脱氢表雄酮（DHEA）在维生素D转化代谢中的作用机制研究

发布时间：2018-04-04 14:45

本文选题：人骨髓间充质干细胞　切入点：梯度密度离心　出处：《广西医科大学》2015年博士论文

【摘要】：第一部分人骨髓间充质干细胞体外培养与鉴定[目的]运用实验室已经成功创建的人骨髓间充质干细胞培养方案,分离、培养和鉴定hMSCs,为后续研究提供充足的干细胞来源。[方法]采用密度梯度离心法分离出单个核细胞,细胞贴壁法在α-MEM培养基中获得原代hMSCs,随后进行传代培养。对成骨细胞分化采用碱性磷酸酶活性检测和Alizarin Red S染色进行鉴定；对脂肪细胞分化应用Oil Red-O染色方法进行鉴定；应用Alcian blue染色对软骨细胞分化进行鉴定。[结果]骨髓细胞培养6-24小时,胞体形状由圆形逐步变为三角型或不规则形状。培养第2-4天间充质细胞形态发生改变,细胞形态逐渐变为成纤维细胞样的梭型,并聚集成集落；培养7-14天,细胞长满培养瓶底面积的70%-80%,细胞排列更加紧密,此时的骨髓间充质干细胞为单层细胞。对细胞进行成脂诱导分化,可检测不同形状的Oil Red-O阳性染色细胞形成；成骨细胞诱导分化后,碱性磷酸酶活性检测,以及Alizarin Red S染色的阳性细胞形成；诱导成软骨细胞分化后可见Alcian blue染色阳性细胞形成。[结论]本试验采用的方法可以成功分离、培养及扩增人骨髓间充质干细胞,并鉴定其具有成骨、成脂、成软骨分化能力。第二部分DHEA上调IGF-I基因表达并促进hMSCs向成骨分化[目的]本研究验证DHEA是否通过调节IGF-I从而促进骨髓间充质干细胞向成骨细胞转化。[方法]采用不同时间点、不同浓度DHEA药物刺激人骨髓间充质干细胞,进行时间和浓度依赖试验,并采用生物化学和小分子酶抑制剂去阐明DHEA在人骨髓间充质干细胞中调节IGF-I基因表达并促进其向成骨分化的机制。在本实验研究,应用PKA信号通路抑制剂H-89, PKC信号通路抑制剂CHE,JNK信号通路抑制剂SP600125分别阻断PKA信号通路,PKC信号通路,JNK信号通路。应用P13K信号通路抑制剂LY294002,P38 MAPK信号通路阻断剂SB203580,P42/44信号通路阻断剂PD098059, IGF-IR受体阻断剂AG1024分别阻断PI3K、P38 MAPK、P42/44 MAPK、IGF-IR信号通路。[结果]在骨髓间充质干细胞中,DHEA可上调IGF-I基因表达,并且存在时间和浓度依赖。IGF-I基因表达高峰期是在DHEA刺激后4-6小时；在刺激时间为6小时情况下,最佳药物浓度为10nM。阻断PI3K,P38 MAPK, P42/44 MAPK信号通路,DHEA上调IGF-1基因表达的作用被抑制。随后样本经特殊酶抑制剂作用后,对成骨分化相关基因和碱性磷酸酶检测均出现表达降低。[结论]DHEA可能经过IGF-I信号通路,包括PI3K, P38 MAPK, P42/44 MAPK信号通路来上调人骨髓间充质干细胞中IGF-I基因表达并促进其向成骨方向分化。第三部分[目的]研究老龄化与1 α-羟化酶关系,并阐明DHEA在维生素D代谢中的作用机制。[方法]运用RT-PCR方法研究在人骨髓间充质干细胞中维生素D代谢关键酶1α-羟化酶的表达,并切分析年龄对其表达的影响。应用IGF-IR受体阻断剂AG1024, CERB阻滞剂KG501分别阻断IGF-1, CERB信号通路,从蛋白水平研究DHEA在维生素D转化代谢中的作用机制。[结果]一组包括19例患者的样本中,年龄从40到87岁,平均年龄56±11岁,研究发现随着年龄的增长,CYP27B1/1α-羟化酶的表达逐渐下降。老年组(年龄55岁,n=12)CYP27B1/1α-羟化酶表达是年轻组CYP27B1/1α-羟化酶表达(年龄50岁,n=7)的64.4%。在老年组和年轻组中,我们通过RT-PCR分别对250HD3或1,25(OH)2D3±DHEA对人骨髓间充质干细胞成骨分化的影响进行研究。在年轻组中,成骨诱导培养3d后,我们发现25OHD3,1,25(OH)2D3, DHEA均可以促进成成骨相关标志基因的表达,如碱性磷酸酶(alkaline phosphatase, ALP)和骨涎蛋白(bone sialoprotein, BSP)。但在老年组中,250HD3对相关成骨标志基因刺激作用较弱。在老年组中,经DHEA预处理后,250HD3和1,25(OH)2D3均可以诱导人骨髓基质干细胞向成骨方向分化。在人骨髓基质干细胞中,通过不同的小分子抑制剂的应用,发现cAMP反应元件结合蛋白(cAMP response element binding protein, CREB)和[GF-I分别介导了脱氢表雄酮调节CYP27B1的作用。通过应用小分子抑制剂AG-1024(一种IGF-IR的抑制剂),发现脱氢表雄酮上调CYP27B1的表达都是通过IGF-I的激活。而通过应用小分子抑制剂KG-501(特殊抑CREB的下游基因),发现脱氢表雄酮上调CYP27B1的表达同样需要通过CREB的激活。由此推测CYP27B1上调则是由于DHEA激活的IGF-I信号系统,继发激活CREB所介导的。[结论]1α-羟化酶/CYP27B1的表达及活性随着人年龄的增长而下降。脱氢表雄酮可能上调IGF-I,从而激活CREB,最终上调CYP27B1,增强人骨髓间充质干细胞对于25OHD3的反应能力,从而增强其成骨分化能力。
[Abstract]:The first part of human bone marrow mesenchymal stem cells in vitro culture and identification of [Objective] by laboratory has successfully created human bone marrow mesenchymal stem cell culture solution, separation, culture and identification of hMSCs, provide sufficient dry by density gradient centrifugation to isolate mononuclear cells. Methods for the follow-up study source, cell the adherent method in a -MEM medium primary hMSCs medium, then were cultured on the differentiation of osteoblasts. Alkaline phosphatase activity assay and Alizarin Red S staining; on adipocyte differentiation using Oil Red-O staining method was used to identify the application of Alcian; blue staining of chondrocytes were identified. Results: bone marrow cells after 6-24 hours, the cell body shape gradually into triangular or irregular shape. The culture of 2-4 days between the morphology of mesenchymal cells changed, the cells gradually turned into fiber The spindle cell type, and gathered into the colony; 7-14 days in culture, cells covered with 70%-80% culture bottle bottom area, cells arranged more closely, the bone marrow mesenchymal stem cells into single cells. The cells were adipogenicdifferentiation, can detect the different shapes of Oil Red-O positive staining cell formation; osteoblast differentiation, detection of alkaline phosphatase activity, and Alizarin Red S staining positive cells formation; the chondrogenic differentiation was seen after Alcian blue staining positive cells. Conclusion] forming method used in this experiment can be successfully isolated, cultured and amplified in human bone marrow mesenchymal stem cells, and identify its osteogenic adipogenic, chondrogenic differentiation ability, part DHEA. Up regulation of IGF-I gene second expression and promotes the osteoblast differentiation of hMSCs [Objective] this study tested whether DHEA by adjusting IGF-I to promote bone marrow mesenchymal stem cells into Bone cell transformation. By using different time points, DHEA concentrations stimulate human bone marrow mesenchymal stem cells, the time and concentration dependent tests, and the biochemical and molecular enzyme inhibitors to elucidate DHEA in human bone marrow mesenchymal stem cells in regulating IGF-I gene expression and its mechanism to promote osteogenesis differentiation. In this study, the application of PKA signaling pathway inhibitor H-89, PKC inhibitor of CHE signaling pathway, JNK signaling pathway inhibitor SP600125 were used to block PKA signaling pathway, PKC signaling pathway, JNK signaling pathway. P13K signaling pathway inhibitor LY294002, P38 MAPK signal pathway inhibitor SB203580 P42/44 signal pathway inhibitor PD098059, IGF-IR blocking PI3K receptor antagonist AG1024, P38 MAPK, P42/44 MAPK, IGF-IR signaling pathway. Results in bone marrow mesenchymal stem cells, DHEA can upregulate the expression of IGF-I gene, and the presence of time And concentration dependent expression of.IGF-I gene in DHEA is the peak 4-6 hours after stimulation; the stimulation time was 6 hours, the best concentration was 10nM. PI3K P38 MAPK, P42/44 block, MAPK signaling pathway, the expression of DHEA gene suppressed the upregulation of IGF-1. Then the samples by special enzyme inhibitors after the action of bone differentiation related gene and alkaline phosphatase assay showed decreased expression. Conclusion]DHEA may be through the IGF-I pathway, including PI3K, P38, MAPK, P42/44, MAPK signaling pathway to increase human bone marrow mesenchymal stem cells in IGF-I gene expression and promote osteogenic differentiation. The third part to study the aging and the relationship between the 1 alpha hydroxylase the expression, and clarify mechanism. Methods DHEA in vitamin D metabolism in the use of RT-PCR method in human bone marrow mesenchymal stem cells in vitamin D metabolism key enzyme 1 alpha hydroxylase, and Analysis of the effect of age on its expression. The application of IGF-IR receptor antagonist AG1024, CERB inhibitor KG501 were used to block IGF-1, CERB signaling pathway, from the study of DHEA protein level conversion mechanism. Results in the metabolism of vitamin D in a group of samples including 19 patients, aged from 40 to 87 years old, the average age of 56. At the age of 11, the study found that with the increase of age, the expression of CYP27B1/1 alpha hydroxylase decreased gradually. The elderly group (age 55, n=12 CYP27B1/1) alpha hydroxylase expression is CYP27B1/1 hydroxylase expression in young group (aged 50, n=7 64.4%.) in the elderly group and the young group, we separately by RT-PCR on 250HD3 or 1,25 (OH) 2D3 + DHEA to study the effect of human bone marrow mesenchymal stem cell into osteogenic differentiation. In young group, osteoblast induced 3D, we found that 25OHD3,1,25 (OH) 2D3, DHEA can promote osteoblast related gene expression signature, Such as alkaline phosphatase (alkaline phosphatase, ALP) and bone sialoprotein (bone sialoprotein, BSP). But in the old group, 250HD3 of osteoblast marker gene stimulation is weak. In the older group, treated with DHEA, 250HD3 and 1,25 (OH) 2D3 can stem cells to differentiate into osteoblasts induction of human bone marrow stromal. In human bone marrow stromal stem cells, through the application of small molecule inhibitors of different cAMP response element binding protein (cAMP response element binding protein, CREB and [GF-I respectively) mediated by dehydroepiandrosterone regulates CYP27B1 function. Through the application of small molecule inhibitors of AG-1024 (an inhibitor of IGF-IR), found that the expression of dehydroepiandrosterone up-regulated the expression of CYP27B1 is through the activation of IGF-I. Through the application of small molecule inhibitors of KG-501 (gene special inhibition of CREB), found that the expression of dehydroepiandrosterone up-regulated CYP27B1 also need to pass After the activation of CREB. The results indicated that CYP27B1 increase is due to the activation of DHEA IGF-I signaling system, the expression and activity of secondary activation mediated by CREB. Conclusion]1 hydroxylase /CYP27B1 decreased with age. DHEA may upregulate IGF-I, which activates CREB, the upregulation of CYP27B1, enhancement of human bone marrow mesenchymal stem cells for the reaction ability of 25OHD3, so as to enhance the capability of osteogenic differentiation.

【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R68

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