BACKGROUND： Studies have shown that the transplantation of mesenchymal stem cells （MSCs） improves dystrophin expression in muscle cell membrane of mdx mice and plays a role in ameliorating sport injuries to the myo...BACKGROUND： Studies have shown that the transplantation of mesenchymal stem cells （MSCs） improves dystrophin expression in muscle cell membrane of mdx mice and plays a role in ameliorating sport injuries to the myocyte. In addition, dystrophin gene plasmid injection exhibits therapeutic effect in mdx mice. However, these two methods exhibit shortcomings, such as low rate of post-transplantation expression. Therefore, the present study determined the combinatorial effects of these two methods. OBJECTIVE： To transfect and observe effects of pSL139 plasmid carrying the micro-dystrophin gene into MSCs, as well as in vitro micro-dystrophin gene expression in transfected MSCs. DESIGN, TIME AND SETTING： A comparative, molecular biology study was performed at the Laboratory of Tissue Engineering, West China Medical Center, Sichuan University from March 2007 to February 2008. MATERIALS： The pSL139 plasmid was cloned and provided by the Department of Neurology, Washington University, USA. Lipofectamine 2000 was purchased from Invitrogen, USA. Mouse anti-human dystrophin N-based terminal monoclonal antibody was purchased from Chemicon, USA. METHODS： Differential velocity adherent technique and density gradient centrifugation were combined to separate and culture MSCs from C57/BL10 mice. The cells were induced to trans-differentiate into osteoblasts. Subsequently, the Lipofectamine 2000 method was used to mediate transfection of plasmid pSL139 into third generation MSCs. MAIN OUTCOME MEASURES： Semi-quantitative reverse transcription polymerase-chain reaction and immunofluorescence were respectively employed to detect micro-dystrophin mRNA and protein expressions in MSCs. RESULTS： At 48 hours after MSC transfection with plasmid pSL139, a 379-kb target band was observed by agarose gel electrophoresis. Immunofluorescence revealed micro-dystrophin expression up to 45%-55%. CONCLUSION： Micro-dystrophin mRNA and protein were highly expressed in pSL139-transfected MSCs, which provided a method for efficient expression of dystrophin.展开更多
The restriction fragment length polymorphisms distribution and frequency of dystrophin gene in Chinese were studied by using 14 subclones of the entire 14kb cDNA for the dystrophin as hybridization probes. Allelic fra...The restriction fragment length polymorphisms distribution and frequency of dystrophin gene in Chinese were studied by using 14 subclones of the entire 14kb cDNA for the dystrophin as hybridization probes. Allelic fragments were detected in hybridization patterns of PvuII/1a, Taq I/2b-3, Taq I/5b-7, and Xba I/10. Among them, the allelic fragments (26kb and 3.8kb) in PvuII/2b-3 pattern and the allelic fragments (10.0kb and 8.4kb) in Taq I/5b-7 patterns had never been reported previously. Compared with the data from Caucasians and Japanese, it indicated that there was a significant difference (P<0.01) of the allelic fragment frequency in Taq I/2b-3 and Xba I/10 patterns between Chinese and Caucasians. The frequencies of allelic fragments A2 (5.6kb) in Taq I/8 and A2 (10.7kb) in EcoR V/9 were high in Caucasians, yet had not been detected in Chinese, the differences were also highly significant. But in Chinese and Caucasians, the B1B2 allelic frequencies in Taq I/5b-7 are the same. As to the frequency of the allelic fragments A1A2 and B1B2 in Pvu II/1a, there was no significant difference between Chinese and Japanese.展开更多
Objective To study the human dystrophin gene molecular deletion mechanism, we analyzed breakpoint regions within junction fragments of deletion-type patients and investigated whether the dystrophin gene's intron s...Objective To study the human dystrophin gene molecular deletion mechanism, we analyzed breakpoint regions within junction fragments of deletion-type patients and investigated whether the dystrophin gene's intron structure might be related to intron instability.Methods Junction fragments corresponding to exon 46 and 51 deletions were cloned. The breakpoint regions were sequenced, and the features of introns with available Genebank sequences were analyzed.Results An analysis of junction fragment sequences corresponding to exon 46 and 51 deletions showed that all 5' and 3' breakpoints are located within repeat sequences. No small insertions, small deletions, or point mutations are located near the breakpoint junctions. By analyzing the secondary structure of the junction fragments, we demonstrated that all junction fragment breakpoints are located in non-matching regions of single-stranded hairpin loops. A high concentration of repetitive elements is found to be a key feature of many dystrophin introns. In total, 34. 8% of the overall dystrophin intron sequences is composed of repeat sequences.Conclusion Repeat elements in many dystrophin gene introns are the key to their structural bases and reflect intron instability. As a result of the primary DNA sequences, single-stranded hairpin loops form, increasing the instability of the gene, and forming the base for breaks in the DNA. The formation of the single-stranded hairpins can result in reattachment of two different breakpoints, producing a deletion.展开更多
OBJECTIVE： To identify global research trends in stem cell transplantation for treating Duchenne muscular dystrophy using a bibliometric analysis of Web of Science. DATA RETRIEVAL： We performed a bibliometric analys...OBJECTIVE： To identify global research trends in stem cell transplantation for treating Duchenne muscular dystrophy using a bibliometric analysis of Web of Science. DATA RETRIEVAL： We performed a bibliometric analysis of studies on stem cell transplantation for treating Duchenne muscular dystrophy from 2002 to 2011 retrieved from Web of Science. SELECTION CRITERIA： Inclusion criteria： （a） peer-reviewed published articles on stem cell transplantation for treating Duchenne muscular dystrophy indexed in Web of Science; （b） original research articles, reviews, meeting abstracts, proceedings papers, book chapters, editorial material, and news items; and （c） publication between 2002 and 2011. Exclusion criteria： （a） articles that required manual searching or telephone access; （b） documents that were not published in the public domain; and （c） corrected papers. MAIN OUTCOME MEASURES： （1）Annual publication output; （2） distribution according to subject areas; （3） distribution according to journals; （4） distribution according to country; （5） distribution according to institution; （6） distribution according to institution in China; （7） distribution according to institution that cooperated with Chinese institutions; （8） top-cited articles from 2002 to 2006; （9） top-cited articles from 2007 to 2011. RESULTS： A total of 318 publications on stem cell transplantation for treating Duchenne muscular dystrophy were retrieved from Web of Science from 2002 to 2011, of which almost half derived from American authors and institutes. The number of publications has gradually increased over the past 10 years. Most papers appeared in journals with a focus on gene and molecular research, such as Molecular Therapy, Neuromuscular Disorders, and PLoS One. The 10 most-cited papers from 2002 to 2006 were mostly about different kinds of stem cell transplantation for muscle regeneration, while the 10 most-cited papers from 2007 to 2011 were mostly about new techniques of stem cell transplantation for treating Duchenne muscular dystrophy. CONCLUSION： The publications on stem cell transplantation for treating Duchenne muscular dystrophy were relatively few. It also needs more research to confirm that stem cell therapy is a reliable treatment for Duchenne muscular dystrophy.展开更多
基金the National Natural Science Foundation of China for Young Scholars, No. 0040205401040
文摘BACKGROUND： Studies have shown that the transplantation of mesenchymal stem cells （MSCs） improves dystrophin expression in muscle cell membrane of mdx mice and plays a role in ameliorating sport injuries to the myocyte. In addition, dystrophin gene plasmid injection exhibits therapeutic effect in mdx mice. However, these two methods exhibit shortcomings, such as low rate of post-transplantation expression. Therefore, the present study determined the combinatorial effects of these two methods. OBJECTIVE： To transfect and observe effects of pSL139 plasmid carrying the micro-dystrophin gene into MSCs, as well as in vitro micro-dystrophin gene expression in transfected MSCs. DESIGN, TIME AND SETTING： A comparative, molecular biology study was performed at the Laboratory of Tissue Engineering, West China Medical Center, Sichuan University from March 2007 to February 2008. MATERIALS： The pSL139 plasmid was cloned and provided by the Department of Neurology, Washington University, USA. Lipofectamine 2000 was purchased from Invitrogen, USA. Mouse anti-human dystrophin N-based terminal monoclonal antibody was purchased from Chemicon, USA. METHODS： Differential velocity adherent technique and density gradient centrifugation were combined to separate and culture MSCs from C57/BL10 mice. The cells were induced to trans-differentiate into osteoblasts. Subsequently, the Lipofectamine 2000 method was used to mediate transfection of plasmid pSL139 into third generation MSCs. MAIN OUTCOME MEASURES： Semi-quantitative reverse transcription polymerase-chain reaction and immunofluorescence were respectively employed to detect micro-dystrophin mRNA and protein expressions in MSCs. RESULTS： At 48 hours after MSC transfection with plasmid pSL139, a 379-kb target band was observed by agarose gel electrophoresis. Immunofluorescence revealed micro-dystrophin expression up to 45%-55%. CONCLUSION： Micro-dystrophin mRNA and protein were highly expressed in pSL139-transfected MSCs, which provided a method for efficient expression of dystrophin.
文摘The restriction fragment length polymorphisms distribution and frequency of dystrophin gene in Chinese were studied by using 14 subclones of the entire 14kb cDNA for the dystrophin as hybridization probes. Allelic fragments were detected in hybridization patterns of PvuII/1a, Taq I/2b-3, Taq I/5b-7, and Xba I/10. Among them, the allelic fragments (26kb and 3.8kb) in PvuII/2b-3 pattern and the allelic fragments (10.0kb and 8.4kb) in Taq I/5b-7 patterns had never been reported previously. Compared with the data from Caucasians and Japanese, it indicated that there was a significant difference (P<0.01) of the allelic fragment frequency in Taq I/2b-3 and Xba I/10 patterns between Chinese and Caucasians. The frequencies of allelic fragments A2 (5.6kb) in Taq I/8 and A2 (10.7kb) in EcoR V/9 were high in Caucasians, yet had not been detected in Chinese, the differences were also highly significant. But in Chinese and Caucasians, the B1B2 allelic frequencies in Taq I/5b-7 are the same. As to the frequency of the allelic fragments A1A2 and B1B2 in Pvu II/1a, there was no significant difference between Chinese and Japanese.
基金the National Natural Science Foundation of China ( Nos. 39700048 and 30271378) the Guangdong Natural Science Foundation (Nos. 980066, 021866 and 974151)+1 种基金and as a key project of the Guangdong Provincial Committee on Science Technology (No. 2002C306
文摘Objective To study the human dystrophin gene molecular deletion mechanism, we analyzed breakpoint regions within junction fragments of deletion-type patients and investigated whether the dystrophin gene's intron structure might be related to intron instability.Methods Junction fragments corresponding to exon 46 and 51 deletions were cloned. The breakpoint regions were sequenced, and the features of introns with available Genebank sequences were analyzed.Results An analysis of junction fragment sequences corresponding to exon 46 and 51 deletions showed that all 5' and 3' breakpoints are located within repeat sequences. No small insertions, small deletions, or point mutations are located near the breakpoint junctions. By analyzing the secondary structure of the junction fragments, we demonstrated that all junction fragment breakpoints are located in non-matching regions of single-stranded hairpin loops. A high concentration of repetitive elements is found to be a key feature of many dystrophin introns. In total, 34. 8% of the overall dystrophin intron sequences is composed of repeat sequences.Conclusion Repeat elements in many dystrophin gene introns are the key to their structural bases and reflect intron instability. As a result of the primary DNA sequences, single-stranded hairpin loops form, increasing the instability of the gene, and forming the base for breaks in the DNA. The formation of the single-stranded hairpins can result in reattachment of two different breakpoints, producing a deletion.
基金supported by the Key TechnologiesR & D Program of Liaoning Province,No.2008225009.
文摘OBJECTIVE： To identify global research trends in stem cell transplantation for treating Duchenne muscular dystrophy using a bibliometric analysis of Web of Science. DATA RETRIEVAL： We performed a bibliometric analysis of studies on stem cell transplantation for treating Duchenne muscular dystrophy from 2002 to 2011 retrieved from Web of Science. SELECTION CRITERIA： Inclusion criteria： （a） peer-reviewed published articles on stem cell transplantation for treating Duchenne muscular dystrophy indexed in Web of Science; （b） original research articles, reviews, meeting abstracts, proceedings papers, book chapters, editorial material, and news items; and （c） publication between 2002 and 2011. Exclusion criteria： （a） articles that required manual searching or telephone access; （b） documents that were not published in the public domain; and （c） corrected papers. MAIN OUTCOME MEASURES： （1）Annual publication output; （2） distribution according to subject areas; （3） distribution according to journals; （4） distribution according to country; （5） distribution according to institution; （6） distribution according to institution in China; （7） distribution according to institution that cooperated with Chinese institutions; （8） top-cited articles from 2002 to 2006; （9） top-cited articles from 2007 to 2011. RESULTS： A total of 318 publications on stem cell transplantation for treating Duchenne muscular dystrophy were retrieved from Web of Science from 2002 to 2011, of which almost half derived from American authors and institutes. The number of publications has gradually increased over the past 10 years. Most papers appeared in journals with a focus on gene and molecular research, such as Molecular Therapy, Neuromuscular Disorders, and PLoS One. The 10 most-cited papers from 2002 to 2006 were mostly about different kinds of stem cell transplantation for muscle regeneration, while the 10 most-cited papers from 2007 to 2011 were mostly about new techniques of stem cell transplantation for treating Duchenne muscular dystrophy. CONCLUSION： The publications on stem cell transplantation for treating Duchenne muscular dystrophy were relatively few. It also needs more research to confirm that stem cell therapy is a reliable treatment for Duchenne muscular dystrophy.
