Di 1 jun yi da xue xue bao = Academic journal of the first medical college of PLA最新文献

Objective: To investigate the effects of histamine on the proliferation, apoptosis and differentiation of human keratinocytes (HKC) and the mechanisms.

Methods: The effect of histamine on the growth of HKC in vitro was examined by MTT assay and trypan blue exclusion assay. Cell cycle analysis and early apoptosis analysis by double staining with Annexin V-FITC and PI were carried out by flow cytometry. DNA ladder assay was performed for the detection of cell apoptosis. HKC free calcium concentration ([Ca(2+)](i)) was measured by laser scanning confocal microscopy in combination with calcium fluorescence probe Fluo-3/AM. HKC differentiation markers keratin 10 (K10) and involucrin was detected by streptavidinbiotin complex immunocytochemical assay.

Results: Histamine at high concentration inhibited the proliferation of HKC with cell viability ratio of 65.6% at 1 x 10(-4) mol/L, while histamine at low concentrations promoted proliferation of HKC with the cell viability ratio of 130.7% at 1 x 10(-8) mol/L. Histamine at 1 x 10(-4) mol/L altered cell cycle distribution of HKC with an increase in G0/G1-phase cell population to 30.97%, a decrease in S-phase population to 73.81% and inhibition of G1/S switching. Histamine at 1 x 10(-4) mol/L induced obvious apoptosis of HKC with early apoptosis ratio of 18.64% as compared with the control (5.60%, P<0.05). Histamine 1 x 10(-4) mol/L induced an increase of HKC [Ca(2+)](i) up to 58.9% and cimetidine (an H(2) receptor antagonist) decreased HKC [Ca(2+)](i) down to 25.4%. Histamine at this concentration down-regulated the expressions of K10 and involucrin of HKC but these changes were not significantly different from those of the control (P>0.05).

Conclusions: Histamine at high concentrations inhibits cell cycle progress of HKC, mediates cell apoptosis and induces the increase of [Ca(2+)](i), which might be a partial explanation for growth arrest of HKC elicited by histamine. Histamine may regulate epidermal tissue turnover under physiological conditions, whereas under pathological circumstances of the skin as in trauma and inflammation, histamine at high concentrations may inhibit the regeneration of epidermis and the differentiation of HKC.

Objective: To observe the excitation and sensitization of dorsal cutaneous primary afferent fibers induced by P2X agonist alpha, beta-methylene ATP (alphabetame-ATP) in rats.

Methods: By means of single fiber electrophysiological recordings on the nerve filaments isolated from the dorsal cutaneous branches of the T(9)-T(13) spinal nerves, the effects of alphabetame-ATP (100 micromol/L, 10 microl) injection into the cutaneous receptive field on the mechanical threshold and spontaneous discharge of rat primary sensory afferent units were observed.

Results: The mean mechanical threshold of Adelta and C fibers prior to alphabetame-ATP injection were 0.384+/-0.018 and 0.943+/-0.102 mN, and lowered to 0.304+/-0.013 and 0.659+/-0.071 mN after the injection, respectively (P<0.05, P<0.01). The mechanical threshold of the Abeta fibers before alphabetame-ATP injection was 0.301+/-0.019 mN, and slightly lowered to 0.288+/-0.018 mN after the injection (P>0.05). Injection of alphabetame-ATP (10 microl) into the receptive fields evoked spontaneous discharge in 7.7% of the Abeta fibers, 66.7% of Adelta fibers and 75.0% of C fibres, respectively, and the proportions of Adelta and C fibers with spontaneous discharge evoked by alphabetame-ATP were significantly greater than that of Abeta fibers (P<0.05). In the control experiments, injection of saline did not significantly affect spontaneous discharge or excite the nerve fibers. The mean discharge frequency of Adelta and C fibers increased from 0.73+/-0.24 and 0.54+/-0.21 impulses/min before injection to 3.05+/-0.65 and 8.53+/-2.04 impulses/min during the injection, with subsequent reduction to 2.40+/-0.60 and 6.68+/-1.68 impulses/min in the following 5 min (P<0.05). In contrast to Adelta and C fibers, Abeta fibers exhibited no significant changes in the mean discharge frequency in response to alphabetame-ATP (0.23+/-0.09 impulses/min before injection, 0.28+/-0.09 impulses/min during injection and 0.22+/-0.14 impulses/min after injection, P>0.05). The excitatory effects of alphabetame-ATP on the discharge rate in Adelta and C primary afferent terminal could be observed in the entire course of experiment.

Conclusion: Peripheral application of alphabetame-ATP, an ATP analogue, excites and sensitizes a subpopulation of Adelta and C fibers but not Abeta fibers of rat dorsal cutaneous primary afferent fibers.

Objective: To investigate the relationship between QT dispersion and torsades de pointes (TDP) in idiopathic long QT syndrome (LQT1).

Methods: Seventy-nine carriers of the gene on the chromosome 11p15.5 were divided into 3 groups according to the severity and frequency of syncope episodes, namely the mild (0-4 episodes, n=59), moderate (5-100 episodes, n=14) and severe (with history of cardiac arrest, n=5). The differences in QT dispersion in the initial ECGs recorded were compared between the 3 groups, and the QT dispersion changes after chronic beta-blocker therapy in 19 moderate to severe cases under 35 years, along with the changes during exercise in 9 cases before and after beta-blocker therapy were evaluated and compared.

Results: QT dispersion was significantly different between the 3 groups (P<0.01), and after chronic beta-blocker therapy, QT dispersion decreased in 10 of the 13 cases (P<0.05). During the exercise test, the QT dispersion was positively correlated with the exercise load before beta-blocker treatment but inversely correlated after the treatment (P<0.01).

Conclusions: QT dispersion is a sensitive and reliable marker to predict TDP and beta blocker should be given to moderate or severe LQT1 cases under 35 years, especially in the asymptomatic cases at risk of TDP.

Objective: To study the PCR amplification, cloning and protein expression of interferon-inducible transmembrane protein-1 (IFITMP-1) gene.

Methods: With the cDNA fragment containing IFITMP-1 gene as template, IFITMP-1 gene was amplified using Pfu enzyme by means of PCR. After EcoRI and HindIII digestion, the target gene fragment was linked to pUCm-T plasmid and sequenced. The IFITMP-1 gene was cloned into pET-Trx protein expression plasmid, and the condition for protein expression was optimized.

Results: The length of the PCR product of IFITMP-1 gene-containing cDNA fragment was about 1000 bp. The IFITMP-1 gene was successfully inserted into pUCm-T plasmid with correct sequence and cloning of the IFITMP-1 gene into the pET-Trx protein expression plasmid was achieved. Expression of the fusion protein of pUCm-T plasmid and IFITMP-1 gene was detected after IPTG induction.

Conclusion: Successful amplification and cloning of the IFITMP-1 gene and its protein expression may facilitate further study of the role of IFITMP-1 gene in colorectal cancer.

Objective: To clone human epidermal growth factor receptor (HER2) gene and establish a MCF-7 cell line with stable co-expression of HER2 and estrogen receptor (ER).

Methods: The full-length HER2 gene fragment was amplified by RT-PCR from the total RNA extracted from human breast cancer cell line SKBR-3 which over-expressed HER2 protein. The gene fragment was then inserted into the vector pcDNA3.1 to construct the recombinant expression plasmid pcDNA3.1-HER2, which was identified by sequence analysis and transfected into ER-expressing MCF-7 cells via lipofectamine. The positive cell clones were obtained after G418 selection. The MCF-7 cells transfected with HER2-cDNA was assayed by immunocytochemistry, flow cytometry and Western blotting.

Results: DNA sequencing results showed that HER2 gene was exactly consistent with the sequence reported in GenBank. The MCF-7 cells transfected with HER2 could highly express HER2 protein as shown by immunocytochemistry, flow cytometry and Western blotting.

Conclusion: HER2 gene has been cloned successfully and the MCF-7 cell line co-expressing HER2 and ER is obtained.

Objective: To establish a 6-10B cell line with stable expression of KIAA1173 gene and study the biological behaviors of the cells.

Methods: The total RNA was extracted from normal skeletal muscular tissues for cloning of KIAA1173 gene by means of RT-PCR which was subsequently introduced into pcDNA3.1 (+) vector. The recombinant eukaryotic expression vector pcDNA 3.1(+)-KIAA1173 was constructed and identified by endonuclease digestion and sequencing before transfection into 6-10B cells via lipofectamine with the empty vector as the control. The positive cell clones were obtained by G418 selection. Stable expression of KIAA1173 gene in the transfected 6-10B cells was determined by RT-PCR, in situ hybridization and immunocytochemistry, and the biological behaviors of the transfected cells were observed by MTT assay, cell invasion assay and tumorigenesis assay in nude mice.

Results: High expression of KIAA1173 at both mRNA and protein levels was observed in the transfected 6-10B cells. The capability of proliferation, invasion and tumorgenicity of the KIAA1173-transfected cells in nude mice was lowered in comparison with those of the cells transfected with pcDNA3.1 (+) vector (P<0.05).

Conclusions: KIAA1173 genes may function as a potential tumor suppressor of nasopharyngeal carcinoma both in vitro and in vivo. The 6-10B cell line expressing KIAA1173 has been obtained, which can be helpful for further study of KIAA1173 gene.

Objective: To study the changes of heat shock protein 70 (HSP70) in the brain of rabbits with craniocerebral missile injury (CMI) and their impact on the injury in a hot and humid environment (HHE).

Methods: Twenty-four New Zealand white rabbits were randomized into 3 equal groups, namely normal temperature (NT) control group (subjected to treatment at temperature of 22.0+/-0.5 degrees C with relative humidity of 50%), NT gunshot group with CMI and HHE gunshot group with CMI. The rabbits in the latter two groups were subjected to normal temperature of 22.0+/-0.5 degrees C with relative humidity of 50% and environmental temperature of 39.0+/-0.5 degrees C with relative humidity of 80% to 85%, respectively, after establishment of CMI models. HSP70 in the brain tissues and lymphocytes was detected by Western blotting and visualized using chemoluminescence and X-ray films, followed by quantitative gel image analysis.

Results: Expressions of HSP70 in the cortex, hypothalamus and lymphocytes increased apparently in HHE gunshot group, and the changes of HSP70 in the lymphocytes were consistent with those in the cerebral cortex and hypothalamus.

Conclusion: HSP70 expression in the brain tissues and the lymphocytes increase evidently after MCI in HHE.

Objective: To observe the effect of lactone I from Atractylodes macrocephala Koidz (LAMK I) on the cytokines and proteolysis-inducing factors (PIF) in cachectic cancer patients.

Method: Sixty-four cachectic cancer patients were randomized into two groups, namely LAMK I group and FOE (fish oil-enriched nutritional supplementation) treatment group. The appetite, body weight changes, mid-arm muscle circumference (MAMC), and KPS scores were recorded 3 and 7 weeks after the treatment. Immunohistochemistry was used to analyze the changes in the cytokines interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-alpha and Western blotting employed to examine the changes of PIF after the treatment.

Results: The patients' appetite and MAMC in LAMKI group was better than those of patients in FOE group, and the serum IL-1 and TNF-alpha levels and urine PIF level were significantly lower than those of FOE group. Body weight and serum IL-6 level were not significantly different between the two groups.

Conclusion: Lactone I from Atractylodes macrocephala Koidz can be beneficial for treating cancer cachexia.

Objective: To study the effect of deoxycholate in inducing apoptosis of human normal esophageal mucosal epithelial cells in vitro and investigate the molecular mechanism.

Methods: Cultured normal human esophageal mucosal epithelial cells were treated with deoxycholate, and the cell apoptosis were evaluated with TUNEL, DNA ladder, flow cytometry with PI-staining, Annexin V-FITC conjugated with PI staining, and Western blotting.

Results: Flow cytometry, TUNEL and DNA ladder demonstrated that deoxycholate could induce apoptosis in normal human esophageal mucosal epithelial cells in a dose- and time-dependent manner. A percentage of 21.3% of the cell population treated with deoxycholate at 500 micromol/L for 30 min exhibited detectable caspase-3 activity shown by flow cytometry, which was significantly higher than the control level (1.5%, P<0.01). Western blotting suggested that deoxycholate down-regulated Bcl-2 protein expression and up-regulated Bax expression, but Fas was negative in the cells before and after deoxycholate treatment.

Conclusions: Deoxycholate could induce apoptosis in cultured human esophageal mucosal epithelial cells. Aaspase-3 activation, Bcl-2 protein down-regulation and Bax up-regulation are involved in deoxycholate-induced apoptosis, which does not involve Fas-L/Fas.

Objective: To evaluate the biocompatibility of polybutylcyanoacrylate nanoparticles (PBCA-NP) manufactured by emulsion polymerization technique.

Methods: MTT assay was performed for evaluation of the cytotoxicity of PBCA-NP (e.g. cell proliferation and hemolytic test) experiments of long-term subcutaneous implantation and implantation in the muscular tissues of PBCA-NP was conducted in rabbits to observe the inflammatory reactions due to the implantation.

Results: No obvious cytotoxicity or hemolytic reactions were observed (with the hemolytic rate at different concentrations of PBCA-NP(5%). Three months after PBCA-NP implantation, obvious lymphocyte infiltration was not observed in the tissues around the implants which underwent gradual degradation.

Conclusion: PBCA-NP possesses good biocompatibility.