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1.
人胎肝干细胞的体外培养及诱导分化 总被引:8,自引:0,他引:8
为体外分离培养和诱导分化人胎肝干细胞 ,从原代分离培养人胎肝干细胞集落 ,免疫细胞化学鉴定细胞集落分子标志物的表达 ;在体外特定细胞因子作用下诱导干细胞定向分化为成熟肝脏细胞 ,对其生物学特性进行初步鉴定。结果 ,从人胎肝组织中成功分离表达AFP、Albumin、Cytokeratin等标志物的胎肝干细胞集落 ,在体外特定细胞因子作用下肝干细胞可定向分化为成熟肝脏细胞。因此人胎肝中同样存在具有干细胞特性的原始细胞 ,体外可定向分化为成熟肝细胞。人胎肝干细胞的分离培养对于生物型人工肝的制备及其深入研究奠定了良好的基础 相似文献
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目的探讨在由HGF介导的人胎儿肝干细胞增殖分化过程中核因子κB(NF-κB)的变化情况及其意义。方法通过ELASA、免疫细胞化学染色及RT-PCR方法,检测在不同浓度HGF作用条件下,人胎儿肝干细胞中NF-κB活性改变情况。结果HGF可促进人胎儿肝干细胞的增殖与分化,在此过程中,HGF浓度与NF-κB活性呈现正相关;免疫细胞化学染色显示,随着HGF剂量的增加,hFHSCs细胞胞核染色程度及出现核染色的细胞的数量亦逐渐增加。RT-PCR结果显示,HGF可促进人胎儿肝干细胞P65及P50mRNA表达,下调IκBmRNA表达,从而使IκB生成减少,促进NF-κB核移位。结论NF-κB信号转导途径可能在肝再生过程中发挥重要作用。 相似文献
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胎肝来源间充质干细胞的分离、培养与多向分化 总被引:4,自引:0,他引:4
目的从胎肝中分离培养间充质下细胞,并研究其牛物学特性。方法用优化的方法从胎肝中分离获得间充质干细胞。利用流式细胞仪分析细胞表型和细胞周期分布,并体外诱导成骨、成脂肪和成肝组织细胞分化。并用染色方法鉴定成骨、成脂肪分化结合形态学方法和RT-PCR方法鉴定成肝组织分化结果。结果从胎肝中分离培养的细胞为成纤维样,贴壁生长.表型相对均一,表面标志为CD90,CD44,CD147,而CD34,CD45,HLA-DR,具有向肝组织分化的潜能,并可向成骨和成脂肪分化。结论从胎肝中可分离培养得到具有多向分化潜能的间充质干细胞。 相似文献
4.
人肛瘘管壁成纤维细胞的体外分离培养和鉴定 总被引:1,自引:0,他引:1
通过组织块法分离并原代培养人肛瘘管壁组织,用胰蛋白酶和乙二胺四乙酸(EDTA)消化成纤维细胞、上皮细胞,获得更加纯化的成纤维细胞;以DMEM培养液为基础培养蒸,添加胎牛血清(体积分数10%)、青霉素(100U/L)和硫酸链霉素(100U/L),置37℃、5%CO2培养箱中培养。倒置相差显微镜和细胞爬片HE染色。观察成纤维细胞形态结构,并对培养成纤维细胞行角蛋白、波形蛋白免疫细胞化学鉴定.结果分离后的成纤维细胞在第4~12h于体外快速贴壁,第2~4天进入对数期生长、增殖期。细胞起源鉴定波形蛋白免疫细胞化学染色为阳性,角蛋白免疫细胞化学染色为阴性。提示该方法所获得的肛瘘管壁成纤维细胞可在体外稳定培养,不含有杂质细胞。可为在细胞分子水平上研究肛瘘瘘管愈合的机制提供可靠的细胞模型。 相似文献
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目的探讨人肝内胆管癌相关成纤维细胞(iCAF)的分离、纯化和鉴定,并初步探究其对肿瘤细胞的影响作用。方法肝内胆管癌标本通过酶消化法(Ⅰ型胶原酶与RPMI 1640混合消化液)分离获得原代细胞,并通过密度梯度离心法进行纯化,iCAF的鉴定通过免疫化学、荧光染色及流式细胞检测进行。通过CCK8及Transwell实验观察iCAF条件培养基对胆管癌细胞的影响作用。结果获得的iCAF以长梭形为主,细胞生长密集相互交织形成网状或漩涡状;免疫细胞化学染色及荧光染色显示:α-SMA阳性、FSP-1阳性;流式细胞检测FSP-1阳性率为98.2%。iCAF条件培养基明显促进了肿瘤细胞增殖和迁移。结论成功分离并纯化了人iCAF,并初步证明iCAF可促进胆管癌细胞增殖和迁移能力,为进一步研究iCAF在肿瘤发生发展中的具体作用机制奠定了重要基础。 相似文献
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目的探索人脂肪间充质干细胞(ADMSCs)分离、培养的方法,以证明人脂肪组织中含有多向分化潜能的ADMSCs,为临床应用提供实验基础。方法采用Ⅰ型胶原酶消化法及贴壁法分离培养腹部手术患者皮下脂肪组织,取第3代细胞,以磷酸盐缓冲生理盐水为空白对照,进行CD13、CD34、CD44、CD45、CD105、HLA-DR、Ⅷ因子表面抗原的免疫细胞化学鉴定。结果人脂肪中含有大量长梭形细胞,免疫化学鉴定显示与空白对照相比较CD44、CD13、CD105抗原呈阳性反应,其余标志物CD45、CD34、HLA-DR、Ⅷ因子抗原均为阴性反应。结论初步证明了人脂肪组织中含有间充质干细胞,为今后ADMSCs的分离培养提供了更简单有效的方法。 相似文献
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目的对人皮肤成纤维细胞进行分离、纯化、培养及细胞鉴定,探讨一种高效的分离及纯化方法,为细胞移植提供种子细胞。方法使用酶消化法原代提取人成纤维细胞,快速贴壁法纯化细胞,对细胞进行形态学观察、HE染色,免疫细胞化学鉴定细胞标志物Vimentin。结果利用倒置显微镜及HE染色,可见细胞为散在分布的梭形贴壁细胞,当细胞汇流时,呈鱼群样或漩涡状排列,且细胞在15代内形态保持不变。结论成功地发现快速提取成人成纤维细胞的方法,为成人自体细胞移植的研究奠定了基础。 相似文献
10.
目的 通过机械划痕模拟机械损伤,探讨机械损伤对人气道上皮细胞生长及细胞因子分泌的影响。方法 通过机械划痕对气道上皮细胞进行损伤造模,将造模后细胞培养液加入正常培养液构建条件培养液;气道上皮细胞分两组,分别用正常细胞培养液及条件培养液进行孵育5天;CCK8法检测细胞增殖情况及活性;HE染色分析细胞形态;免疫细胞化学检测处理后气道上皮细胞中TGF-β、β-catenin、HIF-1表达水平。结果 利用损伤细胞培养液进行培养后,对比正常培养细胞,造模组气道上皮细胞数量降低,活性下降(P<0.05);HE染色显微镜下观察细胞变化情况,造模后细胞形态未变,胞核染色未变,胞质淡染;免疫细胞化学测定两组细胞因子表达水平,结果提示造模组TGF-β、β-catenin、HIF-1表达水平升高(P<0.05)。结论 机械损伤可抑制气道上皮细胞生长;缺氧及TGF-β、Wnt/β-catenin通路可能参与了气道机械损伤后的修复过程。 相似文献
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Liver stem cells: a scientific and clinical perspective 总被引:3,自引:0,他引:3
The promise of liver stem cells lie in their potential to provide a continual and readily available source of liver cells that can be used for gene therapy, cellular transplant, bioartificial liver-assisted devices, drug toxicology testing and use as an in vitro model to understand the developmental biology of the liver. Both the rodent and human embryonic stem cell, bone marrow hematopoietic stem cell, mesenchymal stem cell, umbilical cord blood cell, fetal liver progenitor cell, adult liver progenitor cell as well as the mature hepatocyte have been reported to be capable of self-renewal, giving rise to daughter hepatocytes both in vivo and in vitro. These cells can repopulate livers in animal models of liver injury and seemingly improve liver function. However, significant challenges still exist before these cells can be used in humans. These include lack of consensus in immunophenotype of liver progenitor cells, uncertainty of the physiological role of reported candidate stem/progenitor cell, practicality in obtaining sufficient quantity of cells for clinical use and concerns over ethics, long-term efficacy and safety. Current molecular techniques of stem cell identification are confounded by cell fusion, horizontal gene transfer, incomplete differentiation and fetal microchimerism. Reports of stem cell transplantation and phase 1 trials of bone marrow transplantation in humans for liver diseases are exciting but require more robust verification. We review the evidence for various candidate stem cells, human clinical trials reported to date and highlight the challenges facing clinicians in their quest to use liver stem cells to save lives. 相似文献
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Mukaddes Esrefoglu 《World journal of gastroenterology : WJG》2013,19(40):6757-6773
Although the liver has a high regenerative capacity,as a result of massive hepatocyte death,liver failure occurs. In addition to liver failure,for acute,chronic and hereditary diseases of the liver,cell transplantation therapies can stimulate regeneration or at least ensure sufficient function until liver transplantation can be performed. The lack of donor organs and the risks of rejection have prompted extensive experimental and clinical research in the field of cellular transplantation. Transplantation of cell lineages involved in liver regeneration,including mature hepatocytes,fetal hepatocytes,fetal liver progenitor cells,fetal stem cells,hepatic progenitor cells,hepatic stem cells,mesenchymal stem cells,hematopoietic stem cells,and peripheral blood and umbilical cord blood stem cells,have been found to be beneficial in the treatment of liver failure.In this article,the results of experimental and clinical cell transplantation trials for liver failure are reviewed,with an emphasis on regeneration. 相似文献
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Transplantation of wheat germ agglutinin-positive hematopoietic cells to prevent or induce systemic autoimmune disease. 下载免费PDF全文
E E Sardi?a K Sugiura S Ikehara R A Good 《Proceedings of the National Academy of Sciences of the United States of America》1991,88(8):3218-3222
Hematopoietic stem cell defects are thought to be involved in the etiopathogenesis of systemic autoimmune disease. Positively selected, stem cell-enriched populations of wheat germ agglutinin-positive (WGA+) low-density bone marrow and fetal liver cells from normal and autoimmune-prone mice were used to determine whether reciprocal transplantation of stem cells between normal and autoimmune-prone mice inhibits or causes development of autoimmune disease. NZB recipients of DBA/2 stem cell populations analyzed greater than 100 days after bone marrow or fetal liver cell transplantation showed decreased levels of anti-DNA antibodies and decreased glomerular lesions when compared with nontreated NZB mice or NZB recipients of NZB stem cell preparations. Female DBA/2 recipients of WGA+ NZB bone marrow cell or fetal liver cell transplants exhibited elevated serum autoantibody levels and developed glomerular lesions characteristic of NZB mice when analyzed greater than 100 days after transplantation. These pathological disturbances were not observed in DBA/2 recipients of DBA/2 stem cell preparations. The data indicate that WGA+ stem cells from autoimmune-prone NZB mice contain the genetic defects responsible for the development of systemic autoimmune disease. 相似文献
15.
Rationale for combined use of fetal liver and thymus for immunological reconstitution in patients with variants of severe combined immunodeficiency 下载免费PDF全文
Rajendra Pahwa Savita Pahwa Robert A. Good Genevieve S. Incefy Richard J. O''''Reilly 《Proceedings of the National Academy of Sciences of the United States of America》1977,74(7):3002-3005
Bone marrow cells from a patient with severe combined immunodeficiency were studied in vitro for thymus-dependent lymphocyte (T cell) differentiation by using, at varying times, thymic epithelial monolayers and culture supernatants, thymopoietin, ubiquitin, and thymic extract as inducing agents. On initial evaluation, with thymopoietin or human thymic extract, only a partial differentiation of marrow cells was achieved into cells bearing the human T cell antigenicity without the capacity to form rosettes with sheep erythrocytes, suggesting that the stem cells were defective. Two fetal liver transplantations aimed at reconstitution were unsuccessful, despite evidence of chimerism. Induction studies at that time demonstrated rosetting capacity (with sheep erythrocytes) of the patient's bone marrow cells after coculture with thymic epithelial monolayers but not with their supernatants. An 18-week fetal thymus (irradiated) was then transplanted, but the transplantation was unsuccessful and no clear evidence of chimerism was demonstrated. Subsequently, transplantation of another fetal liver resulted in chimerism and immunologic reconstitution. Serum thymic factor activity rose from 1:2 before transplantation to 1:16 after reconstitution. The combined use of fetal thymus and liver may provide effective immunological reconstitution in some variants of severe combined immunodeficiency. 相似文献
16.
Oertel M 《Journal of gastroenterology》2011,46(8):953-965
Because organ shortage is the fundamental limitation of whole liver transplantation, novel therapeutic options, especially
the possibility of restoring liver function through cell transplantation, are urgently needed to treat end-stage liver diseases.
Groundbreaking in vivo studies have shown that transplanted hepatocytes are capable of repopulating the rodent liver. The
two best studied models are the urokinase plasminogen activator (uPA) transgenic mouse and the fumarylacetoacetate hydrolase
(FAH)-deficient mouse, in which genetic modifications of the recipient liver provide a tissue environment in which there is
extensive liver injury and selection pressure favoring the proliferation and survival of transplanted hepatocytes. Because
transplanted hepatocytes do not significantly repopulate the (near-)normal liver, attention has been focused on finding alternative
cell types, such as stem or progenitor cells, that have a higher proliferative potential than hepatocytes. Several sources
of stem cells or stem-like cells have been identified and their potential to repopulate the recipient liver has been evaluated
in certain liver injury models. However, rat fetal liver stem/progenitor cells (FLSPCs) are the only cells identified to date
that can effectively repopulate the (near-)normal liver, are morphologically and functionally fully integrated into the recipient
liver, and remain viable long-term. Even though primary human fetal liver cells are not likely to be routinely used for clinical
liver cell repopulation in the future, using or engineering candidate cells exhibiting the characteristics of FLSPCs suggests
a new direction in developing cell transplantation strategies for therapeutic liver replacement. This review will give a brief
overview concerning the existing animal models and cell sources that have been used to restore normal liver structure and
function, and will focus specifically on the potential of FLSPCs to repopulate the liver. 相似文献
17.
Oppenheim SM Muench MO Gutiérrez-Adán A Moyer AL BonDurant RH Rowe JD Anderson GB 《Blood cells, molecules & diseases》2001,27(1):296-308
Both allogeneic and xenogeneic hematopoietic chimera models have been developed, including fetal sheep models that demonstrated high levels of stable, multilineage engraftment created by in utero hematopoietic stem cell transplantation. The aim of this study was to test the efficacy of in utero transplantation to create xenogeneic sheep-goat hematopoietic chimeras. Fetal liver cells and T-cell-depleted adult bone marrow were tested as sources of hematopoietic stem cells. Donor cells were injected intraperitoneally into 130 recipient fetuses between 49 and 62 days of gestation. Groups 1 and 2 received crude fetal liver cell preparations. Group 3 received fetal liver cells that were incubated overnight in a phytohemagglutinin-stimulated lymphocyte-conditioned medium (PHA-LCM). In Group 4, hematopoietic stem cells were concentrated by using additional density separations. Group 5 fetal recipients received low-density, T-cell-depleted adult bone marrow cells. In Group 1, fetuses were accessed via hysterotomy. Hematopoietic stem cells were injected into Groups 2, 3, 4, and 5 without cutting through the uterine wall. Fetal survival in the five groups ranged from 56 to 100%. The percentage of chimeras from injected fetuses ranged from 43 to 92% by FACS and PCR analyses; however, levels of chimerism were low (<1%). The highest rates of chimerism were found among recipients of low-density fetal liver cells. Despite the pre-immunocompetent status of the fetal recipients and the genetic similarities between sheep and goats, high levels of engraftment were not observed. The consistently low levels of chimerism observed in this study, as well as the poor results recently reported by others using these procedures, indicate that significant barriers exist to transplanting hematopoietic stem cells in utero. 相似文献
18.
Troeger C Surbek D Schöberlein A Schatt S Dudler L Hahn S Holzgreve W 《Swiss medical weekly》2006,136(31-32):498-503
Early prenatal diagnosis and in utero therapy of certain fetal diseases have the potential to reduce fetal morbidity and mortality. The intrauterine transplantation of stem cells provides in some instances a therapeutic option before definitive organ failure occurs. Clinical experiences show that certain diseases, such as immune deficiencies or inborn errors of metabolism, can be successfully treated using stem cells derived from bone marrow. However, a remaining problem is the low level of engraftment that can be achieved. Efforts are made in animal models to optimise the graft and study the recipient's microenvironment to increase long-term engraftment levels. Our experiments in mice show similar early homing of allogeneic and xenogeneic stem cells and reasonable early engraftment of allogeneic murine fetal liver cells (17.1% donor cells in peripheral blood 4 weeks after transplantation), whereas xenogeneic HSC are rapidly diminished due to missing self-renewal and low differentiation capacities in the host's microenvironment. Allogeneic murine fetal liver cells have very good longterm engraftment (49.9% donor cells in peripheral blood 16 weeks after transplantation). Compared to the rodents, the sheep model has the advantage of body size and gestation comparable to the human fetus. Here, ultrasound-guided injection techniques significantly decreased fetal loss rates. In contrast to the murine in utero model, the repopulation capacities of allogeneic ovine fetal liver cells are lower (0.112% donor cells in peripheral blood 3 weeks after transplantation). The effect of MHC on engraftment levels seems to be marginal, since no differences could be observed between autologous and allogeneic transplantation (0.117% donor cells vs 0.112% donor cells in peripheral blood 1 to 2 weeks after transplantation). Further research is needed to study optimal timing and graft composition as well as immunological aspects of in utero transplantation. 相似文献
19.
Allain JE Dagher I Mahieu-Caputo D Loux N Andreoletti M Westerman K Briand P Franco D Leboulch P Weber A 《Proceedings of the National Academy of Sciences of the United States of America》2002,99(6):3639-3644
Liver regeneration after partial hepatectomy results primarily from the simple division of mature hepatocytes. However, during embryonic and fetal development or in circumstances under which postnatal hepatocytes are injured, organ regeneration is believed to occur from a compartment of epithelial liver stem or progenitor cells with biliary and hepatocytic bipotentiality. The ability to identify, isolate, and transplant epithelial liver stem cells from fetal liver would greatly facilitate the treatment of hepatic diseases currently requiring orthotopic liver transplantation. Here we report the identification and immortalization by retrovirus-mediated transfer of the simian virus 40 large T antigen gene of primate fetal epithelial liver cells with a dual hepatocytic biliary phenotype. These cells grow indefinitely in vitro and express the liver epithelial cell markers cytokeratins 8/18, the hepatocyte-specific markers albumin and alpha-fetoprotein, and the biliary-specific markers cytokeratins 7 and 19. Bipotentiality of gene expression was confirmed by clonal analysis initiated from single cells. Endogenous telomerase also is expressed constitutively. After orthotopic transplantation via the portal vein, approximately 50% of the injected cells integrated into the liver parenchyma of athymic mice without tumorigenicity. Three weeks after transplantation, cells having seeded in the liver parenchyma expressed both albumin and alpha-fetoprotein but had lost expression of cytokeratin 19. These results provide strong evidence for the existence of a bipotent epithelial liver stem cell in nonhuman primates. This unlimited source of donor cells also should enable the establishment of a model of allogenic liver cell transplantation in a large animal closely related to humans and shed light on important questions related to liver organogenesis and differentiation. 相似文献
20.
N. J. Rencricca D. Howard B. Kubanek F. Stohlman 《European journal of haematology》1976,16(3):189-195
Erythroid regeneration was studied in lethally irradiated mice given transplants containing equivalent numbers of haemopoietic stem cells (i.e. CFU) from fetal liver, neonatal marrow or adult marrow. Adult marrow was taken from normal control mice, whose CFU for the most part were not in active cell cycle, as well as from phenylhydrazine-treated groups whose CFU were in similar state of proliferation (i.e. ?40–50% in DNA synthesis) as those derived from fetal liver and neonatal marrow. Splenic and femoral radioiron (59Fe) incorporation were measured at intervals after transplantation and were found to begin earliest in mice given fetal liver, then in animals given neonatal marrow and latest in recipients of adult marrow. Peripheral reticulocytes showed a similar pattern of recovery. The data reported herein suggest that the differences in erythroid regeneration evoked by transplants of fetal liver, neonatal marrow or adult marrow, are not solely attributed to the degree of proliferation in the pluripotential stem cell compartment. These data may, however, suggest a shorter doubling time for cells comprising the fetal and newborn committed erythroid compartments. 相似文献