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>

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.

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.

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.

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>