摘要
目的探讨交感神经递质去甲肾上腺素(NE)对小鼠骨髓间充质干细胞(BMSC)迁移的影响及其机制。方法(1)取20只3周龄雄性C57BL/6小鼠,处死后取出股骨和胫骨,分离培养BMSC并鉴定。取第2代或3代细胞,分为磷酸盐缓冲液(PBS)组、1μmol/L NE组、10μmol/L NE组及100μmol/L NE组,每组8孔。1μmol/L NE组、10μmol/L NE组及100μmol/L NE组细胞分别在含体积分数1%胎牛血清的低糖DMEM培养基(以下简称低血清培养基)内分别加入终物质的量浓度为1、10、100μmol/L的NE培养,PBS组细胞在低血清培养基内加入等体积的PBS培养。在刺激前(0 d)及刺激1、3、5 d,采用细胞计数试剂盒8法检测细胞增殖活性(以吸光度值表示)。(2)细胞划痕试验1中,取细胞分为PBS组和单纯NE组,行划痕试验后,单纯NE组细胞采用低血清培养基+终物质的量浓度为10μmol/L的NE培养,PBS组细胞采用低血清培养基+等体积PBS培养。细胞划痕试验2中,取细胞分为PBS组、普萘洛尔+NE组、酚妥拉明+NE组,行划痕试验后,普萘洛尔+NE组细胞每天采用低血清培养基+终物质的量浓度为1μmol/L的普萘洛尔、酚妥拉明+NE组细胞每天采用低血清培养基+终物质的量浓度为10μmol/L的酚妥拉明预处理30 min后,均再采用低血清培养基+终物质的量浓度为10μmol/L的NE培养;PBS组采用低血清培养基+等体积PBS培养。细胞划痕试验3中,取细胞分为单纯NE组、单纯(2E,6E)-2,6-二(4-吡啶基亚甲基)环己酮(SC-66)组、SC-66+NE组,行划痕试验后,单纯NE组采用低血清培养基+终物质的量浓度为10μmol/L的NE培养;单纯SC-66组细胞每天采用终物质的量浓度为30 mmol/L的SC-66预处理30 min后,再采用低血清培养基培养;单纯SC-66+NE组每天采用终物质的量浓度为30 mmol/L的SC-66预处理30 min后,再采用低血清培养基+终物质的量浓度为10μmol/L的NE培养。以上3个细胞划痕试验,每组样本数均为6,均计算划痕后24、48、72 h的划
Objective To investigate the effects and mechanism of sympathetic neurotransmitter norepinephrine(NE)on the migration of bone marrow mesenchymal stem cells(BMSCs)in mice.Methods(1)Twenty 3-week-old male C57BL/6 mice were sacrificed for isolating,culturing,and identifying BMSCs from the femur and tibia.Cells of the second or third passages were divided into phosphate buffer solution(PBS)group,1μmol/L NE group,10μmol/L NE group,and 100μmol/L NE group,with 8 wells in each group.Cells in 1μmol/L NE group,10μmol/L NE group,and 100μmol/L NE group were cultured in low-sugar Dulbecco′s modified eagle medium containing 1%volume fraction of fetal bovine serum(hereinafter referred to as low-serum medium)added with NE in final molarity of 1μmol/L,10μmol/L,and 100μmol/L,respectively.Cells in PBS group were cultured in low-serum medium added with the same volume of PBS.Before stimulation(0 d)and on stimulation day 1,3,5,cell counting kit 8 method was used to detect cell proliferation activity(expressed as the absorbance value).(2)In cell scratch test 1,cells were divided into PBS group and simple NE group.After the scratch test,cells in simple NE group were cultured with low-serum medium+NE in final molarity of 10μmol/L,and cells in PBS group were cultured with low-serum medium+the same volume of PBS.In cell scratch test 2,cells were divided into PBS group,propranolol+NE group,and phentolamine+NE group.After the scratch test,cells in propranolol+NE group were pretreated with low-serum medium+propranolol in final molarity of 1μmol/L for 30 minutes each day,cells in phentolamine+NE group were pretreated with low-serum medium+phentolamine in final molarity of 10μmol/L for 30 minutes each day,and then they were cultured with low-serum medium+NE in final molarity of 10μmol/L.Cells in PBS group were cultured with low-serum medium+the same volume of PBS.In cell scratch test 3,cells were divided into simple NE group,simple(2E,6E)-2,6-bis(4-pyridylmethylene)cyclohexanone(SC-66)group,and SC-66+NE group.After the scratch t
作者
孔亚男
金晶
程飚
Kong Yanan;Jin Jing;Cheng Biao(Department of Plastic Surgery,the First Affiliated Hospital of Anhui Medical University,Hefei 230022,China;Department of Burns and Plastic Surgery,General Hospital of Southern Theater Command of PLA,Guangzhou 510010,China)
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2020年第12期1173-1182,共10页
Chinese Journal of Burns
基金
国家重点研发计划(2017YFC1103301)
军事医学创新工程专项(18CXZ029)
国家自然科学基金面上项目(81171812、81272105、81671924)
广东省科技计划(2014B020212010、2015B020233012)
广州市健康医疗协同创新重大专项(201508020253)。
