背景:目前,冲击波疗法被广泛的用于治疗骨不连、骨折延迟愈合和股骨头坏死以及运动系统慢性劳损性疾病,取得了很好的疗效,但其作用机制并不明确.目的:观察体外冲击波作用于骨髓间充质干细胞后c-Fos、c-Jun蛋白表达的变化情况.设计、时间及地点:随机分组设计,对比观察,于2007-03/2008-03在吉林大学基础医学院病理学国家重点实验室完成.材料:骨髓来源于健康志愿者.方法:体外培养人骨髓间充质干细胞,取P4代细胞,加入2.5 g/L胰蛋白酶消化制成细胞悬液,用无血清低糖DMEM培养基调整细胞密度在1.0×10~9L~(-1)的水平,然后取6 mL细胞悬液,分装入6个1.5 mL.的Eerrendorf管内,1管为对照组,5管为诱导组.将诱导组Eerrendorf管置入体外液电冲击波碎石机中,采用最佳冲击波强度(工作电压8.5 kV,脉冲次数为120次)作用于细胞,分别在作用后5,15,30 min,1,2 h提取蛋白质.主要观察指标:倒置显微镜观察骨髓间充质干细胞形态变化:四甲基偶氮唑盐法测定细胞生长曲线;Western Blot检测细胞内c-Fos、c-Jun蛋白表达的变化.结果:①骨髓间充质干细胞接种于培养板,细胞呈圆形,24 h后,细胞开始缓慢分裂、增殖,半量换液后见贴壁细胞形态 呈梭形.第3天后细胞增殖加快,呈团簇集落样生长.10～14 d各个细胞克隆增大,直至铺满培养板底面达到融合状态.传代培养的骨髓间充质干细胞呈圆形,24 h内完全贴壁,形态与原代培养的细胞类似,1周左右达到完全融合,呈漩涡状排列.传至10代以后,细胞分裂增殖速度明显减慢,细胞逐渐老化.②四甲基偶氮唑盐结果显示:原代及P2、P3代的人骨髓间充质干细胞生长曲线呈S型,在第3-5天为增殖旺盛期.⑨体外冲击波诱导后,人骨髓间充质干细胞中c-Fos、c-Jun的磷酸化水平开始增高,分别于30,15 min后达高峰,分别为对照组的2.56倍,1.68倍(P<0.01),之后逐渐下降;c-Fos、c-Jun的总量未见明显变化(P>0.05).结论:体外冲击波作用于体外培养的人骨髓间充质干细胞后,呈现了细胞内c-Fos、c-Jun蛋白活性增高的变化.
揹景:目前,遲擊波療法被廣汎的用于治療骨不連、骨摺延遲愈閤和股骨頭壞死以及運動繫統慢性勞損性疾病,取得瞭很好的療效,但其作用機製併不明確.目的:觀察體外遲擊波作用于骨髓間充質榦細胞後c-Fos、c-Jun蛋白錶達的變化情況.設計、時間及地點:隨機分組設計,對比觀察,于2007-03/2008-03在吉林大學基礎醫學院病理學國傢重點實驗室完成.材料:骨髓來源于健康誌願者.方法:體外培養人骨髓間充質榦細胞,取P4代細胞,加入2.5 g/L胰蛋白酶消化製成細胞懸液,用無血清低糖DMEM培養基調整細胞密度在1.0×10~9L~(-1)的水平,然後取6 mL細胞懸液,分裝入6箇1.5 mL.的Eerrendorf管內,1管為對照組,5管為誘導組.將誘導組Eerrendorf管置入體外液電遲擊波碎石機中,採用最佳遲擊波彊度(工作電壓8.5 kV,脈遲次數為120次)作用于細胞,分彆在作用後5,15,30 min,1,2 h提取蛋白質.主要觀察指標:倒置顯微鏡觀察骨髓間充質榦細胞形態變化:四甲基偶氮唑鹽法測定細胞生長麯線;Western Blot檢測細胞內c-Fos、c-Jun蛋白錶達的變化.結果:①骨髓間充質榦細胞接種于培養闆,細胞呈圓形,24 h後,細胞開始緩慢分裂、增殖,半量換液後見貼壁細胞形態 呈梭形.第3天後細胞增殖加快,呈糰簇集落樣生長.10～14 d各箇細胞剋隆增大,直至鋪滿培養闆底麵達到融閤狀態.傳代培養的骨髓間充質榦細胞呈圓形,24 h內完全貼壁,形態與原代培養的細胞類似,1週左右達到完全融閤,呈漩渦狀排列.傳至10代以後,細胞分裂增殖速度明顯減慢,細胞逐漸老化.②四甲基偶氮唑鹽結果顯示:原代及P2、P3代的人骨髓間充質榦細胞生長麯線呈S型,在第3-5天為增殖旺盛期.⑨體外遲擊波誘導後,人骨髓間充質榦細胞中c-Fos、c-Jun的燐痠化水平開始增高,分彆于30,15 min後達高峰,分彆為對照組的2.56倍,1.68倍(P<0.01),之後逐漸下降;c-Fos、c-Jun的總量未見明顯變化(P>0.05).結論:體外遲擊波作用于體外培養的人骨髓間充質榦細胞後,呈現瞭細胞內c-Fos、c-Jun蛋白活性增高的變化.
배경:목전,충격파요법피엄범적용우치료골불련、골절연지유합화고골두배사이급운동계통만성로손성질병,취득료흔호적료효,단기작용궤제병불명학.목적:관찰체외충격파작용우골수간충질간세포후c-Fos、c-Jun단백표체적변화정황.설계、시간급지점:수궤분조설계,대비관찰,우2007-03/2008-03재길림대학기출의학원병이학국가중점실험실완성.재료:골수래원우건강지원자.방법:체외배양인골수간충질간세포,취P4대세포,가입2.5 g/L이단백매소화제성세포현액,용무혈청저당DMEM배양기조정세포밀도재1.0×10~9L~(-1)적수평,연후취6 mL세포현액,분장입6개1.5 mL.적Eerrendorf관내,1관위대조조,5관위유도조.장유도조Eerrendorf관치입체외액전충격파쇄석궤중,채용최가충격파강도(공작전압8.5 kV,맥충차수위120차)작용우세포,분별재작용후5,15,30 min,1,2 h제취단백질.주요관찰지표:도치현미경관찰골수간충질간세포형태변화:사갑기우담서염법측정세포생장곡선;Western Blot검측세포내c-Fos、c-Jun단백표체적변화.결과:①골수간충질간세포접충우배양판,세포정원형,24 h후,세포개시완만분렬、증식,반량환액후견첩벽세포형태 정사형.제3천후세포증식가쾌,정단족집락양생장.10～14 d각개세포극륭증대,직지포만배양판저면체도융합상태.전대배양적골수간충질간세포정원형,24 h내완전첩벽,형태여원대배양적세포유사,1주좌우체도완전융합,정선와상배렬.전지10대이후,세포분렬증식속도명현감만,세포축점노화.②사갑기우담서염결과현시:원대급P2、P3대적인골수간충질간세포생장곡선정S형,재제3-5천위증식왕성기.⑨체외충격파유도후,인골수간충질간세포중c-Fos、c-Jun적린산화수평개시증고,분별우30,15 min후체고봉,분별위대조조적2.56배,1.68배(P<0.01),지후축점하강;c-Fos、c-Jun적총량미견명현변화(P>0.05).결론:체외충격파작용우체외배양적인골수간충질간세포후,정현료세포내c-Fos、c-Jun단백활성증고적변화.
BACKGROUND: Now, Shock Wave Therapy is used to cure the ununion and delayed union of bone, the avascular necrosis of the femoral head and the chronic injury of locomotor system, what has got a good curative effect. But, the mechanism is not clear. OBJECTIVE: To investigate the expression of c-Fos, c-Jun protein in human bone marrow mesenchymal stem cells (BMSCs) Influenced by shock wave.DESIGN, TIME AND SETTING: Randomized group, the controlled study was performed at the State Key Laboratory of Pathology, College of Basic Medicine, Jilin University, from March 2007 to March 2008. MATERIALS: Bone marrow was obtained from healthy volunteers.METHODS: Human BMSCs were cultured in vitro. And the fourth generation cells were digested into cell suspension with 2.5 g/L trypsin and adjusted at a density of 1.0×10~9/L with DMEM-LG. Then, the cells were divided into 6 1.5-mL Eerrendorf tubes, one was control group and the other five were experimental groups. The experimental groups were treated with an optimal dose of shock wave (8.5 kV, 120 times) by liquid-electric shock wave lithotripsy. Then the protein was extracted at different time points (5, 15, 30 minutes, 1, 2 hours) after treatment.MAIN OUTCOME MEASURES: Inverted microscope was used to observe the morphologic feature of BMSCs. The growth curves were charted by MTT method. The change of activations of c-Fos, c-Jun were tested by western blot. RESULTS: ①BMSCs were seeded in culture plate. The cells began to divide and proliferate slowly 24 hours later, and became fusiform shape after adhering to the wall. 3 days later, the speed of proliferation quickened, and cells accumulated colony. At day 10-14, the number of hMSCs grew till they covered the bottom of the culture plate. The round passage hMSCs adhered to the wall completely in 24 hours, which were similar to primary cells. The cells connected together during one week, and showed vortex-like. The speed of proliferation became slower and the cells became older when hMSCs were passaged to the tenth generation. ②MTT method showed that the growth curve of original, P2, P3 hMSCs looked like S shape, and the third to fifth days were growing period. ③The phosphorylation level of c-Fos and c-Jun began to increase after induced by shock wave and reached a peak at 30 and 15 minutes, respectively. And their phosphorylation level were 2.56-fold and 1.68-fold (P < 0.01) compared with the average level in control groups, respectively. Then they began to decrease. There was no apparent change in the total dose (P > 0.05). CONCLUSION: Following the treatment of shock wave, the activations of c-Fos and c-Jun in BMSCs increased.
