Acanthoma ; metabolism ; pathology ; surgery ; Adenoma ; metabolism ; pathology ; Adult ; Aged ; Diagnosis, Differential ; Female ; Humans ; Keratin-14 ; metabolism ; Keratin-5 ; metabolism ; Keratin-6 ; metabolism ; Keratosis ; metabolism ; pathology ; surgery ; Male ; Poroma ; classification ; metabolism ; pathology ; surgery ; Sweat Gland Neoplasms ; classification ; metabolism ; pathology ; surgery

The most effective sequence of small interfering RNA (siRNA) silencing STAT3 of psoriatic keratinocytes (KCs) was screened out,and the effects of the most effective siRNA combined with ultrasonic irradiation and SonoVue microbubbles on the expression of STAT3 of KCs and the dose-and time-response were investigated.Three chemically-synthetic siRNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs,and the effects on STAT3 expression were detected,then the most effective siRNA was selected for the subsequent experiments.The negative controls of siRNA (siRNA-NC) labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles were transfected into KCs,then the optimal parameters of ultrasonic irradiation were determined.The most effective siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and SonoVue microbubbles was transfected into KCs,and the dose-and time-response of RNA interference was determined.The effect of RNA interference by the most effective siRNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group) was compared with that only carried by Lipofectamine 3000 (L group).The results showed that siRNA-3 achieved the highest silencing efficacy.0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation.The siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue mierobubbles could effectively knock down the STAT3 expression at mRNA and protein levels in dose-and time-dependent manners determined at 100 nmol/L with maximum downregulation on mRNA at 48 h,and on protein at 72 h after transfection.The LUS group achieved the highest silencing efficacy.It was concluded that siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down the STAT3 expression in psoriatic KCs,and the optimized transfection condition and the sequence of siRNA-3 could serve for further research on gene therapy of psoriasis.

OBJECTIVETo determine if alteration in TP63 is responsible for a Chinese patient with ectrodactyly-ectodermal dysplasia clefting (EEC) syndrome, but without cleft palate/lip.

METHODSScreening of TP63 gene was performed in the patient with EEC syndrome and his family members using PCR-single strand conformational polymorphism (SSCP) analysis, then performed by direct sequencing of the coding region.

RESULTSA C > T substitution at nucleotide position 838 in exon 7 was detected in the patient, and the change predicted a heterozygous missense mutation, Arg280Cys. His parents showed the wild type.

CONCLUSIONSThe results indicate that the de novo mutation Arg280Cys of the TP63 gene observed in the patient maybe contribute to his EEC syndrome.

Asian Continental Ancestry Group ; Cleft Lip ; genetics ; Cleft Palate ; genetics ; Ectodermal Dysplasia ; genetics ; Exons ; Genotype ; Heterozygote ; Humans ; Mutation, Missense ; Transcription Factors ; genetics ; Tumor Suppressor Proteins ; genetics

BACKGROUND:Previous studies have confirmed that ethanol can promote adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and up-regulate the expression of PPARγ and aP2 in the tumor necrosis factor α (TNF-α) signaling pathway. As a member of the ZIP protein family, Zrt/Irt-like protein 1 (ZIP1) is closely related to bone metabolism and osteogenic differentiation.OBJECTIVE:To study the effect of BMSCs transfected by ZIP1 on TNF-α signaling pathway in the process of adipogenic differentiation.METHODS:The BMSCs from rabbits were isolated and cultured under different concentrations of alcohol (0.03, 0.09,0.15, 0.21 mol/L), followed by transfection by ZIP1 siRNA and ZIP1 expression vector.RESULTS AND CONCLUSION:After culture in alcohol, the expression levels of aP2 and PPARγ proteins were both significantly increased (P < 0.05), and the level of triglyceride was increased in all alcohol groups except for 0.03 mol/L alcohol group (P < 0.05). After siRNA transfection, the expression levels of aP2 and PPARγ as well as the level of triglyceride were increased significantly in all the alcohol groups (P < 0.05); however, ZIP1 transfection decreased the expression levels of aP2 and PPARγ proteins (P < 0.05). To conclude, ZIP1 siRNA could promote the adipogenic differentiation of BMSCs through the activation of TNF-α signaling pathway.

OBJECTIVETo elucidate the molecular mechanism of Wendan Tang in prevention of lipid metabolism disorder in adult rats.

METHODOn the basis of hyperlipidemia rat models, triglycerides (TG), total cholesterol (TC) in serum, activities of lipase (LA), lipoprotein lipase (LPL), hepatic lipase (HL) in liver, parts of hemogram and hepatic LDLR mRNA levels were investigated 21 days after the feeding of atherogenic diet.

RESULTWendan Tang significantly reduced the serum TG, TC and increased the activity of LPL and LA, but caused no chang in HL. The result of RT-PCR test showed that high fat and high cholesterol feeding could significantly induce the reduction of LDLR mRNA levels, while Wendan Tang could increase hepatic LDLR density.

CONCLUSIONWendan Tang can prevent disorder of lipid metabolism by regulating TC, TG, LDL-c through upregaulation of LDLR transcription level and improving antioxidant ability.

Animals ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Female ; Hyperlipidemias ; blood ; metabolism ; prevention & control ; Lipid Metabolism ; Liver ; metabolism ; Male ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Receptors, LDL ; biosynthesis ; genetics

Background Corneal neovascularization (CNV) is an important cause of visual impairment and graft rejection after allograft corneal transplantation in inflammatory corneal diseases. The mechanisms and therapy relating to CNV are intensely investigated at all times. Objective This study was to evaluate the effect of eicosapentaenoic acid (EPA) on CNV induced by alkali cauterization and its mechanism. Methods The animal models of corneal neovasculation were induced in the right eyes in 72 Sprayue-Dawley rats by putting a piece of 3 mmfilter paper with 1 mol/L NaOH at the center of the cornea for 30 seconds. The rats were then divided randomly into the 0.02 mg EPA treatment group (24 rats) ,0.03 mg EPA treatment group (24 rats) ,model group (24 rats) and normal group (6 rats). EPA of 0.04 ml with doses of 0.02 mg or 0. 03 mg or saline solution of 0. 04 ml was injected subconjunctivally in model rats and immediately after cauterization. The presence of CNV and corneal edema were observed daily by slit lamp biomicroscope. 1,4,7 and 14 days after operation, corneal histopathological examination was performed by hematoxylin and eosin staining. The vascular endothelial cells were stained with CD34 by immunohistochemistry,and the expression of IL-1α,IL-6 mRNA and the nuclear factor-κBp65 ( NF-κBp65 ) proteins was measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The use of animals complied with the Regulations for the Administration of Affair Concerning Experimental Animals by Hebei Province( version 1998 ). Results Under the slit lamp, CNV grew slowly from days 2-4 with obvious corneal edema and defect of epithelium. Larger CNV area and less edema were seen from days 7-10. Maximal vessel growth was observed 14 days after injury with thinner vessels in the model group. Histological examination showed that part of the corneal epithelium was damaged;serious corneal edema, more inflammatory cells and a lot of CNV in the stroma were presented in the model group. However, repairing of the corneal epithelium without CNV ,light corneal edema and less inflammatory cells were found in both the 0. 02 mg EPA and 0. 03 mg EPA treatment groups 7 days after alkali cauterization. The relative area of CNV in the 0. 02 mg EPA treatment group was ( 15.80±6.43 )% and ( 11.06±2. 14)% ,and that in the 0. 03 mg EPA treatment group was (16. 10±7.41 )% and (11.06±2. 51 )%, showing significant reduction in comparison with the model group [ (84. 74±7.77)% and (89.63±7.50) % ] 7 days and 14 days after operation ( P＜0. 05 ). Stronger expression of CD34 in the vascular endothelial cells of the cornea stroma was observed in the model group and an absence of CD34 was observed in the EPA-treated groups on the 7th day. RT-PCR revealed that the expression of IL-1α mRNA and IL-6 mRNA was lower in the EPA treatment groups than the model group ( P＜0. 05 ), and Western blot analysis showed that the expression of NF-κB/p65 in the corneas in the EPA treatment groups was significantly lower than that in the model group on the 4th day after operation (P＜0.05).Conclusion Topical application of EPA suppresses CNV induced by alkali burn possibly by inhibiting the expression of NF-κB,IL-1α and IL-6.

OBJECTIVETo explore the biological effect on NB4 cells proliferation, all-trans retinoic acid (ATRA) inducing differentiation and resistance to apoptosis by vascular endothelial growth factor (VEGF)-C cDNA transfection.

METHODSThe recombinant eukaryotic expression plasmid pcDNA3.1-VEGF-C and the vacant pcDNA3.1 vector were introduced separately into NB4 cells by lipofectamine mediation. The positive clones were screened by G418 and identified by reverse transcriptase-PCR (RT-PCR) and Western blotting. The proliferation capacity of NB4/VEGF-C cells was analysed by MTT assay and colony forming assay in vitro. After NB4/VEGF-C cells were induced by ATRA, the expression level of C/EBPalpha gene, CD11b on cells surface and morphological alteration were analysed by real-time quantitative PCR (RQ-PCR), flow cytometry (FCM), and Wright-Giemsa staining, respectively. FCM Annexin V-FITC/PI dual labeling technique was performed to investigate the etoposide (Vp16) induced NB4/VEGF-C cells apoptosis and bcl-2 gene expression level in these cells was analysed by RQ-PCR. The NB4/pcDNA3.1 cells was used as control in the above experiments.

RESULTSA stable NB4 cell line that secrets VEGF-C and its control lines were established. The proliferation capacity of the former was stronger than that of the latter. The expression level of C/EBPalpha gene of NB4/VEGF-C cells on ATRA treatment was only 1/32 that of NB4/pcDNA3.1 cells. The CD11b level and the degree of differentiation of NB4/VEGF-C were weaker than that of NB4/pcDNA3.1 cells. The percentage of apoptotic NB4/VEGF-C cells induced by Vp16 [(7.20 +/- 2.52)%] was significantly lower than that of NB4/pcDNA3.1 cells [(16.07 +/- 3.58)%] (P = 0.005), but the bcl-2 gene expression level of NB4/VEGF-C cells is 2.28-fold that of NB4/pcDNA3.1 cells.

CONCLUSIONThe VEGF-C via VEGFR-3 signaling pathway could promote the proliferation of leukemic cells by autocrine pathway and inhibit the cell differentiation mediated by ATRA and chemotherapy-induced apoptosis. VEGF-C/VEGFR-3 signaling loops might play an important role in disease progression and be potential therapeutic target for the treatment of leukemias.

Apoptosis ; drug effects ; genetics ; physiology ; Blotting, Western ; CCAAT-Enhancer-Binding Protein-alpha ; genetics ; metabolism ; CD11b Antigen ; genetics ; metabolism ; Cell Differentiation ; genetics ; physiology ; Cell Line, Tumor ; Cell Proliferation ; DNA, Complementary ; genetics ; Drug Resistance, Neoplasm ; Flow Cytometry ; Genetic Vectors ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; metabolism ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Tretinoin ; pharmacology ; Vascular Endothelial Growth Factor C ; genetics ; metabolism ; physiology

OBJECTIVETo establish NB4/VEGF-C cells xenograft in nude mice model, and explore the effect of VEGF-C on hematological malignancies

METHODSNB4/VEGF-C or NB4/pcDNA3.1 cell lines were established by transfecting the recombinant pcDNA3.1-VEGF-C plasmid and the vacant pcDNA3.1 vector into NB4 cells. The recombinant VEGF-C was identified by RT-PCR and Western blotting. Eighteen male BALB/c nude mice aged 4 - 5 weeks were equally divided into two groups. Mice irradiated by 4 Gy ⁶⁰Co were subcutaneously injected with 1 × 10⁷NB4/VEGF-C or NB4/pcDNA3.1 cells into one side of axilla. The volumes of xenograft tumor was evaluated according to L × t² × 0.52. Microvessel density (MVD) on the xenograft tumor section was detected by IHC with VWF antibody.

RESULTSNB4 cell xenograft tumors were developed in all mice of both the two groups. The growth of NB4/VEGF-C cells in nude mice was faster than in controls. There were statistically significant differences in the volume and weight of xenograft tumor between NB4/VEGF-C and NB4/pcDNA3.1 cell groups \[(631.44 ± 114.42) mm³ vs (491.22 ± 70.05) mm³\] (P = 0.006) and \[(321.78 ± 27.84) mg vs (288.57 ± 40.12) mg\] (P = 0.031), respectively. MVD in xenograft tumor of NB4/VEGF-C cells \[(50.8 ± 11.7)/mm²\] was higher than that in controls \[(18.9 ± 7.0)/mm²\] (P = 0.021). The Bcl-2 protein level in NB4/VEGF-C cells xenografts was higher than that in controls.

CONCLUSIONVEGF-C could promote proliferation of NB4 cells by inducing angiogenesis and inhibit cells apoptosis by upregulating antiapoptotic Bcl-2 protein expression in NB4 cells xenograft tumor.

Animals ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neovascularization, Pathologic ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Vascular Endothelial Growth Factor C ; metabolism ; Xenograft Model Antitumor Assays

OBJECTIVESTo investigate the effects of FAK-related non-kinase (FRNK) on expressions of type I collagen and matrix metalloproteinase-2 (MMP-2) mRNA and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA in rat hepatic stellate cells (HSC).

METHODSUsing in vitro cell culture technique, FRNK plasmids were transfected into HSC mediated by cationic liposome. Type I collagen synthesis capability in HSC was examined by 3H-Pro incorporation assay. The levels of FRNK in HSC were assayed by Western blot, and the expressions of MMP-2 and TIMP-2 were assayed by RT-PCR on mRNA levels.

RESULTSThe exposure of HSC to FRNK caused the expression of FRNK protein to be up-regulated, and the FRNK protein contents reached the highest point at 48 h after the transfection, P less than 0.05. The expressions of MMP-2 mRNA were up-regulated by FRNK; the expressions of TIMP-2 mRNA were down-regulated by FRNK; the ratios of MMP-2 mRNA/TIMP-2 mRNA were enhanced by FRNK.

CONCLUSIONAfter FRNK was transfected, the capability of type I collagen synthesis in HSC was inhibited, which may be related to the up-regulation of MMP-2 mRNA/TIMP-2 mRNA.

Animals ; Cells, Cultured ; Collagen Type I ; metabolism ; Hepatic Stellate Cells ; cytology ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Plasmids ; Protein-Tyrosine Kinases ; genetics ; RNA, Messenger ; genetics ; Rats ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Transfection

BACKGROUND: Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been widely used on clinic; however, there are still few reports addressing rhBMP-2-induced osteogenesis in intervertebral disc.OBJECTIVE: To verify the effect of rhBMP-2 to induce interbody fusion in rabbits.DESIGN, TIME AND SETTING: Randomized controlled animal study and multi-level evaluation, which was performed in Affiliated Hospital of Medical College of Qingdao University from February to July 2007.MATERIALS: 24 adult New-Zealand rabbits weighing 3.5-4.5 kg were used to expose L4-5 and L5-6 intervertebral disc; rhBMP-2 (1 mg/ampoule, purity≥95%) was provided by Beijing Bailingke Biological Products Co., Ltd.METHODS: 24 rabbits were randomly divided into experimental group and control group with 12 rabbits for each. In the experimental group, saline (20 μL, containing 200 μg rhBMP-2) was injected into nucleus pulposus of L4-5 intervertebral disc; equivalent saline was inserted into nucleus pulposus of L5-6 intervertebral disc as controls. Rabbits in the control group were injected with saline (20 μL) into nucleus pulposus of L4-5 intervertebral disc.MAIN OUTCOME MEASURES: Morphological changes of injected segments were observed by hand-feeling check together with histological and imaging tests at 10, 30, 60, and 90 days postoperatively.RESULTS: 24 rabbits were included in the final analysis. ①In the experimental group, the motion range of L4-5 segment was not limited at 10 days postoperatively, and lightly limited at 30 days, but severely limited at 60 days postoperatively; L4-5 segment was fixed tightly at 90 days postoperatively. Moreover, motion range of L5-6,segment and articular motion range in the control group were not changed remarkably. ② L4-5 interbedy space was narrowed at 10 days or even disappeared at 90 days postoperatively, and then osteogenesis fusion was formed. Transmittance of intervertebral space in the L5-6 segment and in the control group was not changed obviously. ③ Nucleus pulposus was gradually shrunk at 10 days postoperatively; partial cartilage endplate transformed into mature woven bone, and collagen fiber structure of annulus fibrosus gradually disappeared at 90 days postoperatively. A lot of mesenchymal cells were aggregated surrounding annulus fibrosus at 10 and 30 days postoperatively. Moreover, mature woven bone was formed in annulus fibrosus near to cartilage endplate at 90 days postoperatively. However, histological and morphological changes were not found in the control group at those four time points.CONCLUSION: rhBMP-2 can induce intervertebral disc osteogenesis so as to achieve interbody fusion.