ABSTRACT
BACKGROUND:Telomerase reverse transcriptase (TERT) plays an important role in telomerase activation, however there is rare report addressing the construction of the lentivirus targeted its genes to inhibit its expression in the spinal cord astrocytes. OBJECTIVE:To construct recombinant lentivirus vector expressing smal interfering RNA against TERT gene and to evaluate its potential for inhibiting the TERT expression. METHODS:After shRNA-TERT sequence was designed and synthesized, the sequence was amplified by PCR and then connected to plasmid pLentilox3.7U6-hTERT to construct recombinant plasmid. The recombinant plasmid was then transfected to DH5αcel s to screen positive colony, and the sequence was identified. The recombinant plasmid pLentilox3.7U6-TERT was transfected in 293T cel s, generating recombinant lentivirus Le-TERT. The titer of recombinant lentivirus was determined and Le-TERT was transfected into the rat spinal cord astrocytes. The expression of TERT in astrocytes was detected by RT-PCR, western blot and immunofluorescence assay. RESULTS AND CONCLUSION:The gene sequencing analysis confirmed that, recombinant plasmid pLentilox3.7U6-TERT was successful y constructed. The real-time quantitative PCR, western blot analysis and immunofluorescence assay indicated that, after Le-TERT was transfected in the astrocytes for 4 days, the inhibition rate of TERT mRNA was (63.98±2.6)%, and Le-TERT was lowly expressed in the transfected astrocytes. Recombinant expression vector pLentilox3.7U6-TERT can produce the lentivirus at high titer and effectively inhibit TERT expression in the transfected astrocytes.
ABSTRACT
BACKGROUND:Sensorineural hearing loss is mainly caused by missing or damaged hair cells in the inner ear. Application of adipose-derived mesenchymal stem cells to regenerate inner ear hair cells is an effective treatment for hearing loss. OBJECTIVE:To explore the feasibility of in vitro inducing adipose-derived mesenchymal stem cells to differentiate into inner ear hair cel-like cells in guinea pigs. METHODS:Adipose-derived mesenchymal stem cells from guinea pigs were isolated and cultured to the 3rd generation. cellphenotype was detected using flow cytometry. Cytokines were added for induction and differentiation by stages, including epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor-1, al-trans retinoic acid, brain-derived neurotrophic factor, neurotrophin 3. RESULTS AND CONCLUSION:Adipose-derived mesenchymal stem cells of guinea pigs cultured in vitro were fusiform and showed a swirled adherent growth. Passage 3 cells were positive for CDCD29 and CD44, but negative for CD34 and CD45. After induction, the cells were positive for nestin and GFAP positive at early stage;after 10-day continuous induction, the cells expressed Myosin VIIa and Math1, specific markers of hair cells, indicating that cytokines can directly induce adipose-derived mesenchymal stem cells to differentiating into inner ear hair cells in guinea pigs.
ABSTRACT
BACKGROUND:Adipose-derived mesenchymal stem cels are a group of pluripotent stem cels, and under certain conditions can differentiate into neural stem celsin vitro. OBJECTIVE:To investigate the proliferative and differentiation ability of different passage mesenchymal stem cellfrom adipose tissue into neurospheres. METHODS:The adipose-derived mesenchymal stem cels from Sprague-Dawley rats were separated and culturedin vitro, and morphology and proliferation rate of cels were observed and compared respectively at passages 3, 6, 10 and 20. The cellsurface antigens and cels cycle were identified by flow cytometry. Furthermore, adipose-derived mesenchymal stem cels were induced into neurospheres, and the neurosphere rate was counted. RESULTS AND CONCLUSION: Adipose-derived mesenchymal stem cels were mainly in long spindle shape, and cels at different passages had highly proliferative capacity in vitro. Except passage 3, adipose-derived mesenchymal stem cels strongly expressed CD29, CD44, CD73 and lowly expressed CD45 and CD34. The proportion of G0/G1 phase in cellcycle was 93.4% at passage 3, 92.7% at passage 6, 92.4% at passage 10, 86.0% at passage 20. Adipose-derived mesenchymal stem cels at passages 6 and 10 were easier to differentiate into neurospheres than those at passage 20 (P < 0.05), but cels at passage 3 were difficult to differentiate into neurospheres. Therefore, when using adipose-derived mesenchymal stem cels as seed cels, we should pay attention to choose the appropriate amplification passage in order to obtain the cels with best differentiation potential and cellpurity.
ABSTRACT
BACKGROUND:A large number of studies have confirmed that anterior approach and posterior approach for multilevel cervical spondylotic myelopathy were effective, but there is stil no conclusion in which one is better. OBJECTIVE:To systematical y assess the clinical effectiveness and safety of anterior approach versus posterior approach for multilevel cervical spondylotic myelopathy. METHODS:The databases such as The Cochrane Library (Issue 3, 2013), PubMed (from 1966 to March 2013), OVID (from 1950 to March 2013), EMbase (from 1966 to March 2013), Chinese Biomedical Literature Database (from 1978 to March 2013), WanFang Database (from 1998 to March 2013), China National Knowledge Infrastructure (from 1999 to March 2013) were electronical y searched and five relevant journals were searched by hand to col ect the randomized control ed trials or non-randomized control ed trials about the clinical effectiveness and safety of anterior approach versus posterior approach for multilevel cervical spondylotic myelopathy. Two reviewers independently screened the literature according to the inclusive and exclusive criteria, extracted the data, and assessed the methodological quality of included studies. Then the meta-analysis was performed by using RevMan5.2 software. RESULTS AND CONCLUSION:A total of 11 control ed trials involving 814 patients were included. Meta-analysis results showed that, compared with posterior approach, postoperative Japanese Orthopaedic Association scores were better (P<0.000 01), improvement rate of neurological function was higher (P=0.000 3), the incidence of C5 root palsy was lower (P=0.007), but operation time was longer (P<0.000 01), amount of intraoperative bleedin g was larger (P=0.000 7), incidence of adjacent segments degeneration was higher (P=0.01), incidence of postoperative complications was higher (P<0.000 01) and the rate of secondary surgical procedures was higher (P=0.003) after anterior approach. Additional y, there were no differences between the two groups in the cervical range of motion (P=0.56). For quantity limitation and low methodological quality of included studies, this conclusion stil needs to be further proved by performing more high-quality and large-scale randomized control ed trials.