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
8944-8948
,共5页
袁桂仪%伍卫%周淑娴%张玉玲%雷娟
袁桂儀%伍衛%週淑嫻%張玉玲%雷娟
원계의%오위%주숙한%장옥령%뢰연
HCN4%腺病毒载体%骨髓间充质干细胞%基因转染
HCN4%腺病毒載體%骨髓間充質榦細胞%基因轉染
HCN4%선병독재체%골수간충질간세포%기인전염
背景:超极化激活及环化核苷酸门控阳离子通道(hyperpolarization-activated cyclic nucleotide-gated cation channel,HCN)基因由于具有不增加诱发心律失常的风险、能够接受自主神经系统调节等优势,成为目前最受关注的生物起搏备选基因.目的:构建携带人HCN4基因的重组腺病毒载体,并测定其对大鼠骨髓间充质干细胞的感染效率.设计、时间及地点:细胞-基因学体外实验,于2008-02/09在中山大学附属第二医院林百欣实验中心完成.材料:SD大鼠10只,由中山大学实验动物中心提供.携带目的基因人HCN4 cDNA的质粒pcDNA3.1-HCN4、人胚肾293细胞、大肠杆菌DH5α由中山大学附属第二医院林百欣实验中心保存.腺病毒穿梭质粒pShuttle-CMV、骨架质粒pAdxsi购自北京诺赛基因组研究中心有限公司.方法:质粒pcDNA3.1-HCN4用Hind Ⅲ+Xba Ⅰ双酶切后回收HCN4片段,亚克隆至pShuttle-CMV中,得到重组穿梭质粒;1-Ceu Ⅰ+1-Sce Ⅰ双酶切处理pShuttle-CMV-HCN4,回收CMV-HCN4片段,亚克隆至腺病毒骨架载体pAdxsi,得到重组腺病毒质粒;重组腺病毒质粒酶切线性化后,应用脂质体法转染293细胞进行包装扩增,得到重组腺病毒AdHCN4;应用AdHCN4转染大鼠骨髓间充质干细胞.主要观察指标:重组腺病毒质粒载体的鉴定,重组腺病毒的鉴定及滴度测定,重组腺病毒的感染效率.结果:构建的重组穿梭质粒pShuttle-CMV-HCN4用Hind Ⅲ+Xho Ⅰ双酶切,得到大小为3 600 bp(HCN4)和5 100 bp(pshutIle-CMV)两个片段,DNA测序结果证实人HCN4基因的全长序列已正确插入到pShuttle-CMV穿梭质粒中;重组腺病毒质粒pAdxsi-CMV-HCN4用Xho Ⅰ酶切得到7个片段,而作为对照的空腺病毒质粒只得到6个片段;重组腺病毒质粒在293细胞中包装后产生的重组腺病毒对293细胞有致病作用;重组腺病毒AdHCN4 PCR鉴定可见657 bp的阳性扩增条带;经多次重复感染后,病毒滴度检测达2.5×10~(11) PFU/mL.成功转染AdHCN4的大鼠骨髓间充质干细胞可表达绿色荧光蛋白,当病毒感染复数值为800,转染效率最高,达90%.结论:实验成功构建携带人HCN4基因的重组腺病毒载体,并可在体外有效转染大鼠骨髓间充质干细胞.
揹景:超極化激活及環化覈苷痠門控暘離子通道(hyperpolarization-activated cyclic nucleotide-gated cation channel,HCN)基因由于具有不增加誘髮心律失常的風險、能夠接受自主神經繫統調節等優勢,成為目前最受關註的生物起搏備選基因.目的:構建攜帶人HCN4基因的重組腺病毒載體,併測定其對大鼠骨髓間充質榦細胞的感染效率.設計、時間及地點:細胞-基因學體外實驗,于2008-02/09在中山大學附屬第二醫院林百訢實驗中心完成.材料:SD大鼠10隻,由中山大學實驗動物中心提供.攜帶目的基因人HCN4 cDNA的質粒pcDNA3.1-HCN4、人胚腎293細胞、大腸桿菌DH5α由中山大學附屬第二醫院林百訢實驗中心保存.腺病毒穿梭質粒pShuttle-CMV、骨架質粒pAdxsi購自北京諾賽基因組研究中心有限公司.方法:質粒pcDNA3.1-HCN4用Hind Ⅲ+Xba Ⅰ雙酶切後迴收HCN4片段,亞剋隆至pShuttle-CMV中,得到重組穿梭質粒;1-Ceu Ⅰ+1-Sce Ⅰ雙酶切處理pShuttle-CMV-HCN4,迴收CMV-HCN4片段,亞剋隆至腺病毒骨架載體pAdxsi,得到重組腺病毒質粒;重組腺病毒質粒酶切線性化後,應用脂質體法轉染293細胞進行包裝擴增,得到重組腺病毒AdHCN4;應用AdHCN4轉染大鼠骨髓間充質榦細胞.主要觀察指標:重組腺病毒質粒載體的鑒定,重組腺病毒的鑒定及滴度測定,重組腺病毒的感染效率.結果:構建的重組穿梭質粒pShuttle-CMV-HCN4用Hind Ⅲ+Xho Ⅰ雙酶切,得到大小為3 600 bp(HCN4)和5 100 bp(pshutIle-CMV)兩箇片段,DNA測序結果證實人HCN4基因的全長序列已正確插入到pShuttle-CMV穿梭質粒中;重組腺病毒質粒pAdxsi-CMV-HCN4用Xho Ⅰ酶切得到7箇片段,而作為對照的空腺病毒質粒隻得到6箇片段;重組腺病毒質粒在293細胞中包裝後產生的重組腺病毒對293細胞有緻病作用;重組腺病毒AdHCN4 PCR鑒定可見657 bp的暘性擴增條帶;經多次重複感染後,病毒滴度檢測達2.5×10~(11) PFU/mL.成功轉染AdHCN4的大鼠骨髓間充質榦細胞可錶達綠色熒光蛋白,噹病毒感染複數值為800,轉染效率最高,達90%.結論:實驗成功構建攜帶人HCN4基因的重組腺病毒載體,併可在體外有效轉染大鼠骨髓間充質榦細胞.
배경:초겁화격활급배화핵감산문공양리자통도(hyperpolarization-activated cyclic nucleotide-gated cation channel,HCN)기인유우구유불증가유발심률실상적풍험、능구접수자주신경계통조절등우세,성위목전최수관주적생물기박비선기인.목적:구건휴대인HCN4기인적중조선병독재체,병측정기대대서골수간충질간세포적감염효솔.설계、시간급지점:세포-기인학체외실험,우2008-02/09재중산대학부속제이의원림백흔실험중심완성.재료:SD대서10지,유중산대학실험동물중심제공.휴대목적기인인HCN4 cDNA적질립pcDNA3.1-HCN4、인배신293세포、대장간균DH5α유중산대학부속제이의원림백흔실험중심보존.선병독천사질립pShuttle-CMV、골가질립pAdxsi구자북경낙새기인조연구중심유한공사.방법:질립pcDNA3.1-HCN4용Hind Ⅲ+Xba Ⅰ쌍매절후회수HCN4편단,아극륭지pShuttle-CMV중,득도중조천사질립;1-Ceu Ⅰ+1-Sce Ⅰ쌍매절처리pShuttle-CMV-HCN4,회수CMV-HCN4편단,아극륭지선병독골가재체pAdxsi,득도중조선병독질립;중조선병독질립매절선성화후,응용지질체법전염293세포진행포장확증,득도중조선병독AdHCN4;응용AdHCN4전염대서골수간충질간세포.주요관찰지표:중조선병독질립재체적감정,중조선병독적감정급적도측정,중조선병독적감염효솔.결과:구건적중조천사질립pShuttle-CMV-HCN4용Hind Ⅲ+Xho Ⅰ쌍매절,득도대소위3 600 bp(HCN4)화5 100 bp(pshutIle-CMV)량개편단,DNA측서결과증실인HCN4기인적전장서렬이정학삽입도pShuttle-CMV천사질립중;중조선병독질립pAdxsi-CMV-HCN4용Xho Ⅰ매절득도7개편단,이작위대조적공선병독질립지득도6개편단;중조선병독질립재293세포중포장후산생적중조선병독대293세포유치병작용;중조선병독AdHCN4 PCR감정가견657 bp적양성확증조대;경다차중복감염후,병독적도검측체2.5×10~(11) PFU/mL.성공전염AdHCN4적대서골수간충질간세포가표체록색형광단백,당병독감염복수치위800,전염효솔최고,체90%.결론:실험성공구건휴대인HCN4기인적중조선병독재체,병가재체외유효전염대서골수간충질간세포.
BACKGROUND:The hyperpoladzation-activated cyclic nucleotide-gated cation channel (HCN) gene is not increase the risk of induced cardiac arrhythmia,and can accept the modulation function of autonomic nervous system.Therefore,it is the first candidate gene for biological pacemakers.OBJECTIVE:To construct recombinant adenovirus vector containing human HCN4 gene and evaluate its transfection efficency in rat bone marrow mesenchymal stem cells (MSCs).DESIGN,TIME AND SETTING:The in vitro cytology-gene experiment was performed at the Lin Bai-xin Experimental Center,Second Hospital of Sun Yat-sen University from February to September 2008.MATERIALS:Ten SD rats were supplied by the experimental animal center of Sun Yat-sen University.Rlasmid pcDNA3.1-HCN4 containing target gene human HCN4,human embryo kidney 293 cells and Escherichia coll DH5α were preserved by our experimental center;Shuttle plasmid pShuttle-CMV containing green fluorescent protein gene and adenoviral backbone plasmid pAdxsi were bought from SinoGenoMax Co.,Ltd.METHODS:HCN4 cDNA segment was liberated from the cloning vector of pcDNA3.1-HCN4 via Hind Ⅲ+Xba Ⅰ,and subcloned into pShuttle-CMV,which was digested by I-Ceu Ⅰ +I-Sce Ⅰ double enzyme and subcloned into adenoviral plasmid to form recombinant adenovirus plasmid.Recombinant adenovirus plasmid was transfected into 293 cell lines by liposome,and the recombinant adenovirus AdHCN4 was packaged and transfected into rat MSCs.MAIN OUTCOME MEASURES:The identification of recombinant adenovirus plasmid vector,identification of recombinant adenovirus and its titration test;the transfection efficiency of recombinant adenovirus.RESULTS:Cloned sequence about 3.6kbp was obtained by Hind Ⅲ+Xho Ⅰ digestion after HCN4 cDNA segment was cloned into pShuttle-CMV.DNA sequencing results indicated that the clone location was correct.Recombinant adenovirus plasmid was cut into seven fragments while empty vector gained only six fragments after digested by Xho Ⅰ.The recombinant adenovirus was pathogenic to 293 cells after recombinant adenovirus plasmid was packaged in it.HCN4 cDNA (657bp) was amplified by PCR with virus titer of 2.5×10~(11) PFU/mL after transfected 293 cells with supematant.The efficiency of recombinant adenovirus infecting rat MSCs was about 90% when multiplicity of infection was 800.Rat MSCs expressed green fluorescence after transfection.CONCLUSION:The adenovirus vector with human HCN4 cDNA was established successfully,which can effectively transfect rat MSCs.
