【摘要】：【目的】 將銀屑病患者骨髓間充質(zhì)干細(xì)胞(Bone marrow Mesenchymal Stem Cells,BMMSCs)體外分化為血管內(nèi)皮細(xì)胞(VEC),并對定向分化的VEC活性進(jìn)行初步研究,為銀屑病患者BMMSCs生物特性的深入研究提供依據(jù)。 【方法】 ①采用密度梯度離心法分離銀屑病患者與對照組骨髓單個(gè)核細(xì)胞,通過貼壁法培養(yǎng)BMMSCs,用流式細(xì)胞儀鑒定細(xì)胞表面標(biāo)志;②加入血管內(nèi)皮細(xì)胞生長因子(VEGF)、堿性成纖維細(xì)胞生長因子(bFGF)定向誘導(dǎo)BMMSCs向VEC分化,流式細(xì)胞儀進(jìn)行細(xì)胞表型鑒定;③免疫細(xì)胞熒光技術(shù)鑒定VEC標(biāo)志物KDR、VWF的表達(dá);④體外血管成形試驗(yàn)檢測誘導(dǎo)的內(nèi)皮細(xì)胞體外成形能力;⑤用transwell小室測誘導(dǎo)的VEC對T細(xì)胞的遷移能力。⑥并對在培養(yǎng)分化過程中,發(fā)現(xiàn)1例雙親均患病的銀屑病患者BMMSCs在原代即開始向VEC分化,采用了高通量RNA測序技術(shù)對其差異表達(dá)基因進(jìn)行了篩選。 【結(jié)果】 ①流式測定顯示兩組培養(yǎng)的BMMSCs高表達(dá)CD44、CD29,而HLA-DR、CD45、CD34陰性表達(dá),說明所培養(yǎng)的BMMSCs純度較高;②銀屑病組CD34、CD45、ICAM-1、HLA-R陽性表達(dá),且明顯高于對照組(p㩳0.05);③免疫細(xì)胞化學(xué)顯示兩組都可分化為VEC;④銀屑病患者誘導(dǎo)的VEC具有體外成形能力;⑤銀屑病組誘導(dǎo)的VEC對T細(xì)胞的具有遷移作用;⑥1例自分化患者高通量測定結(jié)果,共篩選了475個(gè)差異表達(dá)基因,其中298個(gè)基因表達(dá)呈增高趨勢,177個(gè)基因呈降低趨勢,其中,差異表達(dá)基因在2個(gè)term上有顯著富集(P0.05),分別為類前列腺素的代謝過程和前列腺素的代謝過程。 【結(jié)論】 銀屑病患者BMMSCs生物學(xué)活性異常,且易于轉(zhuǎn)分化為VEC。銀屑病患者BMMSCs參與了銀屑病的發(fā)病,其向VEC的異常分化可能是導(dǎo)致銀屑病患者皮損真皮乳頭層微血管異常增生的根源。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R758.63

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