骨髓间充质干细胞联合来氟米特对小鼠淋巴细胞增殖的抑制  

Inhibitory effects of bone marrow mesenchymal stem cells combined with leflunomide on the proliferation of mouse lymphocytes

作　　者：裘影影[1] 李晶[1] 殷玉俊[1] 汤郁[1] 尤海燕[1] 朱伟[2] 焦志军[1] 

机构地区：[1]江苏大学附属医院风湿科,江苏省镇市212001 [2]江苏大学医学技术学院血液学实验室,江苏省镇江市212000

出　　处：《中国组织工程研究与临床康复》2008年第29期5658-5662,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：江苏省科技厅社会发展项目(BS2005043)~~

摘　　要：背景：来氟米特是一种新型的免疫抑制剂，其活性产物A771726在体外能抑制增生分裂细胞的二氢乳清酸脱氢酶和酩氨酸激酶的活性，抑制活化淋巴细胞的增殖。目的：观察骨髓间充质干细胞在体外联合来氟米特对小鼠淋巴细胞增殖的抑制效应。设计、时间及地点：随机对照，细胞学免疫调控实验，于2007—06／11在江苏大学附属医院中心实验室完成。材料：清洁级5-6周龄BALB／c雌性小鼠42只，18只分离制备骨髓间充质干细胞，传至第3代用于实验；24只用于制备脾淋巴细胞。来氟米特活性代谢产物A771726由美国欣凯公司肖飞博士惠赠。非特异性有丝分裂原ConA、脂多糖为sigma公司产品。方法：将制备的T、B淋巴细胞悬液分为骨髓间充质干细胞组、来氟米特组、骨髓间充质干细胞＋来氟米特组，其中骨髓间充质干细胞浓度为2×10^8L^-1，骨髓间充质干细胞数：淋巴细胞数=1：30，来氟米特终浓度为15mg／L，此外各组加入5mg／L非特异性有丝分裂原ConA或25mg／L脂多糖。主要观察指标：MTF法检测T、B淋巴细胞增殖抑制率。通过表面标志检测T、B淋巴细胞活化情况。流式细胞仪测定T淋巴细胞凋亡程度。RT-PCR法检测T淋巴细胞干扰素γ、白细胞介素10、白细胞介素4mRNA表达水平。结果：与阳性对照比较，骨髓间充质干细胞组、来氟米特组、骨髓间充质干细胞＋来氟米特组T淋巴细胞体外增殖抑制率均明显提高（P〈0．05），且后者变化幅度大于前两组（P〈0．05）；各组B淋巴细胞增殖抑制情况与上述结果相似。与阳性对照比较，各组T淋巴细胞CD3^＋CD69^＋、CD3^＋CD28^＋的表达以及B淋巴细胞CD19^＋CD69^＋、CD19^＋CD86^＋的表达均基本相似；骨髓间充质干细胞组、骨髓间充质干细胞＋来氟米特组T淋巴细胞凋亡率明显下降（P〈0．05）。与阳性对照比较，骨髓间充质干细胞�BACKGROUND： Leflunomide is a new immunodepressant. Its active product A771726 can inhibit the activities of dihydroorate dehydrogenase and the proliferation of activated lymphocytes in vitro. OBJECTIVE： To investigate the inhibitory effects of bone mesenchymal stem cells （BMSCs） combined with leflunomide （LEF） on lymphocyte proliferation in mice in vitro. DESIGN, TIME AND SETTING： The randomized control cytology immunoregulation experiment was performed at the Central Laboratory, Affiliated Hospital of Jiangsu University from June to November 2007. MATERIALS： Forty-two BALB/c female mice aged 5-6 weeks were used in this study. Eighteen of them were used to prepare BMSCs. At the third passage, BMSCs were used in this study. Twenty-four of them were used to prepare splenic lymphocytes. LEF activated metabolic product A771726 was presented by Doctor Xiao from Cinkate, USA. Non-specific mitogen ConA and lipopolysaccharide were obtained from Sigma, USA. METHODS： T and B lymphocyte suspension was divided into BMSCs group, LEF group, BMSCs＋LEF group. The concentration of BMSCs was 2×10^8 L^-1. Number of BMSCs： number of lymphocytes = 1： 30. The final concentration of LEF was 15 mg/L. In addition, 5 mg/L non-specific mitogen ConA and 25 mg/L lipopolysaccharide were used in each group. MAIN OUTCOME MEASURES： MTT was used to detect the inhibitory rate of T and B lymphocyte proliferation. Activation of T and B lymphocytes was measured by surface markers. Flow cytometry was applied to determine the apoptosis rate of T lymphocytes. Reverse transcription-polymerase chain reaction （RT-PCR） was employed to examine interferon γ, interleukin 10 and interleukin 4 mRNA expression of T lymphocytes. RESULTS： Compared with the positive control, the inhibitory rate of T lymphocyte proliferation was significantly increased in the BMSCs group, LEF group, BMSCs＋LEF group （P 〈 0.05）, and the changes was greater in the BMSCs＋LEF group compared with that in the BMSCs group and LEF group �

关 键 词：骨髓间充质干细胞 来氟米特 淋巴细胞 免疫调节 

分 类 号：R394.2[医药卫生—医学遗传学]