神经肽P物质及BMP信号通路在ST2细胞成骨分化过程中的作用研究

发布时间：2018-05-04 02:37

  本文选题：神经肽P物质 + 骨髓间充质干细胞　； 参考：《山东大学》2017年硕士论文

【摘要】：第一部分神经肽P物质对ST2细胞增殖分化的影响目的:初步探讨神经肽P物质(Neuropeptide substance,SP)对ST2细胞成骨分化过程中增殖和分化的影响,并筛选出合适的促增殖分化的SP作用浓度和时间。方法:将第三代(P3)ST2以5×103/孔铺板于96孔板,用含有不同浓度SP(10-10Mol/L、10-8Mol/L、10-6Mol/L、10-5Mol/L)的成骨条件培养基(Osteoblast Medium,OBM)进行刺激,以仅含2%胎牛血清(FBS)的OBM作为对照组(control),每组设5个复孔和1个调零孔。分别以SP刺激ST2细胞24、48、72h,CCK-8检测细胞增殖活性,筛选出合适的药物浓度。然后取第三代(P3)的ST2按8×104/孔密度铺板于6孔板,加入含有10-6Mol/L浓度SP 的 OBM,培养 1,3,5,7 天,酶联免疫吸附实验(enzyme-linked immune sorbent assay,ELISA)检测细胞培养上清液中碱性磷酸酶(alkaline phosphatase,ALP)、Ⅰ型胶原蛋白(CollaⅠ)和骨钙素(osteocalcinv,OCN)的表达,实验重复3次。并采用免疫荧光染色检测细胞ALP的活性。结果:(1)神经肽 P 物质(Neuropeptide substance,SP)可促进小鼠 BMSCs(ST2细胞)的增殖活性,且与SP的浓度和作用时间有一定的相关关系,当SP浓度为10-6Mol/L时,细胞增殖效应最明显。(2)神经肽 P 物质(Neuropeptide substance,SP)可促进小鼠 BMSCs(ST2细胞)的成骨分化,早期成骨分化标记物ALP的表达在第5天时达峰值,中晚期标志物Colla Ⅰ、OCN的表达在第7天时最高。SP可增强ST2细胞ALP的活性。结论:yL经肽 P 物质(Neuropeptide substance,SP)可促进小鼠 BMSCs(ST2 细胞)的增殖和成骨分化,该效应具有剂量依赖性和时间依赖性。第二部分BMP信号通路对SP促ST2细胞成骨分化过程的调控作用研究目的:探讨BMP信号通路在SP促ST2细胞成骨分化过程的作用。方法:取P3的ST2以8×104/孔的密度铺板于6孔板,设3个复孔。实验分组为SP 组:10-6Mol/L SP;SP+BMP 通路抑制剂(Noggin)组:10-6Mol/L SP 和 100ng/ml Noggin;抑制剂组:100ng/mlNoggin;对照组:等体积2%FBS,培养5天或7天后采用ELISA法检测细胞上清液中ALP,ColIa Ⅰ和OCN的表达量,并采用免疫荧光染色检测细胞的ALP活性。结果:(1)SP可明显促进ST2细胞成骨分化标记物ALP、ColIa Ⅰ和OCN的表达,加入BMP信号通路抑制剂Noggin后,标记物的表达显著减少,且仅加入Noggin也可抑制ALP、ColIa Ⅰ和OCN的表达。(2)免疫荧光染色结果显示SP可促进ST2细胞ALP的表达活性,加入BMP信号通路抑制剂Noggin后,ALP活性受到抑制,单纯加入Noggin也可抑制细胞ALP的活性表达。结论:SP可促进ST2的成骨分化和ALP活性表达,而BMP信号通路抑制剂Noggin可抑制其效应的发挥,表明BMP信号通路可能在SP促ST2细胞成骨分化中产生了一定的调节作用。
[Abstract]:Part I effects of neuropeptide substance P on proliferation and differentiation of ST2 cells objective: to investigate the effects of neuropeptide substance P on proliferation and differentiation of ST2 cells during osteogenic differentiation. The appropriate SP concentration and time for proliferation and differentiation were selected. Methods: the third generation P3CST2 was incubated with 5 脳 10 3 / hole plate on 96 well plate. The osteogenic condition medium containing 10 ~ (-10) mol / L ~ (-10 ~ (-8) mol / L ~ (10 ~ (-6) mol / L) was used to stimulate the osteoblast medium. The OBM containing only 2% fetal bovine serum (FBS) was used as the control. Each group was given five multiple holes and one zero adjusting hole. The proliferation activity of ST2 cells stimulated by SP was determined by CCK-8 at 24 ~ 48 ~ 72 h, and the appropriate concentration of CCK-8 was screened out. Then the ST2 of the third generation of P3 was added to the 6-hole plate at the density of 8 脳 10 4 / pore, and the 10-6Mol/L containing SP was added to the plate, and cultured for 5 days. Enzyme linked immune sorbent Assay (Elisa) assay was used to detect the expression of alkaline phosphatase (ALP) and osteocalcinvan (OCNs) in the supernatant of cell culture. The activity of ALP was detected by immunofluorescence staining. Results (1) Neuropeptide substance P (Neuropeptide substance1) could promote the proliferation of mouse BMSCs(ST2 cells, which was related to the concentration of SP and the time of action. When SP concentration was 10-6Mol/L, The proliferation of BMSCs(ST2 cells was enhanced by neuropeptide substance P (NPP), and the expression of ALP, the marker of early osteogenic differentiation, reached its peak on the 5th day. The expression of Colla 鈪，

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