Objective To investigate the effects of integrin β1 gene expression inhibited by short hairpin RNA (shRNA) on invasion of pancreatic carcinoma PANC1 cells in vitro,and investigate the mechanism.Methods The eukaryotic expression plasmid of shRNA targeting integrin β1 gene ( integrin β1 shRNA) and control eukaryotic expression plasmid shRNA (c-shRNA) was constructed and was transfected into PANC1 cells.The cells without plasmid transfection were used as control.The expressions of integrinβ1,MMP 2,MMP 9 mRNA and protein were detected by real-time PCR and Western blotting.The invasive ability of PANC1 cells was observed with Transwell cell culture chamber.Results Integrinβ1 mRNA expressions in integrinβ1 shRNA group,c-shRNA group and control group were 0.0029 ± 0.0004,0.0131 ± 0.0009,0.0138 ± 0.0005 ; the expressions of integrinβ1 protein were 0.0159 ± 0.0062,0.3215 ± 0.0126,0.3107 ±0.0094; the inhibitory rate of integrinβ1 mRNA and protein expression in integrinβ1 shRNA group was (78.6 ±7.2 ) ％ and (92.9 ± 3.2) ％ ( P ＜ 0.01 ).But there was no difference between the c-shRNA group and control group (P =0.2999).Number of penetrating cells in integrinβ1 shRNA group decreased from 52 ±5 to 21 ±4( P ＜ 0.01 ) ; the expression of MMP 2 and MMP 9 mRNA decreased from 0.592 ± 0.073,0.847 ± 0.069 to 0.102 ± 0.034,0.273 ± 0.071 ; the expression of M MP2 and MMP 9 protein decreased from 0.225 ± 0.046,0.416 ±0.081 to 0.059 ±0.013,0.106 ±0.022(P ＜0.05).Conclusions Recombinant integrinβ1 shRNA expression plasmid can effectively inhibit the expression of integrinβ1 gene and suppress the invasion of PANC1 cells in vitro by down-regulating MMP 2 and MMP 9 gene expression.

OBJECTIVEThe three-dimensional (3D) craniofacial measurements were studied through the quantitative computed tomography (CT). The dynamic database of quantitative measurement of three-dimensional craniofacial bone was established as mandible in physiological position.

METHODS170 aesthetics female were examined by spiral volumetric CT (GE SR-7000). 3D craniofacial bone images were reformatted and 3D measurements were performed in SUN Workstation respectively. 33 points were defined in the 3-d craniofacial structure in screen, 14 distances and 11 angles were measured, and 12 ratios were calculated in each case. All data were transferred into the database based on the SPSS software. There is all information of one case (such as number, sex, age, distances, angers) in one row; each column is a measurement item. The mean, standard deviation, standard error, medium, coefficient of variation and 95% confidence interval of data can be calculated and the correlation, regression between several groups of measurement item can be proceeded by computer automatically in the dynamic database.

RESULTS3D craniofacial bone imagings were displayed in arbitrary views without disturbing superposition by using cutting, rotating and 3D measurement procedures. The large data volume provides more information of special relationship of skull base, zygomatic bone, maxilla, mandible and vertebra. The coefficient of variation of skull base is less than them of maxilla and mandible. The standard deviation of ratios is further smaller than the standard deviation of distances and angles. With stepwise regression, the equation is (Go - Go) Y = 0.578X1 + 0.754X2 + 0.228X3 - 0.579X4 - 14.672; (Tz- Tz) : Y = 0.775X1 + 0.161X2 + 0.348X3 + 0.201X4 + 27.730.

CONCLUSIONSThe database offers reference of the studying of growth rule of craniofacial bone of aesthetics female. It will help improve diagnostic accuracy, staging of reconstruction, precision of corrective surgery, and follow-up patients.

Adult ; Asian Continental Ancestry Group ; Databases, Factual ; Female ; Humans ; Imaging, Three-Dimensional ; Skull ; anatomy & histology ; diagnostic imaging ; Tomography, X-Ray Computed ; Young Adult

OBJECTIVETo investigate the effect of baicalin on liver fatty acid binding protein in oxidative stress model in vitro.

METHODS(1) Cellular oxidative stress in vitro was induced by incubating cells with 400μmol/L hydrogen peroxide (H₂O₂) for 20 minutes at 37 degrees C in the dark. After Chang liver cell line was treated with different dose of baicalin for 24, 48 and 72 hours. MTT assay was employed to detect cell viability, and then the hydrogen peroxide (TC50) of the different dose of baicalin was calculated. (2) Based on MTT assay, cells were treated with three different doses of baicalin (25, 50, 100 μmol/L) for 24 and 48 hours before being exposed to 400 μmol/L H₂O₂ for 20 minutes at 37 degrees C. Then, reactive oxygen species (ROS) assay and activity assays of superoxide dismutase (SOD) and reduced glutathione hormone (GSH) were evaluated. (3) Realtime PCR and Western blotting were applied to explore the influence of baicalin on the expression level of L-FABP. (4) One-way ANOVA was used for results statistical analysis.

RESULT(1) MTT assay showed baicalin treatment at 25, 50, 100 μmol/L for 24 and 48 hours was feasible (83.60% ± 3.47%, 72.36% ± 2.18%, 70.16% ± 2.04% for 24 hours; 84.93% ± 3.11%, 76.16% ± 2.45%, 72.72% ± 2.31% for 48 hours, P > 0.05, F = 386.24, 475.92 respectively). Meanwhile, we found by the linear regression model that the median toxic concentration of baicalin for 48 hours was 170.6 μmol/L, and the median toxic concentration of baicalin for 24 hours was 153.2 μmol/L. (2) ROS assay showed dichlorofluorescin in all baicalin-treated cells after stress was significantly reduced (37.0 ± 3.30, 22.90 ± 3.84, 29.60 ± 2.52 for 24 hours respectively, P < 0.05, F = 70.06; 35.77 ± 2.35, 21.80 ± 3.10, 23.87 ± 1.98 for 48 hours respectively, P < 0.05, F = 110.92) as compared with the H₂O₂-treated cells. Moreover, 50 μmol/L baicalin treatment for 48 hours was the optimal condition against ROS generation (21.80 ± 3.10, P < 0.01, F = 110.92). Furthermore, the activities of intracellular SOD and GSH was increased significantly (51.53 ± 1.91 μg/mg for SOD, P < 0.05, F = 93.81; 49.85 ± 1.45 U/mg for GSH, P < 0.05, F = 92.51). (3) Although realtime PCR analysis indicated 50 μmol/L baicalin treatment for 48 hours could have no changes of the level of L-FABP expression under the oxidative stress condition, western blotting analysis indicated 50 μmol/L baicalin treatment for 48 hours could increase up to about 80% for the level of L-FABP expression.

CONCLUSIONBaicalin was suggested to be able to enhance both L-FABP expression and activity of intracellular SOD and GSH, and therefore protected hepatocytes from oxidative stress.

Catalase ; metabolism ; Cell Line ; Fatty Acid-Binding Proteins ; metabolism ; Flavonoids ; pharmacology ; Glutathione ; metabolism ; Glutathione Peroxidase ; metabolism ; Hepatocytes ; metabolism ; Humans ; Hydrogen Peroxide ; Oxidative Stress ; drug effects ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism

Objective To investigate the flexibility of both the covered stents specially designed for use in intracranial vasculature and the delivering system in passing through the bone tube and the physiological curves of the cranial internal carotid artery(CICA)to reach the targeted area,the performance (adherence)of the covered stents in occluding vascular wall diseases and the impact on the vascular branches of the covered segment.Methods The covered stents specially designed for use in intracranial vaseulature were used to treat 13 patients with CICA diseases using endovascular techniques.There were 4 huge pseudoaneurysms,4 giant aneurysms,3 small wide-necked aneurysms,1 giant pseudoaneurysm with concurrent internal carotid cavernous fistula(CCF),and 1 CCF.Prior to the detachment of the covered stents,balloon occlusion test(BOT)of the internal carotid artery on the diseased side and whole-brain digital subtraction angiography(DSA)were performed in all the patients.Three to 16 months following procedure,DSA and clinical follow-ups were performed.Results Thirteen patients all tolerated the BOT well with the DSA demonstrating well-opened anterior and posterior communicating arteries.The covered stents and the delivering systems all successfully passed CICA to reach the targeted diseased area,with the diseased segments of the internal carotid artery including C3—C4 in 4 cases,C4—C5 in 4 and C6—C7 in 5.Immediately following the detachment of the covered stents,DSA demonstrated that 7 aneurysms were completely occluded,4 aneurysms had slight endoleak,and 1 CCF had markedly-decreased blood flow through the fistula.In the patient with concurrent pseudoaneurysm and CCF,the pseudoaneurysm disappeared and the blood flow through the fistula was markedly-reduced immediately following the stenting procedure.Apart from one patient with aneurysmal subarachnoid hemorrhage who died due to extensive vascular spasm on the 9th day following the stenting procedure,all the other 12 patients had unobstructed stented vessels on the follow-up DSA images,with 2 demonstrating slight stenosis.In the 6 patients with post-procedure endoleak,DSA showed that the endoleak in 4 patients had disappeared,one endoleak disappeared following the second stenting,and one CCF remained low-flow fistula.There was no sequela related to the occlusion of branches in the covered arterial segment.Conclusion The covered stents specially designed for use in the intracranial vasculature and the delivering system are both flexible enough to pass the tortuous CICA to reach the intracranial diseased artery,and are effective in managing CICA diseases.Further follow-up is still needed to determine the long-term effect of the covered stents,and the adherence of the covered stents needs further investigation.

Cell Adhesion ; Cell Movement ; Cell Proliferation ; Hep G2 Cells ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; genetics ; Transfection

OBJECTIVETo ascertain whether connexin 26 (Cx26) gene was a nuclear modifier gene in an extensive family with matrilineal nonsyndromic deafness associated with A1555G mutation in Huaiyin, China.

METHODSFollowing PCR-restriction fragment length polymorphism (PCR-RFLP) with ApaI restriction enzyme, Cx26 genes from 26 cases, with A1555G mitochondrial mutations in this family, and 62 controls (including 2 patrilineal relatives, 10 spouse controls and 50 unrelated controls), were sequenced.

RESULTSCompared with the reference sequence of Cx26 gene, totally four kinds of nucleotide changes,79G -->A, 109G-->A, 341G-->A and 235delC, were detected in a heterozygous form. However, the former three were previously reported polymorphisms, and only the 235delC was a previously described recessive mutation associated with most autosomal nonsyndromic sensorineural hearing loss in Japan and China. Further study showed that the heterozygous 235delC mutation existed in both one individual with mild hearing loss and two individuals with normal hearing. Clinical characterization showed that 235delC mutation did not seem to modify the deafness phenotype due to the A1555G mutation. Moreover, this 235delC mutation was deduced to derive from a married-in control. Finally, there were no co-segregation between the phenotypes of hearing loss and the genotypes for Cx26 genes based on the four kinds of nucleotide changes.

CONCLUSIONSThe heterozygous 235delC mutation of the Cx26 gene may not modulate the severity of hearing loss associated with A1555G mutation and Cx26 gene is unlikely to be a modifier gene for hearing loss due to A1555G mitochondrial mutation in this Chinese family.

Adolescent ; Adult ; Case-Control Studies ; Child ; Child, Preschool ; China ; epidemiology ; Connexin 26 ; Connexins ; genetics ; Deafness ; epidemiology ; ethnology ; genetics ; Female ; Genotype ; Humans ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Mutation ; Pedigree ; Phenotype ; Polymorphism, Restriction Fragment Length ; Sequence Analysis ; Young Adult

Objective To investigate the effects of liver X receptor (LXR) agonist on adiposederived mesenchymal stem cells (AD-MSCs) implantation into infarcted hearts of mice.Method AD-MSCFluc+ which stably expressed firefly luciferase (Fluc) were isolated from β-actin-Fluc transgenic mice and characterized by flowcytometry.Male FVB mice were randomly allocated into the following four groups (n=10each):(1) sham group; (2) MI + PBSgroup; (3) MI + AD-MSCFluc+ group; (4) MI + AD-MSCFluc + LXR agonist (T0901317) group.AD-MSCFluc+ or PBS were injected intramyocardially into periinfarcted region of mice heart after permanent left anterior descending ( LAD ) artery ligation.Bioluminensence imaging (BLI)was performed for quantification of injected cells retention and survival.Cardiac function was evaluated by echocardiography.Results The AD-MSCFluc+ were positive for CD44 and CD90 by flowcytometry.BLI evidenced the firefly luciferase expression of AD-MSCFluc+ which was positively correlated with cell numbers ( r2 =0.98 ).The results of BLI in vivo revealed that LXR agonist could improve the survival of AD-MSCFluc+ at day 7,14 and 21 after transplantation compared with AD-MSCFluc+ alone group.Cardiac function was further improved in combination therapy group compared with AD-MSCFluc+ alone group (P＜0.05).Conclusions LXR agonist T0901317 can improve the retention and survival of intramyocardial injected AD-MSCFluc+ post-MI,and the combination therapy of T0901317 and AD-MSCFluc+ has a synergetic effect on improving cardiae funetion in this model.

Aim To investigate the effect of luteolin on M1 macrophages polarization through HIF-1α-mediated glycolytic pathway. Methods RAW264.7 cells were divided into control groups(M0)and LPS+IFN-γ groups(M1). M1 groups were further divided into luteolin group, 2-DG(glycolysis inhibitor)group, luteolin+2-DG group,luteolin+DMOG(HIF-1α agonist)group. The protein expression levels of iNOS, Arg-1 and HIF-1α were detected by Western blot. Macrophage phenotype was detected by flow cytometry. In addition, the expression levels of IL-6 and IL-10 were measured by ELISA. The gene expression levels of GLUT1, HK2, PFK1, PK and HIF-1α were quantified by quantitative real-time PCR. Results Compared with M1 groups, luteolin and luteolin+2-DG treatment groups decreased the expression levels of GLUT1, HK2, PFK1, PK and HIF-1α related to glycolysis. In addition, luteolin and luteolin+2-DG treatment group significantly inhibited the expression of M1 macrophage markers such as iNOS, CD86 and IL-6, whereas up-regulated M2 macrophage markers Arg-1, CD206 and IL-10. Notably, the inhibitory effects of luteolin on M1 macrophages were restored by DMOG. Conclusion Luteolin regulates M1 macrophage polarization by inhibiting the glycolytic pathway induced by HIF-1α.

A subset of patients of amyotrophic lateral sclerosis (ALS) present with mutation of Cu/Zn superoxide dismutase 1 (SOD1), and such mutants caused an ALS-like disorder when expressed in rodents. These findings implicated SOD1 in ALS pathogenesis and made the transgenic animals a widely used ALS model. However, previous studies of these animals have focused largely on motor neuron damage. We report herein that the spinal cords of mice expressing a human SOD1 mutant (hSOD1-G93A), besides showing typical destruction of motor neurons and axons, exhibit significant damage in the sensory system, including Wallerian-like degeneration in axons of dorsal root and dorsal funiculus, and mitochondrial damage in dorsal root ganglia neurons. Thus, hSOD1-G93A mutation causes both motor and sensory neuropathies, and as such the disease developed in the transgenic mice very closely resembles human ALS.
Amyotrophic Lateral Sclerosis/enzymology/*pathology ; Animals ; Axons/*pathology ; Disease Models, Animal ; Ganglia, Spinal/pathology ; Humans ; Mice ; Mice, Transgenic ; Mitochondria/pathology ; Motor Neurons/metabolism/pathology ; Mutation ; Nerve Degeneration/*pathology ; Sensory Receptor Cells/*pathology ; Spinal Cord/*pathology ; Superoxide Dismutase/genetics/*physiology

BACKGROUNDNatural articular cartilage has a limited capacity for spontaneous regeneration. Controlled release of transforming growth factor-beta1 (TGF-beta1) to cartilage defects can enhance chondrogenesis. In this study, we assessed the feasibility of using biodegradable chitosan microspheres as carriers for controlled TGF-beta1 delivery and the effect of released TGF-beta1 on the chondrogenic potential of chondrocytes.

METHODSChitosan scaffolds and chitosan microspheres loaded with TGF-beta1 were prepared by the freeze-drying and the emulsion-crosslinking method respectively. In vitro drug release kinetics, as measured by enzyme-linked immunosorbent assay, was monitored for 7 days. Lysozyme degradation was performed for 4 weeks to detect in vitro degradability of the scaffolds and the microspheres. Rabbit chondrocytes were seeded on the scaffolds containing TGF-beta1 microspheres and incubated in vitro for 3 weeks. Histological examination and type II collagen immunohistochemical staining was performed to evaluate the effects of released TGF-beta1 on cell adhesivity, proliferation and synthesis of the extracellular matrix.

RESULTSTGF-beta1 was encapsulated into chitosan microspheres and the encapsulation efficiency of TGF-beta1 was high (90.1%). During 4 weeks of incubation in lysozyme solution for in vitro degradation, the mass of both the scaffolds and the microspheres decreased continuously and significant morphological changes was noticed. From the release experiments, it was found that TGF-beta1 could be released from the microspheres in a multiphasic fashion including an initial burst phase, a slow linear release phase and a plateau phase. The release amount of TGF-beta1 was 37.4%, 50.7%, 61.3%, and 63.5% for 1, 3, 5, and 7 days respectively. At 21 days after cultivation, type II collagen immunohistochemical staining was performed. The mean percentage of positive cells for collagen type II in control group (32.7% +/- 10.4%) was significantly lower than that in the controlled TGF-beta1 release group (92.4% +/- 4.8%, P < 0.05). Both the proliferation rate and production of collagen type II in the transforming growth factor-beta1 microsphere incorporated scaffolds were significantly higher than those in the scaffolds without microspheres, indicating that the activity of TGF-beta1 was retained during microsphere fabrication and after growth factor release.

CONCLUSIONChitosan microspheres can serve as delivery vehicles for controlled release of TGF-beta1, and the released growth factor can augment chondrocytes proliferation and synthesis of extracellular matrix. Chitosan scaffolds incorporated with chitosan microspheres loaded with TGF-beta1 possess a promising potential to be applied for controlled cytokine delivery and cartilage tissue engineering.

Animals ; Cartilage ; metabolism ; Cell Proliferation ; Chitosan ; administration & dosage ; Chondrocytes ; cytology ; Drug Carriers ; Microspheres ; Rabbits ; Tissue Engineering ; methods ; Transforming Growth Factor beta1 ; administration & dosage ; chemistry