【作者】 何晓霞；

【导师】 白海； 王存邦；

【作者基本信息】 兰州大学 ， 内科学， 2009， 硕士

【摘要】 目的研究健康供体骨髓源性间充质干细胞(BM-MSC)中Toll样受体(TLRs)的表达特征;研究POLY(I:C)(TLR3激活剂)与脂多糖(LPS,TLR4激活剂)分别对BM-MSC增殖的影响;研究POLY(I:C)与LPS分别对BM-MSC分泌IL-6、IL-10和IL-12的影响。方法Ficoll法分离培养健康供体的BM-MSC;用流式细胞仪(FCM)检测BM-MSC中CD34、CD45、HLA-DR、CD44和CD71的表达;免疫细胞化学法检测爬片BM-MSC细胞中CD71的表达;半定量RT-PCR法检测TLR1-10mRNA的表达;MTT法检测POLY(I:C)和LPS对BM-MSC增殖的影响;EHSA法检测POLY(I:C)、LPS对BM-MSC分泌IL-6、IL-10和IL-12的影响。结果FCM发现体外分离培养的BM-MSC中CD34、CD45和HLA-DR表达呈阴性,CD44和CD71表达呈阳性;爬片BM-MSC细胞中CD71的表达呈阳性。BM-MSC中TLR1-10 mRNA均有表达,其中TLR2、TLR3、TLR4、TLR7、TLR8、TLR9均高表达,TLR1、TLR5、TLR6、TLR10均低表达,BM-MSC中有较高水平的TLR3和TLR4表达。体外培养第5天时,各浓度POLY(I:C)组存活的BM-MSC细胞数均少于10%FBS组(分别p=0.000),而多于DMEM组(分别p＜0.05),浓度(concentration,Conc)和存活细胞数(viable count,VC)间存在半对数直线相关关系(r=-0.998,p=0.039)。体外培养第7天时,中浓度和高浓度LPS组存活细胞数均少于10%FBS组(p=0.013,p=0.003),而多于DMEM组(p=0.001,p=0.002)。与对照组相比,用不同剂量的POLY(I:C)刺激时,BM-MSC分泌IL-6明显增多(P=0.000,P=0.021),且25ug╱mL组明显高于2.5ug╱mL组(P=0.000);但分泌IL-10明显减少(P=0.000),且250ug╱mL组明显低于25ug/mL组;而对IL-12的分泌无明显影响。与对照组相比,用不同剂量的LPS刺激时,BM-MSC分泌IL-6明显增多(P=0.000),但各剂量组间无显著性差异(P＞0.05),而对IL-12和10的分泌无明显影响。结论从健康供体骨髓中分离培养的BM-MSC表达不同水平的TLR1-10 mRNA。BM-MSC中TLR3和TLR4的表达是功能性的。更多还原

【Abstract】 Objective The aim of this study was to dectect the expression of Toll-like receptors of mesenchemal stem cells derived from bone marrow of healthy donor(BM-MSC) and to study effects of TLR3 and TLR 4 activator POLY(I:C) and LPS on proliferation of BM-MSC and on cytokines secretion by BM-MSC respectively.Methods BM-MSC was isolated from bone marrow of healthy donor by FicoU method.Expression of CD34,CD45,HLA-DR,CD44 and CD71 in BM-MSC were shown by flow cytometry.CD71 was detected in BM-MSC by immunocytochemistry.TLR1-10 mRNA levels in BM-MSC were evaluated by semiquantitative RT-PCR.Effects of POLY(I:C) and LPS on proliferation of BM-MSC were studied by MTF method.Effects of POLY(I:C) and LPS on secretion of IL-10,IL-12 and IL-6 by BM-MSC were studied by ELISA method.Results The Results showed that expression of CD34,CD45 and HLA-DR in BM-MSC was negative while expression of CD44 and CD71 was positive by FCM.CD71 was localized in BM-MSC by immunocytochemistry.All of TLR1-10 mRNA was found in BM-MSC with high expression level of TLR2,TLR3,TLR4,TLR7,TLR8,TLR9 and low expression level of TLR1,TLRS,TLR6,TLR10. Viable counts of different concentration teams of POLY(I:C) were less than that of 10%FBS team (p=0.000,respectively),but more than that of DMEM team(p＜0.05,respectively) 5 days after incubation while a semilogarithm linear correlation was found between concentration and viable count(r=-0.998,p =0.039).However,viable counts of moderate and high concentration teams of LPS were less than that of 10%FBS team(p=0.013,p=0.003),but more than that of DMEM team(p=0.001,p=0.002) 7 days after incubation.Expression of CD34,CD45 and HLA-DR in BM-MSC was negative while expression of CD44 and CD71 was positive by FCM.CD71 was localized in BM-MSC by immunocytochemistry.High levels of TLR3 and 4 mRNA was found to be expressed in BM-MSC by RT-PCR.Compared with the control,the amout of IL-6 secreted by BM-MSC incubated with different doses of TLR3 activator POLY(I:C) was increased significantly(P=0.000,P=0.021),and the amount of IL-6 secreted was more in 25ug/mL group than that in 2.5ug/mL group(P=0.000).But when compared with the control,the amout of IL-10 secreted incubated with different doses of TLR3 activator POLY(I:C) was decreased significantly (P=0.000) and the amount of IL-10 secreted was less in 250ug/mL group than that in 25ug/mL group (P=0.000) while the amount of IL-12 secreted was not affected.Compared with the control,the amout of IL-6 secreted by BM-MSC incubated with different doses of TLR4 activator LPS was increased significantly(P=0.000),and there was no difference among the three groups(P＞0.05) while the amount of IL-10 an IL-12 secreted were not affected.:Conclusion Different levels of TLR1-10 mRNA were expressed in BM-MSC of healthy donor.It is suggested that expression of TLR3 and 4 on BM-MSC be functional.更多还原