AIM: To investigate the effect of glibenclamide (Glib) on the viability and acid-base equilibrium of glioblastoma cells.METHODS: U251 cells and U87 cells were treated with Glib at different concentrations.The inhibitory rates were detected by CCK-8 assay.The effective dose was screened and the experiment was divided into control group and drug treatment groups.The migration ability was monitored by wound healing assay, and intracellular pH was detected by pH indicator fluorescent probe.The protein expression levels of inwardly-rectifying potassium channel 4.1 (Kir4.1) and monocarboxylate transport protein 1 (MCT1) were determined by Western blot.RESULTS: The half maximal inhibitory concentrations (IC50) of Glib for 48 h exposure of U251 cells and U87 cells were 400.20 μmol/L and 553.70 μmol/L, respectively.The effective inhibition doses of Glib for U251 cells were from the ranges of 100 μmol/L to 1 600 μmol/L, and those for U87 cells were from 50 μmol/L to 1 600 μmol/L in a concentration-dependent manner (P<0.05).Glib not only inhibited the migration (P<0.05) of U251 cells and U87 cells, which was negatively correlated with drug concentration (P<0.05), but also reduced the intracellular fluorescence intensity in experimental group (P<0.05), suggesting that with the increase in drug concentration, the intracellular pH decreased gradually (P<0.05).The protein expression of Kir4.1 and MCT1 was down-regulated by treatment with Glib, and was negatively correlated with concentration of Glib.CONCLUSION: Glib, a kind of potassium channel blocker, induces intracellular acidification via down-regulating the expression of Kir4.1 and MCT1, thus inhibiting the growth of glioblastoma in a certain dose range.

ObjectiveTo investigate the application of rapid immunohistochemical staining technique in intraoperative frozen section diagnosis of thyroid neoplasm.Methods MaxVision one-step rapid immunohistochemical staining technique was used to detect the expression of CK19,HBME-1,and Gal-3 in frozen section of papillary thyroid carcinoma(PTC)andthyroid benign lesions.MaxVision conventional immunohistochemistry of frozen remaining tissue was served as control.ResultsMaxVision one-step rapid immunohistochemical staining technique could be completed in 20 minutes.The positive localizations of three markers detected by rapid immunohistochemistry were similar to conventional immunohistochemistry, in general.The expression of CK19 was located in cytoplasm and cellular membrane.Gal-3 and HBME-1 were mainly detected in follicular luminal border and/or surface of papilla. The staining intensity in rapid immunohistochemistry was stronger than that in conventional immunohistochemistry. The positive rates of CK19,HBME-1,and Gal-3 by rapid immunohistochemistry in frozen sections were: 0 (0/28),10.7 ％ (3/28),0 (0/28),respectively,for benign lesions (nodular goiter,Hashimoto thyroiditis,thyroid adenoma); and 94.9 ％(37/39),92.3 ％ (36/39),92.3 ％ (36/39),respectively,for PTC.The expression of three markers between thyroid benign lesions and PTC had a significant difference (x2 =59.326,55.861,44.605,all P ＜ 0.001).In benign lesions,the rate of same case with two and more positive markers was 0,while in PTC it was 100 ％ and significantly different (x2 =67.000,P ＜ 0.05).ConclusionMaxVision one-step rapid immunohistochemical staining technique could be applied in intraoperative frozen section diagnosis.Detecting CK19,HBME-1,and Gal-3 expression in intraoperative frozen section has an auxiliary value for diagnosis of PTC.

Objective To observe the expression of fibronectin in bone of fluorosis rats and in vitro cultured osteoblast,and to study the role of fibronectin in pathogenesis of chronic fluorosis.Methods Male and female Wistar rats 144 were randomly divided into four groups,which were designated as the control group(normal diets,n =36),fluoride group(normal diets + 100 mg/L fluoride,n =36),lower calcium monophagia group (synthetic diets,n =36) and lower calcium monOphagia with fluoride group(synthetic diets + 100 mg/L fluoride,n =36).Rats were sacrificed 4 and 8 months after beginning of the experiment,respectively,and femur tissue was fixated and paraffin-embedded.The osteoblast isolated from calvaria of neonatal rats was treated with different dose of fluoride(0,1,2,4 mg/L fluoride,respectively) for 48 and 72 h,cell culture supernatant and cells were collected,respectively.The cranial osteoblasts were cultured in vitro and divided into four groups according to different concentration of fluoride added,which were 0(control group),0.01,1.00,and 10.00 mg/L groups.These cells were treated with mineralized induced medium at day 2 and cultured for 3 more weeks whereafter,and then the slides were fixed in alcohol.The expression of fibronectin in rat femur tissue was detected by immunohistochemistry (IHC),and fibronectin mRNA expression was determined by in situ hybridization; the fibronectin levels in supernatant of cultured osteoblast was examined by enzyme-linked immunosorbent assay(ELISA),and the expression of fibronectin mRNA in osteoblasts was detected with RT-PCR; skull mineralized nodule formation of osteoblasts was observed under a light microscopy after stained with 0.1％ red alizarin liquid.Results Little expression of fibronectin (brown granules under light microscope) could be seen in femur tissue of fluorosis rats of control group and lower calcium monophagia group; but abundantly expressed in fluoride group and lower calcium monophagia with fluoride group; the fibronectin was also expressed in osteoblasts,bone cells and bone marrow cells with less red particles in the control group and lower calcium monophagia group,but more in the fluoride group and lower calcium monophagia with fluoride group.The expression of fibronectin protein in supernatant of cultured osteoblasts was significantly increased in the group of 4 mg/L fluoride at 48 h(0.108 ± 0.042,t =0.764,P＜ 0.05) compared with control group(0.081 ± 0.010); the value was also significantly increased in 1,2,4 mg/L groups at 72 h(0.089 ± 0.010,0.087 ± 0.012,0.098 ± 0.023; t =0.765,0.704,0.996; all P ＜ 0.05) compared with control group (0.070 ± 0.014) ; the expression of fibronectin mRNA was much higher in 1,2,4 mg/L groups at 48 h (0.61 ±0.06,0.77 ± 0.07,0.77 ± 0.07) and 72 h(1.61 ± 0.14,2.54 ± 0.20,2.75 ± 0.22) compared with control group [0.48 ± 0.04(t =0.111,0.182,0.182,all P ＜ 0.05),0.97 ± 0.08(t =0.093,0.109,0.108,all P＜ 0.05) ].A lot of mineralized nodules could be seen under light microscope in 1.00 and 10.00 mg/L groups.Conclusions The expression of fibronectin in bone of fluorosis rats and in vitro cultured osteoblasts are increased,and fluoride also promotes the mineralization nodules formation of osteoblasts.These results suggest that fibronectin may regulate the process of bone mineralization,and possibly play a role in the development of skeletal fluorosis.

The 125 strains of the symbiotic and epiphyte microorganisms were isolated from marine organisms (Sea cucumber, Sea urchin, Sea anemone, Sea actinia, Ulra, Sargassum, Undaria). Among them,21 strains of bacteria,8 strains of actinomycetes and 2 strains of fungi have shown to have antagonistic activity on bacterial or fungal growth. In the 21 strains of bacteria, 7 strains belong to Bacillus sp., 11 strains belong to Vibro sp., and 3 strains belong to Pseudomonas sp.. In the 8 strains of actinomycetes, 5 strains belong to Streptomyces sp., 3 strains belong to Micromonospora sp.. 2 strains of fungi belong to Penicillum sp..

OBJECTIVETo study the therapeutic mechanism of Zhizhu Pill (ZP) for treating functional dyspepsia (FD) rats.

METHODSTotally 30 ten-day-old male rats were randomly divided into the normal control group (n =10) and the model group (n = 20). The FD rat model was induced using gastric administration of 0.1% iodoacetamide (IA) combined tail clamping. The model was evaluated when rats were 8-week old. Successfully modeled rats were randomly divided into the model group (n = 10) and the ZP group (n = 10). Rats in the normal group and the model group were administered with normal saline by gastrogavage, while those in the ZP group were administered with ZP Decoction (2 mL/100 g) by gastrogavage. All medication lasted for 7 successive days. The contractile activity in in vitro longitudinal gastric muscle was recorded using Power Lab biological signal collecting system. The expression of growth hormone secretagogue receptor (GHSR) in stomach of FD rats was detected using Western blot and immunohistochemistry (IHC).

RESULTSCompared with the normal group, average frequencies of gastric contraction and changing rates of amplitude obviously decreased in the model group (P < 0.05). Results of Western blot and IHC showed that the expression of GHSR decreased in the model group (P < 0.01). Compared with the model group, average frequencies of gastric contraction and changing rates of amplitude obviously increased in the ZP group (P < 0.05). Results of Western blot and IHC showed that the expression of GHSR increased in the ZP group (P < 0.01).

CONCLUSIONZP could promote the gastric motility in FD rats induced by gastric administration of IA combined tail clamping, and its mechanism might be related to up-regulating GHSR protein level.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Dyspepsia ; drug therapy ; Gastrointestinal Motility ; Male ; Muscle Contraction ; drug effects ; Muscle, Smooth ; drug effects ; metabolism ; Random Allocation ; Rats ; Receptors, Ghrelin ; metabolism

Neuritin is a new neurotrophic factor found recently. In order to identify the function of Neuritin clearly, the coding sequence of human neuritin was amplified by PCR from neuritin cDNA , this fragment digested by NocI and NotI was inserted into pET32a by T4 ligase and transformed into E. coli BL21 then the recombinant plasmid named pET32a-neuritin was constructed successfully . Neuritin was expressed distinctly after inducing by EPTG. The product was identified as neuritin by SDS-PAGE and Western blot analysis . The expression production was purified on Ni2+-NTA column.

Objective To explorethe effect of Bailingcapsule on epithelial-mesenchymal transition(EMT) inrats withadenine-in-duced tubulointerstitial fibrosis .Methods Tubulointerstitial fibrosis ani mal models were established and SDrats were dividedinto mo-del group (n=30) ,treatment group (n=30) andcontrol group(n=30) ,randomly .Experi mental rats were harvested at 7 w,12 w,17 wafter onset of experi ment and functional evaluations were performed. Histology ,i mmunohistology were examined to investigateboth histolopathology changes and the expression of bone morphogenic protein-7 (BMP-7) ,transforming growth factor-?1(TGF-?1)and a-smooth muscle actin (?-SMA) in kidneys at three ti me points mentioned above ,respectively .Results Compared with controlgroup ,24 h urinary proteinin model grouplost increasingly and significantly difference appeared at three ti me points relative to controlgroup(P0 .05) rel-ative to control group.There was significant difference at 12 wand 17 w(P

Objective To explore the effect of Bailing capsule on epithelial-mesenchymal transition( EMT) in rats with adenine-in-duced tubulointerstitial fibrosis. Methods Tubulointerstitial fibrosis animal models were established and SD rats were divided into mo-del group ( n = 30), treatment group ( n = 30) and control group( n = 30), randomly. Experimental rats were harvested at 7 w, 12 w,17 w after onset of experiment and functional evaluations were performed. Histology, immunohistology were examined to investigateboth histolopathology changes and the expression of bone morphogenic protein-7 (BMP-7), transforming growth factor-β1 (TGF-β1 )and a-smooth muscle actin (α-SMA) in kidneys at three time points mentioned above, respectively. Results Compared with controlgroup, 24 h urinary protein in model group lost increasingly and significantly difference appeared at three time points relative to controlgroup ( P ＜ 0.01 ). Urinary NAG in model group was markedly higher than that in control group from 7 w after onset (P ＜ 0.01 ) andwas increasingly raised at 12 w and 17 w (P＜0.01). The value of blood BUN and Cr in model group increased at 7 w (P＞0.05) rel-ative to control group. There was significant difference at 12 w and 17.w (P ＜ 0.01 ). Histologically, kidneys in model group, at 7 w,exhibited tubular casts and gently tubular dilation, granuloma in cortex, mononuclear cells infiltration in tubulointerstitial areas, andmild interstitial fibrosis. At 12 w, the degree of tubular injury and tubulointerstitial fibrosis gradually aggravated. Up to 17 w, diffusetubular dilation or atrophy was observed and focal tubules disappear. Diffuse interstitial fibrosis was exhibited. In normal kidneys, im-munohistochemistry suggested that the light expression of BMP-7 was detected in proximal renal tubular epithelial cells and marked ex-pression was identified in distal tubule, collecting duct, and renal tubular epithelial in junction area between cortex and medulla. How-ever, the expression of BMP-7 in kidneys of model group significantly decreased with increasing tubulointerstitial fibrosis and was nega-tive correlation with the expression of TGF-β1(r = -0. 981 P＜0.01) and α-SMA (r= -0.975 P＜0.01). Bailing capsule ad-ministration protected the expression of BMP-7 and reduced TGF-β1 and α-SMA expression before 12 w(P＜ 0.01 ). Conclusions Ourstudy shows an anti-fibrotic reno-protective function of Bailing capsule in rats with tubulointerstitial fibrosis via prevention of epithelial-mesenchymal transition at early stage. However, the beneficial effect lost with increasing tubulointerstitial fibrosis.

Objective To study the prevalence of the mtDNA A1555G gene mutation in Chinese population with nonsyndromic hearing impairment.Methods PCR-RFLP,directional sequencing of PCR products were applied in 325 patients with nonsyndromic hearing impairment and 50 normal controls.Results The mutation rate of the mtDNA A1555G was 14.5% (47/325),28 of 47 cases were homozygosis,19 of 47 cases were heterozygosis.The same mutation was not detected in the control subjects.Conclusion The mutation rate of the mtDNA A1555G is relatively high in the Chinese NSHI patients,the mutation type includes both heterozygosis and homozygosis.