中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2009年
45期
8811-8816
,共6页
脐带间充质干细胞%分化%肝样细胞
臍帶間充質榦細胞%分化%肝樣細胞
제대간충질간세포%분화%간양세포
背景:成纤维生长因子可促进间充质干细胞增殖、贴壁生长,但对其诱导间充质干细胞向肝细胞分化的实验报道为数不多.当肝细胞生长因子质量浓度达1 μg/L时,可促进肝细胞有丝分裂,它是正常肝细胞最强的促有丝分裂剂.目的:体外分离培养人脐带间充质干细胞,拟揭示其生物学特性及在细胞因子联合诱导下向肝样细胞分化的能力.设计、时间及地点:细胞学体外观察,于2008-08/2009-04在暨南大学血研所完成.材料:脐带取自健康足月胎儿,产妇对实验知情同意,由广州华侨医院提供.肝细胞生长因子、成纤维生长因子为美国Peprotech产品.方法:Ⅳ型胶原酶消化+差速贴壁法分离培养人脐带间充质干细胞,取传至第3代细胞,进行细胞表面抗原分析、细胞周期测定,检测其成脂、成骨能力.取第5代细胞.调整细胞密度为5×10~9 L~(-1),分为2组:对照组用含体积分数为5%胎牛血清的DMEM/F12培养液培养;诱导组在其基础上,添加20 μg/L肝细胞生长因子、10 μg/L成纤维生长因子联合诱导其向肝样细胞分化.主要观察指标:人脐带间充质干细胞的生物学特性,人脐带间充质干细胞体外向肝样细胞的分化情况.结果:成功从人脐带中分离并纯化得到间充质干细胞,第3代细胞92.2%处在G_0/G_1期;表达CD29,CD44,CD105,不表达造血细胞标志CD34,CD45;油红O染色后胞浆中呈现红色颗粒,碱性磷酸酶染色后细胞质呈黑色,具有成脂、成骨能力.经肝细胞生长因子、成纤维生长因子联合诱导10 d后,RT-PCR及Western blot检测结果显示细胞表达肝细胞特异性抗原甲胎蛋白、白蛋白,对照组均呈阴性表达.结论:人脐带中含有丰富的间充质干细胞,其具有较强的多向分化潜能,经肝细胞生长因子与成纤维生长因子联合诱导后,易向肝样细胞分化.
揹景:成纖維生長因子可促進間充質榦細胞增殖、貼壁生長,但對其誘導間充質榦細胞嚮肝細胞分化的實驗報道為數不多.噹肝細胞生長因子質量濃度達1 μg/L時,可促進肝細胞有絲分裂,它是正常肝細胞最彊的促有絲分裂劑.目的:體外分離培養人臍帶間充質榦細胞,擬揭示其生物學特性及在細胞因子聯閤誘導下嚮肝樣細胞分化的能力.設計、時間及地點:細胞學體外觀察,于2008-08/2009-04在暨南大學血研所完成.材料:臍帶取自健康足月胎兒,產婦對實驗知情同意,由廣州華僑醫院提供.肝細胞生長因子、成纖維生長因子為美國Peprotech產品.方法:Ⅳ型膠原酶消化+差速貼壁法分離培養人臍帶間充質榦細胞,取傳至第3代細胞,進行細胞錶麵抗原分析、細胞週期測定,檢測其成脂、成骨能力.取第5代細胞.調整細胞密度為5×10~9 L~(-1),分為2組:對照組用含體積分數為5%胎牛血清的DMEM/F12培養液培養;誘導組在其基礎上,添加20 μg/L肝細胞生長因子、10 μg/L成纖維生長因子聯閤誘導其嚮肝樣細胞分化.主要觀察指標:人臍帶間充質榦細胞的生物學特性,人臍帶間充質榦細胞體外嚮肝樣細胞的分化情況.結果:成功從人臍帶中分離併純化得到間充質榦細胞,第3代細胞92.2%處在G_0/G_1期;錶達CD29,CD44,CD105,不錶達造血細胞標誌CD34,CD45;油紅O染色後胞漿中呈現紅色顆粒,堿性燐痠酶染色後細胞質呈黑色,具有成脂、成骨能力.經肝細胞生長因子、成纖維生長因子聯閤誘導10 d後,RT-PCR及Western blot檢測結果顯示細胞錶達肝細胞特異性抗原甲胎蛋白、白蛋白,對照組均呈陰性錶達.結論:人臍帶中含有豐富的間充質榦細胞,其具有較彊的多嚮分化潛能,經肝細胞生長因子與成纖維生長因子聯閤誘導後,易嚮肝樣細胞分化.
배경:성섬유생장인자가촉진간충질간세포증식、첩벽생장,단대기유도간충질간세포향간세포분화적실험보도위수불다.당간세포생장인자질량농도체1 μg/L시,가촉진간세포유사분렬,타시정상간세포최강적촉유사분렬제.목적:체외분리배양인제대간충질간세포,의게시기생물학특성급재세포인자연합유도하향간양세포분화적능력.설계、시간급지점:세포학체외관찰,우2008-08/2009-04재기남대학혈연소완성.재료:제대취자건강족월태인,산부대실험지정동의,유엄주화교의원제공.간세포생장인자、성섬유생장인자위미국Peprotech산품.방법:Ⅳ형효원매소화+차속첩벽법분리배양인제대간충질간세포,취전지제3대세포,진행세포표면항원분석、세포주기측정,검측기성지、성골능력.취제5대세포.조정세포밀도위5×10~9 L~(-1),분위2조:대조조용함체적분수위5%태우혈청적DMEM/F12배양액배양;유도조재기기출상,첨가20 μg/L간세포생장인자、10 μg/L성섬유생장인자연합유도기향간양세포분화.주요관찰지표:인제대간충질간세포적생물학특성,인제대간충질간세포체외향간양세포적분화정황.결과:성공종인제대중분리병순화득도간충질간세포,제3대세포92.2%처재G_0/G_1기;표체CD29,CD44,CD105,불표체조혈세포표지CD34,CD45;유홍O염색후포장중정현홍색과립,감성린산매염색후세포질정흑색,구유성지、성골능력.경간세포생장인자、성섬유생장인자연합유도10 d후,RT-PCR급Western blot검측결과현시세포표체간세포특이성항원갑태단백、백단백,대조조균정음성표체.결론:인제대중함유봉부적간충질간세포,기구유교강적다향분화잠능,경간세포생장인자여성섬유생장인자연합유도후,역향간양세포분화.
BACKGROUND:Fibroblast growth factor can promote proliferation of mesenchymal stem cells (MSCs).grew along the wall.However,there are few reports on the differentiation of MSCs into hepatocytes following fibroblast growth factor induction.When mass concentration of hepatocyte growth factor was 1 μg/L,it can promote mitosis of hepatocytes,and is the strongest mitogenic agent for normal hepatocytes.OBJECTIVE:To explore the biological characteristics of human umbilical cord MSCs/n vitro and their differentiation ability to hepatocyte-like cells under the induction of chemical factors.DESIGN,TIME AND SETTING:The cytological in vitro study was conducted at the Institute of Blood,Jinan University from August 2008 to April 2009.MATERIALS:Umbilical cord was obtained from healthy full-term fetus,which was provided by the Guangzhou Huaqiao Hospital.The parturient signed informed consent.Hepatocyte growth factor and flbroblast growth factor were bought from Peprotech,USA.METHODS:The MSCs from human cord were isolated and cultured by type Ⅳ colagenase digestion+differential adherence.At the third passage,MSCs received cell surface antigen analysis and cell cycle determination to detect their ability to differentiate into adipocytes and osteoblasts.At the fifth passage,MSCs were adjusted into 5×10~9/L and assigned into 2 groups.BMSCs in the control group were incubated in DMEM/F12 containing 5% fetal bovine serum.BMSCs in the induction group were treated with above-mentioned medium supplemented with 20 μg/L hepatocyte growth factor and 10 μg/L flbroblast growth factor.MAIN OUTCOME MEASURES:The following parameters were measured:biological characteristics of human umbilical cord MSCs and differentiation of human umbilical cord MSCs into hepatocyte-like cells in vitro.RESULTS:At the third passage,MSCs derived from human cord expressed CD29,CD44,CD105,but not antigens of hematopoietic CD34,CD45,and 92.2% of them were in G_0/G_1 phase.Oil red O staining showed cytoplasm presented red granules.Alkaline phosphatase staining demonstrated that cytoplasm was black,with the differentiation ability into adipocytes and osteoblasts.Following 10 days of combined induction of hepstocyte growth factor and fibroblast growth factor,RT-PCR and Western blot results confirmed that cells expressed alpha fetoprotein and albumin.Negative expression was found in the control group.CONCLUSION:Human umbilical cord contained plenty of MSCs,with strong potential of multi-differentiation.Umbilical cord MSCs can differentiate into hepatocyte-like cells following combined induction of hepatocyte growth factor and fibroblast growth factor.