Objective To study the effect of selective cyclooxygenase-2 inhibitor celecoxib on cell apoptosis of human colorectal cancer cell line HT-29 and the probable mechanism involved by detecting the expressions of cytochrome C, Caspase-9 and poly ADP-ribose polymerase(PARP) at protein level. Methods Apoptosis was determined by Acridine orange and Ethidium bromide staining under fluorescence microscope and flow cytometry. The protein expression of cytochrome C, Capsase-9 and PARP were examined by Western blotting.Results Celecoxib induced apoptosis of HT-29 human colorectal cancer cells in a concentration and time-dependent manner from 0 to 120 ?mol/L. Sub-G 1 peak was detected by flowcytometry, and the apoptotic rate was between(7.31?2.37)%-(48.30 ?2.86)%. Celecoxib induced cytochrome C release into the cytosol from mitochondria, then activated Caspase-9 and consequently triggered PARP cleavage.Conclusion Celecoxib can induce apoptosis through a cytochrome C-dependent pathway in human colorectal cancer cell line HT-29.

Objective To design and prepare recombinant mutant human high mobility group box 1 protein (HMGB1s) that can combine with HMGB1 receptors but cannot produce inflammatory effect, and accordingly lead to the creation of a new potential agent for anti-inflammatory therapy. Methods This experiment was based on successful clone and expression of human HMGB1.Six mutant HMGB1 cDNA were designed and constructed by one step inverse PCR. They were cloned into prokaryotic expressive vector pQE80L and followed with production of mutant HMGB1s and identification by Western blotting. Results Six mutant proteins were designed and constructed into prokaryotic expressive vector pQE80L. The recombinant HMGB1 proteins were obtained and identified by Western blotting. Conclusion Human HMGB1 mutants have been successfully constructed and the expression and characterization of intent proteins are identified. It will lay a foundation for further study on biological functions of HMGB1.

Objective To study the effects of early enteral feeding on the preservation of intestinal mucosal barrier in severely burned patients. Methods Twenty-two patients with severe burn were randomly divided into early enteral feeding group (EF) and delayed enteral feeding group (DF). The levels of serum endotoxin and TNF-? were dynamically detected in the patients of both groups, and two unmetabolized sugars (lactose and mannitol) were orally administered in these patients on 1d, 3d and 5d of postburn. The concentrations of lactose and mannitol in urinary and the L/M ratio were observed. Intestinal permeability was assessed by the L/M ratio. Results The levels of serum endotoxin and TNF-? in severely burned patients were significantly higher than in normal (P

Objective To study the effect of a selective COX-2 inhibitor Celecoxib on cell proliferation and apoptosis of human coloretal cancer cell line HT-29 to seek an effective and safe drug for colon cancer chemoprevention. Methods Using MTT assay, flow cytometry(FCM), acridine orange and ethidium bromide staining, the effect of celecoxib on the proliferation and apoptosis of HT-29 cells were investigated. Results The growth of HT-29 cells was inhibited by celecoxib in a dose- and time- dependent manners. FCM analysis showed that the treated HT-29 cells had typical Sub-G 1 peak, the apoptotic rate of which was (7 31?2 37)%～(48 3?2 86)%. The cell ratio of G 0/G 1 phase increased, whereas the cell ratio of S and G 2/M phases decreased after treatment, which was in a dose-dependent manner as well. The treated HT-29 cells exhibited some morphological features of apoptosis, including cell shrinkage, nuclear condensation, DNA fragmentation, and the formation of apoptosis bodies, the apoptotic index of which was in a dose- and time- dependent manners. Conclusions Celecoxib inhibited proliferation and induced apoptosis of human colorectal cancer cell line HT-29, which may be related to blocking the cell cycle progress of HT-29 cells.

Objective To establish a method for in vitro induction and amplification of immature dendritic cells(DCs) with maturation resistance from human peripheral blood. Methods Mononuclear cells separated from peripheral blood were cultured with rhGM-CSF and rhIL-4 for 9 days, and rhIL-10 was added into medium at the 7th day. The suspending cells were examined with scanning electronic microscope and flow cytometry, and their ability for stimulating non-sensitized T lymphocyte proliferation was observed by mixed lymphocyte reaction(MLR). Cultured cells were stimulated with LPS and TNF-? for additional 2 days, respectively, and MLR was performed again. Results rhGM-CSF+rhIL-4-induced and IL-10-treated peripheral blood mononuclear cells(PBMC) exhibited typical morphological characteristics and immunological phenotype of DCs with high expression of CD1a and no expression of CD 83 on the cellular surface. Costimulating molecules CD 40 and CD 86 expressions were down-regulated.The capability of cultured cells for stimulating the proliferation of non-sensitized T lymphocyte was weak, and the same result was observed in cultured cells stimulated with LPS or TNF-?. Conclusion Immature dendritic cells with maturation resistance were obtained by culturing with IL-10,which might be a useful in the induction of immune tolerance of allogenic skin grafting for the major burn patients with deep burn wounds.

Objective To investigate the effect of c-fos antisense oligonucleotide on the phenotypes of vascular smooth muscle cells in venous autograftt. Methods The external jugular veins were grafted into abdominal aortic arteries in 20 Wistar rats which were divided into test group and control group randomly. The anastomosis and transplanted veins were coated with c-fos antisense oligonucleotide glue gel in the test group while the control group were merely coated with glue gel. The autografted veins were removed and measured by means of pathology and immunohistochemistry two weeks later. Meanwhile the conversion status of the vascular smooth muscle cells were observed with electron microscope. Results The myo-endothelial structure was observed clearly in test group while it was obscure in control group; the expression of c-fos、c-myc、PCNA in vascular smooth muscle cells was significantly decreased in test group. Conclusions The c-fos antisense oligonucleotide can influence the phenotypes of the vascular smooth muscle cells in autografted veins and inhibit the cells’ proliferation. All these indicate that it is a prospective genetic prophylactic therapy for the restenosis of autografted veins.

Objective To observe the effects of low frequency electrical stimulation (LFES) on the proliferation of endogenous brain neural stem cells (NSCs) and on the expression of basic fibroblast growth factor (bFGF)and epidermal growth factor (EGF) in rats with acute cerebral infarction; to explore the therapeutic mechanism of LFES in improving neural function. Methods Fifty-four rats were randomly divided into a LFES group, a placebo stimulation group and a sham-operated group. Each group was further divided into 3rd day, 7th day and 14th day subgroups, with 6 rats in each subgroup. An acute cerebral infarction model was induced in the rats of the LFES and placebo stimulation groups by middle cerebral artery occlusion (MCAO). Three days after the operation, rats in the LFES group began LFES treatment (frequency 30 Hz, pulse width 250 μs, current intensity 3 mA, 10 min/d) ,while the placebo stimulation group was treated identically but without electricity. The rats in the sham-operated group had no special treatment. The expression of nestin positive cells in the subgranular zone of the hippocampus and the subventricular zone was detected by immunohistochemical staining. The expression of bFGF, EGF proteins and mRNA in the ischemic hemisphere was detected by Western blotting and RT-PCR analysis. A screen test was applied to evaluate motor function. Results Nestin-positive cells in the subgranular and subventricular zones of rats in the LFES group increased significantly more than in the placebo stimulation group at the 7th and 14th day. The expression of bFGF, EGF proteins and mRNA in the ischemic hemisphere was up-regulated compared to the placebo stimulation group at the 7th and 14th day. At the 14th day a difference in motor function was observed in rats in the LFES group compared with the placebo stimulation group. Conclusion LFES can promote the proliferation of endogenous brain NSCs and the expression of bFGF and EGF in rats with acute cerebral infarction. It can also improve motor function and enhance neural plasticity in the brain.

Objective:To investigate the expression and clinical significance of growth differentiation factor 15 (GDF-15) in papillary thyroid carcinoma (PTC).Methods:The tumor tissues and metastatic lymph node tissues of 3 PTC patients who underwent radical surgery of thyroid cancer in the First Hospital of Hunan University of Chinese Medicine from January to February 2019 were collected, and the differential expressed genes were screened by high-throughput sequencing; 20 cases of primary tumor tissues and metastatic lymph node tissues were collected to verify the sequencing results. Another 20 cases of primary PTC tissues and adjacent tissues (>2 cm away from the tumor edge) were collected to verify the expression of target genes in tumor tissues and adjacent tissues. Sixty-four pathological specimens of PTC patients who underwent radical surgery of thyroid cancer in the First Hospital of Hunan University of Chinese Medicine from January to December 2014 were collected, of which 31 patients had lymph node metastasis. The real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of GDF-15 and verify the sequencing results; immunohistochemistry was used to detect the expression of GDF-15 protein in the primary PTC tissues, adjacent tissues and metastatic lymph node tissues. According to the expression of GDF-15 protein in the primary tumor tissues of PTC patients, the patients were divided into high-expression group (35 cases) and low-expression group (29 cases), and the relationship between GDF-15 expression level and clinicopathological characteristics of patients was analyzed; Kaplan-Meier method was used to analyze the 5-year tumor-free survival rate of the two groups.Results:The mRNA high-throughput sequencing results of 3 cases of PTC primary and metastatic tissues showed that the top 10 differential expressed genes were CDH2, CDF15, DKK1, GLIPR1, PCDH7, ID3, FBN1, MYPN, UBASH3B and CCDC80. The expression of GDF-15 mRNA in 20 cases of PTC primary tumor tissues and adjacent tissues were 4.1±0.5 and 2.8±0.3, and the difference was statistically significant ( t = 2.220, P = 0.032). The expression of GDF-15 mRNA in another 20 cases of PTC primary tumor tissues and metastatic lymph node tissues were 3.1±0.4 and 5.8±0.7, and the difference was statistically significant ( t = 3.556, P = 0.001). The results of immunohistochemistry showed that GDF-15 had the immunohistochemical scores of (4.0±0.3) points, (6.1±0.3) points and (9.0±0.4) points in PTC adjacent tissues, primary tumor tissues and metastasis tissues. The expressions of GDF-15 protein between PTC primary tumor tissues and adjacent tissues, metastatic tissues and adjacent tissues, and metastatic tissues and primary tumor tissues were significantly different (all P < 0.01). The differences in composition ratios of tumor long-axis diameter, tumor T stage and N stage between GDF-15 high-expression group and low-expression group were statistically significant (all P<0.05). The 5-year tumor-free survival rates in GDF-15 high-expression group and low-expression group were 60% and 83%, and the difference was statistically significant ( P = 0.033). Conclusions:The expressions of GDF-15 in PTC adjacent tissues, tumor tissues and metastatic lymph node tissues gradually increase, and its expression level is related to tumor progression, recurrence and metastasis. It can be used as a potential clinical prognostic warning molecule and therapeutic target.

Objective To study the changes of endothelin-1(ET-1) and calcitonin gene-related peptide(CGRP) in plasma of cerebral vasospasm(CVS) after resection of skull base tumors and the effect of the two factors on cerebral vasospasm. Methods Totally 34 cases were divided into symptomatic cerebral vasospasm group,asymptomatic cerebral vasospasm group and nonvasospasm group after resection of skull base tumors.The blood specimens were obtained from the 34 patients on days 1,3,5,7 and 14 after the resection.The concentration of ET-1 and CGRP was detected by radioimmunoassay;meanwhile,transcranial doppler was recorded.Another 10 normal adult served as control group. Results ① Concentration of ET-1 in plasma elevated from the 1st day after resection of skull base tumors,reaching peak levels on day 5 to day 7,then decreased gradually and nearly recoverd on day 14.Concentration of CGRP in plasma decreased from day 3 after resection of skull base tumors,with the lowest concentration on day 7,then increased gradually and recoverd on day 14.② Concentration of ET-1 in plasma of the three groups was higher than that of normal adult group,while concentration of CGRP of the three groups was lower than that of normal adult group.③ Concentration of ET-1 in plasma in vasospasm groups was higher than that in nonvasospasm group(P

Objective To analyze the prevalent feature and variant tendency of Scrub Typhus in Guangdong Province from 2006 to 2012, and provide a basis of strategy and measure on prevention and protection of Scrub Typhus. Methods Clinical information of all individual cases was gained from China Information System for Disease Control and Prevention. Cochran-Armitage trend χ2 test was used to describe the variance of Scrub Typhus incidence and the clinical cases of 2013 were predicted by autoregressive integrated moving average (ARIMA) model, while descriptive epidemiological analysis was adopted to describe the endemic distribution, seasonal characteristics and population distribution of Scrub Typhus. Results A total of 8 163 Scrub typhus cases were reported in Guangdong Province from 2006 to 2012. Annual mean incidence of the disease was 1.15/100 000 with the incidence showing an upward tendency (χ2 = 3 191.976, P < 0.01). There were 14 dead clinical cases reported with a fatality rate to be 1.72‰(14/8 163). Totally 3 166 people were predicted to infect Scrub Typhus in 2013 by ARIMA model. The disease was prevalent from the end of May to early October and the peak time was in late August (Z = 2 303.71, P < 0.01). The top five cities developed Scrub Typhus were Zhaoqing, Guangzhou, Yunfu, Shaoguan and Qingyuan. Male-female ratio was 1.00 ∶ 1.08 in all reported cases in which 52.78%(4 309/8 163) of them aged from 40 to 65 years old and 59.30%(4 841/8 163) of them were farmers. Conclusions The incidence of Scrub Typhus is rising in Guangdong Province, with the prevalent peak in summer and autumn, and rural populations are at high risk. The most effective preventions are strengthening health education , enhancing individual protection and protecting key populations in special districts.