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>

Clinical and neuroimaging features of enterovirus71 related acute flaccid paralysis in patients with hand-foot-mouth disease

OBJECTIVE@#To investigate clinical and neuroimaging features of enterovirus71 (EV71) related acute flaccid paralysis in patients with hand-foot-mouth disease.@*METHODS@#Nine patients with acute flaccid paralysis met the criterion of EV71 induced hand-foot-mouth disease underwent spinal and brain MR imaging from May 2008 to Sep 2012.@*RESULTS@#One extremity flaccid was found in four cases (3 with lower limb, 1 with upper limb), two limbs flaccid in three cases (2 with lower limbs, 1 with upper limbs), and four limbs flaccid in two cases. Spinal MRI studies showed lesion with high signal in T2-weighted images (T2WI) and low signal T1-weighted images (T1WI) in the spinal cord of all nine cases, and the lesions were mainly in bilateral and unilateral anterior horn of cervical spinal cord and spinal cord below thoracic 9 (T9) level. In addition, the midbrain, pons, and medulla, which were involved in 3 cases with brainstem encephalitis, demonstrated abnormal signal. Moreover, spinal cord contrast MRI studies showed mild enhancement in corresponding anterior horn of the involved side, and strong enhancement in its ventral root.@*CONCLUSIONS@#EV71 related acute flaccid paralysis in patients with hand-foot-mouth disease mainly affected the anterior horn regions and ventral root of cervical spinal cord and spinal cord below T9 level. MR imaging could efficiently show the characteristic pattern and extent of the lesions which correlated well with the clinical features.

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>

Effect of notch signaling on differentiation of rat marrow mesenchymal stem cells into neurons induced by fasudil hydrochloride / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effect of notch signaling on differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons induced by fasudil hydrochloride.</p><p><b>METHODS</b>The experiments were divided into non-transfected group, transfected group (transfected with Rn-Notch1-siRNA), positive control group (transfected with Rn-MAPK-1 Control siRNA) and negative control group (transfected with negative control siRNA). Fasudil hydrochloride induced MSCs differentiating into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The expression of notch1 mRNA, Hes1 mRNA and MAPK1 mRNA in MSCs was detected by RT-PCR. The expression of Notch1 protein, NSE, neurofilament M (NF-M) and glial fibrillary acidic protein(GFAP)was detected by immunocytochemical method. The viability of MSCs was detected by MTT.</p><p><b>RESULTS</b>(1) The fluorescence of MSCs was mostly displayed after transfection for 72 h and the efficiency of transfection was up to 91.3% +/- 4.2%. Meanwhile, the notch1 mRNA and Hes1 mRNA expressed by MSCs of transfected group were significantly decreased (P < 0.05) and MTT displayed that the viability of MSCs was also significantly reduced (P < 0.05). (2) Fasudil hydrochloride could induce MSCs differentiate into neurons and the best efficiency of induction was observed in the transfected group. There was higher expression of NSE and neurofilament-M (NF-M) than the other groups (P < 0.05).</p><p><b>CONCLUSION</b>There may be notch1 signaling and Rho/Rho GTPase signaling synergy on differentiation of rat bone marrow stromal cell into neurons induced by fasudil hydrochloride and they jointly promote the differentiation of MSCs into neurons.</p>

Diagnostic value of diffusion tensor magnetic resonance imaging in patients with cerebral infarction / 中国脑血管病杂志

Objective: To analyze the characteristics of diffusion tensor magnetic resonance imaging in patients with cerebral infarction and to explore the values of diagnosis and predicting prognosis of diffusion tensor imaging (DTI) in patients with cerebral infarction in different stages. Methods: Forty patients with cerebral infarction in different stages and 40 healthy volunteers were examined by magnetic resonance imaging (MRI), including conventional T1 and T2-weighted imaging, diffusion-weighted imaging and DTI. Fractional anisotropy (FA) images were reconstructed. The values of FA and apparent diffusion coefficient (ADC) were measured in the infarcted regions, corresponding contralateral normal regions and corresponding normal regions in normal control group. Results: DTI showed that the size of infarction foci was more accurate and clearer than that of the conventional MRI. The FA values of the infarcted regions, infarcted ipsilateral posterior limb of internal capsule, cerebral peduncle, and corticospinal tract in cerebral infarction group were 0.12 ± 0.01, 0.29 ± 0.03, 0.36 ± 0.12 and 0.35 ± 0.04, respectively. They were lower than those in the contralateral corresponding regions 0.35 ± 0.08, 0.50 ± 0.13, 0.53 ± 0.14 and 0.56 ± 0.07, and they all had significant differences (P 0.05). The FA and ADC values in brain tissues changed regularly with the time of infarction after cerebral infarction. The FA values in the affected sides had no consistent changes as compared with the contralateral sides in the superacute phase. They increased or decreased slightly, then (during acute stage, subacute stage and chronic stage) decreased irreversibly; the ADC values in the affected sides changed with time regularly; they decreased significantly; then gradually returned to normal, and after that increased again. Conclusion: DTI examination contributes to the diagnosis of cerebral infarction. The combination of the ADC and FA values may more accurately conduct clinical stage and evaluate the time of the occurrence of cerebral infarction.

A related analysis for alpha, beta fibrinogen gene haplotypes and nucleotide polymorphisms associated with the ischemic stroke in Hainan Han population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the association of that the polymorphisms and haplotypes of Taq I site in beta fibrinogen gene and the single nucleotide sites -455 G/A, -249 C/T, -148 C/T, +1689T/G, Bsm A I G/C, 448 G/A, Bcl I G/A, Hinf I A/C in beta-fibrinogen gene are linked up with the ischemic stroke(IS).</p><p><b>METHODS</b>Turbidmetric assay was used to measure the plasma fibrinogen level of one hundred and sixty cases with ischemic stroke and one hundred and thirty healthy individuals from Hainanese Han population. The polymorphisms and genotypes were characterized by PCR-RFLP. Hardy-Weinberg equilibrium and statistical differences of allelic, genotype and haplotype frequencies were obtained by Chi-square test. Pairwise linkage disequilibrium was calculated and haplotypes of nine or four polymorphisms were estimated by the EH + program.</p><p><b>RESULTS</b>There were highly significant differences in genotype frequencies and allelic frequencies of the polymorphisms -455 G/A, -148 C/T, 448 G/A, which happened between the IS group and control subjects (P< 0.01). However, the significant differences of the allelic frequencies in the other six polymorphisms were not found between the IS group and the control (P> 0.05). The odds ratio(OR) with the rare alleles of A -455, T -148 and A 448 is 2.46, 2.30 and 2.08 (95% confidence interval 1.153%-3.924%, 1.429%-3.694% and 1.298%-3.329%) respectively. No definite haplotype block was found by linkage disequilibrium analysis in the control group and the IS group. Association of haplotypes constructed from the nine polymorphisms with IS was not found. Among the haplotypes constructed from four polymorphisms including -455 G/A, -148 C/T, 448 G/A alleles, haplotype differences were found between the control group and the IS group. Haplotypes with G -455, C -148, G448 alleles appeared more frequently in control group(P< or = 0.01), whereas haplotypes with A -455, T -148, A 448 occurred more frequently in the IS group(P< 0.01).</p><p><b>CONCLUSION</b>The results of multi-allele and haplotype analysis indicated that the polymorphisms -455 G/A, -148 C/T, 448 G/A in beta fibrinogen gene were the possible risk factors associated with the occurrence of ischemic stroke in Hainan Han population.</p>

Clinical application of diffusion tensor imaging in cognitive impairment of patients with acute cerebral infarction / 中华神经科杂志

Objective To evaluate the value of diffusion tensor imaging(DTI)in cognitive impairment of patients with acute cerebral infarction.Methods Diffusion tensor images were obtained from 30 volunteers who underwent clinical MR imaging and were found to have no abnormalities on conventional MR images and 30 patients who were clinically diagnosed cerebral infarction and were found to have infarction lesions on conventional MR images.Color-coded FA images and three-dimensional color-coded tensor images were reconstructed.For volunteers,average apparent diffusion coefficient(ADC)and fractional anisotropy(FA)were measured in some main white matter structures of peripheral white matter, basal ganglia,and cerebral peduncle,etc.For infarction patients,ADC and FA were measured and compared between infarction lesions and corresponding contralateral normal regions.Pearson correlation analysis was used to determine correlation with cognitive impairment.Results In infarction patients group, FA and ADC of lesions unrecovered declined.Change in ADC and FA had positive correlation with cognitive impairment of patients with acute cerebral infarction.Conclusion DTI has positive correlation with cognitive impairment of patients with acute cerebral infarction.