmik J Soukiasian Lawrence S C Czer Itzhak Avital Takeshi Aoki Yong-Hwan Kim Yutaka Umehara Jennifer Pass Robert Tabrizi Kathy Magliato Gregory P Fontana Wen Cheng Achilles A Demetriou Alfredo Trento 《The Journal of heart and lung transplantation》2004,23(7):873-880
BACKGROUND: Several studies have identified beta2-microglobulin-negative (beta2M(-)) cells as a potential stem cell fraction in the bone marrow of rats and humans. We studied the ability of bone marrow-derived beta2M(-) cells to differentiate into cardiomyocytes and reconstitute the myocardium in a model of myocardial infarction. METHODS: beta2M(-) cells were purified from bone marrow of Lewis rats using a magnetic activated cell-sorting technique. beta2M(-) cells, 2.5 x 10(6) cells in 100 microl of phosphate-buffered saline (PBS), were transplanted 7 days after infarction into a transmural myocardial scar induced by cryoinjury in Lewis rats (n = 9). Control Group 1(n = 10) received a 100-microl injection of PBS, and Control Group 2 (n = 15) received no injection. The beta2M(-) cells were labeled before transplantation, using the membrane fluorescent intercalated dye, PKH26. Repopulation was examined at 6 and 8 weeks after transplantation. Differentiation of beta2M(-) cells into cardiac myocytes was determined by the colocalization of troponin and PKH26 to the same cell, utilizing immunohistochemistry, ultraviolet photomicroscopy and fluorescence microscopy on 6-microm serial sections. Area of engraftment within the scar was calculated by planimetry. RESULTS: The treatment group had multiple islands of de novo-formed myocardium within the fibrous matrix of the transmural scar (mean area 35 +/- 4.2% of scar area at 6 and 8 weeks). These cells colocalized cardiac-specific troponin and PKH26. Using these techniques, no myocardial islands were seen in the control groups. Before transplantation, beta2M(-) cells were troponin-negative. CONCLUSIONS: This study demonstrates that beta2M(-) cells represent a novel sub-population of bone marrow-derived stem cells capable of successful and substantial engraftment in areas of transmural myocardial scar, with de novo formation of cardiac myocytes. The functional significance of this observation is being studied. 相似文献
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目的 对比研究骨髓单个核细胞经门静脉移植治疗大鼠肝硬化的疗效,为临床应用提供依据.方法 采用四氯化碳(CCl4)综合法制作Wisrar大鼠肝硬化模型,8周后经病理检查证实成模者共20只进入实验.随机分为治疗组和对照组,每组10只,两组大鼠分别经门静脉注入骨髓单个核细胞和等体积的肝素生理盐水.各组大鼠继续皮下注射CCl4(CCl4使用方法与前述造模方法相同),在原环境下继续饲养4周后处死.切取肝组织分别作HE及Masson染色.疗效评价指标包括:①一般情况;②肝脏羟脯氨酸和胶原纤维含量;③肝纤维化病理学分级.结果 (1)治疗组大鼠一般情况改善;对照组无明显改变.(2)治疗组与对照组中肝脏羟脯氨酸含量分别为(0.50±0.12)g、(0.59±0.34)g;胶原纤维含量百分数分别为(3.75±0.98)%、(5.02±0.44)%.两组之间各指标均有统计学差异(P<0.05).(3)治疗组肝纤维化病理分级也有下降,治疗前后差异明显(P<0.05),而对照组无明显变化.表明治疗组行骨髓单个核细胞移植后大鼠肝纤维化程度减轻.结论 骨髓单个核细胞肝内移植能改善大鼠肝纤维化程度,为临床肝硬化的治疗带来了新的手段. 相似文献
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目的 观察异体骨髓单个核细胞和胰岛细胞通过肝脏和静脉途径移植后对糖尿病大鼠的治疗作用.方法 密度梯度离心法分离胰岛细胞,淋巴细胞分离液分离骨髓单个核细胞,28只糖尿病大鼠模型随机分为A、B、C、D组,A组在肝脏被膜下多点注射1000个胰岛细胞,B组在体外将1000个胰岛细胞和1×107个骨髓单个核细胞混合后在肝脏被膜下多点注射,C组通过尾静脉注射1000个胰岛细胞,D组在体外将1000个胰岛细胞和1×107个骨髓单个核细胞混合后通过尾静脉注射,移植后于不同时间点尾静脉测定随机血糖,比较不同细胞组合和移植途径之间对糖尿病的治疗作用.结果 A、B组血糖于术后3 d内开始下降,A组血糖可降至正常水平(7.98±2.28)mmol/L,血糖维持正常水平(3.71±0.95)d,B组降至(7.72±1. 75)mmol/L可维持(4.86±1.06)d,静脉移植组血糖于术后4 d内降至正常(7.35±1.40)mmol/L,可维持(7.85±1.46)d,D组静脉注射胰岛于4 d起效(7.00±0.83)mmol/L,血糖可降至正常水平可维持(14.10±1.21)d,各组间血糖随时间变化的趋势及维持正常水平的时间具有统计学意义(P<0.05).结论 骨髓单个核细胞和胰岛混合细胞通过尾静脉移植对大鼠血糖维持正常时间最长,血糖控制水平最理想. 相似文献
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Masato Komori MD PhD ; Shingo Tsuji MD PhD ; Masahiko Tsujii MD PhD ; Hiroaki Murata MD PhD ; Hideki Iijima MD PhD ; Masakazu Yasumaru MD PhD ; Tsutomu Nishida ; Takanobu Irie ; Sunao Kawano MD PhD ; Masatsugu Hori MD PhD 《Wound repair and regeneration》2005,13(1):109-118
Bone marrow is reported to contain hematopoietic stem cells and other adult somatic stem cells that have phenotypes of cells composing tissues other than bone marrow. To explore the implication of bone marrow-derived cells in the treatment of inflammatory bowel diseases, experimental colitis was induced in wild-type rats after transplantation of bone marrow from transgenic rats expressing green fluorescence protein (GFP). Chronic colitis was induced 21 days later using 30 mg 2,4,6-trinitrobenzenesulfonic acid (TNBS). Control rats received saline. At 28, 56, and 224 days after TNBS administration, rats were euthanized, and tissues were removed and processed for paraffin-embedded sections. Cells derived from bone marrow were identified by immunohistochemistry using anti-GFP antibody. To identify the phenotypes of the cells expressing GFP, we conducted serial-section analysis and double-staining analysis using antibodies against cytokeratin (epithelial cells) or vimentin (interstitial cells). In the present study, GFP-positive, bone marrow-derived cells occupied 37.6% and 4.25% of the colonic epithelium at 28 days and 56 days after the induction of TNBS-colitis, respectively. Also, significant amounts of mucosal and submucosal interstitial cells were derived from the bone marrow. These findings showed that a large amount of bone marrow-derived cells were involved in regeneration of the colon after experimental colitis in rats. 相似文献
17.
《中华实验外科杂志》2009,26(10)
目的 探讨经门静脉途径输注供体骨髓细胞后,大鼠小肠移植中受体基因嵌合率的改变及可能机制.方法 建立大鼠异位节段小肠移植模型(供体为雄性BN,受体为雌性Lewis,各15只),并将其随机分为三组,对照组、FK506组、PV组(喂养FK506及经门静脉注射供体骨髓细胞组).应用原位荧光杂交定位检测以及实时定量聚合酶链反应技术分析供体雄性细胞的SRY基因在移植术后受体大鼠血液、肝脏及脾脏中的嵌合情况.结果 PV组受体雌性大鼠血液(3.03±0.16)%、肝脏(0.86±0.05)%、脾脏(4.08±0.12)%中供体SRY基因嵌合率均较FK506组(1.15±0.07)%、(0.21±0.02)%、(1.23±0.05)%、对照组(1.10±0.07)%、(0.22±0.02)%、(1.17±0.06)%明显增高(P<0.01).结论 经门静脉系统输注供体骨髓细胞可建立稳定的混和嵌合体状态. 相似文献
18.
【摘要】 目的 观察在生理和病理情况下,骨髓来源的干细胞(BMSC)能否分化成肾小管上皮细胞。 方法 绿色荧光蛋白(GFP)标记的C57BL/6转基因小鼠提供骨髓细胞。同种无荧光标记的C57BL/6小鼠100只分为正常对照组、全身照射组、缺血再灌注组、骨髓移植组、骨髓移植+缺血再灌注组。受体鼠的骨髓重建经血液常规检查和流式细胞仪检测确认,并采用荧光组织化学、免疫组织化学等方法观察绿色荧光标记的BMSC在受体鼠肾脏的分布及数量。 结果 全身致死剂量γ射线照射未造成小鼠肾脏组织结构和生理功能的明显改变。骨髓移植后第56、84天的受体鼠肾小管中有少量GFP阳性细胞的存在[(78.75±5.99)%、(79.58±4.60)%],激光共聚焦显微镜进一步证实这些细胞位于肾小管,并表达肾小管上皮细胞特异性的功能蛋白megalin。 结论 在生理和病理情况下,骨髓干细胞均可以向肾小管上皮细胞转分化,参与肾小管上皮细胞的更新,并且在急性肾小管坏死的病理条件下,骨髓干细胞的肾向转化率与肾脏受损程度有关。 相似文献