OBJECTIVETo study possible mechanisms of Shangjuxu (ST37) and the large intestine.

METHODSTotally 40 SD rats were selected. The distension of end colon was used as injured afferent stimulus. Activities of locus coeruleus (LC) neurons were recorded by extracellular microelectrode technique. Shangjuxu (ST37) and Hegu (L14) were needled to observe general features of discharge reactions, distention of colon induced discharge reactions of LC, and its effects on distention of colon induced discharge reactions of LC.

RESULTSDistention of colon could induce incrased discharge of LC neurons by 127.33% ± 45.48%. But needling at Shangjuxu (ST37) and Hegu (L14) could inhibit this injured response by 38.24% ± 7.69% and 21.29% ± 13.16% respectively (all P < 0.01).

CONCLUSIONSNeedling at Shangjuxu (ST37) and afferent signals of colon distension converged and interacted with each other. Needling at Shangjuxu (ST37) could significantly inhibit colon distension induced discharge of LC neurons, which might be one of mechanisms for Shangjuxu (ST37) and the large intestine relationship.

Acupuncture Therapy ; Animals ; Colon ; Intestine, Large ; Locus Coeruleus ; physiology ; Neurons ; Rats ; Rats, Sprague-Dawley

The purpose of the present study was to explore the effects of hypoxic exposure on mitochondrial adenine nucleotide translocator (ANT) activity and its characteristics. Male Wistar rats were exposed to hypoxia in a hypobaric chamber simulating high altitude at 5 000 m for 1, 5, 15 and 30 d. Control rats were fed outside the hypobaric chamber. Rats were sacrificed by decapitation and mitochondria from the cerebral cortex were isolated by differential centrifugation at each time point. The ANT activity was detected by the atractyloside (ATR)-inhibitor stop technique. Mitochondria was initiated by addition of (3)H-ADP and terminated after 12 s by quick addition of ATR. The radioactivity was measured in a liquid scintillation counter. Nonspecific binding of (3)H-ADP to mitochondria was estimated by incubation of mitochondrial samples with ATR prior to the addition of (3)H-ADP. This blank was substracted from the measured radioactivities. The activity of ANT was expressed as nanomoles (3)H-ADP per minute per milligram protein. The ANT density was determined by titrating the rate of state 3 respiration with increasing concentrations of carboxyatractyloside (CAT). Mitochondria were pre-incubated with CAT in a respiratory medium before ADP addition to initiate state 3 respiration. Plots of O2 consumption versus CAT appeared biphasic with an increasing inhibitory segment followed by a steady respiration, indicating that state 3 respiration was completely inhibited. The density of ANT was determined by the amount of CAT required to completely inhibit state 3 respiration, assuming a 1:1 binding stoichiometry, which was expressed as ANT density per milligram mitochondria protein. (ATP+ADP) in mitochondria was measured by high performance liquid chromatography (HPLC). The results showed that there was an obvious decrease in the ANT activity during hypoxic exposure. The lowest ANT activity was seen in 5 d group. Partial recovery of ANT activity was observed in 15 and 30 d groups, but ANT activity was still lower than that in the control group (P<0.01). Compared with that in normoxic control group, no change of ANT density in mitochondria was observed in hypoxia group (P>0.05). The turnover number of ANT in control, 1, 5, 15, 30 d groups were 16.67, 1.90, 0.40, 1.81, 4.73 pmol ADP/(min.pmol ANT), respectively. However, (ATP+ADP) in mitochondria in 1, 5, 15, 30 d groups were 63.37%, 48.44%, 52.73%, 60.16% of control group respectively. Therefore, the turnover number of energy production and expenditure were reduced. These observations suggest that the change of ANT activity may be one of the mechanisms of cellular oxidative phosphorylation dysfunction during hypoxic exposure.
Altitude ; Animals ; Atractyloside ; pharmacology ; Cerebral Cortex ; metabolism ; physiopathology ; Hypoxia ; metabolism ; physiopathology ; Male ; Mitochondria ; metabolism ; Mitochondrial ADP, ATP Translocases ; metabolism ; Rats ; Rats, Wistar

Objective To observe the effect of iodine excess(HI),polyinosinic-polycytidylic acid[Poly(I:C),Poly]and thyroglobulin(TG)on the thyroid of mice by the expression of Toll-like receptor 3(TLR3)to reveal the functional role of TLR3 in autoimmune thyroiditis.Methods Forty-two non-obese diabetic mice,body weight (20±3)g,were divided into six groups:control group,HI group,Poly group,TG group,HI+TG group,HI+Poly group. Fed with deionized water and injected intraperitoneally with physiological saline 0.1 ml each day for a week, the mice in control group were injected with physiological saline every other day at the same dose for 1 week before they were sacrificed; HI group drank 0.05% NaI water and were injected intraperitoneally with physiological saline same as control group; Poly group drank deionized water and were injected intraperitoneally with poly 0.1 ml (1 g/L)each day of the week, then the mice were injected with Poly every other day at the same dose for 1 week before they were sacrificed; TG group drank deionized water and were injected intraperitoneally with physiological saline same as control group, immunized with 0.1 mg TG by subcutaneously injecting and the immunization was enhanced after they were fed half dose for 4 and 8 weeks separately. In HI + Poly group, the treatment was the same as HI group and Poly group; HI + TG group: the treatment was the same as HI group and TG group. Eight weeks later, mice were sacrificed and thyroids were taken to make frozen sections, Hematoxylin-Eosin (HE) staining was employed to observe the morphological change of the thyroids. The expression of TLR3 of thyroids was observed under fluorescence microscope after Immumofluorescence using TLR3 antibody and TR3-positive cells were analyzed in the thyroid density. Results HE staining showed thyroids of Poly group had no inflammation under microscope.There were different degrees of inflammatory cell infiltration in HI group and TG group. The inflammatory cell infiltration and the damage of follicular thyroid of HI + TG group and HI + Poly group were serious, and the degrees of inflammation were higher over "++". Thyroid follicular epithelial cell with TLR3 expression could be seen in Poly group and HI group, meanwhile, there were TLR3 strong positive inflammatory cells in HI group under fluorescent microscope. Using stereological analysis of TLR3-positive cell density in the thyroid, the difference between groups was statistically significant(F=7.870, P<0.01 ). TLR3-positive cell density in the thyroid of HI + Poly group was higher[ (9.287 ± 0.522)mm2] than control group[ (0.062 ± 0.025)mm2, P < 0.01] significantly, meanwhile, the density in HI + Poly group was higher than HI group [ (2.574 ± 0.257 )mm2] and Poly group[ (1.361 ± 0.148 )mm2, all P < 0.01]. The density in HI + TG group[ (4.843±0.405)mm2] was higher than HI group and TG group[(1.601 ±0.268)mm2, all P < 0.01 )]. Conclusions Excessive iodine and thyroglobulin can induce thyroiditis, and stimulate the expression of TLR3 in the thyroid follicular epithelial, Poly aggravated thyroiditis induced by iodine excess in NOD mice; TLR3 positive inflammatory cells also appeared in inflammatory region, suggesting that TLR3 is involved in the pathogenesis of autoimmune thyroiditis

Objective To investigate the influence of iodine excess on expression of TRAIl/TRAIL-sR1 in NOD and Balb/c mice and to study the effect of TRAIl/TRAIL-sR1 on the pathogenesis of experimental autoimmune thyroiditis(EAT). Methods Both Balb/c and NOD mice were divided randomly into control and iodine excess group by feeding with water containing no NaI or 0.05% Nal. The mice were sacrificed after 8 weeks. TRAIL and TRAIL-sR1 mRNA levels were detected by RT-PCR. The function, morphology and apoptosis of thyroids were also observed by ELISA and Tunnel stain. Results Treated by HI, enlarged follicles and flattened epithelium by accumulation of colloid were found in thyroids of both NOD and Balb/c mice. But significant lymphoid cell infiltration and local fibrosis were only found in thyroids of NOD HI group. The relative weight of thyroids of NOD mice in HI group[(104.8±14.5)mg/kg]was heavier than that of control group [(71.8±20.4)mg/kg]. The level of TT4 declined in HI group[(30.77±3.59)mmol/L]compared with control group[(36.43±2.66)mmol/L], meanwhile, the level of TSH was higher in HI group[(6.98±0.66)μg/L]than that in control group [(5.55±0.56)μg/L]. The difference being statistically significant(t=7.773,-9.526,-4.458, all P < 0.05). The relative weight of thyroids of Balb/c mice of HI group[(155.8±20.8)mg/kg]also heavier than that of control group [(105.1±22.0) mg/kg]. The level of TT4 droped in HI group [(19.75±3.32) mmoL/L]was higher than that in control group[(23.46±6.21)mmoL/L], the level of TSH in HI group[(4.14±1.71)μg/L]was higher than that in control group[(3.55±1.41)μg/L], the difference being statistically significant(t=7.554,-7.239,3.140, all P< 0.05). A great deal of apoptotie ceils observed in NOD (3.97±0.91) and Balb/c mice (1.05±0.45) by Tunnel stain were greater than control groups (0.21±0.15, 0.10±0.03), the difference being statistically significant in beth of the two species(t=-7.167,-17.772, both P < 0.05). The apoptosis index of thyroid follicular epithelium in NOD was obviously higher than Balb/c(t=-7.625, P<0.05). The level of TRAIL mRNA did not remarkably change in Balb/c between control group(0.000 59±0.000 39) and HI group(0.001 24±0.000 46, t=-1.940, P>0.05), but it increased apparently in NOD mice HI group(0.018 88±0.005 77) than that of control group(0.009 61± 0.00591, t=-2.71, P<0.05). The level of the expression of TRAIL-sR1 mRNA increased in HI groups of NOD (0.000 53±0.000 15) and Balb/c mice(0.000 42±0.000 09) than that in control groups of NOD(0.000 28± 0.000 05) and Balb/c mice (0.000 17±0.000 06) and the differences were statistically significant between the two species(t=3.050,3.990, all P<0.05). The differences of the expression of TRAIL and TRAIL-sR1 mRNA between the two species were significant(t=-3.37,-4.76, all P<0.05). Conclusions Iodine excess induces colloid goiter in beth species of mice and thyroiditis in NOD mice. The increase of TRAIL and TRAIL-sR1 influenced by iodine excess is one of the molecular bases of follicular epithelium apoptosis and inflammation in thyroids. Genetic factor is a key factor in the pathogenesis of thyroiditis.

Aim To improve the rate of melanoma metastasis animal model and provide a reliable experimental modeling meth-od for the study of melanoma metastasis mechanism. Methods Five immunosuppressants were selected and then their targets were screened by network pharmacology. Intraperitoneal injection of immunosuppressants and intravenous injection of B16F10 cells were performed on C57BL/6 mice, then the mice were observed, and body weight were recorded daily. The contents of TNF-α, IL-6, IL-10, VEGF and MMP-9 in serum were measured with ELISA kit. The mice were dissected, then the metastasis situa-tion and the number of metastatic nodules in the lung and other organs were evaluated. HE-staining was involved to determine the morphology of metastatic tissues. Results The closeness centrality of cyclosporine and dexamethasone ranked the top and the targets were concentrated in lung and liver. No significant difference in body weight were observed after animal madel in-duced. The contents of TNF-α, IL-6 IL-10, VEGF and MMP-9 in the model group increased. The number of metastatic nodules in the lung significantly augmented with some kind of liver metas-tasis. HE-staining in the lung tissues showed that tumor presen-ted invasive growth. Conclusion By intraperitoneal injection of dexamethasone, the success rate of mice melanoma metastasis model can be greatly increased.

Adult ; Cytokines ; Humans ; Lymphocyte Subsets ; Lymphohistiocytosis, Hemophagocytic ; Lymphoma ; T-Lymphocyte Subsets ; Th1 Cells ; Th2 Cells

OBJECTIVETo study the apoptotic effect on the squamous cell carcinoma cell line TCa83 induced by recombined adenovirus vector containing TRAIL gene and CMV promoter.

METHODSThe TCa83 cell line was firstly infected with different titre of AdCMV-EGFP containing enhanced green fluorescence protein gene (EGFP) as control, and investigated the transducing rate through fluorescence to obtain the definite titre. Then TCa83 cell line was infected with AdCMV-TRAIL in proper titre, and TRAIL gene was detected by means of RT-PCR. After TCa83 cell line was infected with AdCMV-TRAIL and AdCMV-EGFP at day 1, 3, 5, 7, the activity of TCa83 cell line were evaluated by MIT and the apoptosis were detected by flow cytometer.

RESULTSProper titre was of 1,000 particles/cell, and TCa83 cell line could be infected 100% in this titre. TRAIL gene was detected by RT-PCR after infected with AdCMV-TRAIL. The activity of TCa83 decreased in both groups, but the AdCMV-TRAIL group decreased more sharply than AdCMV-EGFP group (P < 0.001). Both AdCMV-TRAIL and AdCMV-EGFP could lead to apoptosis of TCa83 cells, but the AdCMV-TRAIL, function stronger than AdCMV-EGFP. Especially there was remarkable statistic difference between two groups (P < 0.0001).

CONCLUSIONAdCMV-TRAIL could effectively decrease the activity of TCa83 cell line and induce apoptosis.

Adenoviridae ; Apoptosis ; Carcinoma, Squamous Cell ; Cell Line ; Genetic Vectors ; Green Fluorescent Proteins ; Humans ; Promoter Regions, Genetic

OBJECTIVETo investigate whether phenotypic modulation of bladder smooth muscle occurs in diabetic rats.

METHODSThirty-two male SD rats were randomly assigned into diabetic group and control group. Diabetic rat models were established by a single intraperitoneal injection of streptozotocin (60 mg/kg). Nine weeks later, the bladder tissues of the rats were examined for structural changes using HE and Masson's trichrome staining , and the expressions of myocardin, α-SMA, and SMMHC in bladder smooth muscles were detected with RT-PCR and Western blotting.

RESULTSCompared with the control group, the diabetic rats showed obvious polydipsia and polyuria with significantly increased collagenous fibers and lowered expressions of myocardin, α-SMA, and SMMHC in the bladder tissue (P<0.05).

CONCLUSIONs In rats at 9 weeks after diabetic model establishment, phenotypic transition of the bladder smooth muscles occurs to cause bladder contractile dysfunction, which may play an important role in the pathology of diabetic bladder dysfunction.

Actins ; metabolism ; Animals ; Diabetes Mellitus, Experimental ; physiopathology ; Male ; Muscle Contraction ; Muscle, Smooth ; physiopathology ; Myosin Heavy Chains ; metabolism ; Nuclear Proteins ; metabolism ; Phenotype ; Rats ; Rats, Sprague-Dawley ; Streptozocin ; Trans-Activators ; metabolism ; Urinary Bladder ; physiopathology

This study was aimed to investigate the cytogenetic changes of MDS cell line (MUTZ-1) with chromosome 5q deletion. R-banding analysis was used to identify chromosome aberrations in MDS cell line and Vysis Spectra Vysion M-FISH was used to further characterize chromosomal complex karyotype. The results indicated that M-FISH exhibited obvious chromosomal aberrations with high frequency including translocation, insertion, breakage and rearrangement, deletion and increasement of chromosome number, the complex karyotype of MUTZ-1 was shown as 50, xx, der (1) t (1;2), ins (1;14), +der (2) t(2;19), der (3) t (3;5), der (3) (3::5::22), 5q-, der (6) t (3;6), der (7) (18::7::17), +8, +der (9) t (1;9), der (10) t (1;10), +11, +12, der (?13) (10::13::5::8), der (14) t (8;14), der (14) t (14, 15), der (15) t (15;21) x 2, +17, +18, -21, -22. It is concluded that M-FISH analysis revealed obvious changes in complex karyotype of MDS cell line MUTZ-1, and the M-FISH technique can increase accuracy of detection for chromosomal complex karyotype, and help diagnosis and prognostic evaluation of MDS.
Child, Preschool ; Chromosome Aberrations ; Chromosome Deletion ; Chromosomes, Human, Pair 5 ; Female ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Karyotyping ; Myelodysplastic Syndromes ; genetics ; Translocation, Genetic ; Tumor Cells, Cultured

OBJECTIVETo prepare pneumolysin as a new protein carrier of vaccine against otitis media with genetic engineering technology and establish the base of the study on pneumococcal conjugative vaccines.

METHODSGenomic DNA was isolated from streptococcus pneumoniae. A pair of primers which included two restriction sites was designed based on the published pneumolysin gene sequence. The pneumolysin gene was amplified from pneumococcal DNA with PCR technology. The restriction enzyme digested fragment was linked into the cloning vector PET-28a and the recombinant plasmid DNA containing pneumolysin was then transfected into host cell E. coli JM109 (DE3).

RESULTSDNA fragments were subcloned to construct the complete pneumolysin gene by a conventional coning and PCR. The inserted pneumolysin gene sequence was confirmed by DNA sequencing and the pneumolysin protein was successfully expressed. The relative molecular mass of the expressed product was 52 000. The expressed product amounted to 8% of the total host cell protein.

CONCLUSIONSThe pneumolysin gene was successfully cloned into host cell using genetic engineering technology. The recombinant pneumolysin was expressed and purified for preparation. This work laid a foundation of the preparation of pneumococcal conjugative vaccines.

Bacterial Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Genetic Engineering ; Genetic Vectors ; Plasmids ; Pneumococcal Vaccines ; genetics ; Streptococcus pneumoniae ; genetics ; Streptolysins ; biosynthesis ; genetics