Capsaicin inhibits experimental autoimmune neuritis in rats through inhibition of autophagy activity / 中国药理学通报

Aim To observe the effect of capsaicin on the experimental autoimmune neuritis (EAN) in rats and explore the mechanism.Methods To induce EAN,male Lewis rats were immunized with peripheralnerve myelin sheath antigen (P257481) peptide and complete Freund's adjuvant (CFA) mixed liquor.Rapamycin (RAPA,2.5 mg · kg-1) was administered by intraperitoneal injection 0.5 h after immunization and capsaicin (1 mg · kg-1 · d-1) was administered by intragastric administration 1.0 h after immunization for 15 days.The incidence and clinical characteristics of EAN were observed.The clinical scores of neurological signs were completed and body weight was measured.Pathological morphology of sciatic nerve was observed by HE staining and Lauck fast blue staining.Ultrastructure of sciatic nerve was observed by transmission electron microscope.Levels of serum tumor necrosis factor alpha (TNF-α),interferon gamma (IFN-γ),interleukin 1β (IL-1β) and intedeukin-6 (IL-6) were tested by enzyme linked immunosorbent assay (ELISA).Expressions of autophagy related protein were measured by Western blot.Results Compared with EAN group,the clinical scores of neurological signs significantly decreased from day 7 to day 16 of post-immunization (P ＜ 0.05),body weight significantly increased from day 3 to day 16 of post-immunization (P ＜ 0.05),demyelination obviously decreased,inflammatory cell infiltration number obviously decreased (P ＜ 0.05),the levels of TNF-α,IFN-γ,IL-1β and IL-6 significantly decreased (P ＜ 0.05),the number of autophagosome in axon of sciatic nerve significantly decreased (P ＜ 0.05),and expressions of Beclin-1 and LC3-Ⅱ and the ratio of LC3-Ⅱ and LC3-Ⅰ were significantly down-regulated,and the expression of p62 was significantly up-regulated (P ＜ 0.05) in EAN + capsaicin group.Rapamycin partially reversed the action of capsaicin.Conclusions Capsaicin inhibits EAN in rats,and the mechanism may be related with the inhibition of autophagy activity.

Linkage analysis of susceptibility loci in 2 target chromosomes in pedigrees with paranoid schizophrenia and undifferentiated schizophrenia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship of susceptibility loci in chromosomes 1q21-25 and 6p21-25 and schizophrenia subtypes in Chinese population.</p><p><b>METHODS</b>A genomic scan and parametric and non-parametric analyses were performed on 242 individuals from 36 schizophrenia pedigrees, including 19 paranoid schizophrenia and 17 undifferentiated schizophrenia pedigrees, from Henan province of China using 5 microsatellite markers in the chromosome region 1q21-25 and 8 microsatellite markers in the chromosome region 6p21-25, which were the candidates of previous studies. All affected subjects were diagnosed and typed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revised (DSM-IV-TR; American Psychiatric Association, 2000). All subjects signed informed consent.</p><p><b>RESULTS</b>In chromosome 1, parametric analysis under the dominant inheritance mode of all 36 pedigrees showed that the maximum multi-point heterogeneity Log of odds score method (HLOD) score was 1.33 (α = 0.38). The non-parametric analysis and the single point and multi-point nonparametric linkage (NPL) scores suggested linkage at D1S484, D1S2878, and D1S196. In the 19 paranoid schizophrenias pedigrees, linkage was not observed for any of the 5 markers. In the 17 undifferentiated schizophrenia pedigrees, the multi-point NPL score was 1.60 (P= 0.0367) at D1S484. The single point NPL score was 1.95(P= 0.0145) and the multi-point NPL score was 2.39 (P= 0.0041) at D1S2878. Additionally, the multi-point NPL score was 1.74 (P= 0.0255) at D1S196. These same three loci showed suggestive linkage during the integrative analysis of all 36 pedigrees. In chromosome 6, parametric linkage analysis under the dominant and recessive inheritance and the non-parametric linkage analysis of all 36 pedigrees and the 17 undifferentiated schizophrenia pedigrees, linkage was not observed for any of the 8 markers. In the 19 paranoid schizophrenias pedigrees, parametric analysis showed that under recessive inheritance mode the maximum single-point HLOD score was 1.26 (α = 0.40) and the multi-point HLOD was 1.12 (α = 0.38) at D6S289 in the chromosome 6p23. In nonparametric analysis, the single-point NPL score was 1.52 (P= 0.0402) and the multi-point NPL score was 1.92 (P= 0.0206) at D6S289.</p><p><b>CONCLUSION</b>Susceptibility genes correlated with undifferentiated schizophrenia pedigrees from D1S484, D1S2878, D1S196 loci, and those correlated with paranoid schizophrenia pedigrees from D6S289 locus are likely present in chromosome regions 1q23.3 and 1q24.2, and chromosome region 6p23, respectively.</p>

Isolation, culture and cryopreservation of cells derived from fetal appendages / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the optimal method for isolation, culture and cryopreservation of cells from fetal appendages, for the purpose of providing viable cells for tissue engineering, cell therapy and gene therapy.</p><p><b>METHODS</b>Trypsin dispersion method was used to isolate cells from human umbilical cord and placenta. The tissues from umbilical cord and placenta were cryopreserved with dimethylsulfoxide (DMSO) in different concentrations. Then the percentage of living cells in thawed tissues, and their micro-structure were observed and compared with fresh tissues under transmission electron microscope. The expression of cell immune phenotype before and after cryopreservation were detected with immuno-histochemistry method.</p><p><b>RESULTS</b>The percentage of living cells in human fresh umbilical cord was 67.0%, while that in cryopreserved umbilical cord was 23.4%, 55.5%, 48.8%, 31.8%, respectively in 5%, 10%, 15%, 20% of DMSO. The percentage of living cells in cryopreserved tissues was similar to that of fresh tissues when the volume percentage of DMSO was 10% (P > 0.05), and it was significantly different with that when volume percentage of DMSO was 5% and 20% (P < 0.01). The result by transmission electron microscope was coincident with the results shown above. The results were similar between placenta and umbilical cord. There was no obvious changes in immune phenotype of the tissue and cells after cryopreservation.</p><p><b>CONCLUSION</b>Cryopreservation with this method can isolate a large amount of cells from fetal appendages, with no changes in immune phenotype after cryopreservation, and the effect was best when the volume percentage of DMSO was 10%.</p>

Mapping of pathogenic genes in a pedigree with autosomal dominant ichthyosis vulgaris / 中国医学科学院学报

<p><b>OBJECTIVE</b>To elucidate the pathogenic genes in a pedigree with autosomal dominant ichthyosis vulgaris (IV).</p><p><b>METHODS</b>Linkage analysis was performed by using STR markers in chromosome 1, and mutation detection was used to screen for FLG gene mutation.</p><p><b>RESULTS</b>A maximum two-point Lod score of 3.46 (theta=0) was obtained at D1S2696. Haplotype analysis placed the critical region in a 15-CM interval defined by D1S2726 and D1S305, but no mutation of FLG was found in our IV patients.</p><p><b>CONCLUSION</b>The pathologic gene of the IV family locates near D1S2696, and the FLG gene may not ruled out from the pathologic genes.</p>

Azoospermia factor microdeletion on Y chromosome in patients with idiopathic azoospermia or severe oligozoospermia / 中南大学学报(医学版)

OBJECTIVE@#To investigate the correlation between male infertility and Y chromosome microdeletions of azoospermia factor (AZF) regions, and to establish a reliable genetic diagnosis in idiopathic infertile male patients with azoospermia or severe oligozoospermia.@*METHODS@#Multiplex PCR amplification of 6 sequence-tagged sites in AZF regions of the Y chromosome was examined among 100 normal karyotype male patients with azoospermia or oligozoospermia.@*RESULTS@#Four patients (4%) had Y chromosome microdeletions, the microdeletions of 3 patients were idiopathic azoospermic and those of the other 1 patient were secretory azoospermia.@*CONCLUSION@#The PCR-based Y chromosome microdeletion screening is simple and effective in the diagnosis of patients with severe male infertility. Microdeletion of Y chromosome is one of the major causes of severe dyszooospermia.

Preimplantation genetic diagnosis of Duchenne muscular dystrophy by single cell triplex PCR / 中南大学学报(医学版)

OBJECTIVE@#To detect two exons of Duchenne muscular dystrophy (DMD) gene and a gender discrimination locus amelogenin gene by single cell triplex PCR, and to evaluate the possibility of this technique for preimplantation genetic diagnosis (PGD) in DMD family with DMD deletion mutation.@*METHODS@#Single lymphocytes from a normal male, a normal female, two DMD patients (exon 8 and 47 deleted, respectively) and single blastomeres from the couples treated by the in vitro fertilization pre-embryo transfer (IVF-ET) and without family history of DMD were obtained. Exons 8 and 47 of DMD gene were amplified by a triplex PCR assay, the amelogenin gene on X and Y chromosomes were co-amplified to analyze the correlation between embryo gender and deletion status.@*RESULTS@#In the normal single lymphocytes, the amplification rate of exons 8 and 47 of DMD and amelogenin gene were 93.8%, 93.8%, and 95.3% respectively. The false positive rate was 3.3%. In the exon 8 deleted DMD patient, the amplification rate of exon 47 of DMD and amelogenin gene was 95.8%, and the false positive rate was 3.3%. In the exon 47 deleted DMD patient, the amplification rate of exon 8 of DMD and amelogenin gene was 95.8%, and the false positive rate was 0. In the single blastomeres, the amplification rate of exons 8 and 47 of DMD and amelogenin gene was 82.5%, 80.0% and 77.5%, respectively, and the false positive rate was 0.@*CONCLUSION@#The single cell triplex PCR protocol for the detection of DMD and amelogenin gene is highly sensitive, specific and reliable, and can be used for PGD in those DMD families with DMD deletion mutation.

Identification of the origin of marker chromosome by comparative genomic hybridization / 中南大学学报(医学版)

OBJECTIVE@#To identify the origin of the marker chromosome in a patient with chromosome aberration, and to provide the precise genetic diagnosis.@*METHODS@#Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) were performed to detect the known small marker chromosome in this patient.@*RESULTS@#The small marker chromosome originated from chromosome 13 pter->q12.@*CONCLUSION@#CGH and FISH can be used to detect the small marker chromosome, which is convenient and quick in detecting the origin of small marker chromosome.

Biological characteristics and safety evaluation of endothelial progenitor cells from the umbilical cord blood / 中南大学学报(医学版)

OBJECTIVE@#To investigate the biological characteristics of endothelial progenitor cells (EPCs) from the umbilical cord blood (UCB), and to evaluate their oncogenicity after long-term culture in vitro.@*METHODS@#The mononuclear cells (MNCs) were isolated from the UCB and cultured in MCDB131 medium supplemented with 20% FBS, VEGF and other growth factors. Morphology of the EPCs was observed, and the growth curve of the EPCs was investigated. Surface antigens of the EPCs were analyzed by the flow-cytometer. The capability of intaking the acetylated low-density lipoprotein (acLDL) of the EPCs was detected using fluoresencent chemical method. The vasoformative capability and genetic stability of EPCs were cultured in matrigel, and examined by karyotype analysis. The oncogenicity of EPCs was verified by the tumorigenesis test in athymic mouse and soft agar.@*RESULTS@#EPCs were successfully derived from the UCB, and could be passaged to at least 42(nd) generation and had strong abilities of proliferation, acLDL intake and vasoformation, but there was not oncogenicity. They expressed endothelial cell-surface antigens and maintained normal karyotype.@*CONCLUSION@#The EPCs with proliferative potential can be isolated from the UCB. They can be passaged in long-term cultures without oncogenicity, and can maintain normal karyotype. The EPCs can be served as a new type of cells in cell and gene therapy.

Novel mutation of Y271H in EXT1 gene causes multiple exostoses / 中南大学学报(医学版)

OBJECTIVE@#To explore the disease associated gene mutation of multiple exostoses by family analysis.@*METHODS@#Polymerase chain reaction and DNA sequencing were used to detect the mutation hot spot regions of EXT1 and EXT2 gene, while restriction fragment length polymorphism was performed to screen the mutation.@*RESULTS@#We found a novel heterozygous mutation c.811T ->C in EXT1 gene of patients, which resulted in the substitution of histidine for tyrosine at codon 271 in this hereditary multiple exostoses family. The mutation was not found in the unaffected family members, nor in the 100 unrelated normal individual, which was unreported before.@*CONCLUSION@#The novel mutation Y271H is the disease-causing mutation in the hereditary multiple exostoses family.

Two novel mutations of the LDL receptor gene associated with familial hypercholesterolemia in a Chinese family / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Familial hypercholesterolemia (FH) is a type of dominant autosomal disease that causes high levels of plasma low-density lipoprotein cholesterol (LDL-C). In the past years, molecular data related to FH were limited in China. Now, to gain more information about FH, we analyzed one proband with a severe FH phenotype as well as his relatives.</p><p><b>METHODS</b>After the entire coding sequence and the intron-exon junctions of the low-density lipoprotein receptor (LDLR) gene were amplified using PCR, we sequenced the LDLR gene of a Chinese FH family. RT-PCR was used to detect changes in the mRNA.</p><p><b>RESULTS</b>Two novel mutations were identified in the LDLR gene of this family. One, W165X, was a G > A substitution at the third nucleotide of codon 165. The other, IVS5-1G > A, was also a G > A substitution at the acceptor splice site of intron 5. The most striking discovery is that the proband was heterozygous for W165X but homozygous for IVS5-1G > A. The cDNA sequencing showed that the IVS5-1G > A mutation caused the insertion of 10 nucleotides, namely GCTCTCACAA, between exon 5 and exon 6.</p><p><b>CONCLUSIONS</b>The two nucleotide variations are thought to be the FH-causing mutations because the co-segregation of the mutant allele with the phenotype of FH has been shown in this Chinese family. These data show an increase in the mutational spectrum of FH in China and verify a scarce mutational form in the LDLR gene.</p>

A mutation 1633-26(C-->A) in EXT1 gene causes multiple exostoses / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the gene mutation in a patient with multiple exostoses, identify the disease-causing gene mutation.</p><p><b>METHODS</b>Polymerase chain reaction and DNA sequencing were used to screen the EXT1 or EXT2 gene mutation, while mismatch primer amplification and restriction endonuclease digestion were performed to confirm the mutation.</p><p><b>RESULTS</b>By DNA sequencing, a mutation in the seventh intron was detected and located at 26 bp of 3' splice site upstream in EXT1 gene, which was unreported before. Mismatch primer amplification and restriction fragment length polymorphism analysis suggested that this mutation was not detected in the normal control.</p><p><b>CONCLUSION</b>The mutation 1633-26(C-->A) may be the disease-causing mutation in this patient with multiple exostoses.</p>

Fetal Membrane Derived Adherent Cells: a Novel Source for Mesenchymal Stem Cells / 中国生物工程杂志

Objective: To establish a method for isolation and purification of fetal membrane derived adherent cells (FMDACs) , and investigate their biological characteristics. Method: FMDACs were isolated with trypsin inducing and cultured in vitro. FMDACs were induced to differentiate into osteoblasts and adipocytes. FACS and immunocytochemistry technique were used to examine the cell surface antigen. The genetic stability was verified by karyotype analysis. Results: FMDACs were successfully isolated and expanded in vitro. They had strong proliferative ability. FMDACs were positive for CD44 and CD29, but negative for CD34, CD14 and CD45. FMDACs were differentiated into osteoblasts and adipocytes after inducement. The karyotype was stable in the sixth-passaged FMDACs and the tumorigenicity was not found. Conclusion; FMDACs have the possibility of multipotent stem cells, which have strong capacities of self-renewal and multidirectional differentiation. The genetic background of FMDACs is stable. FMDACs may be used as a kind of novel seed cells for tissue engineering.

Biotoxicology and biodynamics of silica nanoparticle / 中南大学学报(医学版)

OBJECTIVE@#To investigate the toxicology and biodynamics of silica nanoparticle.@*METHODS@#The silica nanoparticles were injected into mice through tail vein, and the mice were amphimixised, the urine was collected in different time, variations of pathology in organs and tissues of the mice were detected. At the same time, the silica nanoparticles' distribution in the tissues was observed through electron microscope.@*RESULTS@#The silica nanoparticles were detected in all tissues and urine of the mice. The injected mice can reproduce as normal.@*CONCLUSION@#The silica nanoparticles do not have toxicity and can be used in vivo.

Identification and clone of human Alzheimer's disease related gene nicastrin promoter / 中南大学学报(医学版)

OBJECTIVE@#To identify the promoter of human nicastrin (NCT) gene, a major component of gamma-secretase which is closely related with pathogenesis of Alzheimer's disease.@*METHODS@#Promoter of human Alzheimer's disease related gene, nicastrin, a 1768 bp fragment was firstly isolated from human genomic DNA by PCR. This fragment's 3 flanking end was 4 bp upstream to the start codon ATG (+1) of the gene. This fragment was used as template, a series of deleted fragments were amplified and constructed to the pGL3-Enhancer plasmid with the artificial designed linkers. The relative activity of their promoter in Hela cells was studied by dual-luciferase assay.@*RESULTS@#The 420 bp fragment showed the strongest activity, and the 237 bp fragment was the minimal fragment in length with activity.@*CONCLUSION@#The promoter of NCT is located at -432/-133 region upstream the translational start codon, while its basal promoter is between -359/-90 that drives the transcription of reporter gene in Hela cells.

Confirmation of the extra small chromosome in abnormality karyotype by PCR and FISH / 中南大学学报(医学版)

OBJECTIVE@#To investigate the source of the extra small chromosome in a patient with karyotype 45,X[115]/46,X + mar[45]/46,XY[29].@*METHODS@#The SRYgene was detected by PCR, and the chromosome Y probe that labeled with biotin was detected by fluorescence in situ hybridization.@*RESULTS@#SRY gene is detected positive and the mar chromosome showed positive signal with FISH in human chromosome Y probe pool.@*CONCLUSION@#The extra small chromosome is part of the chromosome Y.

Stable suppression of beta-catenin expression in prostate cancer cell line by retrovirus mediated RNAi / 中南大学学报(医学版)

OBJECTIVE@#To set up a prostate cancer cell line in which beta-catenin expression is stably suppressed and to investigate the role of Wnt/beta-catenin signaling pathway in prostate tumorgenesis.@*METHODS@#We select 3 sites in the complete coden sequence region of beta-catenin gene as the RNAi targets, ligated the annealed double pre-DNA strands into the retroviral vectors pSUPER-retro and transfected them into the packaging cells PA317, and then collected supernatant with retrovirus to infect DU145. After selection by puromycin and culture expansion, the stable cell clones were attained. Expression of the 2 target genes of Wnt/beta-catenin signaling pathway cyclinD1 and c-myc, was detected in the beta-catenin RNAi cells by Western blot. The effect of suppressing beta-catenin by RNAi on cell proliferation was quantified by methylthiazoletetrazolium (MTT) assay.@*RESULTS@#Western blotting and RT-PCR showed that the expression level of beta-catenin in the 2 stable cell clones apparently decreased. CyclinD1 and c-myc expression decreased in the beta-catenin RNAi cells. MTT showed that the cell number of beta-catenin expression suppression cell clones decreased significantly (P < 0. 05), suggesting the cell proliferation was prevented.@*CONCLUSION@#The beta-catenin gene stable suppression cell line was successfully established.

Genetic linkage analysis in localizing a gene of autosomal dominant familial dilated cardiomyopathy with conduction defect / 中南大学学报(医学版)

OBJECTIVE@#To localize the gene of autosomal dominant familial dilated cardiomyopathy with conduction defect.@*METHODS@#A Chinese family which was diagnosed as dilated cardiomyopathy with conduction defect was studied. Venous blood (3 - 5 mL) from some family members was collected, and genomic DNA was extracted from the blood. Then whole genome wide scan was performed after excluding the known markers on the candidate loci (CMD1A, CMD1 E, CMD1F, and CMD1H) by two-point linkage analysis.@*RESULTS@#No significant evidence for linkage was found in the two point linkage analyses to the known markers in the analyzed family. And the whole genome wide scan showed the maximum LOD score reached 2.68 at marker D3S1614 ( at recombination fraction theta = 0).@*CONCLUSION@#The related gene in this kindred is located on 3q26 other than on CMD1A, CMD1H, CMD1E, and CMD1F.

Hotspot of the mutations of keratin 9 gene in a diffuse palmoplantar keratoderma family / 中南大学学报(医学版)

OBJECTIVE@#To identify the gene causing diffuse palmoplantar keratoderma in a Chinese pedigree.@*METHODS@#Four normal individuals and 3 patients in a diffuse palmoplantar keratoderma family and 10 unrelated control samples were recruited. The hotspot of the mutations of keratin 9 gene was analyzed by polymerase chain reaction and direct sequencing.@*RESULTS@#We found a G485A transition in ke ratin 9 gene, resulting in the substitution of glutamine for arginine at codon 162 in this diffuse palmoplantar keratoderma family. The mutation was not found in the 10 unrelated control samples and 4 normal individuals.@*CONCLUSION@#The mutation G485A found in keratin 9 gene is the disease-causing mutation in the diffuse palmoplantar keratoderma family.

Molecular analysis of SLC26A4 gene in a Chinese deafness family / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the pathogenic gene for a non-syndromic hearing loss family.</p><p><b>METHODS</b>Mutation analysis was carried out by polymerase chain reaction and direct sequencing of all exons of SLC26A4 (solute carrier family 26, member 4) gene.</p><p><b>RESULTS</b>Compound heterozygous mutations N392Y and S448X were detected in the proband of the family, heterozygous mutation S448X was detected in the father, heterozygous mutation N392Y was detected in the mother.</p><p><b>CONCLUSION</b>The proband's hearing loss resulted from the compound heterozygous mutations N392Y and S448X for SLC26A4 gene.</p>

A minidystrophin-EGFP fusion gene expressed in Cos-7 cells mediated by human source vector / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To construct a human source vector containing minidystrophin-EGFP fusion gene and investigate its expression in Cos-7 cells.</p><p><b>METHODS</b>The recombinant human source vector named pHrnDysG was constructed with PCR-clone methods. Three fragments of dystrophin gene were PCR amplified from normal human dystrophin gene cDNA (GenBank NM04006). These three fragments were ligated to generate a minidystrophin gene. The enhanced green fluorescent protein (EGFP) gene was fused to the C terminal of the minidystrophin gene, and then the pHrnDysG was finally obtained by cloning the fusion gene to pHrneo. Fluorescence microscope and RT-PCR were used to detect the expression of minidystrophin-EGFP fusion gene after the recombinant construct was transfected into Cos-7 cells by lipofectamine.</p><p><b>RESULTS</b>Restrictive enzyme digestion analysis and sequencing confirmed that pHrnDysG vector was constructed successfully. After the recombinant pHrnDysG was transfected to Cos-7 cells, RT-PCR demonstrated that the fusion gene was successfully transcribed, and the green fluorescence was observed at the cell membrane.</p><p><b>CONCLUSION</b>The minidystrophin-EGFP fusion gene mediated by pHrneo vector could express in Cos-7 cells and its products' localization in the cell membrane was the same as that of full length dystrophin. These results suggested that the recombinant human source vector pHrnDysG might be potentially used in studies on the gene therapy of Duchenne muscular dystrophy.</p>