A preliminary study on the construction of double suicide gene delivery vectors by mesenchymal stem cells and the in vitro inhibitory effects on SKOV3 cells
The aim of the present study was to investigate the efficacy of using human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) as gene delivery vectors in the treatment of ovarian cancer. Lentivectors overexpressing cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-...
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| Veröffentlicht in: | Oncology reports 2014-02, Vol.31 (2), p.781-787 |
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| creator | JIANG, JINGYAN WEI, DEE SUN, LI WANG, YUXIA WU, XIHAI LI, YING FANG, ZHENGHUI SHANG, HUI WEI, ZENGTAO |
| description | The aim of the present study was to investigate the efficacy of using human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) as gene delivery vectors in the treatment of ovarian cancer. Lentivectors overexpressing cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-tk) (pGC-FU-CD-TK) were constructed, and confirmed by enzyme digestion, DNA sequence and western blotting. Quantitative PCR (PCR) was used to verify the overexpression of the fusion gene (CD and HSV-tk). SKOV3 cells were co-cultured with MSCs/tk+CD+ at a 1:1 ratio, and were then treated with the prodrugs (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the cytotoxic effects were evaluated using MTT assay and flow cytometry. DNA sequencing demonstrated that the sequence of HSV-tk and CD genes were consistent with the objective sequence and western blotting verified that the constructed lentivector could produce the HSV-tk/CD gene. The packed titer was 2.00e+8 TU/ml. The pGC-FU-CD-TK could be stably transferred to hUCB-MSCs, and the infection efficiency was almost 80%. RT-PCR demonstrated that the expression levels of the HSV-tk/CD fusion gene in MSCs/tk+CD+ group was 75 times that in the negative control (P |
| doi_str_mv | 10.3892/or.2013.2898 |
| format | Article |
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Lentivectors overexpressing cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-tk) (pGC-FU-CD-TK) were constructed, and confirmed by enzyme digestion, DNA sequence and western blotting. Quantitative PCR (PCR) was used to verify the overexpression of the fusion gene (CD and HSV-tk). SKOV3 cells were co-cultured with MSCs/tk+CD+ at a 1:1 ratio, and were then treated with the prodrugs (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the cytotoxic effects were evaluated using MTT assay and flow cytometry. DNA sequencing demonstrated that the sequence of HSV-tk and CD genes were consistent with the objective sequence and western blotting verified that the constructed lentivector could produce the HSV-tk/CD gene. The packed titer was 2.00e+8 TU/ml. The pGC-FU-CD-TK could be stably transferred to hUCB-MSCs, and the infection efficiency was almost 80%. RT-PCR demonstrated that the expression levels of the HSV-tk/CD fusion gene in MSCs/tk+CD+ group was 75 times that in the negative control (P<0.05). Compared with GCV or 5-FC alone, the growth inhibition rate (GIR) was significantly higher in the combined treatment (F=85.35, P<0.05). The reconstructed MSCs/tk+CD+ vectors were capable of slowing down the growth of human SKOV3 cells in the presence of prodrugs in vitro. The use of combination chemotherapy exhibited a more significant inhibitory effect than using a single prodrug.</description><identifier>ISSN: 1021-335X</identifier><identifier>EISSN: 1791-2431</identifier><identifier>DOI: 10.3892/or.2013.2898</identifier><identifier>PMID: 24317390</identifier><language>eng</language><publisher>Greece: D.A. Spandidos</publisher><subject>Adenoviridae - genetics ; Analysis ; Antimetabolites - pharmacology ; Base Sequence ; Brain cancer ; Cancer ; Cancer therapies ; Care and treatment ; Cell Proliferation - drug effects ; Cloning ; Coculture Techniques ; Cytosine Deaminase - biosynthesis ; Cytosine Deaminase - genetics ; Cytosine Deaminase - therapeutic use ; Deoxyribonucleic acid ; DNA ; Drug resistance ; Enzymes ; Female ; Fetal Blood - cytology ; Flow cytometry ; Flucytosine - pharmacology ; Gene expression ; Gene therapy ; Gene Transfer Techniques ; Genes, Transgenic, Suicide - genetics ; Genetic Therapy - methods ; Genetic Vectors ; Health aspects ; Humans ; Kinases ; lentivector ; mesenchymal stem cells ; Mesenchymal Stromal Cells - cytology ; Mesenchymal Stromal Cells - drug effects ; Microscopy ; Ovarian cancer ; Ovarian Neoplasms - genetics ; Ovarian Neoplasms - therapy ; Plasmids ; Prodrugs - therapeutic use ; Recombinant Fusion Proteins ; Sequence Analysis, DNA ; SKOV3 cells ; Stem cells ; suicide cancer gene therapy ; Thymidine Kinase - biosynthesis ; Thymidine Kinase - genetics ; Thymidine Kinase - therapeutic use ; Tumors ; Vectors (Biology)</subject><ispartof>Oncology reports, 2014-02, Vol.31 (2), p.781-787</ispartof><rights>Copyright © 2014, Spandidos Publications</rights><rights>COPYRIGHT 2014 Spandidos Publications</rights><rights>Copyright Spandidos Publications UK Ltd. 2014</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c486t-a26c3fc50c28ff1be22a2f6cbc5682bff7484d7ec39110b4f2b120bdaeafd8fd3</citedby><cites>FETCH-LOGICAL-c486t-a26c3fc50c28ff1be22a2f6cbc5682bff7484d7ec39110b4f2b120bdaeafd8fd3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24317390$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>JIANG, JINGYAN</creatorcontrib><creatorcontrib>WEI, DEE</creatorcontrib><creatorcontrib>SUN, LI</creatorcontrib><creatorcontrib>WANG, YUXIA</creatorcontrib><creatorcontrib>WU, XIHAI</creatorcontrib><creatorcontrib>LI, YING</creatorcontrib><creatorcontrib>FANG, ZHENGHUI</creatorcontrib><creatorcontrib>SHANG, HUI</creatorcontrib><creatorcontrib>WEI, ZENGTAO</creatorcontrib><title>A preliminary study on the construction of double suicide gene delivery vectors by mesenchymal stem cells and the in vitro inhibitory effects on SKOV3 cells</title><title>Oncology reports</title><addtitle>Oncol Rep</addtitle><description>The aim of the present study was to investigate the efficacy of using human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) as gene delivery vectors in the treatment of ovarian cancer. Lentivectors overexpressing cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-tk) (pGC-FU-CD-TK) were constructed, and confirmed by enzyme digestion, DNA sequence and western blotting. Quantitative PCR (PCR) was used to verify the overexpression of the fusion gene (CD and HSV-tk). SKOV3 cells were co-cultured with MSCs/tk+CD+ at a 1:1 ratio, and were then treated with the prodrugs (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the cytotoxic effects were evaluated using MTT assay and flow cytometry. DNA sequencing demonstrated that the sequence of HSV-tk and CD genes were consistent with the objective sequence and western blotting verified that the constructed lentivector could produce the HSV-tk/CD gene. The packed titer was 2.00e+8 TU/ml. The pGC-FU-CD-TK could be stably transferred to hUCB-MSCs, and the infection efficiency was almost 80%. RT-PCR demonstrated that the expression levels of the HSV-tk/CD fusion gene in MSCs/tk+CD+ group was 75 times that in the negative control (P<0.05). Compared with GCV or 5-FC alone, the growth inhibition rate (GIR) was significantly higher in the combined treatment (F=85.35, P<0.05). The reconstructed MSCs/tk+CD+ vectors were capable of slowing down the growth of human SKOV3 cells in the presence of prodrugs in vitro. The use of combination chemotherapy exhibited a more significant inhibitory effect than using a single prodrug.</description><subject>Adenoviridae - genetics</subject><subject>Analysis</subject><subject>Antimetabolites - pharmacology</subject><subject>Base Sequence</subject><subject>Brain cancer</subject><subject>Cancer</subject><subject>Cancer therapies</subject><subject>Care and treatment</subject><subject>Cell Proliferation - drug effects</subject><subject>Cloning</subject><subject>Coculture Techniques</subject><subject>Cytosine Deaminase - biosynthesis</subject><subject>Cytosine Deaminase - genetics</subject><subject>Cytosine Deaminase - therapeutic use</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>Drug resistance</subject><subject>Enzymes</subject><subject>Female</subject><subject>Fetal Blood - cytology</subject><subject>Flow cytometry</subject><subject>Flucytosine - pharmacology</subject><subject>Gene expression</subject><subject>Gene therapy</subject><subject>Gene Transfer Techniques</subject><subject>Genes, Transgenic, Suicide - genetics</subject><subject>Genetic Therapy - methods</subject><subject>Genetic Vectors</subject><subject>Health aspects</subject><subject>Humans</subject><subject>Kinases</subject><subject>lentivector</subject><subject>mesenchymal stem cells</subject><subject>Mesenchymal Stromal Cells - cytology</subject><subject>Mesenchymal Stromal Cells - drug effects</subject><subject>Microscopy</subject><subject>Ovarian cancer</subject><subject>Ovarian Neoplasms - genetics</subject><subject>Ovarian Neoplasms - therapy</subject><subject>Plasmids</subject><subject>Prodrugs - therapeutic use</subject><subject>Recombinant Fusion Proteins</subject><subject>Sequence Analysis, DNA</subject><subject>SKOV3 cells</subject><subject>Stem cells</subject><subject>suicide cancer gene therapy</subject><subject>Thymidine Kinase - biosynthesis</subject><subject>Thymidine Kinase - genetics</subject><subject>Thymidine Kinase - therapeutic use</subject><subject>Tumors</subject><subject>Vectors (Biology)</subject><issn>1021-335X</issn><issn>1791-2431</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><recordid>eNptkktrFTEUxwdRbK3uXEtAKC6cax7zXF6KVbHQhQ_chTxOOikzyTXJXJjv4odtxltbK5JFwsnv_z8nOacoXhK8YV1P3_mwoZiwDe367lFxTNqelLRi5HE-Y0pKxuofR8WzGK8xpi1u-qfF0Xrfsh4fF7-2aBdgtJN1IiwoplkvyDuUBkDKu5jCrJLNAW-Q9rMcAcXZKqsBXYEDpLN2D1m5B5V8iEguaIIITg3LJMZsCBNSMI4RCad_21qH9jYFnw-DlTarFgTGZH1cM3_5fPmdHSTPiydGjBFe3O4nxbfz91_PPpYXlx8-nW0vSlV1TSoFbRQzqsaKdsYQCZQKaholVd10VBrTVl2lW1CsJwTLylBJKJZagDC6M5qdFG8Ovrvgf84QE59sXCsQDvwcOal63FY9beuMvv4HvfZzcLk6TnpGm7bGTXdPXYkRuHXGpyDUasq3FanrhmY2U5v_UHlpmGz-fDA2xx8ITv8SDCDGNEQ_zmuD4kPw7QFUwccYwPBdsFNuMCeYr1PDfeDr1PB1ajL-6vZRs5xA38F_xuQ-cdzlLlrt4x3jQ8lIiWmJ246wG_nnyqM</recordid><startdate>20140201</startdate><enddate>20140201</enddate><creator>JIANG, JINGYAN</creator><creator>WEI, DEE</creator><creator>SUN, LI</creator><creator>WANG, YUXIA</creator><creator>WU, XIHAI</creator><creator>LI, YING</creator><creator>FANG, ZHENGHUI</creator><creator>SHANG, HUI</creator><creator>WEI, ZENGTAO</creator><general>D.A. Spandidos</general><general>Spandidos Publications</general><general>Spandidos Publications UK Ltd</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AN0</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope></search><sort><creationdate>20140201</creationdate><title>A preliminary study on the construction of double suicide gene delivery vectors by mesenchymal stem cells and the in vitro inhibitory effects on SKOV3 cells</title><author>JIANG, JINGYAN ; WEI, DEE ; SUN, LI ; WANG, YUXIA ; WU, XIHAI ; LI, YING ; FANG, ZHENGHUI ; SHANG, HUI ; WEI, ZENGTAO</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c486t-a26c3fc50c28ff1be22a2f6cbc5682bff7484d7ec39110b4f2b120bdaeafd8fd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Adenoviridae - genetics</topic><topic>Analysis</topic><topic>Antimetabolites - pharmacology</topic><topic>Base Sequence</topic><topic>Brain cancer</topic><topic>Cancer</topic><topic>Cancer therapies</topic><topic>Care and treatment</topic><topic>Cell Proliferation - drug effects</topic><topic>Cloning</topic><topic>Coculture Techniques</topic><topic>Cytosine Deaminase - biosynthesis</topic><topic>Cytosine Deaminase - genetics</topic><topic>Cytosine Deaminase - therapeutic use</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>Drug resistance</topic><topic>Enzymes</topic><topic>Female</topic><topic>Fetal Blood - cytology</topic><topic>Flow cytometry</topic><topic>Flucytosine - pharmacology</topic><topic>Gene expression</topic><topic>Gene therapy</topic><topic>Gene Transfer Techniques</topic><topic>Genes, Transgenic, Suicide - genetics</topic><topic>Genetic Therapy - methods</topic><topic>Genetic Vectors</topic><topic>Health aspects</topic><topic>Humans</topic><topic>Kinases</topic><topic>lentivector</topic><topic>mesenchymal stem cells</topic><topic>Mesenchymal Stromal Cells - cytology</topic><topic>Mesenchymal Stromal Cells - drug effects</topic><topic>Microscopy</topic><topic>Ovarian cancer</topic><topic>Ovarian Neoplasms - genetics</topic><topic>Ovarian Neoplasms - therapy</topic><topic>Plasmids</topic><topic>Prodrugs - therapeutic use</topic><topic>Recombinant Fusion Proteins</topic><topic>Sequence Analysis, DNA</topic><topic>SKOV3 cells</topic><topic>Stem cells</topic><topic>suicide cancer gene therapy</topic><topic>Thymidine Kinase - biosynthesis</topic><topic>Thymidine Kinase - genetics</topic><topic>Thymidine Kinase - therapeutic use</topic><topic>Tumors</topic><topic>Vectors (Biology)</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>JIANG, JINGYAN</creatorcontrib><creatorcontrib>WEI, DEE</creatorcontrib><creatorcontrib>SUN, LI</creatorcontrib><creatorcontrib>WANG, YUXIA</creatorcontrib><creatorcontrib>WU, XIHAI</creatorcontrib><creatorcontrib>LI, YING</creatorcontrib><creatorcontrib>FANG, ZHENGHUI</creatorcontrib><creatorcontrib>SHANG, HUI</creatorcontrib><creatorcontrib>WEI, ZENGTAO</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>British Nursing Database</collection><collection>ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><jtitle>Oncology reports</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>JIANG, JINGYAN</au><au>WEI, DEE</au><au>SUN, LI</au><au>WANG, YUXIA</au><au>WU, XIHAI</au><au>LI, YING</au><au>FANG, ZHENGHUI</au><au>SHANG, HUI</au><au>WEI, ZENGTAO</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A preliminary study on the construction of double suicide gene delivery vectors by mesenchymal stem cells and the in vitro inhibitory effects on SKOV3 cells</atitle><jtitle>Oncology reports</jtitle><addtitle>Oncol Rep</addtitle><date>2014-02-01</date><risdate>2014</risdate><volume>31</volume><issue>2</issue><spage>781</spage><epage>787</epage><pages>781-787</pages><issn>1021-335X</issn><eissn>1791-2431</eissn><abstract>The aim of the present study was to investigate the efficacy of using human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) as gene delivery vectors in the treatment of ovarian cancer. Lentivectors overexpressing cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-tk) (pGC-FU-CD-TK) were constructed, and confirmed by enzyme digestion, DNA sequence and western blotting. Quantitative PCR (PCR) was used to verify the overexpression of the fusion gene (CD and HSV-tk). SKOV3 cells were co-cultured with MSCs/tk+CD+ at a 1:1 ratio, and were then treated with the prodrugs (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the cytotoxic effects were evaluated using MTT assay and flow cytometry. DNA sequencing demonstrated that the sequence of HSV-tk and CD genes were consistent with the objective sequence and western blotting verified that the constructed lentivector could produce the HSV-tk/CD gene. The packed titer was 2.00e+8 TU/ml. The pGC-FU-CD-TK could be stably transferred to hUCB-MSCs, and the infection efficiency was almost 80%. RT-PCR demonstrated that the expression levels of the HSV-tk/CD fusion gene in MSCs/tk+CD+ group was 75 times that in the negative control (P<0.05). Compared with GCV or 5-FC alone, the growth inhibition rate (GIR) was significantly higher in the combined treatment (F=85.35, P<0.05). The reconstructed MSCs/tk+CD+ vectors were capable of slowing down the growth of human SKOV3 cells in the presence of prodrugs in vitro. The use of combination chemotherapy exhibited a more significant inhibitory effect than using a single prodrug.</abstract><cop>Greece</cop><pub>D.A. Spandidos</pub><pmid>24317390</pmid><doi>10.3892/or.2013.2898</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Oncology reports, 2014-02, Vol.31 (2), p.781-787 |
| issn | 1021-335X 1791-2431 |
| language | eng |
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| source | MEDLINE; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection |
| subjects | Adenoviridae - genetics Analysis Antimetabolites - pharmacology Base Sequence Brain cancer Cancer Cancer therapies Care and treatment Cell Proliferation - drug effects Cloning Coculture Techniques Cytosine Deaminase - biosynthesis Cytosine Deaminase - genetics Cytosine Deaminase - therapeutic use Deoxyribonucleic acid DNA Drug resistance Enzymes Female Fetal Blood - cytology Flow cytometry Flucytosine - pharmacology Gene expression Gene therapy Gene Transfer Techniques Genes, Transgenic, Suicide - genetics Genetic Therapy - methods Genetic Vectors Health aspects Humans Kinases lentivector mesenchymal stem cells Mesenchymal Stromal Cells - cytology Mesenchymal Stromal Cells - drug effects Microscopy Ovarian cancer Ovarian Neoplasms - genetics Ovarian Neoplasms - therapy Plasmids Prodrugs - therapeutic use Recombinant Fusion Proteins Sequence Analysis, DNA SKOV3 cells Stem cells suicide cancer gene therapy Thymidine Kinase - biosynthesis Thymidine Kinase - genetics Thymidine Kinase - therapeutic use Tumors Vectors (Biology) |
| title | A preliminary study on the construction of double suicide gene delivery vectors by mesenchymal stem cells and the in vitro inhibitory effects on SKOV3 cells |
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