Effect of p65 gene inhibited by siRNA on differention of rat marrow mesenchymal stem cells into neurons / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effect of p65 gene inhibited by siRNA on neuronic differentiation in the marrow mesenchymal stem cells (MSCs).</p><p><b>METHODS</b>The MSCs were transfected with Rn-p65-siRNA. Fasudil hydrochloride induced MSCs differentiating into neurons. The non-transfected group and negative control group (transfected with negative control siRNA marked by Cy3) were used as controls. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope at 24 h,48 h and 72 h after transfected with negative control siRNA. The viability of MSCs was detected by MTT at 24 h, 48 h and 72 h after transfected with Rn-p65-siRNA. The expressions of p65 mRNA and protein in MSCs were detected by RT-PCR and Western blot respectively. The expressions of p65 protein, NSE, MAP-2 and glial fibrillary acidic protein (GFAP) were detected by immunocytochemical method after transfection for 6 h.</p><p><b>RESULTS</b>The fluorescence of MSCs was mostly displayed after transfection of 72 hours and the efficiency of transfection was up to 83.3% ± 3.8%. Meanwhile, the p65 mRNA and p65 protein expressed by MSCs of transfected group were significantly decreased (P < 0.05); MTT displayed that the viability of MSCs was also significantly reduced (P < 0.05). The best efficiency of induction was observed in the transfected group. There were higher expressions of NSE and MAP-2 than the other group (P < 0.05).</p><p><b>CONCLUSION</b>The p65 gene inhibited by siRNA can promote the marrow mesenchymal stem cells to differentiate into neurons.</p>

The effect of small ubiquitin-like modifier-1 modification on the formation of Lewy body-like inclusions in cytoplasm and apoptosis of HEK293 cell induced by overexpression and mutation of alpha-synuclein / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the effect of small ubiquitin-like modifier (SUMO-1) modification on the formation of Lewy body like inclusions in cytoplasm and apoptosis of HEK293 cell induced by overexpression and mutation of alpha-synuclein.</p><p><b>METHODS</b>cDNA encoding the human alpha-synuclein without the stop codon was cloned into a pGEM T-easy vector. Restriction enzyme mapping and DNA sequencing were performed to analyze the plasmid, which was then subcloned into a pEGFP-N1 vector. The recombinant plasmid alpha-synuclein-pEGFP was transfected into HEK293 cells by lipofectamin method. Inclusions in the cultured cells were identified with HE staining. Apoptosis of the HEK293 cell was measured by Hoechst 33258 staining, MTT and Annexin V-PE flow cytometry.</p><p><b>RESULTS</b>The Lewy-body like inclusions were found in cytoplasm of cultured cells. Hoechst staining showed that the nuclei of cells were enlarged in the wild-type and A53T mutation groups 48 h after transfection, chromatin were accumulated and appeared spot-like. The nucleus stain was equitable in the K96R and K96R-A53T groups. MTT assay showed that the viability of cells transfected with empty plasmid was 96.2%, but it dropped to 53.4% and 56.1% in cells transfected with wild-type alpha-synuclein-pEGFP and A53T mutant group, respectively. The viability was 72.3% and 69.8% in cells transfected with K96R and K96R-A53T, respectively (P<0.05). Forty eight hours after transfection, the apoptosis rate was 3.9% in empty plasmid group, 32.2% and 34.1% in cells transfected with wild-type and mutant alpha-synuclein-pEGFP, 19.4% and 20.3% in the K96R and K96R-A53T transfected cells. There was significant difference between the two groups (P<0.05).</p><p><b>CONCLUSION</b>SUMO-1 modification did not have influence on the Lewy body-like inclusions formation in cytoplasm of HEK293 cell in vitro, but had a toxic effect which could increase the apoptosis induced by wild type overexpression and mutation of alpha-synuclein.</p>

Effect of SUMO-1 on mitochondria subcellular localization of alpha-synuclein and its degradation via ubiquitin-proteasome system / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the effect of sumoylation of alpha-synuclein by SUMO-1 on the mitochondria subcellular localization of alpha-synuclein and its degradation via ubiquitin-proteasome system.</p><p><b>METHODS</b>Primers of wild-type, A53T pathogenic mutant and K96R mutant of human alpha-synuclein were designed to amplify the corresponding cDNAs without stop codon. The cDNAs were cloned into pGEM T-easy vector, analyzed by using enzyme mapping and DNA sequencing, and subcloned into pEGFP-N1 vector. The recombinant plasmids of pEGFP-alpha-synuclein-WT, pEGFP-alpha-synuclein-A53T and pEGFP-alpha-synuclein-K96R were transfected into HEK293 cells by lipofectamine method. The expression of the alpha-synuclein protein was measured by immunofluorescence and confocal microscope. Then mitochondria staining as well as immunofluorescence were utilized to investigate the effect of wild-type, A53T mutant and sumoylation of alpha-synuclein on mitochondria subcellular localization of alpha-synuclein. The effect of sumoylation of alpha-synuclein on its degradation via the ubiquitin-proteasome system in the cells was assayed by Western-blot.</p><p><b>RESULTS</b>The enzyme mapping suggested that the eukaryotic expression plasmids for human wild-type, A53T and K96R mutants of the alpha-synuclein gene were constructed successfully. By immunofluorescence and confocal microscope, it was observed that alpha-synuclein-WT and alpha-synuclein-A53T proteins aggregated in cytoplasm, and alpha-synuclein-K96R protein aggregation was decreased in cytoplasm of cultured cells. The alpha-synuclein proteins of wild-type, A53T and K96R mutants were co-localized with mitochondria. Western-blot analysis revealed that both wild-type and A53T mutant affected the amount of the ubiquitinated proteins.</p><p><b>CONCLUSION</b>Neither overexpression of wild-type and A53T pathogenic mutant alpha-synuclein, nor sumoylation of alpha-synuclein, affected the subcellular localization in the mitochondria. However, overexpression of wild-type and A53T mutant alpha-synuclein affected the amount of the ubiquitinated proteins.</p>