【作者】 张文艳；

【导师】 蔡海波；

【作者基本信息】 华东理工大学 ， 生物化工， 2018， 硕士

【摘要】 体外扩增是解决脐血造血干细胞(hematopoietic stem cells,HSCs)移植过程中数量不足的有效方法。在HSCs的体外扩增过程中添加糖类、细胞因子等外源性物质,导致细胞内活性氧(Reactive oxygen species,ROS)水平急剧增加,破坏细胞内的氧化还原动态平衡,造成细胞功能损伤,影响HSCs的体外增殖。因此,有必要对HSCs体外培养过程中细胞内ROS水平进行调控。黄酮类、多酚类和蒽醌类化合物广泛存在于各种植物中,具有抗氧化活性,在一定的浓度范围内能够有效的清除自由基,有利于细胞的体外增殖。为此,本文以新鲜分离的脐血CD34+细胞为起始细胞,在无血清培养体系中考察了黄酮类抗氧化物(葛根素和金丝桃苷)浓度对CD34+细胞体外扩增的影响。结果发现:0.1μM葛根素能够有效地提高总细胞和CD34+细胞的扩增;与对照组相比,1μM金丝桃苷能够促进总细胞、CD34+细胞和CD34+CD38-细胞的扩增,并且两组之间CD34+细胞的再扩增能力无显著性差异。培养体系中添加1μM金丝桃苷培养14d后,总细胞、CD34+细胞和CD34+CD38-细胞的扩增倍数分别为54.9±9.6、9.1±1.8和8.1±0.9倍,显著高于对照组的42.0±7.7、5.8±1.5和5.8±1.3倍(p<0.05)。进而分析了 1μM金丝桃苷对CD34+细胞内ROS水平和细胞凋亡的影响,发现培养体系中添加1μM金丝桃苷后,培养物中凋亡细胞的比例显著降低,但是对细胞内的ROS水平没有影响。此外,考察了多酚类(儿茶素)和蒽醌类(芦荟大黄素)抗氧化物浓度对CD34+细胞体外扩增的影响。结果发现,培养体系中添加10μM儿茶素培养14d后,总细胞、CD34+细胞的扩增倍数分别为57.2±12.8和9.7±0.8倍,显著高于对照组42.7±9.7和7.1±0.2倍(p<0.05);并且培养14d后CD34+细胞的再扩增能力与对照组相比无显著性差异。进一步考察了 10μM儿茶素对细胞内的ROS水平和凋亡细胞比例的影响,结果发现,在培养体系中,添加10μμM儿茶素培养CD34+细胞,显著降低培养物中凋亡细胞的比例,而对培养过程中细胞内ROS水平没有影响。添加1μM芦荟大黄素培养14d后,CD34+细胞和CD34+CD38细胞的扩增倍数分别为5.4±1.1和5.8±0.9倍,显著高于对照组的3.7±0.9和4.2±0.9倍(p<0.05),但是培养14d后CD34+细胞的再扩增能力显著下降。在培养体系中,添加1μM芦荟大黄素培养CD34+细胞,显著降低培养物中凋亡细胞的比例,对培养过程中细胞内ROS水平没有影响。综上所述,植物来源的抗氧化物降低了 CD34+细胞体外培养时细胞凋亡比例,促进了 CD34+细胞的体外扩增。以上结果为应用植物来源的抗氧化物优化CD34+细胞体外培养条件提供了理论依据。更多还原

【Abstract】 Ex vivo expansion is apotential approach to overcome the limited amount of hematopoietic stem cells(HSCs)in cord blood transplantation.However,recent studies indicate that high levels of intracellular reactive oxygen species(ROS)that are frequently caused by glucose and cytokines weaken the cellular proliferation dramatically.Addition of antioxidants,such as flavonoids,polyphenols,and anthraquinones isolated from plants,would benefit ex vivo expansion were examined comprehensively.In the present study,freshly isolated CD34+ cells from umbilical cord blood were cultured in serum free medium with cytokine cocktails.First of all,the dose effect of flavonoids on ex vivo expansion of CD34+ cells wasstudied.The results showed that an increase of total cells and CD34+ cells was yielded by the addition of 0.1 μM Puerarin;the expansion of total cells,CD34+ cells and CD34+ CD38-cells was promoted significantly by the addition of 1μM hyperoside.The fold expansion of total cells,CD34+ cells and CD34+ CD38" cells could achieve 54.9±9.6,8.94±1.87 and 8.0±0.9 folds respectivelyafter 14 days culture with 1μM hyperoside,which were significantly higher than those in control group.And the cells cultured with 1μM hyperoside showed same re-expansion ability of CD34+ cells as those of the cells cultured without hyperoside.Further,the effect of hyperoside on the level of intracellular ROS and the rate of apoptosis cells were investigated,the result showed that the proportion of apoptosis cells was markedly decreased by the addition of 1μM hyperoside,however,the level of cellular ROS was not affected during culture with hyperoside.Moreover,the dose effect of polyphenols and anthraquinone on ex vivo expansion of CD34+ cellswere studied.The results demonstrated that the fold expansion of total cells and CD34+ cells could achieve57.2±12.8and 9.7±0.8 folds respectively,after 14 days culture with 10μM catechin,which increased significantly compared with those in control group.And the cells cultured with 10μM catechin showed same re-expansion ability of CD34+ cells as those in control group.Furthermore,the effect of catechin on the level of intracellular ROS and the rate of apoptosis cells were investigated,the results showed that the proportion of apoptosis cells was markedly decreased by the addition of 10μM catechin,however,the level of cellular ROS was not affected during culture with catechin.After 14-day culture with 1λM aloe emodin,the fold expansion of CD34+ cells and CD34+ CD38-cells were 5.4±1.1 and 5.8±0.9 folds respectively,which were both much higher than those in control group,but the re-expansion ability of CD34+ cells was decreased obviously compared with control.The proportion of apoptosis cells was markedly decreased by the addition of 1μM aloe emodin,however,the level of cellular ROS was not affected during culture with aloe emodin.The results indicated that the addition of those antioxidants promoted ex vivo expansion of HSCs by reduced the percentage of apoptosis cells.The above results provided a theoretical basis for the application of antioxidants to optimize the ex vivo expansion of hematopoietic stem cells.更多还原