Objective To investigate the role of Ca 2+ signaling in the overexpression of detrusor smooth muscle cell connexin 43 by cyclic stretch in vitro. Methods The detrusor smooth muscle cells (DSMC) grown on collagen-coated silicone membranes were subjected to cyclic stretch-relaxation. The Cx43 mRNA in DSMC were detected with RT-PCR, and the concentration of intracellular free Ca 2+ in DSMC was measured by confocal microscopy in conjunction with the calcium indicator, Fura-3 (Molecular Probes). Results The overexpression of Cx43 mRNA induced by cyclic stretch was significantly inhibited by EGTA.The increase of intracellular free Ca 2+ induced by stretch was also inhibited completely by EGTA, 61.95% by GdCl3, 29.98% by Nifedipine, 87.98% by Ryanodine and Thapsigargin. Conclusion The stretch-induced Ca 2+ entry, via the Ca 2+-induced Ca 2+ release mechanism, may play an important role in DSMC connexin 43 overexpression induced by cyclic stretch.

Objective To investigate the pathological features of sural nerve biopsy in patients with motor neuron disease （MND）. Methods 22 patients with amyotrophic lateral sclerosis (ALS) underwent sural nerve biopsy and routine electrophysiological examination. The transected images were captured and morphometrically analyzed.Results The myelinated fiber density decreased in ALS patients' sural nerves, and larger fibers were involved mostly. The thinly myelinated fibers increased. Conclusion The sural nerves of ALS patients shows mild but definite pathological changes.

Objective To improve the recognition for infantile spinal muscular atrophy (SMA-Ⅰ) and the level of early diagnosis,intervention and treatment for SMA-Ⅰ.Methods The clinical data of 39 patients with SMA-Ⅰ were analyzed retrospectively, including the clinical manifestations, neural electrophysiological characteristics, geno-type, diagnosis,treatment and prognosis of SMA-Ⅰ.Results Of the 39 cases with SMA-Ⅰ , 37 cases (94.9％) had onset in 6 months after birth.The paralyses of the limbs were symmetrical and flaccid.The lower was more severe than the upper , and the proximal was more severe than the distal, hypotonia and tendinous reflex disappears.Thirty-two cases (82.1％) had normal serum creatine kinase, and 7 cases (17.9％) increased slightly.Nerve electrophysiological examination showed that 169 (96.0％) had spontaneous potentials in 176 muscles.Of 160 limb muscles,35 (21.8％) released few motor unit potential (MUP) ,117 (73.1％) extended the duration of MUP and 104 (65.0％) increased the amplitude of M UP.Of 167 peripheral motor nerves, 160 (95.8％)decreased the amplitude of the compound muscle action potential and 162 (97.0％) had normal motor conduction velocity.Of 93 peripheral sensory nerves, 93 (100.0％) had normal range of the conduction.The gene detection showed that 38 cases had homozygous deletion of exon 7,8, and 1 case had homozygous deletion of exon 7 and heterozygous deletion of exon 8.In the effective follow-up of 30 cases,6 cases died in the 2-3 months after birth,4 cases died in 10 months after birth, 12 cases died in 12-18 months after birth.Six cases survived to 2 years old,2 cases survived to 3 years old,and all of them died of pulmonary infection.Conclusions There are typical clinical and nerve electrophysiological characteristics for SMA-I.Nerve electrophysiological examination can be used as an important method for diagnosis and differential diagnosis for SMA-I.Genetic testing can be used to identify the disease and make prenatal diagnosis.Through comprehensive intervention the quality of life can be improved in SMA-Ⅰ children.

After oral administration of Salvia miltiorrhiza (Danshen in Chinese), Panax notoginseng (Sanqi in Chinese) and Danshen Sanqi combination suspensions to Beagle dogs, the plasma concentration-time profiles of danshensu, tanshinone II(A), cryptotanshinone, notoginsenoside R1, ginsenoside Rg1 and Rb1 were analyzed by LC-MS/MS. Pharmacokinetic parameters were calculated and analyzed with BAPP 2.0 software. The results showed that the Cmax and AUC of danshensu, notoginsenoside R1, ginsenoside Rg1 and Rb1 in Danshen Sanqi combination group all decreased in comparison with those of Danshen or Sanqi given alone, while the CLz/F and Vz/F increased to some extent. No significant differences of the pharmacokinetics of tanshinone II(A) and cryptotanshinone were observed between groups.

Functional magnetic resonance imaging(fMRI),as a noninvasive technology of measuring human brain function,has been used in neurolingustic research on normal subjects and on patients with brain damage,to investigate the neural basis underlying language processing.Such studies complement and development the classical knowledge accumulated on aphasia,provide new insights into the neural mechanisms of aphasia and the plasticity of recovery from aphasia,and might be helpful to evaluate the capability of language functional recovery and the efficiency of rehabilitative strategies also.

Microscopy, Electron, Transmission ; methods ; Negative Staining ; methods ; Viruses ; ultrastructure

Objective To observe the effects of Yiqi Huoxue Tongluo Jiedu fang (YHTJF) on pneumocyte apoptosis after lung ischemia/reperfusion (I/R) injury (LIRI) in mice and to investigate whether c-Jun N-terminal protein kinase (JNK) is involved in the mechanism of apoptosis.Methods Seventy C57BL/6J male mice were randomly divided into seven groups:normal control group (C group),carboxyl methyl cellulose-Na+normal control group (CMC-Na+C group),CMC-Na+sham group (CMC-Na+S group),CMC-Na+I/R group (CMC-Na+I/R group) and CMC-Na+YHTJF-low,-middle,-high dose groups (CMC-Na+YL,CMC-Na+YM,CMC-Na+YH groups).C group did not undergo any processing;in CMC-Na+S group,only was chest opened without clipping the lung hilum;in the rest of the four groups,they all underwent opening of the chest and clipping the lung hilum for 30 minutes,then the clipping of artery was relieved and left lung reperfusion was carried out for 3 hours.After operation,the mice were sacrificed,the lung tissues were harvested.Under light and electron microscopes,the lung morphological and ultra-structural changes were observed,and the changes of index of quantitative evaluation for alveolar damage (IQA) were determined.The terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) was applied to evaluate the apoptosis index (AI) of the lung tissues.The protein and mRNA expressions of JNK and glucose regulating protein 78 (GRP78) in lung tissues were detected by Western Blot and reverse transcription-polymerase chain reaction (RT-PCR);the correlations between lung AI and the expressions of mRNA and protein of JNK and GRP78,IQA were analyzed.Results Compared with CMC-Na+S group,IQA,AI and mRNA and the protein expressions of JNK and GRP78 in CMC-Na+I/R group were obviously higher [IQA:(74.00 ± 7.31)％ vs.(7.00 ± 1.23)％,AI:(64.40 ± 11.97)％ vs.(5.60 ± 1.14)％,JNK mRNA (gray value):1.143 ± 0.284 vs.0.152 ± 0.128,GRP78 mRNA (gray value):0.897 ± 0.129 vs.0.284 ± 0.044,JNK protein (A value):0.428 ± 0.074 vs.0.073 ± 0.052,GRP78 protein (A value):1.075 ± 0.145 vs.0.589 ± 0.060].Compared with CMC-Na+I/R group,the IQA,AI,protein and mRNA expressions of JNK and GRP78 in CMC-Na+YL,CMC-Na+YM,CMC-Na+YH groups were all lower,and the degree of reduction in group CMC-Na+YM was the most remarkable,greater than that in CMC-Na+YL or CMC-Na+YH group [IQA:(26.20 ± 3.35)％ vs.(34.00±5.34)％,(41.20±9.18)％,AI:(29.40±3.05)％ vs.(48.20±3.83)％,(39.20±6.14)％,JNK mRNA (gray value):0.681 ± 0.130 vs.0.804 ± 0.153,0.938 ± 0.11,GRP78 mRNA (gray value):0.450 ± 0.105 vs.0.747 ± 0.231,0.566 ± 0.115,JNK protein (A value):0.188 ± 0.049 vs.0.261 ± 0.065,0.209 ± 0.063,all P ＜ 0.01],compared with the CMC-Na+I/R group,the expression of GRP78 protein was obviously higher in CMC-Na+YH,CMC-Na+YL,CMC-Na+YM groups and the most remarkably high was in CMC-Na+YH group (A value:1.429 ±0.226 vs.1.130±0.169,1.128 ±0.177,all P ＜ 0.01).The apoptosis of each group was mainly in the pulmonary vascular endothelial cells and alveolar epithelial cells,and brown particles were positive cells under light microscope.Under transmission electron microscope:nuclear pyknosis and margination under the nuclear membrane,cytoplasm condensed,lamellar bodies decreased and emptying increased,cell membrane microvilli decreased or disappeared,mitochondria swelling,inflammatory cells increased in alveolar septum and adhering onto the capillary walls could be seen in CMC-Na+I/R group.Compared with CMC-Na+I/R group,the lung tissue ultrastructural damage alleviated,ultrastructure of alveoli clearly seen,nuclear chromatin relatively uniform,cytoplasm increased,type Ⅱ alveolar epithelial cell surface microvilli relatively plenty,lamellar corpuscle number increased,mitochondria swelling ameliorated in CMC-Na+YH,CMC-Na+YL,CMC-Na+YM groups and the most remarkable one was CMC-Na+YM group.AI was significantly positive correlated with the mRNA and protein expressions of JNK,GRP78 and IQA (r =0.907,0.928,0.880,0.712,0.911,all P ＜ 0.01).Conclusions YHTJF may effectively alleviate the cell apoptosis in mice LIRI,and its mechanism may be related to the inhibition of JNK pathway.

Objective To explore the cultural method of oral keratinocytes and make preparations for further investigation in using oral keratinocytes as a new choice of seed cells for the reconstruction of tissue-engineered urethra. Methods Oral keratinocytes of rabbits were isolated in vitro and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin(i3T3) or a culture dish without i3T3 respectively. Cell morphology was observed and cell growth was detected at intervals.Meanwhile,oral keratinocytes obtained from in vitro culture were performed immunofluorescence staining with broad-spectrum keratin antibody(AE1/AE3) and keratin 19 antibody(K19).The percentage of positive cells of passage 2 reactive to AE1/AE3 was assessed by flow cytometry. ResultsOral keratinocytes seeded onto a feeder layer of i3T3 exhibited finer morphous,better amplification capability,and could be passed for 7 or 8 generations.However,those cultured without i3T3 took on various morphous and could only be subcultured 2 generations before ageing.It was indicated by immunofluorescence staining that oral keratinocytes obtained from in vitro culture were positive for AE1/AE3 staining and 40% were positive for K19 staining.The result of flow cytometry revealed that the amout of positive keratinocytes reactive to AE1/AE3 was more than 95% of total cellular score. Conclusion Oral keratinocytes of rabbits can be cocultured with i3T3 in vitro and magnitude quantity can be attained,laying a favourable foundation for oral keratinocytes as a new choice of seed cells for urethral reconstruction with tissue engineering.

Background The injury or surgery of cornea cause the proliferation of corneal stromal cells and scar formation.Recent research showed that cureumin can obviously reduce the degree of fibrosis of tissue.But if curcumm play inhibitory effect on corneal keratocytes fibrosis is rarely reported.Objecttve This studv was to investigate the effect of curcumin on the transformation of corneal keratocytes into fibroblasts in vitro and further explore the antifibrotic effect of curcumin on corneal keratocytes.Methods The murine corneal keratocytes from 150 BALB/c mice were isolated and primary culture in DMEM culture medium containing 10% fetal bovine serum and then divided into blank control group(inducer group,CG),low-dose group(CG+7.5 mg/L curcumin),mediumdose group(CG+10.0 mg/L curcumin),high-dose group(CG+12.5 mg/L curcumin),non-inducer group.Seven days following intervention,the expression of cell markers such as keratocan,aldehyde dehydrogenase(ALDH),decorin and fibronectin-1 in keratocytes were analyzed by RT-PCR.The effect of curcumin on cultured murine corneal keratocytes proliferation was evaluated by MTS technique.The expression of fibronectin-1 in murine cornea was investigated by immunofluorescence assay.Results The primarily cultured keratocytes showed tlIe fusiform-like shape with the abundant cytoplasm and big nuclei.In the presence of curcumin,the mRNA levels of keratocan and ALDH were down-regulated and those of CD90 and decorin were up-regulated,showing the significantly differences with the increase of dose(P<0.05),but the expression pf fibronectin-i was not obviously changed with the alteration of dose of curcumin. MTS showed that the inhibitory rates of curcumin on keratocytes in 10.0 mg/L and 2. 5 mg/L groups were enhanced in comparison with 7.5 mg/L group, showing statistically significant difference among three groups( F = 956.00, P<0.05). The expression of fibronectin-1 was found in the corneal keratocytes with the red fluorescence in stroma. Conclusion Curcumin can inhibit the fibrosis of corneal keratoeytes in a dose-dependent manner. These results offer a preliminary theoretical basis for the application of curcumin in controlling corneal scar formation during wound healing.

Background Researches demonstrated that dendritic cells(DCs) are uniformly immature in the central cornea but mature in the peripheral region of cornea.So an important question is which factor impact the maturation of DCs,especially in terms of corneal transplant rejection and the known roles of DCs in the development and persistence of some corneal diseases.Objective This study aimed to examine whether corneal stroma cells (CSCs) inhibit DCs maturation through secreting transforming growth factor beta 2 (TGF-β2) and prostaglandin E2 (PGE2).Methods DCs,T cells and CSCs were isolated and cultured from clean BALB/c and C57BL/6 mice.The level of PGE2 and TGF-β2in CSCs culture supernatant and the fresh RPMI 1640 medium were then analyzed by enzyme linked immunosorbent assay (ELISA).During the DCs maturation stage,the neutralizing TGF-β2 antibody and the EP2 receptor antagonist AH6809 were added in the CSCs culture supernatant respectively.According to the different treatment,cultured cells were assigned to different groups as follows:control group,CSCs culture supernatant group,AH6809 group,TGF-β2 antibody group,AH6809 +TGF-β2 antibody group.Subsequently,the cellular surface markers for DCs,including CD11c,CD80,CD86,and MHC- Ⅱ,were analyzed by flow cytometry.The capability of stimulating the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reactions,and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran(FITC) uptake.Results The data of ELISA showed a higher concentration of TGF-β2 and PGE2 in murine CSCs culture supernatant than in the fresh RPMI 1640 medium.Compared with the CSCs culture supernatant group,the expression of CD80,CD86,and MHC- Ⅱ was up-regulated ( P ＜ 0.05 ),the expression of dextran was down-regulated ( P ＜ 0.05 ),and the stimulate index was increased( P＜ 0.05 ) in the TGF-β2 antibody group; the expression of CD86,and MHC-Ⅱ was up-regulated (P＜0.05),the expression of dextran was down-regulated ( F =13.740,P =0.006 ),and the stimulate index was increased(P＜0.05) in the AH6809 group;the expression of MHC-Ⅱ was up-regulated and the stimulate index was increased with statistical difference in interaction(P＜0.05 ) in the AH6809+TGF-β2 antibody group.Compared with the control group,the expression of CD80 and CD86,and the stimulate index was still lower(P＜0.05 ).Conclusions TGF-β2 and PGE2 contribute to the inhibitory effects on DCs maturation mediated by murine CSCs in vitro and further have additive effect on the immunosuppression of DCs.