Transforming growth factor beta1 gene silenced adipose-derived stem cell transplantation reduces thrombocytopenic purpura

doi:10.3969/j.issn.2095-4344.2017.01.011

Abstract

Abstract:

BACKGROUND: Some studies have shown that stem cell therapy offers new promise for the treatment of thrombocytopenic purpura, but there are still difficulties in the perfect treatment for thrombocytopenic purpura.
OBJECTIVE: To investigate the effect of transplantation of transforming growth factor beta1 (TGF-β1) gene silenced mouse adipose-derived stem cells (ADSCs) in mice with thrombocytopenic purpura.
METHODS: The mouse ADSCs were cultured in vitro and transfected by the siRNA expression plasmids containing TGF-β1 (TGF-β1-siRNA). Mouse model of thrombocytopenic purpura were made by guinea pig anti-mouse platelet serum intraperitoneally injected. Then, model mice were randomly assigned into model, ADSCs, TGF-β1 silenced ADSCs, or prednisone group, followed by intravenous injection of 20 μL normal saline, 2×10⁶/L CM-Dil-labeled ADSCs, 20 μL TGF-β1 silenced ADSCs suspension via the tail vein, or intragastrical administration of 5 mg/kg prednisone, respectively. The mice in each group were sacrificed after 14 days of treatment.
RESULTS AND CONCLUSION: Compared with the other three groups, the TGF-β1 silenced ADSCs group showed a significant increase in the number of CM-Dil positive cells, blood platelet count, and proportion of thromocytogenic megakaryocytes, while a marked decrease in the total number of megakaryocytes, PAIgG values and TGF-β1 level (P < 0.05). To conclude, TGF-β1 gene silenced ADSCs transplantation can effectively reduce the TGF-β1 and PAIgG values in ITP mice, and promote the differentiation and maturation of megakaryocytes in the bone marrow, which can effectively improve the number of platelets, playing a role in the treatment of ITP.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Tissue Engineering, Transforming Growth Factor beta, Blood Platelets

CLC Number:

R394.2

Yin Wan-yi, Shen Yang, Liu Qing-chi, Jia Xiao-hui, Zhang Li-hong. Transforming growth factor beta1 gene silenced adipose-derived stem cell transplantation reduces thrombocytopenic purpura[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(1): 60-65.

Figures/Tables 2

2.1 实验动物数量分析实验共纳入小鼠80只，均造模成功并进入结果分析，期间无死亡、感染及补充和缺失等情况发生。 2.2 动物一般情况各实验组在造模后的第2天均出现明显的皮下出血和紫癜，以注射部位、四肢、腹部、尾部多见，注射后短时间内小鼠精神萎靡、活动量少、行动迟缓、喜扎堆。各组予不同方式干预后，脂肪干细胞组出血明显减轻，泼尼松组及转化生长因子β1沉默组紫癜明显减退、活动量增加；而模型组小鼠的紫癜情况无显著变化。 2.3 脂肪干细胞的形态观察分别在显微镜下观察P4代和M-Dil标记的脂肪干细胞。传3代后的脂肪干细胞，生长旺盛，形态渐趋一致、愈趋向长梭形，以一点为中心向两头呈放射状。细胞呈复层生长，胞膜完整、细胞状态良好，细胞间联系紧密，见图1A。经过CM-Dil标记的脂肪干细胞细胞，荧光显微镜观察，可见红色荧光细胞，标记率达98%，见图1B。 2.4 转染后脂肪干细胞中转化生长因子β1蛋白的表达应用Western blot法于TGF-β沉默脂肪干细胞转染后3 d对各组转化生长因子β1蛋白表达量比较发现，转化生长因子β1呈现低表达，第14天再次对各组转化生长因子β1表达量进行比较，蛋白仍呈现低表达水平(图2)。 2.5 CM-Dil标记转染后脂肪干细胞情况荧光显微镜下，经CM-Dil标记的脂肪干细胞呈现红色荧光。血小板减少性紫癜模型组及泼尼松组未见CM-Dil阳性细胞，脂肪干细胞组较多[(16.69±3.17)%]，转化生长因子β1沉默组[(22.98± 4.06)%]明显增多(P < 0.05，图3)。 2.6 血小板、骨髓巨核细胞计数、比例变化对各组血小板计数进行比较发现：与血小板减少性紫癜模型组比较，脂肪干细胞组、泼尼松组及转化生长因子β1沉默组血小板计数均较高；且转化生长因子β1沉默组最高(P < 0.05)，这提示脂肪干细胞、转化生长因子β1与泼尼松相类似，均能够提升血小板数，对血小板减少性紫癜小鼠的治疗其积极作用(表1)。各组巨核细胞计数进行比较发现，脂肪干细胞组、泼尼松组及转化生长因子β1沉默组计数均较血小板减少性紫癜模型组低，且转化生长因子β1沉默组最低(转化生长因子β1沉默<泼尼松组<脂肪干细胞组)，差异有显著性意义(P < 0.05)。进一步行细胞比例的比较，各治疗组产板巨核细胞均较血小板减少性紫癜模型组增加，且经转化生长因子β1 沉默治疗组增加更明显(转化生长因子β1沉默组>泼尼松组>脂肪干细胞组)(P < 0.05)，见表1，2。说明泼尼松、脂肪干细胞及转化生长因子β1均能够使骨髓巨核细胞分化增加，且产板比例增加，进而提升外周血小板数，对血小板减少性紫癜小鼠进行治疗。 2.7 各组血清PAIgG及骨髓转化生长因子β1水平变化由表3可以看出，血小板减少性紫癜模型组PAIgG水平较脂肪干细胞组、泼尼松组及转化生长因子β1沉默组明显升高，差异有显著性意义(P < 0.05)，且脂肪干细胞组>泼尼松组>转化生长因子β1沉默组(P < 0.05)。各组骨髓转化生长因子β1进行比较发现：血小板减少性紫癜、泼尼松和脂肪干细胞组之间差异无显著性意义(P > 0.05)；而转化生长因子β1沉默组与血小板减少性紫癜模型组相比，转化生长因子β1水平显著下降(P < 0.05)。"

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