黄芩苷体外扩增脐血间充质干细胞过程中核转录因子κB激活及白细胞介素6浓度变化

Changes in nuclear factor kappa B activation and interleukin-6 levels during baicalin-induced amplification of umbilical cord blood mesenchymal stem cells in vitro

背景：课题组前期实验通过广泛筛选，发现中药单体黄芩苷在脐血间充质干细胞体外扩增和向神经元定向诱导中效果良好，且其诱导效果明显优于其他单纯的抗氧化剂，可能有另外的机制在起作用。目的：探讨黄芩苷体外扩增人脐血间充质干细胞过程中核转录因子kB的激活及白细胞介素6浓度变化。设计、时间及地点：细胞学体外观察，于2007—02／07在南昌大学第一附属医院神经内科实验室完成。材料：健康孕妇足月顺产儿的脐带血10份，由南昌大学第一附属医院产科提供。所用黄芩苷相对分子质量为446．35，经高效液相色谱一荧光法测定纯度〉95％。抗氧化剂β-巯基乙醇为北方同正生物技术公司产品。方法：采用明胶沉降加密度梯度离心两步法分离脐血单个核细胞，调整细胞浓度为1．0×10^9L^-1接种，加入含体积分数为0．2胎牛血清、谷氨酰胺、B27、300ug／L粒巨噬细胞集落刺激因子、300ug／L干细胞因子的DMEM液体培养体系进行纯化和扩增培养。按照添加抗氧化剂的不同分为3组：空白对照组，不添加任何抗氧化剂；黄芩苷组向DMEM液体培养体系中加入100umol／L黄芩苷；β-巯基乙醇组向DMEM液体培养体系中加入3mmol／Lβ-巯基乙醇。主要观察指标：不同培养时间点脐血单个核细胞的扩增情况；用发绿色荧光信号的细胞核百分率代表核转录因子kB的激活率，间接免疫荧光染色检测脐血单个核细胞P65核移位细胞率；双抗夹心酶联免疫法测定贴壁细胞生长上清液中白细胞介素6的浓度。结果：培养第1周，β-巯基乙醇组与黄芩苷组脐血单个核细胞数量基本相似（P〉0．05），均明显高于空白对照组（P〈0．01）；培养第2-4周，脐血单个核细胞数量黄芩苷组〉β-巯基乙醇组〉空白对照组（P〈0．01）。培养3d及1～4周，空白对照组与β-巯基乙醇组的脐血单个核细胞P65核移位细胞率基本相似（P〉0．05），但均显著低于黄芩苷组（P〈0．01）。3组细胞培养上清液中白细胞介素6浓度的变化与脐血单个核细胞P65核移位细胞率基本。致。结论：黄芩苷能有效促进人脐血单个核细胞体外增殖，可能与其一定程度上激活即刻早期基因核转录因子kB，进而促使细胞提高分泌白细胞介素6水平有关。

BACKGROUND： Previous studies have found that baicalin has good effects on inducing in vitro amplification and differentiation of umbilical cord blood mesenchymal stem cells （MSCs） into neurons. Its effect is significantly better than other simple antioxidant, and there may be other mechanism of action. OBJECTIVE： To explore nuclear factor- k： B activation and interlenkin-6 concentration during in vitro amplification of human umbilical cord blood MSCs induced by baicalin. DESIGN, TIME AND SETTING： The cytokine in vitro study was performed at the Laboratory of Department of Neurology, First Affiliated Hospital, Nanchang University from February to July 2007. MATERIALS： Ten samples were harvested from healthy pregnants following full-term delivery, which was provided by First Affiliated Hospital, Nanchang University. Relative molecular mass of baicalin was 446.35, whose purity was over 95% detected using high performance liquid chromatogram-fluorometric method. β -mercaptoethanol was purchased from Tongzheng, China. METHODS： Human cord blood mononuclear cells were isolated by the two-step assay of gelatin sedimentation plus density gradient centrifugation separation, and purified and expanded at 1.0×10^9 L^-1 in the DMEM liquid culture medium, supplemented with 0.2 volume fraction fetal bovine serum, glutamine, B27,300 u g/L granulocyte colony-stimulating factor, 300 u g/L stem cell factor. According to different antioxidant, there were 3 groups. Blank control group did not receive any antioxidant. In the baicalin group, 100 u mol/L baicalin was added in the DMEM. 3 mmol/L β -mercaptoethanol was added in the DMEM in the β -mercaptoethanol group. MAIN OUTCOME MEASURES： At different time points, amplification of cord blood mononuclear cells was measured. Activation rate of nuclear factor- k B was represented by percentage of nuclei with green fluorescence. P65 positive rate in the nucleus of cord blood mononuclear cells was measured by indirect immunofluorescence staining. Superuatant interleukin-6 levels were tested by double enzyme-linked immunoassay. RESULTS： Number of cord blood mononuclear ceils was identical between the β -mercaptoethanol group and baicalin group at week 1 （P 〉 0.05）, which was significantly higher than in the blank control group （P 〈 0.01）. At weeks 2-4, number of cord blood mononuclear cells was the most in the baicalin group, followed by 13 -mercaptoethanol group and by the blank control group （P 〈 0.01） At day 3 and weeks 1-4, P65 positive rate in the nucleus of cord blood mononuclear cells was similar between the blank control group and the β -mercaptoethanol group （P 〉 0.05）, but was significantly lower than in the baicalin group （P 〈 0.01）. Interleukin-6 levels were identical to the P65 positive rate in the nucleus of cord blood mononuclear cells in each group. CONCLUSION： Baicalin can effectively promote in vitro proliferation of cord blood mononuclear cells, which may be associated with activation of nuclear factor-k B, resulting in enhancing intedeukin-6 levels.