BACKGROUND:The clinical research have found that the interbervebral disc herniation often occurs in several members or even al the members of a family, and the location, reason and symptom are basical y the same, indicating that genes play an important role in this kind of disease. OBJECTIVE:To analyze the apoptosis Fas gene expression characteristics of lumbar disc in the familial patients with intervertebral disc herniation. METHODS:Semi-quantitative reverse transcription-PCR was used to test Fas gene expression of vertebral pulp and cartilage endplate in the intervertebral disc among 15 familial patients, 21 ordinary patients and five fresh cadavers. RESULTS AND CONCLUSION:Fas gene expression level of endplate of familial and ordinary patients with intervertebral disc herniation was higher than that of fresh cadavers, and there was no significant difference (P<0.05);there was no significant difference in Fas gene expression in endplates between familial patients and ordinary patients with intervertebral disc herniation (P>0.05). Compared with the vertebral pulps of ordinary patients with intervertebral disc herniation and fresh cadavers, there was no significant difference in the Fas expression of vertebral pulps of familial patients with intervertebral disc herniation (P>0.05). The increasing Fas gene expression may be secondary in the endplates of familial patients with intervertebral disc herniation, which can prevent intervertebral disc degeneration through preventing the endplate degeneration.

Objective To detect the expression of CD133 in hepatic cell lines SMMC7721 and bcl-7402, and to investigate the possibility of CD133 as the surface marker of liver cancer stem cells. Methods The cell cycle and expression of CD133 in hepatic cell lines SMMC7721 and bcl-7402 were detected by flow eytometry. Magnetic cell sorting was used to isolate CD133-positive and CD133-negative cells. The differences in morphology, proliferation and differentiation between CD133-positive and CD133-negative cells were observed and analyzed by one-way ANOVA and u test. Results The percentages of CD133-positive cells in SMMC7721 and bcl-7402 cell lines were 0.7% -1.0% and 1.7% -8.9%, respectively. The percentages of CD133-positive cells in G0>/G1> phase in the 2 cell lines were 85.3% and 89.4%, which were significantly higher than CD133-negative cells and unsorted cells (F = 14.49, 38.84, P <0.05). The in vitro proliferation capability of CD133-positive cells was greater than that of CD133-negative cells and unsorted cells, especially during day 1-3 and day 5-7 (F =49.32,784.04, 89.91, 152.83, P < 0. 05). During the cultivation, the proportion of the CD133-positive cells decreased as time passed by, and the proportion of CD133-positive cells was nearly the same as unsorted cells on day 15 (u =O. 271, P <0.05). Conclusions CD133-positive ceils have strong capability of proliferation and differentiation in SMMC7721 and bcl-7402 cell lines in vitro. CD133 is one of the surface markers of liver cancer stem cells.

A Review: A concept of tissue adaptation to hypoxia (i. e. hypoxic preconditioning) was developed and its corresponding animal models were reproduced in 1966s. The methods of model reproduction in rat, rabbit, and mouse in particular and the main results are briefly introduced in this review. The tolerance to hypoxia of preconditioned animals is significantly increased. Regular changes in animals' behavior, neurophysiology, respiratory and circulatory physiology, neuron morphology in vivo and function of brain and spinal cord in vitro are briefly demonstrated. The protective effects in vivo and in vitro of homogenate extract taken from the brain of preconditioned animals, neurochemicals and molecular neurobiological alterations are briefly presented. The essence and significance of tissue adaptation to hypoxia/hypoxic preconditioning are discussed in the review in terms of evolution and practical implication.
Animals ; Brain ; metabolism ; physiology ; Disease Models, Animal ; Hypoxia ; metabolism ; Mice ; Rabbits ; Rats

Actins ; metabolism ; Animals ; Collagen Type III ; metabolism ; Collagen Type IV ; metabolism ; Desmin ; metabolism ; Diabetes Mellitus, Experimental ; complications ; Diabetic Nephropathies ; metabolism ; pathology ; HSP47 Heat-Shock Proteins ; metabolism ; Kidney Glomerulus ; metabolism ; pathology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Vimentin ; metabolism

Objective To establish a method for detection of gene mutations in β-thalassemia by high-resolution melting (HRM) and study its preliminary clinical application.Methods Two common mutations [ IVS-2-654 ( C ＞ T ) and -28 ( A ＞ G ) ]of β-thalassemia in Wenzhou city population were selected.The plasmid DNA fragments of these mutations were constructed by TA clone technology as PCR templates or genotyping controls.A method for detection of β-thalassemia gene mutations based on HRM analysis was established and its specificity,sensitivity and repeatability were methodologically evaluated.One hundred and seventeen patients with clinically suspected β-thalassemia from Second Affiliated Hospital and Yu ying Children's Hospital of Wenzhou Medical College were enrolled into this study.The genomic DNA was extracted from whole blood cells and detected by HRM method.The results were compared with the direct sequencing data.Results HRM method could detect the mutations [ IVS-2-654( C ＞ T) and -28 ( A ＞ G ) ]of β-thalassemia and the results did not show any non-specific amplified fragments.All within-run and between-run coefficients of variation for different DNA types' Tm were smaller than 0.1％.And minimum 103 copies of DNA of each assay and 10％ mutation could be determined by this method.One hundred and seventeen patients with clinically suspected β-thalassemia were detected with HRM and all the results were in accordance with direct DNA sequencing.There were 45 IVS-2-654 ( C ＞ T)heterozygous mutation and 9-28 ( A ＞ G)heterozygous mutation and none homozygous mutation.Conclusion The method of rapid identification of β-thalassemia gene mutations based on HRM analysis is successfully established,which is a convenient,rapid,specific,sensitive and accurate technique for screening gene mutations in β-thalassemia as well as a general technical platform to identify other gene mutations.

Objective To establish a stable cell line secreting human IgE Cε2-4 protein, and in-vestigate the binding capacity of receptor FcεR Ⅰ Methods The E24 gene was derived from SKO-O07 cell line, and was then cloned into pcDNA3.1 (+) (signal peptides were synthesized and fused at the 5'-end of E24 gene) or pCMV-L vector. After transient transfection into 293T cell, the secreted F24 protein was ana-lyzed by sandwich ELISA. The best vector was chosen to be transfected into CHO cells with LipofectAMI-NETM 2000 reagent. After being selected by G418 and subcloned three times by limited-dilution method, two stable cell lines were established. E24 gene was amplified by RT-PCR, and the E24 protein in the superna-tant was identified by ELISA. Besides, the binding capacity of FceR ⅠⅡ was analyzed by flow cytometry method. Results Three mammalian expression vector SP-E24-F3. 1, SP lI-E24-P3.1 and E24-PL were constructed and transient transfected to 293T cells. The output of E24 protein in the supernatant were 19.1, 19.4 and 8.7 μg/ml, respectively. Then the vector SP IX-E24-P3.1 was transfected into CHO cells. Final-ly, two single clones secreting E24 protein were stably obtained. The output of E24 were all at least 25 μg/ml. RT-PCR could detect the E24 gene from one of the two clones. Furthermore, flow cytometry results showed that E24 could bind the receptor in a dose-dependent manner. Conclusion Two stable cell line se- creting E24 protein were obtained, while E24 could specifically bind FcεR Ⅰ.

Objective To study the growth suppressive effect of demethylation drug 5-aza-2'-deoxycytidine on bladder tumor cells. Methods The growth suppressive effect of DAC on 4 transitional cell carcinoma (TCC) cell lines was measured using the Cell Proliferation Reagent WST-1 assay.The effects of DAC on apoptosis induction and cell cycle arrest were analyzed by flow cytometric analysis. Caspase 3, 9 activities were analyzed by APOPCYTO Caspase Colorimetric Assay Kit and PCNA expression was also investigated by Western blot to clarify the mechanism of DAC against TCC. Results DAC inhibited the growth of all TCC cell lines tested in a dose-dependant manner, however,growth suppressive effect of DAC was independent of p53 status in TCC. DAC inhibited proliferation via inducing G2/M cell cycle arrest but not via inducing apoptosis. After treated with 0, 1 and 8 μmol/L DAC, cells of RTl 12 in G2/M phase was (36.3 ± 3.4) %, (46.2 ± 4.6) % and (56.5 ±6.2) %, TCCsup was (37.5 ± 3.8) %, (48.4 ±4.9) % and (60.1 ± 6.7) %, respectively. The expression of PCNA was decreased by DAC, but caspase3, 9 activities were not activated. Conclusion DAC could suppress the growth of TCC cells and might be a new strategy to treat bladder malignancy in the future.

Objective To investigate the effect of sevoflurane anesthesia on the cognitive function and phosphorylation of tau in hippocampal neurons in amyloid precursor protein (APP) transgenic mice.Methods Male APP gene mutation mice,weighing 18-22 g,aged 8-12 weeks,were used in the study.Forty-four APP positive mice were randomly divided into 2 groups (n =10 each):sevoflurane group (group AS,n =28) and control group (group AC,n =16).Forty-four APP negative mice were randomly divided into 2 groups:sevoflurane group (group S,n =28) and control group (group C,n =16).Animals in groups S and AS inhaled 3％ sevoflurane for 4 h.While in groups C and AC,animals inhaled pure oxygen for 4 h.Morris water maze was performed 24 h after sevoflurane or pure oxygen inhalation.The phosphorylation of tau at Ser262 and Ser396 was detected by Western blot on 1 day after pure oxygen inhalation (T1) in groups AC and C,and on 1,3 and 7 days after sevoflurane inhalation in groups AS and S.Results Compared with group C,the escape latency was significantly prolonged and the duration of staying at the original platform quadrant was shortened in groups S and AC,and the phosphorylation of tau at Ser262 in group S and phosphorylation of tau at Ser262 and Ser396 in group AS were increased (P ＜0.05).Compared with group S,the escape latency was significantly prolonged,the duration of staying at the original platform quadrant was shortened and the phosphorylation of tau at Ser262 and Ser396 was increased in group AS (P ＜ 0.05).Compared with group AC,the escape latency was significantly prolonged,the duration of staying at the original platform quadrant was shortened and the phosphorylation of tau at Ser262 and Ser396 was increased in group AS (P ＜ 0.05).Conclusion Sevoflurane anesthesia can aggravate the impairment of cognitive function in APP positive mice and the increase in the phosphorylation of tau at Ser262 and Ser396 is involved in the mechanism.

OBJECTIVETo measure the peripheral serum levels of CC-chemokine ligand-18 (CCL-18) in patients with pneumoconiosis, and to investigate the feasibility of the index asa potential biomarker for pneumoconiosis.

METHODSSeventy-seven male patients with pneumoconiosis (stage 1:40 cases, stage 2:22 cases, stage 3:15 cases), including 42 cases of silicosis and 35 cases of coal worker pneumoconiosis, were enrolled as subjects, and 162 healthy male physical examinees in our hospital were used as controls. A fasting blood sample (3 ml) was collected from the peripheral venous blood of each patient or control. The CCL-18 concentration in serum was measured by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe serum CCL-18 concentrations of the patients with pneumoconiosis were significantly higher than those of the controls [(116.70 ± 82.85) ng/ml vs. (83.34 ± 64.83) ng/ml]; (Z = -2.389, P < 0.05). The serum CCL-18 concentrations of the patients with silicosis were significantly higher than those of the patients with coal worker pneumoconiosis (147.02 ± 93.32 ng/ml vs. 96.43 ± 47.19 ng/ml; Z = -3.030, P < 0.05). There were no significant differences in serum CCL-18 concentration among different stages of pneumoconiosis (P > 0.05). The degree of respiratory impairment was positively correlated with the serum CCL-18 concentration in patients with pneumoconiosis (r = 0.611, P < 0.01).

CONCLUSIONSerum CCL-18 level can be used as a potential biomarker for pneumoconiosis.

AIM:To observe the efficacy of intravitreal conbercept injection for chronic central serous chorioretinopathy (CSC).METHODS: Nine eyes of 9 patients diagnosed as chronic CSC between October 2015 to May 2016 were treated with an intravitreal injection of conbercept (0.5mg/0.05mL) (six patients were given the same does of intravitreal injection again at 1mo after the first injection).Follow-up observation was at 1, 2, and 6mo after injection.Observed indicators included best-corrected visual acuity (BCVA), intraocular pressure, optical coherence tomography (OCT), fundus fluorescein angiography (FFA), choroidal indocyanine green angiography (ICGA), macular fovea thickness (CMT), subfoveal choroidal thickness (SFCT).RESULTS:Seven of the 9 patients responded significantly to the drug, while 2 patients had no response.The CMT was 373.12±72.43μm at baseline, which decreased significantly to 332.05±67.13μm, 282.24±62.30μm and 225.56±71.08μm at 1, 2 and 6mo after the intravitreal injection.The mean thickness of SFCT was 422.11±64.82μm before treatment.The choroidal thickness of non-responsive patients before treatment was below average, respectively 353μm and 365μm.The SFCT of 1, 2, and 6mo after treatment was 391.45±75.24μm, 365.53±63.07μm, 355.40±66.65μm.Before treatment and 1mo after, there was no significant difference (P=0.074), but there was statistically significant (P<0.01) between those of before and 2mo and 6mo after.The mean BCVA of the prior treatment was 0.53±0.32, the after treatment was 0.65±0.20, there was no different between the two(P>0.05).CONCLUSION: Intravitreal conbercept injection in chronic CSC may have some effect in accelerating subertinal fluid resolution and decreasing the CMT.The SFCT within 6mo after treatment was significantly lower than pretreatment.The SFCT may be an indicator of whether patients respond.