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Objective To knockout the MATP gene of mouse melanoma cell line B16F10 using CRISPR/Cas9 system,and to lay foundation for the functional study of MATP gene.Methods Specific primers of MATP were designed according to the report in http://crispr.mit.edu/ website.The primers were linked to pCAS9/gRNA1 vector.Then the positive vector was transfected into mouse melanoma B16F10 cells,and monoclonal cell lines were obtained by the infinite dilution method.After the genomes of different monoclonal cell lines were extracted and sequenced,the cell lines with MATP gene cleavage were screened,and the expression of MATP in these cell lines was verified by Western-blot analysis.Results Three MATP gene knockout cell lines were successfully obtained.The western-blot results showed that the cell lines did not express MATP protein.Conclusions The knockout of MATP gene in B16F10 cell line can be successfully achieved using the pCAS9/gRNA1 vector.
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BACKGROUND:None of the current treatment strategies has been focused on relieving or reversing the disk degeneration process after degenerative disc diseases. In recent years, more and more scientists try to treat degenerative disc diseases using stem cel therapy. OBJECTIVE:To explore the research status and prospects of stem cel therapy for degenerative disc diseases. METHODS: A computer-based online search of PubMed database between January 2004 and December 2014was performed to search related articles with the key words of “stem cel, intervertebral disk” in English. Literatures related to stem cel therapy for degenerative disc diseases were selected; in the same field, the articles published lately in authoritative journals were preferred. RESULTS AND CONCLUSION: A total of 342 articles were primarily selected, and 43 articles were involved in result analysis according to inclusion criteria. Stem cel therapy is a newly treatment for degenerative disc diseases. Cels appropriate for stem cel therapy include embryonic stem cels, induced pluripotent stem cels, mesenchymal stem cels, human umbilical cord mesenchymal stem cels and chondrocytes or nucleus pulposus cels. Although cel leakage, intervertebral disc infection and tumorigenesis are the main chalenges, stem cel therapy for degenerative disc diseases is promising in the future.
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BACKGROUND:At present, the clinical treatment of lumbar disc degeneration mainly includes conservative treatment, traditional surgery and minimal y invasive surgery. The therapeutic purpose is to relieve symptoms, but the long-term effect is not very satisfactory. Therapeutic methods focusing on biological functional recovery have been concerned gradual y, but the clinical application is far in sight. OBJECTIVE:To review the advances in the treatment of lumbar disc degeneration regarding tissue-engineered repair and biomechanics. METHODS:PubMed database was searched by the first author for relevant articles published before December 2014 using the keywords of“intervertebral disc degeneration, clinical treatment, biological treatment, tissue engineering, biomechanics, repair, progress”in English. A total of 100 articles were searched initial y and final y, 40 articles were included in result analysis. RESULTS AND CONCLUSION:Although the therapeutic schemes are varied, the treatment of intervertebral disc degeneration is a great chal enge for clinicians and basic researchers. Currently there is no perfect clinical treatment, and indications corresponding to various therapies should be paid attention as wel as long-term fol ow-up evaluation. For various reasons, the biological treatment for intervertebral disc degenerative disease is becoming more and more popular, providing a promising prospect for the treatment of intervertebral disc degeneration. So far, large amounts of data have been obtained from animal experiments, but there are stil many problems to be solved. Other chal enges also involve the al aspects of general tissue engineering methods, such as cel s, cytokines and scaffolds. In these studies, the nucleus pulposus tissue engineering based on the combination of heparin-functionalized chitosan hydrogel, cytokines and stem cel s exhibits a promising prospect.