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
8865-8870
,共6页
田孝祥%韩雅玲%康建%闫承慧%齐岩梅%陶杰%吴光哲
田孝祥%韓雅玲%康建%閆承慧%齊巖梅%陶傑%吳光哲
전효상%한아령%강건%염승혜%제암매%도걸%오광철
载体%SM22α%纯化%平滑肌细胞%胚胎干细胞
載體%SM22α%純化%平滑肌細胞%胚胎榦細胞
재체%SM22α%순화%평활기세포%배태간세포
背景:胚胎干细胞是平滑肌细胞重要的来源之一,但是胚胎干细胞分化细胞的异质性导致难以获得较纯的平滑肌细胞.目的:为进一步纯化胚胎干细胞来源的平滑肌细胞,拟在体外构建平滑肌特异性SM22α启动子驱动的嘌呤霉素抗性(puromycin acetyltransferase,pac)基因与增强型绿色荧光蛋白(enhanced green fluorescence protein,EGFP)基因双表达载体,即pSM22-PAC-IRES2-EGFP载体,并在胚胎干细胞中检测其有效性及特异性.设计、时间及地点:基因水平细胞观察实验,于2007-05/2008-09在解放军沈阳军区总医院全军心血管病研究所完成.材料:小鼠胚胎干细胞系R1购自美国ATCC公司,编号SCRC-1011~(TM).pSM22α-EGFP载体由本实验室构建;pIRES2-EGFP载体、pSM2C载体、pSuper.basic载体购自Invitrogen公司.方法:用聚合酶链反应方法从pSM22α-EGFP中扩增SM22α启动子,然后用该启动子替换pIRES2-EGFP载体中的CMV启动子,构建pSM22-IRES2-EGFP.再从pSM2C中用HindⅢ/Cla Ⅰ酶切获得pac基因,将pac基因片段亚克隆到pSuper.basic中,构建pSuper-PAC.最后BgⅢ/Acc Ⅰ双酶切pSuper-PAC获得pac基因片段,将其插入到pSM22α-IRES2-EGFP,构建成pSM22α-PAC-IRES2-EGFP.将pSM22α-PAC-IRES2-EGFP用脂质体法转染胚胎干细胞,G418筛选阳性克隆.诱导胚胎干细胞阳性克隆分化,RT-PCR扩增pac基因鉴定阳性克隆.对分化细胞行平滑肌细胞标志物SMα-actin免疫荧光染色.主要观察指标:①pSM22α-PAC-IRES2-EGFP测序结果.②pac基因扩增.③荧光显微镜下同时观察分化细胞EGFP的表达及SMα-actin染色情况.结果:HindⅢ/Cla Ⅰ双酶切得到261 bp,664 bp,5 000 bp 3个片段,与预期结果一致,测序结果证实pSM22α-PAC-IRES2-EGFP构建成功.Pac基因扩增证实有4株胚胎干细胞克隆转染成功.转染成功的胚胎干细胞被诱导分化后,部分细胞表达EGFP,且这些细胞SMα-actin染色呈阳性.结论:实验成功构建了平滑肌细胞筛选载体pSM22α-PAC-IRES2-EGFP.成功转染这一载体的胚胎干细胞表达pac基因及EGFP基因,且EGFP表达具有平滑肌特异性.
揹景:胚胎榦細胞是平滑肌細胞重要的來源之一,但是胚胎榦細胞分化細胞的異質性導緻難以穫得較純的平滑肌細胞.目的:為進一步純化胚胎榦細胞來源的平滑肌細胞,擬在體外構建平滑肌特異性SM22α啟動子驅動的嘌呤黴素抗性(puromycin acetyltransferase,pac)基因與增彊型綠色熒光蛋白(enhanced green fluorescence protein,EGFP)基因雙錶達載體,即pSM22-PAC-IRES2-EGFP載體,併在胚胎榦細胞中檢測其有效性及特異性.設計、時間及地點:基因水平細胞觀察實驗,于2007-05/2008-09在解放軍瀋暘軍區總醫院全軍心血管病研究所完成.材料:小鼠胚胎榦細胞繫R1購自美國ATCC公司,編號SCRC-1011~(TM).pSM22α-EGFP載體由本實驗室構建;pIRES2-EGFP載體、pSM2C載體、pSuper.basic載體購自Invitrogen公司.方法:用聚閤酶鏈反應方法從pSM22α-EGFP中擴增SM22α啟動子,然後用該啟動子替換pIRES2-EGFP載體中的CMV啟動子,構建pSM22-IRES2-EGFP.再從pSM2C中用HindⅢ/Cla Ⅰ酶切穫得pac基因,將pac基因片段亞剋隆到pSuper.basic中,構建pSuper-PAC.最後BgⅢ/Acc Ⅰ雙酶切pSuper-PAC穫得pac基因片段,將其插入到pSM22α-IRES2-EGFP,構建成pSM22α-PAC-IRES2-EGFP.將pSM22α-PAC-IRES2-EGFP用脂質體法轉染胚胎榦細胞,G418篩選暘性剋隆.誘導胚胎榦細胞暘性剋隆分化,RT-PCR擴增pac基因鑒定暘性剋隆.對分化細胞行平滑肌細胞標誌物SMα-actin免疫熒光染色.主要觀察指標:①pSM22α-PAC-IRES2-EGFP測序結果.②pac基因擴增.③熒光顯微鏡下同時觀察分化細胞EGFP的錶達及SMα-actin染色情況.結果:HindⅢ/Cla Ⅰ雙酶切得到261 bp,664 bp,5 000 bp 3箇片段,與預期結果一緻,測序結果證實pSM22α-PAC-IRES2-EGFP構建成功.Pac基因擴增證實有4株胚胎榦細胞剋隆轉染成功.轉染成功的胚胎榦細胞被誘導分化後,部分細胞錶達EGFP,且這些細胞SMα-actin染色呈暘性.結論:實驗成功構建瞭平滑肌細胞篩選載體pSM22α-PAC-IRES2-EGFP.成功轉染這一載體的胚胎榦細胞錶達pac基因及EGFP基因,且EGFP錶達具有平滑肌特異性.
배경:배태간세포시평활기세포중요적래원지일,단시배태간세포분화세포적이질성도치난이획득교순적평활기세포.목적:위진일보순화배태간세포래원적평활기세포,의재체외구건평활기특이성SM22α계동자구동적표령매소항성(puromycin acetyltransferase,pac)기인여증강형록색형광단백(enhanced green fluorescence protein,EGFP)기인쌍표체재체,즉pSM22-PAC-IRES2-EGFP재체,병재배태간세포중검측기유효성급특이성.설계、시간급지점:기인수평세포관찰실험,우2007-05/2008-09재해방군침양군구총의원전군심혈관병연구소완성.재료:소서배태간세포계R1구자미국ATCC공사,편호SCRC-1011~(TM).pSM22α-EGFP재체유본실험실구건;pIRES2-EGFP재체、pSM2C재체、pSuper.basic재체구자Invitrogen공사.방법:용취합매련반응방법종pSM22α-EGFP중확증SM22α계동자,연후용해계동자체환pIRES2-EGFP재체중적CMV계동자,구건pSM22-IRES2-EGFP.재종pSM2C중용HindⅢ/Cla Ⅰ매절획득pac기인,장pac기인편단아극륭도pSuper.basic중,구건pSuper-PAC.최후BgⅢ/Acc Ⅰ쌍매절pSuper-PAC획득pac기인편단,장기삽입도pSM22α-IRES2-EGFP,구건성pSM22α-PAC-IRES2-EGFP.장pSM22α-PAC-IRES2-EGFP용지질체법전염배태간세포,G418사선양성극륭.유도배태간세포양성극륭분화,RT-PCR확증pac기인감정양성극륭.대분화세포행평활기세포표지물SMα-actin면역형광염색.주요관찰지표:①pSM22α-PAC-IRES2-EGFP측서결과.②pac기인확증.③형광현미경하동시관찰분화세포EGFP적표체급SMα-actin염색정황.결과:HindⅢ/Cla Ⅰ쌍매절득도261 bp,664 bp,5 000 bp 3개편단,여예기결과일치,측서결과증실pSM22α-PAC-IRES2-EGFP구건성공.Pac기인확증증실유4주배태간세포극륭전염성공.전염성공적배태간세포피유도분화후,부분세포표체EGFP,차저사세포SMα-actin염색정양성.결론:실험성공구건료평활기세포사선재체pSM22α-PAC-IRES2-EGFP.성공전염저일재체적배태간세포표체pac기인급EGFP기인,차EGFP표체구유평활기특이성.
BACKGROUND:Embryonic stem cells (ESCs) serve as a major cell source for smooth muscle cells,but the heterogeneity of cells derived from ESCs result in difficulty to obtain high purity smooth muscle cells.OBJECTIVE:To construct a double expression vector of puromycin resistance (pac) gene and enhanced green fluorescence protein (EGFP) gene driven by smooth muscle specific SM22α promoter (pSM22α-PAC-IRES2-EGFP),in addition,to detect its availability and specificity in ESCs.DESIGN,TIME AND SETTING:The observational experiment of gene level was performed at the Cardiovascular Institute,General Hospital of Shenyang Military Region from April 2007 to September 2008.MATERIALS:ESCs line R1 with number SCRC-1011TM was purchased from American ATCC Company.The pSM22α-EGFP vector was constructed by our laboratory.And the pIRES2-EGFP,pSM2C and pSuper.basic vectors were purchased from Invitrogen Company.METHODS:SM22α promoter was cloned from pSM22α-EGFP by polymerase chain reaction.CMV promoter of pIRES2-EGFP vector was replaced by SM22 promoter to establish pSM22α-IRES2-EGFP.Pac gene,excised from pSM2C by HindⅢ/Clal digestion,was sub-cloned into pSuper.basic to establish pSuper-PAC.After BgⅢ/Accl enzyme digestion of pSuper-PAC,pac gene fragment was obtained,which was further sub-cloned into pSM22α-IRES2-EGFP to produce pSM22α-PAC-IRES2-EGFP.ESCs were transfected with pSM22α-PAC-IRES2-EGFP using lipofectamine.Positive clones were selected by G418 and induced to differentiate and further identified by amplification of pac gene by RT-PCR.Differentiated cells were immunostained by SM α-actin,and expression of SM α-actin and EGFP was observed simultaneously under fluorescence microscope.MAIN OUTCOME MEASURES:Sequencing result of pSM22α-PAC-IRES2-EGFP;Amplification of pac gene;EGFP expression;as well as SM α-actin immunostaining.RESULTS:Three segments of 261 bp,664 bp,and 5000 bp were obtained by HindⅢ/Clal digestion,which was coincident with expectation,and the sequencing results showed that pSM22α-PAC-IRES2-EGFP vector was successfully constructed.Amplification of pac gene identified 4 ESCs clones successfully transfected.After induction of differentiation,partial portion of differentiated cells expressed EGFP,accompanied by positively stained by SM α-actin antibody.CONCLUSION:pSM22α-PAC-IRES2-EGFP vector was successfully constructed.ESCs clones transfected with this vector expressed pac gene and EGFP gene,and the expression of EGFP is smooth muscle specific.
