CAJ | 학술논문

背景:肌源性干细胞在体外培养过程中存在纯化、扩增、定向分化等难题,碱性成纤维细胞生长因子作为生物活性因子的一员,因其生物活性的多效性而备受关注.目的:探讨碱性成纤维细胞生长因子对大鼠肌源性干细胞体外增殖的影响.设计、时间及地点:细胞学体外观察,于2008-04/07在沈阳医学院完成.材料:健康雄性Wister大鼠5只,由沈阳医学院实验动物中心提供.碱性成纤维细胞生长因子为珠海生物制品有限公司产品.方法:无菌条件下取大鼠左前肢肱三头肌,采用酶消化法分离肌源性干细胞,应用密度梯度离心法和差速贴壁法进行纯化.取第2代肌源性干细胞,分别用终浓度为6.25,12.50,25.00,50.00,100.00μg/L碱性成纤维细胞生长因子进行干预;以单纯加入200 μL生长培养基作为空白对照组,以加入200μL生长培养基+肌源性干细胞作为阴性对照组.分别于培养24,48,72,96 h加入MTT溶液.主要观察指标:免疫细胞化学法鉴定大鼠肌源性干细胞,MTT比色法检测细胞增殖情况.结果:肌源性干细胞多呈Sca-1阳性反应,少数呈Desmin阳性反应.①不同浓度碱性成纤维细胞生长因子对肌源性干细胞的促增殖效应:与阴性对照组比较,各浓度碱性成纤维细胞生长因子组对肌源性干细胞均有明显的促增殖效应(P<0.05);浓度为6.25-50.00 μg/L时促增殖作用随其质量浓度升高而显著增强(P<0.01),至50.00 μg/L时其促增殖作用接近顶峰.②碱性成纤维细胞生长因子在不同培养时间点对肌源性干细胞的促增殖效应:随培养时间的延长肌源性干细胞明显增殖,与阴性对照组比较,碱性成纤维细胞生长因子组出现显著促增殖效应(P<0.01)的时间为培养96 h.结论:碱性成纤维细胞生长因子可促进体外培养的大鼠肌源性干细胞增殖,且呈浓度一时间依赖性增加,50.00 μg/L与96 h为较佳的体外培养质量浓度和时间. 揹景:肌源性榦細胞在體外培養過程中存在純化、擴增、定嚮分化等難題,堿性成纖維細胞生長因子作為生物活性因子的一員,因其生物活性的多效性而備受關註.目的:探討堿性成纖維細胞生長因子對大鼠肌源性榦細胞體外增殖的影響.設計、時間及地點:細胞學體外觀察,于2008-04/07在瀋暘醫學院完成.材料:健康雄性Wister大鼠5隻,由瀋暘醫學院實驗動物中心提供.堿性成纖維細胞生長因子為珠海生物製品有限公司產品.方法:無菌條件下取大鼠左前肢肱三頭肌,採用酶消化法分離肌源性榦細胞,應用密度梯度離心法和差速貼壁法進行純化.取第2代肌源性榦細胞,分彆用終濃度為6.25,12.50,25.00,50.00,100.00μg/L堿性成纖維細胞生長因子進行榦預;以單純加入200 μL生長培養基作為空白對照組,以加入200μL生長培養基+肌源性榦細胞作為陰性對照組.分彆于培養24,48,72,96 h加入MTT溶液.主要觀察指標:免疫細胞化學法鑒定大鼠肌源性榦細胞,MTT比色法檢測細胞增殖情況.結果:肌源性榦細胞多呈Sca-1暘性反應,少數呈Desmin暘性反應.①不同濃度堿性成纖維細胞生長因子對肌源性榦細胞的促增殖效應:與陰性對照組比較,各濃度堿性成纖維細胞生長因子組對肌源性榦細胞均有明顯的促增殖效應(P<0.05);濃度為6.25-50.00 μg/L時促增殖作用隨其質量濃度升高而顯著增彊(P<0.01),至50.00 μg/L時其促增殖作用接近頂峰.②堿性成纖維細胞生長因子在不同培養時間點對肌源性榦細胞的促增殖效應:隨培養時間的延長肌源性榦細胞明顯增殖,與陰性對照組比較,堿性成纖維細胞生長因子組齣現顯著促增殖效應(P<0.01)的時間為培養96 h.結論:堿性成纖維細胞生長因子可促進體外培養的大鼠肌源性榦細胞增殖,且呈濃度一時間依賴性增加,50.00 μg/L與96 h為較佳的體外培養質量濃度和時間.
배경:기원성간세포재체외배양과정중존재순화、확증、정향분화등난제,감성성섬유세포생장인자작위생물활성인자적일원,인기생물활성적다효성이비수관주.목적:탐토감성성섬유세포생장인자대대서기원성간세포체외증식적영향.설계、시간급지점:세포학체외관찰,우2008-04/07재침양의학원완성.재료:건강웅성Wister대서5지,유침양의학원실험동물중심제공.감성성섬유세포생장인자위주해생물제품유한공사산품.방법:무균조건하취대서좌전지굉삼두기,채용매소화법분리기원성간세포,응용밀도제도리심법화차속첩벽법진행순화.취제2대기원성간세포,분별용종농도위6.25,12.50,25.00,50.00,100.00μg/L감성성섬유세포생장인자진행간예;이단순가입200 μL생장배양기작위공백대조조,이가입200μL생장배양기+기원성간세포작위음성대조조.분별우배양24,48,72,96 h가입MTT용액.주요관찰지표:면역세포화학법감정대서기원성간세포,MTT비색법검측세포증식정황.결과:기원성간세포다정Sca-1양성반응,소수정Desmin양성반응.①불동농도감성성섬유세포생장인자대기원성간세포적촉증식효응:여음성대조조비교,각농도감성성섬유세포생장인자조대기원성간세포균유명현적촉증식효응(P<0.05);농도위6.25-50.00 μg/L시촉증식작용수기질량농도승고이현저증강(P<0.01),지50.00 μg/L시기촉증식작용접근정봉.②감성성섬유세포생장인자재불동배양시간점대기원성간세포적촉증식효응:수배양시간적연장기원성간세포명현증식,여음성대조조비교,감성성섬유세포생장인자조출현현저촉증식효응(P<0.01)적시간위배양96 h.결론:감성성섬유세포생장인자가촉진체외배양적대서기원성간세포증식,차정농도일시간의뢰성증가,50.00 μg/L여96 h위교가적체외배양질량농도화시간.
BACKGROUND: Muscle-derived stem cells have some problems in pudflcation, amplification and directional differentiation during in vitro culture. Basic fibroblast growth factor as a membrane of bloactive factor has been paid great attention, due to its biological activity.OBJECTIVE: To explore effects of basic flbroblast growth factor on/n vitro proliferation of rat musde-derived stem cells.DESIGN, TIME AND SETTING: The cytological in vitro study was performed at the Shenyang Medical College from April to July 2008.MATERIALS: Five healthy male Wistar rats were supplied by Experimental Animal Center, Shenyang Medical College. Basic fibroblast growth factor was produced by Zhuhai Biological Product Co., Ltd.METHODS: Left anterior limb triceps brachii was sterilely obtained from rats. Muscle-derived stem cells were harvested by isolation with enzyme digestion, pudfied by density gradient centrifugation and differential attachment. At the second passage,musde-derived stem cells were treated with basic fibroblast growth factor at final concentration of 6.25, 12.50, 25.00, 50.00,100.00 pg/L. Cells received 200 uL growth medium as blank control group, and those treated with 200 μL growth medium +musde-derived stem celts as negative controls. MTT was added at 24, 48, 72 and 96 hours of culture.MAIN OUTCOME MEASURES: Immunocytochomical method was used to determine rat musde-dedved stem cells. MTT assay was utilized to detect cell proliferation.RESULTS: Musde-denved stem cells were mostly positive for Sca-1, but few was positive for Desmin. Compared with the negative control group, vanous concentrations of basic fibroblast growth factor had significant proliferation effects on musde-derived cells (P < 0.05). At 6.25-50.00 μg/L, the proliferation effect became strong with the increased concentration (P <0.01). At 50.00 μg/L, the proliferation promotion effect nearly reached a peak. Musde-derived stem cells significantly proliferated over time. Compared with the negative control group, significant proliferation promotion effect was detected in the basic fibroblast growth factor groups at 96 hours (P < 0.01).CONCLUSION: Basic fibroblast growth factor can promote proliferation of rat muscle-derived stem cells/n vitro in a dose-time dependent fashion. 50.00 μ g/L and 96 hours are separately good concentration and time for in vitro culture.