中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
5期
392-397
,共6页
武晶晶%华宁%东莉洁%李筱荣
武晶晶%華寧%東莉潔%李篠榮
무정정%화저%동리길%리소영
睫状神经营养因子%慢病毒载体%骨髓间充质干细胞
睫狀神經營養因子%慢病毒載體%骨髓間充質榦細胞
첩상신경영양인자%만병독재체%골수간충질간세포
Ciliary neurotrophic factor%Lentiviral vector%Bone marrow mesenchymal stem cell
背景 间充质干细胞作为基因转移的载体,已在多种疾病的研究中发挥作用.该方法可能为外源性神经营养因子在中枢神经系统,包括在视网膜中的应用提供更有效的途径. 目的 构建慢病毒介导的过表达睫状神经营养因子(CNTF)的骨髓间充质于细胞(BMSCs),为眼科视网膜、视神经疾病的治疗提供新的方法.方法 对SD大鼠BMSCs进行培养和传代,第4代细胞用于实验.将全基因合成含信号肽的大鼠CNTF基因编码序列克隆至pHⅣ-dTomato慢病毒载体质粒,构建重组质粒CNTF-dTomato.CNTF-dTomato/pHⅣ-dTomato与慢病毒辅助质粒psPAX2及pMD2.G共转染293T细胞进行慢病毒包装,获得重组慢病毒CNTF-lenti及不含CNTF的control-lenti.以CNTF-lenti或control-lenti感染SD大鼠BMSCs,构建CNTF-BMSCs及空载-BMSCs细胞,根据红色荧光蛋白dTomato表达情况确定病毒感染效率;未感染lenti的BMSCs为空白-BMSCs.用ELISA法测定空白-BMSCs、空载-BMSCs及CNTF-BMSCs细胞培养液中CNTF的质量浓度.将CNTF-BMSCs向成骨和成脂2个方向诱导分化,并用茜素红S(ARS)染色和油红O染色对分化细胞进行鉴定.结果 CNTF-dTomato质粒转化大肠杆菌Stbl3感受态细胞后,菌落PCR产物大小约为1 033 bp,质粒测序结果显示,pHⅣ-dTomato质粒中插入部分的碱基序列与预期序列一致,显示CNTF-dTomato质粒构建成功.重组慢病毒对BMSCs的感染率达95％.ELISA检测结果显示,感染后2、3、4、7d,CNTF-BMSCs组细胞上清液中CNTF蛋白的质量浓度明显高于空白-BMSCs组及空载-BMSCs组,差异均有统计学意义(均P=0.000);CNTF-BMSCs组感染后2、3、7d细胞上清液中CNTF质量浓度均明显高于感染后1d,差异均有统计学意义(P=0.013、0.004、0.042).CNTF-BMSCs经成脂培养基诱导后分化的细胞油红0染色呈红色着染,经成骨培养基诱导后分化的细胞ARS染色可见橘红色钙结节. 结论 本研究成功构建CNTF-dTomato质粒,利用慢病毒载体可成功构建稳定过表达分泌型CNTF的大鼠BMSCs,CNTF-BMSCs具备向脂肪细胞和成骨细胞分化的潜能.
揹景 間充質榦細胞作為基因轉移的載體,已在多種疾病的研究中髮揮作用.該方法可能為外源性神經營養因子在中樞神經繫統,包括在視網膜中的應用提供更有效的途徑. 目的 構建慢病毒介導的過錶達睫狀神經營養因子(CNTF)的骨髓間充質于細胞(BMSCs),為眼科視網膜、視神經疾病的治療提供新的方法.方法 對SD大鼠BMSCs進行培養和傳代,第4代細胞用于實驗.將全基因閤成含信號肽的大鼠CNTF基因編碼序列剋隆至pHⅣ-dTomato慢病毒載體質粒,構建重組質粒CNTF-dTomato.CNTF-dTomato/pHⅣ-dTomato與慢病毒輔助質粒psPAX2及pMD2.G共轉染293T細胞進行慢病毒包裝,穫得重組慢病毒CNTF-lenti及不含CNTF的control-lenti.以CNTF-lenti或control-lenti感染SD大鼠BMSCs,構建CNTF-BMSCs及空載-BMSCs細胞,根據紅色熒光蛋白dTomato錶達情況確定病毒感染效率;未感染lenti的BMSCs為空白-BMSCs.用ELISA法測定空白-BMSCs、空載-BMSCs及CNTF-BMSCs細胞培養液中CNTF的質量濃度.將CNTF-BMSCs嚮成骨和成脂2箇方嚮誘導分化,併用茜素紅S(ARS)染色和油紅O染色對分化細胞進行鑒定.結果 CNTF-dTomato質粒轉化大腸桿菌Stbl3感受態細胞後,菌落PCR產物大小約為1 033 bp,質粒測序結果顯示,pHⅣ-dTomato質粒中插入部分的堿基序列與預期序列一緻,顯示CNTF-dTomato質粒構建成功.重組慢病毒對BMSCs的感染率達95％.ELISA檢測結果顯示,感染後2、3、4、7d,CNTF-BMSCs組細胞上清液中CNTF蛋白的質量濃度明顯高于空白-BMSCs組及空載-BMSCs組,差異均有統計學意義(均P=0.000);CNTF-BMSCs組感染後2、3、7d細胞上清液中CNTF質量濃度均明顯高于感染後1d,差異均有統計學意義(P=0.013、0.004、0.042).CNTF-BMSCs經成脂培養基誘導後分化的細胞油紅0染色呈紅色著染,經成骨培養基誘導後分化的細胞ARS染色可見橘紅色鈣結節. 結論 本研究成功構建CNTF-dTomato質粒,利用慢病毒載體可成功構建穩定過錶達分泌型CNTF的大鼠BMSCs,CNTF-BMSCs具備嚮脂肪細胞和成骨細胞分化的潛能.
배경 간충질간세포작위기인전이적재체,이재다충질병적연구중발휘작용.해방법가능위외원성신경영양인자재중추신경계통,포괄재시망막중적응용제공경유효적도경. 목적 구건만병독개도적과표체첩상신경영양인자(CNTF)적골수간충질우세포(BMSCs),위안과시망막、시신경질병적치료제공신적방법.방법 대SD대서BMSCs진행배양화전대,제4대세포용우실험.장전기인합성함신호태적대서CNTF기인편마서렬극륭지pHⅣ-dTomato만병독재체질립,구건중조질립CNTF-dTomato.CNTF-dTomato/pHⅣ-dTomato여만병독보조질립psPAX2급pMD2.G공전염293T세포진행만병독포장,획득중조만병독CNTF-lenti급불함CNTF적control-lenti.이CNTF-lenti혹control-lenti감염SD대서BMSCs,구건CNTF-BMSCs급공재-BMSCs세포,근거홍색형광단백dTomato표체정황학정병독감염효솔;미감염lenti적BMSCs위공백-BMSCs.용ELISA법측정공백-BMSCs、공재-BMSCs급CNTF-BMSCs세포배양액중CNTF적질량농도.장CNTF-BMSCs향성골화성지2개방향유도분화,병용천소홍S(ARS)염색화유홍O염색대분화세포진행감정.결과 CNTF-dTomato질립전화대장간균Stbl3감수태세포후,균락PCR산물대소약위1 033 bp,질립측서결과현시,pHⅣ-dTomato질립중삽입부분적감기서렬여예기서렬일치,현시CNTF-dTomato질립구건성공.중조만병독대BMSCs적감염솔체95％.ELISA검측결과현시,감염후2、3、4、7d,CNTF-BMSCs조세포상청액중CNTF단백적질량농도명현고우공백-BMSCs조급공재-BMSCs조,차이균유통계학의의(균P=0.000);CNTF-BMSCs조감염후2、3、7d세포상청액중CNTF질량농도균명현고우감염후1d,차이균유통계학의의(P=0.013、0.004、0.042).CNTF-BMSCs경성지배양기유도후분화적세포유홍0염색정홍색착염,경성골배양기유도후분화적세포ARS염색가견귤홍색개결절. 결론 본연구성공구건CNTF-dTomato질립,이용만병독재체가성공구건은정과표체분비형CNTF적대서BMSCs,CNTF-BMSCs구비향지방세포화성골세포분화적잠능.
Background The application of mesenchymal stem cells to transfer specific genes is under investigation in various diseases.Using this strategy may provide a more effective method to supply exogenous neurotrophic factors to the cental nervous system,including retina.Objective This study was to construct ciliary neurotrophic factor(CNTF)-overexpressing bone marrow mesenchymal stem cells (BMSCs) using lentiviral vectors.Methods Rat secreted-CNTF gene cDNA was synthesized and subcloned into a lentiviral vector plasmid pHⅣ-dTomato to construct recombinant vector CNTF-dTomato.CNTF-dTomato/pH Ⅳ-dTomato plasmid were co-transfected into 293T packaging cell line with packaging plasmid psPAX2 and enveloped plasmid pMD.2G to produce recombinant lentivirus CNTF-lenti and control-lenti.Rat BMSCs were infected with CNTF-lenti/control-lenti to produce CNTF-BMSCs and control-BMSCs.Expression of dTomato and efficiency of infection was evaluated under the fluorescence microscope.Uninfected BMSCs(pure BMSCs) served as the blank control.CNTF protein level in the supernate was detected by enzyme-linked immunosorbent assay (ELISA) and compared among the blank-BMSCs group,control-BMSCs group and CNTF-BMSCs group.Adipogenic and osteogenic differentiation of CNTF-BMSCs were induced using adipogenic-inducible medium and osteogenic-inducible medium and identified using oil-red O staining and alizarin red S (ARS) staining.Results After CNTF-dTomato plasmid was transfected into Stbl3 competent cells,the colony PCR product was 1 033 bp.The inserted sequence in the pHⅣ-dTomato plasmid was coincident with the expected one.The results of DNA sequencing showed that CNTF-dTomato plasmid was successfully constructed.The infection rate of CNTF-lenti was approximately 95％.ELISA showed that on the post-infected day 2,3,7,the CNTF protein levels in the supernate were significantly higher in the CNTF-BMSCs group than those in the blankBMSCs group and control-BMSCs group (all at P=0.000).In the CNTF-BMSCs group,the CNTF protein levels in the supernate were significantly increased on post-infected day 2,3,7 compared with day 1 (P =0.013,0.004,0.042).The adipogenic-induced cells showed the red staining to oil red O,and osteogenic-induced cells presented the orange staining to ARS.Conclusions BMSC line with stable expression of CNTF is successfully established by lentiviral vectors.CNTF-BMSCs have the potential to differentiate towards adipocytes and osteoblasts.
