OBJECTIVETo introduce the eukaryotic expression vector pEGFP-C1-PDX-1 into nestin-positive cell derived from bone marrow stromal cells by nucleofection and optimize the conditions for transfection.

METHODSThe recombinant plasmid was transfected into bone marrow stromal cells-derived nestin-positive cells with varied DNA quantities or the serum concentration in the medium. The expression of PDX-1 gene in the transfected cells was detected by RT-PCR.

RESULTSSatisfactory efficiency of transfection was achieved with the DNA quantity of 2-10 microg and medium serum concentration of 20%. PDX-1 expression was detected in the transfected cells by RT-PCR.

CONCLUSIONThe optimized transfection conditions result in enhanced efficiency of PDX-1 gene transfection into nestin-positive cells derived from bone marrow stromal cells, which may serve as the seed cells in tissue-engineering.

Bone Marrow Cells ; metabolism ; Genetic Vectors ; Homeodomain Proteins ; genetics ; Humans ; Intermediate Filament Proteins ; metabolism ; Nerve Tissue Proteins ; metabolism ; Nestin ; Plasmids ; Stromal Cells ; metabolism ; Trans-Activators ; genetics ; Transfection ; methods

Adolescent ; Adult ; Asian Continental Ancestry Group ; Child ; Child, Preschool ; Dermatitis, Atopic ; genetics ; Female ; Genetic Predisposition to Disease ; Humans ; Infant ; Intermediate Filament Proteins ; genetics ; Male ; Mutation ; Young Adult

OBJECTIVETo detect FLG gene mutations in 10 families affected with ichthyosis vulgaris and to explore mutational hot spot of the FLG gene in Chinese Han population.

METHODSPCR and direct sequencing were carried out to identify potential mutations of the FLG gene in above families. One hundred healthy individuals were analyzed as normal controls.

RESULTSThree mutations (3321delA, 5757delCCAG and S2706X) were identified in 7 families. A homozygous mutation 3321delA was also detected in two unrelated patients. No mutations were found in the remaining three families. Neither of the null mutations (5757delCCAG and S2706X) was found in the 100 controls. However, for 3321delA, a heterozygous mutation was also found in two of the controls.

CONCLUSIONThree FLG mutations have been identified in the selected families with ichthyosis vulgaris, and the 3321delA mutation was most prevalent (46.9%). Mutations 5757delCCAG and S2706X were first found in patients with ichthyosis vulgaris. Other candidate genes may underlie the disease in those without a FLG mutation.

Asian Continental Ancestry Group ; Base Sequence ; China ; Female ; Genotype ; Humans ; Ichthyosis Vulgaris ; genetics ; Intermediate Filament Proteins ; genetics ; Male ; Mutation ; Pedigree ; Phenotype

OBJECTIVETo identify the type of the intermediate filament (IF) protein of Angiostrongylus cantonensis and analyze its tissue localization.

METHODSRecombinant pET-IF of antigen IF was expressed in E.coli with IPTG induction, and the expression products were purified by His.Bind column and identified for determining the type of the IF protein by Western blotting. Anti-IF antibody was prepared by multi-spot subcutaneous injection into mouse and used to detect the tissue slices of A. cantonensis by immunohistochemical analysis.

RESULTSThe antigen IF were correctly expressed and purified, and identified as a keratin located in the intestine wall and cytoplusma.

CONCLUSIONThe antigen IF is distributed in the intestine wall of A. cantonensis.

Angiostrongylus cantonensis ; cytology ; metabolism ; Animals ; Cell Nucleus ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Intermediate Filament Proteins ; classification ; genetics ; isolation & purification ; metabolism ; Protein Transport

OBJECTIVETo study the effect of lentivirus-mediated RNA interference (RNAi) of nestin on the metastatic potential of human melanoma cell line UACC903.

METHODSA lentiviral vector for RNAi targeting the coding region of human nestin mRNA (nestin-RNAi-LV) and another control vector containing a nonsense sequence were constructed. The vectors were transfected into UACC903 cells, and nestin expression in the cells was detected by RT-PCR, immunofluorence assay and Western blotting. The invasive ability and migration of the transfected UACC903 cells was evaluated using Transwell and scrape assay, respectively. Fluorescence assay was used to examine the expressions of E-cadherin, N-cadherin and β-catenin in the cells.

RESULTSThe lentiviral vector nestin-RNAi-LV was constructed successfully. Compared with the control vector, nestin-RNAi-LV resulted in obviously reduced expression of nestin mRNA and protein, lowered migration ability of UACC903 cells, and reduced cell adhesion and invasiveness (P<0.05).

CONCLUSIONLentivirus-mediated nestin RNAi can specifically inhibit nestin expression to cause decreased cell migration and invasiveness of human melanoma cell line UACC903.

Cell Line, Tumor ; Cell Movement ; genetics ; Genetic Vectors ; Humans ; Intermediate Filament Proteins ; genetics ; metabolism ; Lentivirus ; genetics ; Melanoma ; pathology ; Neoplasm Metastasis ; genetics ; Nerve Tissue Proteins ; genetics ; metabolism ; Nestin ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics

OBJECTIVETo establish transgenic mice with GFP expression in the vascular endothelium during neovascularization.

METHODSThe vector nestin-hsp68-gfp containing nestin second intron was introduced into U251 cells and the expression level of GFP was detected by fluorescence microscopy. Transgenic mice were produced by microinjection. The genome of the offspring mice was screened by PCR, and GFP expression in the vascular endothelium was detected using immunohistochemistry.

RESULTSThirteen offspring mice were obtained and 2 of them were positive for GFP in the vascular endothelium as detected by PCR. GFP was detected in the offspring mice both at the embryonic stage and after birth.

CONCLUSIONSThe transgenic mice with GFP expression in the vascular endothelium during neovascularization have been successfully established.

Animals ; Animals, Newborn ; Base Sequence ; Endothelium, Vascular ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Intermediate Filament Proteins ; biosynthesis ; genetics ; Mice ; Mice, Transgenic ; Molecular Sequence Data ; Neovascularization, Pathologic ; genetics ; Neovascularization, Physiologic ; genetics ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Nestin

OBJECTIVETo establish a nasopharyngeal carcinoma (NPC) cell line with stable nestin gene silencing induced by short-hairpin RNA (shRNA) interference.

METHODSThe nestin mRNA levels in 8 NPC cell lines were detected by real-time PCR and immunofluorescence assay. The recombinant lentiviral shRNA expression plasmid targeting nestin was packaged into mature lentivirus by 293T cells, and the supernatant containing the virus was harvested, concentrated and titrated. The best target for RNA interference was selected by real-time PCR. 5-8F cells was then infected by the recombinant lentiviral vector, and the expression of nestin in the cells was detected by Western blotting and real-time PCR.

RESULTSThe 8 NPC cell lines showed different nestin expression levels, among which 5-8F cells had the highest nestin expression. The recombinant lentiviral vector was successfully constructed and verified by PCR and sequencing. Nestin mRNA and protein levels was significantly reduced in 5-8F cells infected with shRNA-nestin lentivirus as compared with the negative control and the blank control cells.

CONCLUSIONUsing the recombinant lentiviral vector constructed, we have successfully established a 5-8F NPC cell line with stable nestin gene silencing.

Carcinoma ; Cell Line, Tumor ; Gene Silencing ; Genetic Vectors ; Humans ; Intermediate Filament Proteins ; genetics ; Lentivirus ; genetics ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Nerve Tissue Proteins ; genetics ; Nestin ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics

OBJECTIVETo investigate the expressions of nestin and vascular endothehal growth factor (VEGF) mRNAs in rat brain tissue after cerebral ischemia-reperfusion injury and their changes in response to Tongxinluo (a traditional Chinese herbal preparation) treatment.

METHODSCerebral ischemia was induced in rats by temporary middle cerebral artery occlusion (MCAO) followed by treatment with Tongxinluo at high and low doses. On days 3, 7, 14 and 21 after MCAO, nestin and VEGF mRNA expressions in the ependyma, subventricular zone (SVZ), and hippocampal subdentate gyrus zone (HDG) in the ischemic hemisphere were quantitatively analyzed using immunohistochemistry and RT-PCR.

RESULTSCompared with the sham-operated group, the rats with cerebral ischemia-reperfusion injury showed significantly increased nestin-positive neurons and VEGF mRNA expression in the SVZ and HDG 7, 14 and 21 days after MCAO (P<0.05). Treatment with Tongxinluo, especially at high doses, significantly increased the number of nestin-positive neurons and VEGF mRNA expression in the rats 7, 14 and 21 days after MCAO (P<0.05).

CONCLUSIONFocal cerebral ischemia in rats results in rapid response and proliferation of neural stem cells in the SVZ and HDG in the ischemic hemisphere possibly by increasing VEGF mRNA expression in the adjacent tissues around the ischemic focus. Tongxinluo may enhance the differentiation and proliferation capacity of the neural stem cells after MCAO by inducing the expression of VEGF mRNA.

Animals ; Brain Ischemia ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Female ; Intermediate Filament Proteins ; genetics ; metabolism ; Male ; Nerve Tissue Proteins ; genetics ; metabolism ; Nestin ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Animals ; Desmin ; analysis ; genetics ; Female ; Immunohistochemistry ; Intermediate Filament Proteins ; analysis ; genetics ; Kidney Glomerulus ; chemistry ; Nephrosis ; metabolism ; Nerve Tissue Proteins ; analysis ; genetics ; Nestin ; Podocytes ; chemistry ; RNA, Messenger ; analysis ; Rats ; Rats, Inbred WKY ; Vimentin ; analysis ; genetics

OBJECTIVETo identify potential mutations of the FLG gene in two Chinese families affected with ichthyosis vulgaris.

METHODSAll coding exons and exon-intron boundary of the FLG gene were amplified by polymerase chain reaction (PCR) and analyzed by direct sequencing. The results were compared with those of 100 unrelated healthy controls.

RESULTSTwo novel missense mutations, c.1360A>G (p.T454A) and c.10363G>T (p.D3455Y), were detected in all affected individuals from family 1 and family 2 respectively but none of the controls.

CONCLUSIONThe c.1360A>G (p.T454A) and c.10363G>T (p.D3455Y) of the FLG gene may lead to alteration of the structure and function of the FLG protein and cause ichthyosis vulgaris in the two families.

Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; DNA Mutational Analysis ; Exons ; genetics ; Family Health ; Female ; Genetic Predisposition to Disease ; ethnology ; genetics ; Humans ; Ichthyosis Vulgaris ; ethnology ; genetics ; Intermediate Filament Proteins ; genetics ; Introns ; genetics ; Male ; Mutation, Missense ; Pedigree