Microsoft Word 邢琪6.0.DOC

Size: px

Start display at page:

Download "Microsoft Word 邢琪6.0.DOC"

姬农鞠
5 years ago
Views:

1 f zu{ g } 15 í } Çf Journal of Clinical Rehabilitative Tissue Engineering Research April 2, 2011 Vol.15, No.14 Œ Œ ÎÎÂµ«Œöº Œ«p Œq ƒ Í n*** k 1 É 2 Ê Ÿ Í 3 Inhibitory effect of bone marrow mesenchymal stem cells carrying cytosine deaminase gene on glioma cells in vitro Xing Qi 1, Song Fei 2, Liu Jian 2, Ji Guang-chun 2, Zhang Da-qing 2, Ma Yu-fang 3 Abstract BACKGROUND: The bone marrow mesenchymal stem cells (BMSCs) expressing foreign cytosine deaminase gene (gene CD) can effectively transform 5-fluorouracil (5-FC) that was used before chemotherapy into chemotherapeutics 5-FC with cytotoxicity. BMSCs expressing gene CD significantly inhibited the growth of glioma cells in vitro. OBJECTIVE: To explore the effects of BMSCs as gene therapy vector expressing exogenous gene cytosine deaminase gene on the proliferation of glioma C6 cells. METHODS: Mouse MSCs were isolated and cultured. Lentivirus vector combined with cytosine deaminase gene and green fluorescent protein (GFP) was constructed. Using lentivirus packaging, cytosine deaminase gene and GFP were transferred to mouse BMSCs. BMSCs expressing cytosine deaminase gene and GFP were stably obtained and cocultured with glioma C6 cells. Following 5-FC was added to the medium, effects of cytosine deaminase gene on the proliferation of glioma cells were detected using flow cytometry. RESULTS AND CONCLUSION: Lentivirus-mediated cytosine deaminase gene and GFP gene successfully transfected mouse BMSCs and formed C57BL/6 mmsc-coda/egfp cells. C57BL/6 mmsc-coda/egfp could induce obvious apoptosis of glioma C6 cells following treatment with 5-FC. The apoptotic rate of C6 glioma cells was 60% (P < 0.05) under the action of 5-FC at a concentration of μg/l. It was thus concluded that C57BL/6 mmsc-coda/egfp could convert 5-FC to 5-FU and had significant restriction, even fatal, influence on the growth of C6 glioma cells. Xing Q, Song F, Liu J, Ji GC, Zhang DQ, Ma YF. Inhibitory effect of bone marrow mesenchymal stem cells carrying cytosine deaminase gene on glioma cells in vitro.zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(14): [ ± Œ Œ qîîâµ«œœ äoð 5 ±ä º Œ qäo g 5 ƒ Œ«p Œ qmà Í n rq xîîâµ«œg o±ƒÿ² Œ Œ Œ«p C6 Œ q Éy» ÙÂµ«Œ Œ Œ GFP Šüq o ±ƒ ³² o â Œ Œ ö GFP ± Ž ÙÎÎÂµ«Œ z Ÿ²Œ Œ ö GFP qîîâµ«œ ˆº Œ«p C6 Œº»» fø 5 Œ ý n Œy Œ Œ Œ«p Œq o v qœ Œ ö GFP Ø± ÙÎÎÂµ«Œ C57BL/6 mmsc-coda/egfp Œ C57BL/6 mmsc-coda/egfp 5 Œ q n ù Œ«p C6 Œq Ãr 5 Œ g μg/ L z C6 Œ«p ŒÃrig 60%(P < 0.05) x C57BL/6 mmsc-coda/egfp ù 5 Œ ±ä 5 C6 Œ«p ŒmÀ qâí nmž Ž ºÀ Œ Œ ÎÎÂµ«Œ Œ«p o ±ƒ doi: /j.issn k É ÊŸ Í. Œ Œ ÎÎÂµ«Œöº Œ«p Œqƒ Í n[j].f zu{ g (14): [ 0 Œ«p È¼ q p [1-2] ë È¼ pq40% äo o}o k ug9~12f [3-4] rð ¼ ŒŒ«p ou { Ë Œ Œ p s fq yx m Œ ÿ ù ñ 5 Œ (5-flucytosine 5-FC) x g5 (5-fluorouracil 5-FU)v Ž p ŒŽ r [5] Œ Œ ± p Œ Ê «o p Œ 5-FCg5-FUqg xâƒ r o5-fu ömq Ó n f x f npžœ Ãq äo 5-FU Ä Œ p ¼²Í v ÂÍoäo g Œ pfq n ÎÎÂµ«Œ(bone marrow mesenchymal stem cells BMSCs) yº ýéä Œ Œ ƒ Éy 1 Department of Anesthesia, First Hospital Affiliated to Dalian Medical University, Dalian , Liaoning Province, China; 2 Department of Neurosurgery, Second Hospital Affiliated to Dalian Medical University, Dalian , Liaoning Province, China; 3 Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian , Liaoning Province, China Xing Qi, Master, Professor, Department of Anesthesia, First Hospital Affiliated to Dalian Medical University, Dalian , Liaoning Province, China Correspondence to: Song Fei, Doctor, Professor, Department of Neurosurgery, Second Hospital Affiliated to Dalian Medical University, Dalian , Liaoning Province, China songfei168@ 163.com Supported by: the Scientific Research of Education Committee of Liaoning Province in 2008, No. 2008Z081*; the Scientific Technology Foundation of Dalian City, No. 2008E13SF203*; the Natural Science Foundation of Liaoning Province in 2009, No * Received: Accepted: ISSN CN /R CODEN: ZLKHAH 2577

2 1 {î Þ{ œ þ Þ ñ {î {î Þ{ œ Ò Þ ñ { î {î Þ{ ñ {î æ 1993 {î Þ{ Ì x Ôd songfei168@ 163.com Ì {î Þ{ œ Ò Þ ñ {î songfei168@ 163.com f É ú:r394.2 h u:b ú: (2011) º ( /DeQ) 2578 džp Œz BMSCshgq É ˆ p Ÿ² Ÿj Ç qý p Œ² g ù g Ÿ²±ƒ [6-7] È yœ Œ qbmscs Ë º Œ «p ŒqmÀ Í n Œ ƒ Í Âö Íp200810/ ²çy É mg Í C57BL Ù6ù 4 Ú ÄÄ Â ƒ«18~20 g SPF n ²çy ÍÙgf Ùg«ü ú SCXK(²) Í²zf Ùgq ˆ}ü2006 y µ ºp ÍÙgq q g Ïö g Ï y pdonrtm221 «293T Œ One Shot stbl3 Competent E. coli xö coda q«gateway BP Clonase TM Enzyme Mix Gateway LR Clonase TM Plus Enzyme Mix Lipofectamine TM 2000 Opti-MEM Reduced Serum Medium «Ïr fž DMEM Œ -EDT A Å Ä ºw Ï g²ø rä É «dmg¹ú Invitrogen ¹ú Qiagen ¹ú PAA ¹ú Gibco ¹ú gü Genebank qœ Œ q Ê Œ Œ Â q5ÿ 3ÿ Œ Œ g g É ºÂÍ Éƒ É ² ÉŒ Œ Êq ÂÍ ¼É 5ÿ ÉÌØ attb1 attb2 ƒ géìg 5ÿ- GGG GAC AAG TTT GTA CAA AAA AGC AGG CTG CCA CCA TGT CGA ATA ACG CTT TAC AA -3ÿ 5ÿ- GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT CAA CGT TTG TAA TCG ATG G -3ÿ BMSCsqÉy» ¹ ³²µÉ ²ñ q Í Éy»BMSCs [8-9] Ù È xƒzé g75% q j 2.0~ 3.0 min z öœ Î ŒÎ n1 ml qÿ100 U/mLÅÄ 100 mg/l Ä ƒzé g15% fž q LG-DMEM» À ÎÎ ÄÀ ÎÎ r/miny 5 min ô» Œ Œ Ž L 1 ±»m p37 ƒzé g5%qco 2»~f» 48 hýé 4 dý¼ é 6 dý¹ ÇÅ«Œ ŒmÀ Ž80%~90% ü x1.25 g/lœ ~0.25 g/l EDTA ä~x }3x ŒÍ ê Œ Ø qcd44 CD45 CD90 CD105 ƒý n Œy²ŸŸÅ ¹ PCR codarq f xÿ codaq «g n ² g²ÿpcr PCR rg³²n ý²ÿ ä(º ƒ Îõd TIANgel Midi Purification Kit ³kŒ ÄŒDNA Ïr ) üö pdown-coda±ƒ f dx ÉØ Í1.5 mlep~f â attb1-coda-attb2 PCR r g 100 ng pdonrtm221 «150 ng ŸØTE buffer (ph 8.0) Ž ƒz 8 μl À BP Clonase enzyme mix 2 min u 2 Ø x ö fø 2 μl BP Clonase enzyme mix u â2 u y 25 1 h Ø 1μL q Kf ö min pdown-coda±ƒq±ä BPö g±äí One Shot OmniMAX TM 2 T1 Phage-Resistant Cells E. coli f 1 μl LR ö gø Í50 μl One Shot OmniMAX 2 T1 Phage-Resistant Cells E. colif À 30 min 42 Ç Œ30 s í±zíà 2 min Ø 250 μl S.O.C. medium RPM q 1 h 100 μl ±äg Íÿ 50 mg/l KanqLB 37 ² }²Ç ý «²Ÿ ÉŒ ä«²ÿlrö LR üö plv/expn2-puro-pgk- codaÿ²±ƒ f dx ÉØ Í 1.5 ml EP~f â pup-pgk 10 fmoles pdown-coda 10 fmoles pdes2-puro 20 fmoles ŸØ1X TE buffer(ph8.0)ž8 μl À LR Clonase Plus 2 min u P.O. Box 1200, Shenyang cn.zglckf.com

3 2 Ø x ö fø 2 μl LR Clonase Plus u â2 u y h Ø 1 μl q Kf ö min plv/expn2-puro-pgk-codaÿ²±ƒq±ä LRö g±äí One Shot stbl3 Competent E. coli f 2 μl LRö gø Í50 μl One Shot stbl3 Competent E. colif À 30 min 42 Ç Œ30 s í±zíà 2 min Ø 250 μl S.O.C. medium RPM.q 1 h 100 μl±äg Íÿ 100 mg/l AmpqLB 37 ² }²Ç ý «²Ÿ o ±ƒqâ Ä «plv/expn2-neo-ef1a-egfp plv/expn2-puro-pgk-codaºƒ õd «f Ïr o â ± Ð1 d(}1 ) y 293FT ŒÍ f10 cm»rf y Œ x ± q ŒmÀ²Í90%~95% üg ± (}2 ) v293ft ŒfôÃ» Ø 5 ml Ó qÿž» Â DNA-Lipofectamine 2000 ü g x f10 cm»rqn gx Â q5 mlep~ Ø 1.5 ml ž Opti-MEM I» 3 μg psl3 3 μg psl4 3 μg psl5 3 μg plv/ EXPN2- Neo-EF1A-eGFP( plv/expn2-puro- PGK- coda) ±±È â Â ù q5 ml EP~ Ø 1.5 ml ž Opti-MEM I» 36 μl q Lipofectamine 2000 ±±È â 5 min 5 miný ÿ «DNAq ž Opti-MEM I» Ø Íÿ Lipofectamine 2000q ž Opti-MEM I» ±±È â 20 min 20 miný DNA- Lipofectamine 2000 üg DNA- Lipofectamine 2000 üg ØÍ293FT Œf ±± Ð ý»rx â üg ˆ37 ƒzé 5%CO 2 Ê»~f²» ± ý1 d(}3 ) ôãÿ DNA-Lipofectamine 2000 ügq» Ø 10 ml ÿ m q ¹» (È ) ˆ37 ƒzé 5%CO 2 Ê»~» ± ý 60 h(}5 ) Äÿ o q»» Í q ry ~f y o Ë ôã Œ vf r/miny 15 min n0.45 μm ² o ôã Œvf y o gy 90 min Ø 500 μl Ó» o È É o ˆ80 o BMSCs coda o egfp o ± Ð1 d(}1 ) dü²q Œ Œ yí¹ f ˆ± q Œ ü ² Í30%~50% ± (}2 ) coda o ( egfp o ) ôã» Ø 1 ml C57 ÙÂ«Œ ¹» 100 µlqcoda o ( egfp o ) ŒùØ Polybrene ²o Œ( 6 mg/l) ˆ 37 ƒzé 5%CO 2 Ê»~²» ± 8 hý ôãÿ o q» Ø Óq ¹» ˆ37 ƒzé 5%CO 2 Ê»~²» ± 24 hý ôãÿ o q» Ø ÿ ² qpuromycinq ¹» ( ÿ ² qneomycinq ¹» ) }²Ÿz ± q Œ ˆ37 5% CO 2 Ê»~²» Puromycin G418 C57 ÙÂ«Œq ²}² ²n mg/l Puromycin}²C57 Ù Â«Œ3 d ²n mg/l G418 }²C57 ÙÂ«Œ5 d 5-fluorocytocine C57MSC- coda/egfpq«} ²n mg/l 5-FCŽ C57MSC-codA/eGFP C57MSC-codA/eGFP¹ C57MSC- coda/ egfp C57 MSC yí¹ ŒmÀÍ90% ü ²ŸRNA RT-PCR C57 MSCcodA/eGFP C57MSCfcodA qÿ² PCRö ƒ 30 μl jõ gè 94 3 min s ² 60 1 min 72 1 min j 30f j µé 72 5 min 12 C57MSC-codA/eGFPŠü5-FC C6Œ«p Œƒ Í n ptranswell ö Ø²» ² màg qœ«p Œ Œ äý n1640» Í ê Œ ý C6Œ«p Œ â yptranswell (¹ ) 10 miný C6Œ«p Œ Âý màg qc57bl/6 mmsc-coda/egfp Œ Œ äý ndf12» Í ê Œ ý C6Œ«p Œ â yptranswell ¼» Œ² ˆ Œ«mÀ }2 ±± transwell ¼q» ô Ø ÿ5-fcq» (5-FC ˆ É μg/ L) ˆ37»~» ŒÃr À Â ÀÀ 5-FCq n Â ÄŒ«p Œ Í ê Œ y ýø 195 ml Annexin V-FITC ü (1X)±± Œ Ø 5 μlannexin V-FITC ± ± â (20~25 ) 10 min y ý Ø 190 μl Annexin V-FITC ü (1X)±± Œ Ø 10 μlväd ±± â À ˆ Ãí²Ÿ Œy Annexin V-FITCg PIg g ³² Œy p ŒÃr À ISSN CN /R CODEN: ZLKHAH 2579

É n} nspss 11.0 ±z nt Í P < 0.05Ÿx i 2 2.1 ð C57BL Ù6ù np È öœ Î ŒÎÎÎ Ùg Œ 2.2 ö (Flow Cytometry FCM)ÆgBMSCs Â Î«FITC PE-cy5 ƒqü d 1 CD44Â ig87.73%( 1a) Sca-1Â ig88.04%( 1b) CD117Â ig12.

4 coda¾ ò ÊÉ Ÿ Œ Œ Ê¹À1 284 bp u427f Genbank q Ê²Ÿ ± º Ê 99% Ž ºfq üùœg{ Ž Œ Œ uq427f Genbank ¹ Ž 2.5 C57MSC-codA/eGFP RT-PCRÇ 3 1 2 3 4 5 6 7 bp 12.37% 87.

04% 100 Figure 1 Surface markers of bone marrow mesenchymal stem cells as detected by flow cytometry 1 Œy BMSCs ŒŸÅ g 2.

alizarin red Figure 2 Adipogenic and osteogenic differentiation of bone marrow mesenchymal stem cells and identification ( 100) 2 BMSCs Œö Î Éä¹ ( 100) 1: Negative control (displace DNA

sample; 7: BMSCs (cdna of normal cells).

4 É n} nspss 11.0 ±z nt Í P < 0.05Ÿx i ð C57BL Ù6ù np È öœ Î ŒÎÎÎ Ùg Œ 2.2 ö (Flow Cytometry FCM)ÆgBMSCs Â Î«FITC PE-cy5 ƒqü d 1 CD44Â ig87.73%( 1a) Sca-1Â ig88.04%( 1b) CD117Â ig12.37% ( 1c) CD34Â i²87.50%( 1d) Í hr ü [10-11] BMSCs Œ 72 hý Œ¼ Œ Çj 2 ýœ Ø rp ü ŒnÀ g ² O x Œ«2b BMSCs Î»}3 Œ f q Œ² ü ô Œ ¾ 2d 2.4 coda¾ ò ÊÉ Ÿ Œ Œ Ê¹À1 284 bp u427f Genbank q Ê²Ÿ ± º Ê 99% Ž ºfq üùœg{ Ž Œ Œ uq427f Genbank ¹ Ž 2.5 C57MSC-codA/eGFP RT-PCRÇ bp 12.37% 87.50% a: CD44 positive rate b: Sca-1 positive rate % 88.04% 100 Figure 1 Surface markers of bone marrow mesenchymal stem cells as detected by flow cytometry 1 Œy BMSCs ŒŸÅ g 2.3 BMSCs n Àn Ç c: CD117 positive rate d: CD34 negative rate a: Lipoblasts before oil red O c: Osteoblasts before alizarin red b: Lipoblasts after oil red O d: Osteoblasts after alizarin red Figure 2 Adipogenic and osteogenic differentiation of bone marrow mesenchymal stem cells and identification ( 100) 2 BMSCs Œö Î Éä¹ ( 100) 1: Negative control (displace DNA template with water); 2: BMSCs (cdna of normal cells); 3: BMSCs-CD/eGFP (the transducted cdna with CD and egfp); 4: plv/expn2-puro-ef1a-cd (expression vector of CD); 5: 100-bp marker; 6: no sample; 7: BMSCs (cdna of normal cells). BMSCs: bone marrow mesenchymal stem cells; CD: cytosine deaminase Figure 3 Electrophoresis analyses of C57MSC-codA/eGFP using RT-PCR 3 C57MSC-codA/eGFP RT-PCR n É 2.6 C57MSC-codA/eGFP Puromycin² n2 mg/l g ƒ}² G418²n300 mg/l g ƒ}² egfp neo o ± C57 ÙBMSCs ýn500 mg/l G418}²Ç Œ Ø g nqc57bl ÙBMSCsgÂ d G418 n3 dý Â dr Í Œ r q Œ µéÿ² 3 d r Í Œ q 7 dý Œ ²qj egfp Ø üí Ù Ÿ ² coda puro o ± C57BL Ù BMSCs ýn3 mg/l puromycin}² Ø g q ÙBMSCsgÂ d }²q Í Çj Œ r }²ý q Œ ²qÂ coda üí Ù 2.7 ö ÆgC57MSC-codA/eGFPî 5-FC C6Ê ö º»}5 Í 5-FC g g μg/l C6Œ«p Œ ±g qãr r i μg/l Ãrz q P.O. Box 1200, Shenyang cn.zglckf.com

1 10 2 1 10 3 1 10 4 μg/l Ãrq² d Ì np1 10 2 1 10 3 μg/lãrj xu ºw4 ²Ÿ ¹ 4 ŒmÀq Àr} q C57BL/6 mmsc-coda/egfpù 5-FC±ä C6Œ «p ŒmÀ ÂÍ mž Ž qg«(5-fu) 8.20% 4.46% Control 69.63% 17.72% 7.52 7.

5 μg/l Ãrq² d Ì np μg/lãrj xu ºw4 ²Ÿ ¹ 4 ŒmÀq Àr} q C57BL/6 mmsc-coda/egfpù 5-FC±ä C6Œ «p ŒmÀ ÂÍ mž Ž qg«(5-fu) 8.20% 4.46% Control 69.63% 17.72% % μg/l % 9.39% Control 54.12% 23.25% 2.39% 33.40% μg/l % Figure 4 Apoptotic rate of bone marrow mesenchymal stem cells transfected with cytosine deaminase gene following treatment with 5-flycytosine at various concentrations 4 ü 5-FC kýœ Œ ± qîîâ µ«œ ŒÃri CD/5-FCŽ y±g º g n q o}n rð Œ«pq o Åk Í º ³² ± Œ Œ ± mùg Œ¼ Ê Œù ñ qñ ( s )5-FC±äg Œ qx rg5-fu g5-fc Î mg q mùg n Œ Œ Œ g5-fu ý º Î Œ [1] v q² p Œ [ ] npž o qo «p p Œ± qÿ² iö p Ð x rg q rq ²n±ƒ q± iöºåý gáèqºà f x ž Œ Ãq öˆ ³²ž ² q±ƒ n ÍÂÍ Íöjx Œº ýœ«p ç öšäq Œ Ç²y ý ùœnpœ «p Åý o [14-15] g n x ŒŽ ƒ Â ƒz ³± káè o BMSCsü º Œ«pq ý [16] dù p Žƒz o o r k}áè [17] Øi Ž Ë ƒ» pÿ ± Ÿ g Œ«p ok q±ƒ Œ ºpBMSCsqŒ«p ý qu{ Nakamizo } [18] q Í±g Â²otBMSCs(hM2SCs)ý Œ«pq ý Ÿ ²y ýj Œ¼ž ŒŒ Ù q k yq Œ«p Œ g q tbmscs Œ«pº² ý² z gt Žƒz BMSCs oœ«p o rq Î ± Å Œ ŒÀ z Ÿ ² oq 2005 xý pt o Ãoo u Í q o ±ƒ² ö g yî ¹q ±ƒ rð Ÿ np ¼ ü Í guö¹ú o ±ƒ t o Ãoo q Í Ã ö± o npº n Ÿ²VSVâ q xot o Ãoo â q Îo± i o g Œq Œ qé ÅÉ Œ qrq ù üí g Œ ˆ À z Ÿ² º ¹Î qg ˆ o ±ƒ x ooq of n [19-20] g ± ± ƒ o â Œ ²8 000 bpq Œ { qx Œ À Ÿ²rq È¼ Ÿ²LaeZ q o ù Ÿ²6f [21] rð ƒ¼ Íf öj o ±ƒ Œ Ó n uo q f x ¼ oö p ²g f x pq o f o ±ƒ q nð Í g o u{ n o g± ±ƒ coda q o coda r ± Ù BMSCs mrna ÍcodAq Ÿ² qo ØŒq o ± qbmscsœ qˆrq Ÿ² Ø ² o ocodaäx³²žœ ÃqÁÈ öœ ²mgƒ Ÿ²qŽ Í Í qcoda o ±ƒ± öÿ ²Œ ùx yƒ ƒ ÍqÄ Íf Øq o Œ Œ q ÙBMSCs (C57MSC-codA/eGFP) ² PCR qc57msc-coda/egfp ÙC6Œ«p Œ ºü» Œ p mà» fø 5-FC ý n Œy Œ Œ C6Œ«p Œq öj C57BL/6 mmsc-coda/egfp 5-FCq n ù Œ«pC6 Œq Ãr 5-FC g μg/l z C6Œ «p ŒÃrig60% P < 0.05 ²k C57BL/6 mmsc-coda/egfpù 5-FC±ä 5-FU C6Œ«p ŒmÀ qâí nmž Ž ² ö ISSN CN /R CODEN: ZLKHAH 2581

6 jg ÌnC57BL/6 mmsc-coda/egfp o Ù ŒŒ«p q u 4 õ h [1] Pizzoni C, Sarandria C, Pierangeli E. Clear-cell meningioma of the anterior cranial fossa. Case report and review of the literature. J Neurosurg Sci. 2009;53(3): [2] Lettau I, Hattermann K, Held-Feindt J, et al. Matrix metalloproteinase-19 is highly expressed in astroglial tumors and promotes invasion of glioma cells. J Neuropathol Exp Neurol. 2010;69(3): [3] Biswas T, Okunieff P, Schell MC, et al. Stereotactic radiosurgery for glioblastoma: retrospective analysis. Radiat Oncol. 2009;17: 4:11. [4] Mirimanoff RO, Gorlia T, Mason W, et al. Radiotherapy and temozolomide for newly diagnosed glioblastoma: recursive partitioning analysis of the EORTC 26981/22981-NCIC CE3 phase III randomized trial. J Clin Oncol. 2006;24(16): [5] Lawrence TS, Rehemtulla A, Ng EY, et al. Preferential cytotoxicity of cells transduced with cytosine deaminase compared to bystander cells after treatment with 5-flucytosine. Cancer Res. 1998;58(12): [6] Liang X, Su YP, Kong PY, et al. Human bone marrow mesenchymal stem cells expressing SDF-1 promote hematopoietic stem cell function of human mobilised peripheral blood CD34+ cells in vivo and in vitro. Int J Radiat Biol. 2010; 86(3): [7] Baksh D, Yao R, Tuan RS. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells. 2007;25(6): [8] Sotiropoulou PA, Perez SA, Papamichail M. Clinical grade expansion of human bone marrow mesenchymal stem cells. Methods Mol Biol. 2007;407: [9] Bühring HJ, Treml S, Cerabona F,et al. Phenotypic characterization of distinct human bone marrow-derived MSC subsets. Ann N Y Acad Sci. 2009;1176: [10] Peister A, Mellad JA, Larson BL, et al. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood. 2004;103(5): [11] da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci. 2006;119(Pt 11): [12] Huber BE, Austin EA, Richards CA,et al. Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proc Natl Acad Sci USA. 1994; 91(17): [13] Miller CR, Gustin AN, Buchsbaum DJ, et al. Quantitation of cytosine deaminase mrna by real-time reverse transcription polymerase chain reaction: a sensitive method for assessing 5-fluorocytosine toxicity in vitro. Anal Biochem. 2002;301(2): [14] Aboody KS, Brown A, Rainov NG, et al. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci USA. 2000;97(23): [15] Huang J, Gao J, Lv X, Li G, Hao D, Yao X, Zhou L, Liu D, Wang R. Target gene therapy of glioma: overexpression of BAX gene under the control of both tissue-specific promoter and hypoxia-inducible element. Acta Biochim Biophys Sin (Shanghai). 2010; 42(4): [16] Hamada H, Kobune M, Nakamura K, et al. Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy. Cancer Sci. 2005;96(3): [17] Kan I, Melamed E, Offen D. Autotransplantation of bone marrow-derived stem cells as a therapy for neurodegenerative diseases. Handb Exp Pharmacol. 2007;180: [18] Nakamizo A, Marini F, Amano T, et al. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res. 2005;65(8): [19] Morris KV, Rossi JJ. Lentiviral-mediated delivery of sirnas for antiviral therapy. Gene Ther. 2006;13(6): [20] Li M, Rossi JJ. Lentiviral vector delivery of sirna and shrna encoding genes into cultured and primary hematopoietic cells. Methods Mol Biol. 2008;433: [21] Dresch C, Edelmann SL, Marconi P, Brocker T. Lentiviral-mediated transcriptional targeting of dendritic cells for induction of T cell tolerance in vivo. J Immunol. 2008;181(7): Ž Èq «Ù 2008 ñ Ì Þ í (2008Z081) {î Þ Á ¾Ð(2008E13SF203) ß m CD/TK }c ¾ Ò ñ Ê ö Ôd2009 ñ Þ ¾Ð ( ) ß m ¾ Åp ñ Ê ö Ôd «h Ð Â c c Ð Ð ò À c c ö n c d d Ž {î Þ{ j d ÌrÀ{ ß À jãà ½ ÒÂ Í½ÅÍ ÒÂ½í d k Â «n t 2006 ÞÁ w Çx d Ë Æ CNKI MEDLINE á Æ Å Æ Çw ¾ Åp Ê ö d u ƒ É ¾ «Åp Î«Ï ~ß Ÿ ¾ û ß É ¾ ¾ ¾ Åp jõ {d Åp ¾ ¾ ÝÊ ö½ dî«è BMSCs-CD/eGFP C6 Ê ö ûß{ 86.6% Åp Ï ¾ î ñ 5 Ê ö j d Åp cmifn ö ñ¾ Ýö½ d 2582 P.O. Box 1200, Shenyang cn.zglckf.com

Microsoft Word - 会行党_2014_7号-OK.doc

Microsoft Word - 会行党_2014_7号-OK.doc i k á ÀŒ» ~st v ~x z Ÿ iv roœ ür Ž ç r± ¼~ Ÿd é~ ~ ¾g ~ ˆî ¾s Î õ lý ~ ù g ˆ êdã»â ~ È ~È d ò lý ~ {c Ï»Ò Ð Ÿ»Â ~ È Þ w «g»â ~ È k x «g»â ~ Ú Û Î ï»â d É 1 ƒr c Ç Í Ð cð c n Æ n û cu cf c ß ƒd «Î ï Ð»Â