银屑病患者骨髓间充质干细胞转分化为血管内皮细胞的初步研究
发布时间：2018-11-24 16:14
【摘要】：【目的】 将银屑病患者骨髓间充质干细胞(Bone marrow Mesenchymal Stem Cells,BMMSCs)体外分化为血管内皮细胞(VEC),并对定向分化的VEC活性进行初步研究,为银屑病患者BMMSCs生物特性的深入研究提供依据。 【方法】 ①采用密度梯度离心法分离银屑病患者与对照组骨髓单个核细胞,通过贴壁法培养BMMSCs,用流式细胞仪鉴定细胞表面标志;②加入血管内皮细胞生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)定向诱导BMMSCs向VEC分化,流式细胞仪进行细胞表型鉴定;③免疫细胞荧光技术鉴定VEC标志物KDR、VWF的表达;④体外血管成形试验检测诱导的内皮细胞体外成形能力;⑤用transwell小室测诱导的VEC对T细胞的迁移能力。⑥并对在培养分化过程中,发现1例双亲均患病的银屑病患者BMMSCs在原代即开始向VEC分化,采用了高通量RNA测序技术对其差异表达基因进行了筛选。 【结果】 ①流式测定显示两组培养的BMMSCs高表达CD44、CD29,而HLA-DR、CD45、CD34阴性表达,说明所培养的BMMSCs纯度较高;②银屑病组CD34、CD45、ICAM-1、HLA-R阳性表达,且明显高于对照组(p㩳0.05);③免疫细胞化学显示两组都可分化为VEC;④银屑病患者诱导的VEC具有体外成形能力;⑤银屑病组诱导的VEC对T细胞的具有迁移作用;⑥1例自分化患者高通量测定结果,共筛选了475个差异表达基因,其中298个基因表达呈增高趋势,177个基因呈降低趋势,其中,差异表达基因在2个term上有显著富集(P0.05),分别为类前列腺素的代谢过程和前列腺素的代谢过程。 【结论】 银屑病患者BMMSCs生物学活性异常,且易于转分化为VEC。银屑病患者BMMSCs参与了银屑病的发病,其向VEC的异常分化可能是导致银屑病患者皮损真皮乳头层微血管异常增生的根源。
[Abstract]:[objective] to differentiate bone marrow mesenchymal stem cells (Bone marrow Mesenchymal Stem Cells,BMMSCs) from psoriatic patients into vascular endothelial cells (VEC),) in vitro and to investigate the activity of VEC in vitro. To provide evidence for further study of biological characteristics of BMMSCs in patients with psoriasis. [methods] 1Bone marrow mononuclear cells were isolated from psoriatic patients and control group by density gradient centrifugation. BMMSCs, was cultured by adherent method and cell surface markers were identified by flow cytometry. 2Vascular endothelial growth factor (VEGF),) basic fibroblast growth factor (bFGF) was added to induce the differentiation of BMMSCs into VEC, flow cytometry was used to identify the cell phenotype, and the expression of VEC marker KDR,VWF was identified by immunocytofluorescence technique. (4) the ability of endothelial cells to be formed in vitro was detected by in vitro angioplasty test. (5) the ability of T cell migration induced by VEC was measured by transwell chamber. 6 during the course of culture and differentiation, it was found that BMMSCs of one psoriatic patient with disease of both parents began to differentiate into VEC in primary culture. The differentially expressed genes were screened by high throughput RNA sequencing. [results] 1Flow flow analysis showed that the BMMSCs of the two groups was highly expressed CD44,CD29, and HLA-DR,CD45,CD34 negative, indicating that the purity of the cultured BMMSCs was high. (2) the positive expression of CD34,CD45,ICAM-1,HLA-R in psoriasis group was significantly higher than that in control group (p0. 05). (5) VEC induced by psoriasis could induce the migration of T cells; In 61 patients with self-differentiation, 475 differentially expressed genes were screened, 298 of which showed an increasing trend of expression and 177 of which showed a decreasing trend. The differentially expressed genes were significantly enriched on two term (P0.05), which were the metabolic process of prostaglandin-like and prostaglandin respectively. [conclusion] the biological activity of BMMSCs in psoriatic patients is abnormal, and it is easy to differentiate into VEC.. BMMSCs is involved in the pathogenesis of psoriasis and its abnormal differentiation to VEC may be the cause of abnormal proliferation of dermal papillary layer in psoriatic patients.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R758.63
[Abstract]:[objective] to differentiate bone marrow mesenchymal stem cells (Bone marrow Mesenchymal Stem Cells,BMMSCs) from psoriatic patients into vascular endothelial cells (VEC),) in vitro and to investigate the activity of VEC in vitro. To provide evidence for further study of biological characteristics of BMMSCs in patients with psoriasis. [methods] 1Bone marrow mononuclear cells were isolated from psoriatic patients and control group by density gradient centrifugation. BMMSCs, was cultured by adherent method and cell surface markers were identified by flow cytometry. 2Vascular endothelial growth factor (VEGF),) basic fibroblast growth factor (bFGF) was added to induce the differentiation of BMMSCs into VEC, flow cytometry was used to identify the cell phenotype, and the expression of VEC marker KDR,VWF was identified by immunocytofluorescence technique. (4) the ability of endothelial cells to be formed in vitro was detected by in vitro angioplasty test. (5) the ability of T cell migration induced by VEC was measured by transwell chamber. 6 during the course of culture and differentiation, it was found that BMMSCs of one psoriatic patient with disease of both parents began to differentiate into VEC in primary culture. The differentially expressed genes were screened by high throughput RNA sequencing. [results] 1Flow flow analysis showed that the BMMSCs of the two groups was highly expressed CD44,CD29, and HLA-DR,CD45,CD34 negative, indicating that the purity of the cultured BMMSCs was high. (2) the positive expression of CD34,CD45,ICAM-1,HLA-R in psoriasis group was significantly higher than that in control group (p0. 05). (5) VEC induced by psoriasis could induce the migration of T cells; In 61 patients with self-differentiation, 475 differentially expressed genes were screened, 298 of which showed an increasing trend of expression and 177 of which showed a decreasing trend. The differentially expressed genes were significantly enriched on two term (P0.05), which were the metabolic process of prostaglandin-like and prostaglandin respectively. [conclusion] the biological activity of BMMSCs in psoriatic patients is abnormal, and it is easy to differentiate into VEC.. BMMSCs is involved in the pathogenesis of psoriasis and its abnormal differentiation to VEC may be the cause of abnormal proliferation of dermal papillary layer in psoriatic patients.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R758.63
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4 蔡绥R，
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