Objective To construct a short hairpin RNA (shRNA) targeting LDH-A, and evaluate the effects on growth, apoptosis and the expression of LDH-A in PANC-1 cells. Method Three shRNAs targeting LDH-A were combined to pGCsilencer vector, and transfected into PANC-1 cells. The expression of LDH-A after transfected by the three shRNAs in pancreatic cancer cell lines PANC-1 was detected by quantitative real time PCR. After tranfected by LDH shRNA-3 with the highest inhibited rate, cell growth was analyzed by MTT assay, apoptosis was detected by flow cytometry. The LDH-A expression was detected by reverse transcription polymerase chain reaction, LDH activity was observed with enzyme cytochemical method. Results The 2-AACt of LDH-A shRNA-3 was (0. 47 ± 0. 02), less than the untransfected pancreatic cancer cell(0. 71 ± 0. 01), the LDH-A shRNA-3 could inhibit the expression of the LDH-A most effectively. The growth of the pancreatic cancer cell was inhibited after 12 h transfected by LDH-A shRNA-3, all the absorbance value of transfected cell in 24 h,36 h,48 h,72 h decreased obviously compared to the untreated pancreatic cancer cell(P <0. 01). The apoptosis rate of the transfected cell increased to 61.74%. The inhibition of LDH-A expression in PANC-1 cells transfected by shRNA-3 was significantly and the activity of LDH reduced. Conclusion LDH-A shRNA inhibits the expression of LDH-A, the proliferation of cancer cells inducing the apoptosis of PANC-1 cells.

Objective To investigate the effects of oxamate on concentrations of calcium in pancreatic cancer cells.Methods Pancreatic cancer cell panc-1 was cultured in vitro with oxamate for 48 h,then stained with Fluo-3/AM,and the light density of cells for different concentration of oxamate under confocal laser microscopy was observed.Results Oxamate influenced the concentration of calcium in panc-1,and this effect corresponded to the concentration of oxamate.Conclusions Oxamate can induce the increase of the concentration of calcium of panc-1,and then influence the cellular pathophysiologic process.

In order to study the treatment of the children with attention deficit hyperactivity disorder (ADHD), the integrated visual and auditory continuous performance test (IVA-CPT) was clinically applied to evaluate the effectiveness of electroencephalogram (EEG) biofeedback training. Of all the 60 children with ADHD aged more than 6 years, the effective rate of EEG biofeedback training was 91.6% after 40 sessions of EEG biofeedback training. Before and after treatment by EEG biofeedback training, the overall indexes of IVA were significantly improved among predominately inattentive, hyperactive, and combined subtype of children with ADHD (P<0.001). It was suggested that EEG biofeedback training was an effective and vital treatment on children with ADHD.
Attention Deficit Disorder with Hyperactivity/physiopathology ; Attention Deficit Disorder with Hyperactivity/*therapy ; Biofeedback (Psychology) ; Brain/*physiopathology ; Electroencephalography

BACKGROUND: Recent studies have demonstrated that energy metabolism-related genes play important roles in intervertebral disc (IVD) cell adaptation to negative environmental factors, such as hypoxia and insufficiency of nutrient. But the effects of these genes in pressure-induced intervertebral disc degeneration remain uncertain.OBJECTTVE: To investigate continuous pressure-induced expression of energy metabolism genes: hypoxia-inducible factor 1α(HIF-lo), glucose transporter-1 (GLUT-1) and vascular endothelial growth factor (VEGF) in rabbit annulus fibrosus (AF).DESIGN: A randomized controlled experiment.SETTING: The Central Laboratory and the Laboratory of Department of Orthopaedics, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The experiment was carried out in the Central Laboratory and the Laboratory of Department of Orthopaedics, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from May to October 2005. Twenty-five Japanese white rabbits(about 4 months old, weighting 2.5-3.0 kg, provided by the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology) were selected to establish the animal model.METHODS: A controllable pressure-induced rabbit intervertebral disc degeneration model was adopted to impose various pressured on rabbit IVDs in vivo. The survived animals whose IVDs were compressed successfully were divided randomly into 4 groups. The IVDs were treated with no pressure as control (control group), with 15 kg axial load for 24 hours (24hours group), 72 hours (72 hours group), and 24 hours with 48 hours free for self-reparation (reparation group). Reverse transcriptase-polymerase chain reaction was used to detect the expression of HIF-1α and GLUT-1. Western Blot and immunohistochemical test were carried out for the content and distribution of VEGF.MAIN OUTCOME MEASURES: Expression of HIF-1α and GLUT-1, content and distribution of VEGF.RESULTS: Twenty of 25 rabbits entered result analysis. Two rabbits were missed because of vertebral fracture, while death of 3 rabbits within 1-3 days postoperatively caused another loss. ①HIF-1α. A very low expression was detected in the control group, while the expression in the 24 hours group was raised over 20 times than that in the control group (t=25.022, P＜0.01). The expression in the 72 hours group and reparation group decreased as compared with the 24hours group. ②GLUT-1 expressed weakly in the control group. The expression in the 24 hours group rose a lot as compared with the control group (t=18.314, P＜0.01) and the expression in the 72 hours group rose slightly than that in the 24 hours group (t =2.819, P＜0.05). The expression in the reparation group is close to that in the 24 hours group. ③Littie VEGF content was detected in the control group, while the content rose significantly in the other 3 groups. Immunohistochemical staining showed more VEGF positive stained cells in outer AF than in inner AF.CONCLUSION: Continuous pressure can strongly up-regulate the expression of energy metabolism gene: HIF-1α,GLUT-1 and VEGF in vivo. These genes play important roles in AF adaptation and reparation in over load-caused damage.

Objective: To investigate HDAC5 expression in gastric cancer cell lines and its effects on the proliferation and apoptosis of the gastric cancer line SGC-7901. Methods: The expression patterns of HDAC5 and Twist1 in gastric cancer cell lines and normal gastric mucosal cells were detected by Western blot. The effects of HDAC5 and Twist1 on the proliferation and apoptosis of SGC-7901 cells were analyzed by MTT and flow cytometry, respectively. Results: The expression of HDAC5 and Twist1 in gastric cancer cell lines were significantly higher than that in normal gastric mucosal cells (P<0.05). HDAC5 knockdown significantly down-regulated Twist1 expression,inhibited cell proliferation, and induced apoptosis in SGC-7901 cells, whereas HDAC5 overexpression exhibited an opposite effect (P<0.05). Moreover, Twist1 knockdown significantly inhibited cell proliferation and induced apoptosis in SGC-7901 cells (P<0.05). Conclusion:HDAC5 may promote cell proliferation and inhibit apoptosis in gastric cancer cells by upregulating Twist1 expression, thus promoting the initiation and development of gastric cancer.

BACKGROUND: It has reported that nicotinamide is capable of protecting intervertebral disc (IVD) against interleukin-1β (IL-1β) or tumor necrosis factor-alpha (TNF-α) induced degeneration. However, the protective mechanism of nicotinamide on IVD cells apoptosis and proliferation remains unclear.OBJECTIVE: To investigate regulatory effects of nicotinamide on rabbit nucleus pulposus cell apoptosis and proliferation in vitro.DESIGN, TIME AND SETTING: Randomized control grouping design, which was carried out in the Laboratory of Orthopaedics and Stem Cell Center, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from April to October 2007.MATERIALS: Ten Japanese white rabbits (aged 2-3 months weighing 1.5-2.0 kg) were used in this study. Furthermore, nucleus pulposus cells obtained from L1-6 lumbar spine were harvested and cultured for further experiments.METHODS: The NP cells were divided into 6 groups, including control group (without any drug as control), nicotinamide group (0.5 g/L nicotinamide), IL-1β group (10 μg/L IL-1β), IL-1β + caspase group (10 μg/L IL-1β and non-specific caspase inhibitor Z-VAD-FMK), IL-1β + small-dose nicotinamide group (10 μg/L IL-1β and 0.05 g/L nicotinamide), and IL-1β + large-dose nicotinamide group (10 μg/L IL-1β and 0.5 g/L nicotinamide). After 3 days of culture, the cells were examined with Annexin V-PI staining, caspase-3, 8 and 9 activity staining and MTT assay.MAIN OUTCOME MEASURES: The apoptotic rates, the positive rates of caspase-3, 8 and 9 activity staining and the absorbance of MTT assay of each group.RESULTS: ① As compared to IL-1β group, the apoptotic rates were decreased in the IL-1β + caspase group and IL-1β + large-dose nicotinamide group (P < 0.01). ②As compared to IL-1β group, the positive rates of caspase-3, 8 and 9 activity staining were decreased in the IL-1β + caspase group, IL-1β + large-dose and small-dose nicotinamide groups (P < 0.01 or P < 0.01). ③As compared to IL-1β group, the absorbance was increased in the IL-1β + caspase group and IL-1β + large-dose nicotinamide group (P < 0.01).CONCLUSION: Nicotinamide is capable of promoting cell proliferation and inhibiting IL-1β induced apoptosis of nucleus pulposus cells in vitro. The inhibition of apoptosis mainly acts via inhibition of the mitochondrial pathway.

The protective effect of niacinamide on interleukin-1beta (IL-1beta)-induced annulus fibrosus (AF) type II collagen degeneration in vitro and the mechanism were investigated. Chiba's intervertebral disc (IVD) culture models in rabbits were established and 48 IVDs from 12 adult Japanese white rabbits were randomly divided into 4 groups: normal control group, niacinamide-treated group, type II collagen degneration group (IL-1beta) and treatment group (niacinamide+IL-1beta). After culture for one week, AFs were collected for inducible nitric oxide synthase (iNOS), cysteine containing aspartate specific protease-3 (Caspase-3) and type II collagen immunohistochemical examination, and type II collagen reverse transcription polymerase chain reaction (RT-PCR). The results showed that rate of iNOS positive staining AF cells in the 4 groups was 17.6%, 10.9%, 73.9% and 19.3% respectively. The positive rate in treatment group was significantly lower than in the type II collagen degeneration group (P<0.01). Rate of Caspase-3 positive staining AF cells in the 4 groups was 3.4%, 4.2%, 17.6% and 10.3% respectively. The positive rate in treatment group was lower than in the type II collagen degeneration group (P<0.01). Type II collagen staining demonstrated that lamellar structure and continuity of collagen in treatment group was better reversed than in the degeneration group. RT-PCR revealed that the expression of type II collagen in treatment group was significantly stronger than that in type II collagen degeneration group (P<0.01). It was concluded that niacinamide could effectively inhibit IL-1beta stimulated increase of iNOS and Caspase-3 in AF, and alleviate IL-1beta-caused destruction and synthesis inhibition of type II collagen. Niacinamide is of potential for clinical treatment of IVD degeneration.

Summary: In order to study the treatment of the children with attention deficit hyperactivity disorder (ADHD), the integrated visual and auditory continuous performance test (IVA-CPT) was clinically applied to evaluate the effectiveness of electroencephalogram (EEG) biofeedback training. Of all the 60 children with ADHD aged more than 6 years, the effective rate of EEG biofeedback training was 91.6 % after 40 sessions of EEG biofeedback training. Before and after treatment by EEG biofeedback training, the overall indexes of IVA were significantly improved among predominately inattentive, hyperactive, and combined subtype of children with ADHD (P<0.001). It was suggested that EEG biofeedback training was an effective and vital treatment on children with ADHD.

BACKGROUND:Recent studies have demonstrated that Niacinamide is capable of promoting the proliferation of intervertebral cells and improving intervertebral disc degeneration.Overloading is thought to the main cause of intervertebral disc degeneration.However,the protective effects of Niacinamide in loading induced intervertebral disc degeneration remains uncertain,OBJECTIVE:To investigate the protective effects of Niacinamide against axial loading induced degeneration of rabbit lumbar disc.DESIGN,TIME AND SETTING:A randomized controlled experiment was carded out in the Central Laboratory and the Laboratory of Department of Orthopaedics,Union Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology from November 2008 to April 2009.MATERIALS:Twenty-four Japanese white rabbits (4 months old,weighing 2.0 kg).Niacinamide was supplied by Tianjin Damao Chemical Reagent Factory.METHODS:Twenty-four Japanese white rabbits were randomly divided into 6 groups.The controllable axial loading induced rabbit lumbar disc degeneration model was adopted to impose 98N pressure on the rabbit discs to induce degeneration.Various doses of Niacinamide were given intragastrically to the rabbits in different groups:2 rabbits in group 1,the loading device was installed without pressing,and no Niacinamide was given;2 rabbits in group 2,given 50 mg/kg Niacinamide for 1 week;5 rabbits in group 3,loaded with 98N for 1 week;5 rabbits in group 4,loaded with 98N for 1 week,then the pressure was released for another week's recovery;5 rabbits in group 5,loaded with 98N and given 50 mg/kg Niacinamide for 1 week;5 rabbits in group 6,loaded with 98N for 1 week and then the pressure was released for another week's recovery,50 mg/kg Niacinamide was continually given during the 2 weeks.MAIN OUTCOME MEASURES:Magnetic resonance image and Thompson's grading system were used to assess degeneration degree of the discs;hematoxylin and eosin staining,immunohistochemical staining for type Ⅱ collagen,and Safranin O staining were used to evaluate histological changes;immunohistochemical staining for P161NK4A was used to evaluate cell proliferation and senescence.RESULTS：①According to the Thompson's grading system，there was no disc exhibited degeneration in group 2；5 rabbits graded Ⅱ in group 3；4 rabbits graded Ⅱ and 1 rabbit graded Ⅲ in group 4；2 rabbits graded Ⅰ and 3 rabbits graded Ⅱ in group 5；3 rabbits graded Ⅰ and 2 rabbits graded Ⅱ in group 6.MRI results revealed the alleviated degeneration in Niacinamide given groups.②The content of type Ⅱ collagen of annulus fibrosus of group 6 was 53.2% higher than that of group 4 (P＜0.01).③Safranin O-Fast Green staining density of group 2 was higher than that of group 1；The staining density of nucleus pulposus and annulus fibrosus of groups 5 and 6 was higher than the corresponding parts of group 4，especially that of nucleus pulposus (P＜0.01，P＜0.01)，and group 6 exhibited slightly increased levels than group 5.④P16INK4A positive staining rates decreased with the extension of Niacinamide administration time.CONCLUSION：Niacinamide can help to alleviate overloading-caused damage to intervertebral disc，and can benefit the recovery of damaged intervertebral disc.

To investigate the influence of recombinant adenovirus carrying tissue inhibitor of metalloproteinase-3 (RAdTIMP-3) on the main compositions of rabbits intervertebral discs and to assess its potential in treatment for intervertebral disc degeneration.[Method]RadTIMP-3 and empty adenovims vector with Lac-Z gene (Rad66) was propagated in 293 Cells and was purified, identified and tittered. Thirty Japanese white rabbits were randomly divided into 5 groups. And 25 μl of various reagents were injected to the L4、5 and L5、6 intervertebral discs of the rabbits as follows:normal saline in group 1, 1.0×1010 OPU/ml of RAd66 in Group 2, and 1.0×1010 OPU/ml of RAdTIMP-3 in group 3, 4 and 5. The intervertebral discs of each group were collected after 2, 2, 1, 2 and 4 weeks after injection respectively.Then X-gal staining, And Group 1, RT-PCR for TIMP-3 and aggrecan core protein,TUNEL staining, immunohistochemical staining for TIMP-3 and type I! Collagen and Safranin O-Fast green staining was carried out to assess the effects of RadTIMP-3 transfection.[Result](1)concentration of RAdTIMP-3 reached 1.9×1012 OPU/ml after propagation and purification. (2)RT-PCR shows that the expression of TIMP-3 was significantly raised in group 3, 4, 5, as compared with group 1 or 2. And the expression of core protein gene in group 3, 4, 5 increased slightly than in group 1 and 2. (3) TUNEL staining revealed that there was not significant difference between the positive-staining rates of any two of the groups. (4)TIMP-3 staining exhibited an obvious increase of positive-staining rates in group 3, 4 and 5 as compared with groupi or 2. The staining density of Safranin O-Fast Green staining and immunohistochemical staining for type II collagen of group 5 was obviously higher than that of group 1 or 2.[Conclusion]RAdTIMP-3 can express widely and safely in rabbit intervertebral discs, and improve the quantity and quality of matrix. It has the potential to be used in treatment for intervertabral disc degeneration.