OBJECTIVETo observe the changes of mitochondria stress in locus coeruleus and the tyrosine hydroxylasic projection after long-term sleep deprivation.

METHODSSleep deprivation mice model was set up by employing "novel environments" method. The expression of NAD -dependent deacetylase Sirtuin type 3 (SIRT3), which regulates mitochondrial energy production and oxidative stress, and heat shock protein 60 (HSP60), a major biomarker of mitochondrial stress, and the tyrosine hydroxylasic projection from locus coeruleus were analyzed after a 5-day sleep deprivation.

RESULTSCompared to the control group, the expression of SIRT3 in locus coeruleus was significantly decreased in respouse to long-term sleep deprivation, while the expression of HSP60 was significantly increased. In addition, relative to control group, pereentage area of the tyrosine hydroxylasic projection to anterior cingulate cortex was substantial decreased in long-term sleep deprivation group.

CONCLUSIONLong-term sleep deprivation induced the decreased level of SIRT3 expression and the elevation of mitochondrial stress in locus coenileus, which may further lead to the loss of tyrosine hydroxylasic projection in mice.

Animals ; Chaperonin 60 ; metabolism ; Locus Coeruleus ; metabolism ; physiology ; Mice ; Mitochondria ; metabolism ; Mitochondrial Proteins ; metabolism ; Oxidative Stress ; physiology ; Sirtuin 3 ; metabolism ; Sleep Deprivation ; Tyrosine ; metabolism

AIM: To construct eukaryotic expression vector of human vascular endothelial growth factor (VEGF) gene, and to explore the effects of VEGF gene on proliferation and chemosensitivity of leukemia cells. METHODS: The recombinant eukaryotic express plasmid pEGFP-C1-hVEGF_(165) was constructed with conventional gene engineering methods. The pEGFP-C1-hVEGF_165 was transfected into leukemia cell K562. The proliferation of the recombinant eukaryotic expression plasmid was analyzed by CCK8 method. The level of VEGF mRNA was detected by reverse transcription and quantitative polymerase chain reaction (RT-PCR). The expression level of VEGF protein was determined by ELISA in cell culture medium and cells. The cell cycle was detected by flow cytometry. RESULTS: VEGF_(165) eukaryotic expression vector was successfully constructed, confirmed by enzyme digestion and sequencing. Compared with the K562 cells transfected pEGFP-C1, the K562 cells transfected with pEGFP-C1-hVEGF_(165) grew faster and expressions of VEGF mRNA and protein increased significantly. In addition, K562 cells transfected with pEGFP-C1-hVEGF_(165) had relatively higher cell survival rate to chemotherapy drugs homoharringtonine(HHT) and fluorouracil(FU). CONCLUSION: VEGF_(165) eukaryotic expression vector increases the level of VEGF mRNA and protein expression, accordingly promotes the proliferation of leukemia cells and decreases the sensitivity of leukemia cells to HHT and FU.

Objective To investigate the effects of periplocin from Cortex Periplocae (CPP) on apoptosis of human lung cancer A549 cells and expression of survivin, and demonstrate its anti-tumor effect and the possible mechanism. Methods Inhibitory effect of CPP at different concentrations (1. 25, 2. 50, 5. 00, 10. 00, 20. 00 ng·mL-1 ) and for different time length (24, 48, 72 h) on A549 cell proliferation was tested by MTT method. Apoptosis rate of A549 cells treated with CPP at different concentrations (2. 50, 5. 00, 10. 00 ng·mL-1 ) were measured using flow cytometry (FCM) for 6, 12, 24, 48, 72 h, respectively. The morphological and ultrastructural changes of the apoptosis cells were observed by acridine orange/ ethidium bromide (AO/ EB) staining and transmission electron microscopy (TEM). The effects of CPP on mRNA and protein expression of apoptosis associated gene survivin were assessed by RT-PCR and Western blotting. Results CPP could significantly inhibit the growth of A549, and the inhibition rate reached (93. 46±2. 35)% . The results of FCM showed that the apoptosis rate of A549 cells treated with CPP was increased significantly as compared to the control group ( P<0. 05). Meanwhile, typical apoptotic peaks were detected. The characteristic morphological changes of apoptosis were observed in A549 exposed to CPP, including cell shrinkage, the nuclei became yellow-red by AO/ EB staining, and typical ultrastructural changes, including nuclear chromatin condensation along the nuclear membrane, vacuolar degeneration of cytoplasm observed by TEM. The result of RT-PCR indicated that survivin mRNA expression decreased obviously in A549 cells exposed to CPP. The protein expression of survivin in A549 cells treated with 10. 0 ng·mL-1 CPP(0. 251±0. 012)was weaker than that in control group(0. 928±0. 016). Conclusion CPP can induce apoptosis in human lung cancer cell lines A549, and the probable mechanism is related to the down-regulation of survivin mRNA and protein.

BACKGROUND:The early damaged chondrocytes are susceptible to de-differentiate and exert unstable phenotype during the in vitro culture, thus needing some growth factors.
OBJECTIVE:To observe the promotion effect of insulin-like growth factor 1 on the in vitro proliferation of chondrocytes in adult rabbits with traumatic arthritis.
METHODS:Traumatic arthritis models of adult rabbits were established by using the modified Hulth method. After the models were successful y established, the distal femur and proximal tibia were harvested under sterile conditions, the chondrocytes were cultured. The cultured cells were divided into two groups:control group was cultured with Dulbecco’s modified Eagle’s medium containing 10%fetal bovine serum, while experimental group was cultured with Dulbecco's modified Eagle’s medium containing 100μg/L insulin-like growth factor 1. The effect of insulin-like growth factor 1 on the proliferation of chondrocytes in adult rabbits with traumatic arthritis was determined through the cytomorphology, cellcounting, and cellactivity.
RESULTS AND CONCLUSION:The chondrocytes in adult rabbits with traumatic arthritis were successful y cultured, the majority of cells were mini-cells, presenting smal fusiform, round or polygonal shape. Hematoxylin-eosin staining showed that the number of cells in experimental group was higher than that in control group. MTT assay found that the absorbance of cells in experimental group was greater than that in control group (P<0.01). Our findings indicate that, insulin-like growth factor 1 can promote the in vitro proliferation of chondrocytes in adult rabbits with traumatic arthritis.

Objective: The purpose of this tudy was to dynamicly observe the weight and the bone mineral density (BMD) of the unsexed rabbits with a few self-made standardized phantoms. Methods:The eighteen healthy adult female rabbits were measured for their weight and BMD in preunsexed and postunsexed 5 months, 10 months with quantitative CT(QCT). Results:There were 61.1% of rabbits whose weight and BMD decreased after 5 months of the postunsexed and 100% of rabbits whose weight and BMD decreased after 10 months of the operation. Conclusion:QCT can be used to dynamicly observe curative effect of drugs in various periods as well,and it is a good method to study osteoporosis.

Objective To investigate the inhibitory effects of periplocin from cortex periplocae (CPP) on human lung cancer cell line QG56 and to discuss its mechanism. Methods QG56 cells were cultured in vitro. The final concentrations of CPP in control group were 1.25, 2.50, 5.00, 10.00 and 20.00μg/L. QG56 cells were treated with ascending concentration of CPP for 24 h, 48 h and 72 h. The cell proliferation was measured using MTT method. The morphological changes of QG56 cells were observed under inverted microscope. Flow cytometry (FCM) was used to detect the effects of CPP on cell cycle and cell apoptosis. The expression of apoptosis associated gene bax mRNA in QG56 cells was detected by RT-PCR. The expres-sion of bax protein before and after treatment of CPP was examined by SP immunocytochemistry. Results The inhibitory ef-fect of CPP on the proliferation of QG56 cells was increased with the increasing concentrations of CPP and the prolonged du-ration of treatment. The morphological changes were displayed in QG56 exposed to CPP. The results of FCM showed that CPP caused cell cycle arrest at G0/G1 phase. The apoptotic rate of QG56 cells was significantly increased after CPP treatment for 48 h (P<0.05). The expression of bax mRNA was increased in QG56 exposed to CPP. The result of immunocytochemis-try indicated that CPP up-regulated the expression of bax protein. Conclusion CPP showed significant inhibitory effect on human lung cancer cell lines QG56 through inducing cell cycle arrest and apoptosis.

Objective To improve the radiological diagnosis of breast cancer .Methods The authors collected 15 cases of breast carcinoma confirmed by operation and pathology. All these cases were misdiagnosed by mammography.Mammographic manifestations and data of clinic were analyzed retrospectively. Results 4 cases changed like small fibroadenoma, 3 cases showed microcalcification, 3 cases showed localized mammary gland architecture distrotion,2 cases showed multiple small patchy shadows in the hyperplasic mammary gland, 2 case showed small focus of increased density, 1 case no abnormal sign, 4 cases among of them can not be palpated mass .Conclusion The combination between palpation and radiology is valuable for diagnosis of breast carcinoma and decreasing misdiagnosis.

Objective To discuss the diagnostic value of two-dimensional speckle tracking imaging in the evaluation of left ventricular rotation and torsion in pre-apical hypertrophic cardiomyopathy(PAHCM)patients. Methods A total of 26 patients with PAHCM,26 patients with hyperten-sive left ventricular hypertrophy(HLVH)and 26 healthy volunteers were recruited for the study. Two-dimensional echocardiographs were performed carefully. Two-dimensional images were obtained from the left ventricular apical four-chamber section,two-chamber section and two short-axis sec-tions,including mitral valve and apical levels. The peak subendocardial rotation(endo-rot),epicardial rotation(epi-rot),transmural torsion(mural-tor)and bulk rotation(bulk-rot)of each short-axis section were measured and analyzed using QLAB 9.1 software. Results In the mitral level, there was no statistically significant difference in endo-rot,epi-rot,mural-tor and bulk-rot between the PAHCM group and normal control group (P>0.05). There was statistically significant difference in endo-rot,epi-rot,mural-tor and bulk-rot between the PAHCM group and HLVH group (all P<0.05). There was statistically significant difference in endo-rot,epi-rot,mural-tor and bulk-rot between the HLVH group and normal control group(all P<0.05). In the apical level,there was no statistically significant difference in epi-rot among three groups(P>0.05). There was statisti-cally significant difference in endo-rot,mural-tor and bulk-rot between each two groups(P<0.05). There was statistically significant difference in G-tor between each two groups(P<0.05). The receiver operating characteristic curve analysis showed that the accuracy of differential diagnosis of G-tor PAHCM was high(the area under the receiver operating characteristic curve is 0.89),the sensitivity was 73.08%,and the specificity was 92.31%. Conclusion Two-dimensional speckle tracking imaging could accurately and quantitatively measure the left ventricular rotation and tor-sion in PAHCM patients. The G-tor could accurately screen and identify between PAHCM and HLVH.

Objective: To investigate the effect of combining acupuncture and auricular point sticking on heart rate variability (HRV) in patients with post-stroke depression (PSD). Methods: A total of 80 cases with PSD were randomized into a treatment group and a control group. The control group was intervened by oral administration of paroxetine hydrochloride, whereas the treatment group received acupuncture plus auricular point sticking base on the same oral administration. The Hamilton depression rating scale (HAMD) and HRV were measured before and after treatment in both groups. Results: The individual and global scores of HAMD significantly dropped after 8 weeks of treatment in both groups (all P<0.05). In the treatment group, anxiety/somatization factor, sleep disturbance, hopelessness factor, cognition factor and global score were significantly different from those in the control group (all P<0.05). The 24 h standard deviation of all normal-to-normal R-R interval (SDNN), standard deviation of 5-minute average of normal R-R intervals (SDANN), root mean square of successive differences (RMSSD), percent of differences between adjacent normal R-R intervals >50 ms (PNN50) and high frequency (HF) were increased while low frequency (LF) and LF/HF decreased significantly after 8 weeks of treatment in both groups (P<0.05). All items in the treatment group were significantly different from those in the control group (all P<0.05). Conclusion: Combining acupuncture and auricular point sticking can enhance the conventional medical treatment for HRV in patients with PSD.

OBJECTIVETo improve the method for monitoring sleep state and respiratory rhythm of SD rats, providing a solution for rats' chewing on the wires, signal loss and instability problems in the animal model of sleep apnea syndrome (SAS).

METHODSWe improved monitoring electrodes of both electrocorticogram (ECoG) and electromyogram (EMG), signal circuit and animal operation.

RESULTSOperation time was shortened and wound exposure time was reduced, which made it easier for postoperative recovery. The ECoG and EMG signals were more stable with sharp image, and signal circuit lines had better conductivity and material durability, achieving continuous monitoring for a long time and high success rate. We could precisely distinguish the sleep wake state and the sleep apnea events in rats according to these signals.

CONCLUSIONThe improved method is more reliable and practical to test the small animal model of SAS, and is more easily to operate and the signals are more stable.

Animals ; Electroencephalography ; methods ; Electromyography ; methods ; Models, Animal ; Monitoring, Physiologic ; methods ; Rats ; Respiration ; Sleep ; Sleep Apnea Syndromes ; diagnosis