摘要
目的 探讨P物质（SP）对骨髓基质干细胞（BMSCs）增殖、分化的影响,并从Wnt/ β-链蛋白信号转导通路角度探讨其可能机制. 方法 取SD大鼠第3代BMSCs,根据加入的刺激物不同将其分为4组（n=3）：SP组、SP ＋SP受体拮抗剂组、SP＋ Wnt/ β-链蛋白信号转导通路抑制剂1（DKK1）组、对照组（加入等量磷酸盐缓冲液）.培养1、3、5、7、9 d采用Alarmar Blue法检测各组BMSCs的增殖情况;于培养7、14d应用荧光定量核酸扩增（Q-PCR）法检测各组碱性磷酸酶（ALP）、骨钙素基因的表达;于培养1、3、5、7 d采用Western blot法检测各组Wnt/β-链蛋白信号转导通路相关蛋白β-链蛋白、C-myc的表达;于培养3d应用免疫荧光法检测各组β-链蛋白的核转移情况.结果 于培养l、3、5、7、9d,SP组细胞数量大于SP＋ SP受体拮抗剂组、SP＋ DKK1组和对照组,差异均有统计学意义（P＜0.05）.于培养14d,SP＋ SP受体拮抗剂组、SP＋ DKK1组和对照组的ALP、骨钙素基因表达量均明显低于SP组,差异有统计学意义（P＜0.05）.于培养1、3、5、7 d,SP＋ SP受体拮抗剂组、SP＋ DKK1组及对照组的C-myc蛋白的表达量均低于SP组,差异均有统计学意义（P＜0.05）;培养3、5d,SP＋SP受体拮抗剂组、SP＋ DKK1组和对照组的β-链蛋白的表达量均低于SP组,差异均有统计学意义（P＜0.05）.于培养3d,SP＋ SP受体拮抗剂组、SP＋ DKK1组和对照组β-链蛋白入核量均少于SP组,差异有统计学意义（P＜0.05）.结论 SP可促进BMSCs的增殖与分化,Wnt/β-链蛋白信号转导通路可能参与调控.
Objective To explore the effect of substance P （SP） on the proliferation and differentiation of bone marrow stromal stem cells （BMSCs） and its association with Wnt/β-catenin signaling pathway.Methods The third passage BMSCs from SD rats were divided into 4 groups according to the stimulator added：SP,SP ＋ SP receptor antagonist,SP ＋ DKK1 （dickkopf 1） and phosphate buffered saline （PBS） （control）.BMSCs were identified using the flow cytometry.At 1,3,5,7 and 9 days,proliferation of BMSCs was detected using Alarmar Blue method.At 7 and 14 days,alkaline phosphatase （ALP） and osteocalcin mRNA were detected using quantitative PCR.At 1,3,5 and 7 days,the protein expression of Wnt/β-catenin signaling including β-catenin and C-myc protein was detected using western blotting.At 3 days,nuclear transfer of β-catenin was investigated using immunofluorescence staining.Results Flow cytometry authenticated BMSCs.At 1,3,5,7 and 9 days,the cells were significantly more in SP group than in control group,but significantly less in SP ＋ SP receptor antagonist and SP ＋ DKK1 groups than in SP group （P ＜0.05）.At 14 days,the expression of ALP and osteocalcin gene was significantly higher in SP group than in control group,but significantly lower in SP ＋ SP receptor antagonist and SP ＋ DKK1 groups than in SP group （P ＜ 0.05）.At 1,3,5 and 7 days,the expression of C-myc protein was significantly higher in SP group than in control group,but significantly lower in SP ＋ SP receptor antagonist and SP ＋ DKK1 groups than in SP group （P ＜ 0.05）.At 3 and 5 days,the expression of β-catenin protein was significantly higher in SP group than in control group,but significantly lower in SP ＋ SP receptor antagonist and SP ＋ DKK1 groups than in SP group （P ＜ 0.05）.At β days,the nuclear transfer of β-catenin was more in SP group than in control group,but less in SP ＋ SP receptor antagonist and SP ＋ DKK1 groups than in SP group （P ＜ 0.05）.Conclusion SP may induce proliferation and differentiation of BMSCs probably through the Wnt/β,-catenin signaling pathway.
出处
《中华创伤骨科杂志》
CAS
CSCD
北大核心
2014年第7期611-616,共6页
Chinese Journal of Orthopaedic Trauma
基金
国家自然科学基金（81171723,30872638）
关键词
P物质
骨髓细胞
细胞增殖
细胞分化
Substance P
Bone marrow cells
Cell proliferation
Cell differentiation
