摘要
目的:比较脐血间充质干细胞和骨髓间充质干细胞对植物血凝素诱导的淋巴细胞增殖的抑制作用。方法:实验于2005-07/2006-03在南昌大学第二附属医院血液病研究所及江西省医学分子重点实验室完成。①实验材料:脐带血9份,由南昌大学第二附属医院妇产科提供,产妇及其家属均知情同意。正常骨髓9份,由南昌大学第二附属医院血液科门诊及住院患者提供,均附有捐赠同意书。外周血9份,取自正常自愿者。本实验经医学伦理委员会批准。②实验方法:无菌条件下采集脐血、骨髓和正常人外周血,分离单个核细胞。脐血及骨髓单个核细胞以1×109 L-1接种于培养瓶中,加入含体积分数为0.1的小牛血清、1×10-3mol/L氢化可的松的IMDM培养液,7d后换液,去除悬浮细胞,以后每3～4d换液1次,待细胞90%融合后,胰蛋白酶+乙二胺四乙酸混合消化,传代培养,取第3代细胞用于实验。正常人外周血单个核细胞以1×109 L-1接种于培养瓶中,加入含体积分数为0.1的小牛血清、10mg/L植物血凝素的IMDM液,培养3d后收集细胞用于实验。③实验评估:采用流式细胞术检测脐血间充质干细胞及淋巴细胞表面分子。取第3代的脐血和骨髓间充质干细胞,体外诱导培养后以脂肪染色液鉴定其向脂肪细胞的分化情况。将不同细胞浓度的脐血和骨髓间充质干细胞分别加入到植物血凝素诱导的外周血淋巴细胞增殖体系中,比较两种不同来源的间充质干细胞对淋巴细胞增殖的调控作用。结果:①脐血间充质干细胞和骨髓间充质干细胞的培养及鉴定:第3代脐血间充质干细胞表面分子标记率CD29为85.18%,CD166为72.52%,CD54为33.70%,CD45为6.70%,CD13为1.51%,CD34为0.23%。正常人外周血单个核细胞中的CD3+T细胞为60%～70%,CD20+B细胞为3.5%～4.5%。脐血间充质干细胞诱导培养3周,可见呈桔黄色的脂肪细胞。②脐血间充质干细胞及骨髓间充质干细胞对淋巴细胞增殖的抑制:淋巴细胞:间充质干细胞为1∶1,1∶0.5,1∶0.1,1∶0.05,1∶0.02,1∶0.01时,脐血间充质干细胞对淋巴细胞增殖的抑制率分别为(49.5±4.8)%,(58.4±6.0)%,(38.1±4.0)%,(31.4±3.2)%,(24.3±3.2)%,(12.6±6.7)%,而骨髓间充质干细胞对淋巴细胞增殖的抑制率分别为(52.4±8.4)%,(65.1±9.7)%,(34.7±4.5)%,(13.0±6.4)%,(-10.7±12.6)%,(-43.9±9.4)%。后3种细胞浓度比例脐血间充质干细胞对淋巴细胞的抑制作用明显强于骨髓间充质干细胞(t=7.72,8.14,14.68,P均<0.05)。结论:脐血间充质干细胞与骨髓间充质干细胞一样,也具有抑制植物血凝素诱导淋巴细胞增殖的作用,抑制效果与间充质干细胞数量有关。低浓度的脐血间充质干细胞对淋巴细胞的抑制作用明显强于骨髓间充质干细胞。
AIM： To compare the inhibitory effect of umbilical cord blood mesenchymal stem cells （UCB-MSCs） and bone marrow mesenchymal stem cells （BM-MSCs） on lymphocytes （LCs） proliferation stimulated by phytohemagglutinin （PHA）. METHODS： The experiment was conducted from July 2005 to March 2006 in Hematology Institute of Second Affiliated Hospital of Nanchang University and Jiangxi Medical Molecule Key Laboratory.①Umbilical cord blood samples （n =9） were collected from healthy baby in Department of Gynecology and Obstetrics of Second Affiliated Hospital of Nanchang University. Puerperants and their family members signed the informed consent. Normal bone marrow （n =9） was harvested from inpatients and Out-Patient Clinic patients of Department of Hematology of Second Affiliated Hospital of Nanchang University. All the persons were with donation consent. Peripheral blood （n =9） was collected from normal volunteers. The trial was approved by medical ethics committee. ②Mononuclear cells （MNCs） were isolated from UCB, BM and peripherial blood, respectively. UCB-MNCs and BM-MNCs were cultured in the IMDM culture liquid containing calf serum of 0.1 volume fraction and 1×10^-2 mol/L cetacort in culture flask at concentration of 1×10^9 L^-1. The culture liquid was replaced after 7 days, and then replaced every 3 or 4 days after removing suspension cells. The cells were serial subcultivation by trypase-EDTA after the cells confusion reached 90% and the third generation cells were used in the experiment. Peripheral blood cells （PBCs） were cultured in the IMDM liquid containing calf serum of 0.1 volume fraction and 10 mg/L PHA in culture flask at concentration of 1×10^9 L^-1, and the cells were harvested after 3-day culture. ③The MSCs and LCs surface molecules were identified by flow cytometry. The differentiation capacities of UCB-MSCs and BM-MSCs into the adipocytes were assessed with histochemical solution. UCB-MSCs and BM-MSCs with various concentrations were added to PHA-induced proliferation system, and then the inhibitory effects of UCB-MSCs and BM-MSCs on proliferation and regulation of LCs were compared. RESULTS： ①Culture and identification of UCB-MSCs and BM-MSCs： The expressions of surface molecules about CD29, CD166, CD54, CD45, CD13 and CD34 on MSCs were 85.18%, 72.52%, 33.70%, 6.70%, 1.51% and 0.23%, respectively. The surface molecules expression about CD3^＋ T and CD20^＋B of PBCs were 60%-70% and 3.5%-4.5%, respectively. UCB-MSCs became into adipocyte which color was yellow after cultured 3 weeks.②Inhibitory effect of UCB-MSCs and BM-MSCs： When MSCs and LCs mixed at the ratio of 1：1, 0.5：1, 0.1：1, 0.05：1, 0.02：1, 0.01：1, the inhibitory rates of UCB-MSCs on LCs proliferation were （49.5±4.8）%, （58.4±6.0）%, （38.1±4.0）%, （31.4±3.2）%, （24.3±3.2）%, （12.6±6.7）%, respectively, whereas that of BM-MSCs were （52.4±8.4）%,（65.1±9.7）%, （34.7±-4.5）%,（13.0±6.4）%,（-10.7±12.6）%, （-43.9±9.4）%, respectively. When the ratio was 0.05：1, 0.02：1 and 0.01：1, the inhibitory effect of UCB-MSCs was much better than that of the BM-MSCs （ t =7.72,8.14,14.68, P 〈 0.05）. CONCLUSION： Both UCB-MSCs and BM-MSCs showed inhibitory effect on PHA-induced LCs proliferation and the effect was associated with the number of MSCs. The inhibitory effect of UCB-MSCs was much stronger than that of BM-MSCs at low concentration.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第24期4678-4681,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
江西省科技厅重点课题(2004年)~~
