Objective To establish human neural stem cells(HNSCs) model for further basic research and clinical application. Methods Cells from human fetal cerebral cortex were collected and cultured with serum free midium and then identified for nestin immunocytochemical staning;The cells were induced to differentiate by 5% fetal bovin serum and identified by neurofilament-200(NF-200) and glial fibrillary acidic protein(GFAP) immunocytochemical staning. Results The harvested cells appeared as clusters in suspension and both NF-200 and GFAP positive cells were observed after induction.After 12 generations of culture,these cells retained the main characteristics of NSCs.Conclusions The HNSCs were harvested from human fetal cerebral cortex and this HNSCs model can be used for futher basic research and clinical applications.;

ObjectiveTo investigate the protective effect of tanshinoneⅡA on the expression of S100A1 protein after acute myocardial ischemia injury in rats.Methods Sixty Wistar rats were randomly divided into sham operation group, acute myocardial ischemia model group and tanshinoneⅡA pretreatment group by random number table. The acute myocardial ischemia model was established by thoracotomy and penetration of a thread and occlusion around the root part of the left anterior descending coronary artery, while the sham operation group was established only by thoracotomy and penetration of a thread around the root part of that artery but without occlusion; 3 days before the operation, in the tanshinoneⅡA pretreatment group, intraperitoneal injection of tanshinoneⅡA solution(at a dose of 1.5 mg/kg) was applied, while in the sham and acute myocardial ischemia groups, intraperitoneal injection of an equal volume of saline was given. Myocardial cell apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL), the levels of serum superoxide dismutase (SOD), malondialdehyde(MDA), creatine kinase(CK), lactate dehydrogenase(LDH) and S100A1 protein were examined and the level of expression of S100A1 protein in myocardial tissue was assayed by immunohistochemical staining and Western Blot.Results Compared with the sham operation group, the myocardial cell apoptosis rate, the contents of MDA, CK, LDH, S100A1 and the level of S100A1 expression in myocardial ischemia group and tanshinoneⅡA pretreated group were significantly increased, while SOD activity was decreased obviously; compared with the myocardial ischemia model group, the myocardial cell apoptosis rate, the contents of MDA, CK, LDH, S100A1 and the level of S100A1 protein expression were significantly reduced〔apoptosis rate:(32.1±4.2)% vs.(72.4±5.4)%, MDA(μmol/L): 9.1±2.2 vs. 17.3±5.2, CK(U/L): 83.3±12.2 vs. 107.5±12.4, LDH (μmol·s-1·L-1): 84.0±16.4 vs. 114.4±16.0, S100A1(μg/L): 37.6±6.0 vs. 78.4±8.6,P<0.05 orP<0.01〕, while the activity of SOD was increased markedly in tanshinoneⅡA pretreated group(kU/L:72.8±10.2 vs. 49.6±8.8,P<0.01). TUNEL staining showed that in the myocardial ischemia model group and tanshinoneⅡA pretreated group, the myocardial cells represented positive staining(brown-yellow in color), irregular in shape with nuclear pyknosis, cell detachment from the surrounding tissue and other characteristics. And in sham operation group,the staining of majority of cells was negative. The results of immunohistochemistry showed that S100A1 protein staining was relatively deep in the myocardial ischemia model group and tanshinoneⅡA pretreated group, and in the latter group, the color of S100A1 protein positive staining was not as deep as that in the former group. Western Blot showed that the S100A1 protein expression in myocardial ischemia model group was 2.8 folds of that of the sham operation group, while the S100A1 protein expression in tanshinoneⅡA pretreated group was significantly decreased compared with that of myocardial ischemia model group(bothP<0.05),which was 1.5 folds of that of the sham operation group.ConclusionTanshinoneⅡA may play a role in inhibiting the expression of S100A1 protein to protect against acute myocardial ischemia injury, suggesting that this agent have a potential effect for treatment of myocardial ischemia.

OBJECTIVE: To study the role of nuclear factor-kappa B (NF-kappaB) and cycloxygenase-2 (COX-2) in the onset of phlegm obstruction due to lung-deficiency in rats and the therapeutic mechanism of Huatan Recipe. METHODS: Twenty-four SD rats were randomly divided into normal group, model group and treatment group, with 8 rats in each group. The rats in the model group and treatment group were exposed to sulfur dioxide and cold wind to establish the rat model of phlegm obstruction due to lung-deficiency, and the rats in the treatment group were also treated with Huatan Recipe, a compound traditional Chinese medicine. The expression of NF-kappaB in the bronchial epithelial cells of the rats was tested with the method of immunohistochemistry, and the COX-2 mRNA in the lung tissues of the rats was measured by using reverse transcription-polymerase chain reaction. RESULTS: The expressions of NF-kappaB and COX-2 mRNA in rats of the model group were higher than those of the normal group (P<0.01), and the expressions of NF-kappaB and COX-2 mRNA in rats of the treatment group were obviously lower than those of the model group (P<0.01). CONCLUSION: The NF-kappaB and COX-2 play an important role in the onset of phlegm obstruction in rats. Huatan Recipe may prevent the development of phlegm obstruction by down-regulating the expressions of NF-kappaB and COX-2 mRNA.

Objective To investigate the relationship between single nucleotide polymorphisms (SNPs) of PRKCG gene (rs2547362,rs3745406) and osteosarcoma susceptibility in the osteosarcoma patients and the normal population.Methods Sixtyone patients with osteosarcoma who had been admitted in our hospital from January 2011 to December 2012 and 63 healthy adults were enrolled in this study.A 2-ml peripheral blood sample was taken from each participant.The RT-qPCR method was used to detect the genotype and allele frequency distribution of PRKCG gene at rs2547362 and rs3745406 in osteosarcoma patients and normal population.Osteosarcoma patients were divided into several groups according to the clinical parameters such as age,gender,histology,tumor location,Enneking classification,tumor metastasis and therapy,and then we analyzed the relations between the genetic polymorphism and clinical parameters.Results 1) The genotype of PRKCG gene at rs3745406 included CC,CT and TT.The differences of genotypes (CC,CT,TF) and alleles (C,T) frequency distribution at rs3745406 were not statistically significant between osteosarcoma patients and the normal population (P=0.490,P=0.554).2) The genotype of PRKCG gene at rs2547362 included CC,CT and TT.The differences of genotypes (CC,CT,TT) and the alleles(C,T) frequency distribution at rs2547362 were statistically significant between the osteosarcoma patients and the normal population (P=0.006,P=0.007).3) The differences of genotypes (CC,CT,TT) and alleles (C,T) frequency distribution at rs3745406 were statistically significant between patients with metastasis and patients without metastasis (P=0.000,P=0.000).The CT and TT genotypes and the T allele carrier frequency at rs3745406 were higher in patients with metastasis than in patients without metastasis.SNPs at rs2547362 were not associated with clinical parameters.Conclusion The genetic polymorphism of PRKCG gene at rs2547362 is associated with osteosarcoma susceptibility.The TT genotype and T allele at rs3745406 are associated with metastasis of osteosarcoma,which may be a risk factor for metastasis in the osteosarcoma patients.

Objective: To investigate the protective effect of sulfated Gastrodia elata polysaccharides (GEPS) on the apoptosis of PC12 cells induced by corticosterone (CORT).Methods: After modifying GEP chemically, GEPS was obtained.PC12 cells were pretreated with GEPS (0, 250, 500 and 1 000 μg·ml-1) for 30 min before cultivating with CORT for 48 h.The cellular viability, lactate dehydrogenase (LDH) release and morphological observation were determined respectively by CCK-8 assay, LDH assay, inverted microscope and DAPI fluorescein stain method.Results: With the pretreatment of GEPS, the survival rate of PC12 cells increased significantly (P<0.05) in a dose-dependent manner.Compared with CORT injured group, GEPS attenuated the release of LDH with statistical significance (P<0.05), and LDH leakage decreased with the concentration of increase GEPS.Under an inverted microscope, PC12 cells incubated with CORT became shrinkage, and aggregated with decreased diopter and bad adhesion,and lots of cells floated.However, with the pretreatment of GEPS, the living status of PC12 cells improved markedly.Compared with CORT injured group, GEPS obviously reduced the apoptosis of PC12 cells.Conclusion: GEPS exhibits protective effects on the apoptosis of PC12 cells induced by CORT, while when compared with GEP, GEPS shows the similar effects.The other bioactivities of GEPS need further studies.

Objective To investigate the gene expression of PIWIL2 in the bladder urothelial carcinoma (BTCC) and siRNA interact on PIWIL2 gene expression in human bladder cancer cell line BIU-87.Methods Semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR) was applied to detect the PIWIL2 expressions in tissues of BTCC (46 cases),cystitis glandularis(21 cases),adjacent non-cancerous tissues (17 cases) and normal bladder tissues (7 cases). 3 specific siRNA targeted PIWIL2 gene were synthesized after designed and transferred. After siRNA was transferred into BIU-87 cells, MTI and TUNEL methods were applied to detect the proliferation inhibitory rate (IR) and apoptosis index (AI) in BIU-87 cells,qRT-PCR and Western blot were used to examine effects of siRNA on the expressions of the PIWIL2 gene and protein,respectively.Results The expression rate of PIWIL2 mRNA in BTCC tissues was 76.08 ％(35/46) and significantly higher than those in the cystitis glandularis tissues (42.86 ％,9/21),adjacent non-cancerous tissues (41.17 ％,7/17) and normal tissues (7.14 ％,1/14) (P =0.008,P =0.010,P =0.000).The IR [(37.52±8.84) ％,(64.36±9.64)％] and (62.94±8.43) ％] and AI [(26.18±5.42) ％,(38.75±6.19) ％ and (40.02±5.64) ％] of BIU-87 cells in the siRNA 1～3 groups were respectively significantly higher than those [(1.97±0.02) ％ and (3.35±0.47) ％] in the control group(P=0.000),and expressions of PIWIL2 mRNA and protein in the siRNA groups were both lower than those in the control group. Moreover, the effects of siRNA 2 group and siRNA 3 group on inhibiting PIWIL2 expression, IR and AI of BIU-87 cells were stronger than siRNA 1 group. Conclusion The over-expression of PIWIL2 suggested that it played an important role in the mechanism of development and malignant progression of BTCC. The siRNA of transcription can significantly inhibit its expression, induce cell apoptosis and inhibit the growth of BIU-87 cells which might provide the experimental evidence for the gene targeting therapy of bladder tumor.

Objective To observe the effects of ( - )-epigallocatechin-3-gallate (EGCG) on human prostate cancer xenografted tumor growth and connexin43 expression in nude mice,and explore the mechanism of the EGCG on prevention for prostate cancer.Methods The methyl thiazolyl tetrazolium and annexin-V/PI double-labeled flow cytometry methods were used to observe the growth inhibiting rate (IR)and apoptosis rate (AR) of human prostate cancer cell line PC-3 which was treated by EGCG at different concentration (10,20 and 40 mg/L,respectively).The scrape-loading fluorescence dye transfer method was applied to assess the gap junction intercellular communication (GJIC) through fluorescence microscope.PC-3 cells were subcutaneously transplanted to establish tumor-bearing nude mice model.A total of 32 mice were randomly divided into four groups,both control group and three treatment groups were treated with different doses of EGCG ( 10,20 and 40 mg/kg,respectively).After two weeks,the mice prostate tumor tissues were taken out.The tumor wet weight was measured and tumor growth inhibiting rate was calculated.The tumor microvascular density (MVD) and apoptosis index (AI) were detected by the immunohistochemical techniques and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling techniques,respectively.Semi-quantitative reverse transcription polymerase chain reaction was used to examine the expression level of the Cx43 mRNA.Results EGCG at concentration ( 10 and 20 mg/L) could significantly inhibit the proliferation[(22.33 ±4.62)％,(38.67 ±5.67)％ vs (3.47 ±0.31 )％,P ＜0.01],induce the apoptosis [(7.84 ± 1.37 ) ％,( 24.53 ± 2.28 ) ％ vs ( 2.17 ± 0.70 ) ％,P ＜ 0.01] and enhance the GJIC of PC-3 cells.EGCG of different doses could inhibit prostate cancer xenografted tumor growth,induce tumor cells apoptosis and inhibit angiogenesis.EGCG ( 20 and 40 mg/kg) could effectively up-regulate Cx43 mRNA expression in xenografted tumor (0.58 ± 0.08,0.80 ± 0.07 vs 0.42 ± 0.04,P ＜ 0.0 ).The effects had significant correlation with the dose-dependent of EGCG ( P ＜ 0.05 ).Conclusions EGCG could up-regulate the Cx43 expression and enhance the gap junction intercellular communication mediated by Cx43 in the prostate tumor,which provide the experimental evidence for the mechanism of its effectively inhibiting the prostate cancer growth.

Objective To investigate the clinicalmanifestations,imaging features,diagnosis and differential diagnosis,treatment and prognosis of renal malignant solitary fibrous tumor(SFT).Methods The clinical data in 1 case of rare renal malignant SFT were retrospectively analyzed.Referring to related literatures,the histological origin,pathological features,differential diagnosis,treatment and follow-up of renal malignant SFT were analyzed.Results The patient was preoperatively diagnosed as right renal clear cell carcinoma.Postoperative pathological examination diagnosed as low grade malignant SFT of right kidney.And immunohistochemistry indicated CD34+,BCL-2 +,CD68+,CD99+,vimentin,Ki-67 5％ +,SMA focal weakly positive.No recurrence or metastasis occurred after 4-month follow-up period.Conclusion Malignant SFT of the kidney is very rare,its diagnosis and differential diagnosis depend on postoperative pathological and immunohistochemical examination.Radical nephrectomy is the main option for malignant SFT of the kidney with good prognosis.

Objective To investigate the effect of salinomycin on cancer stem cell formation of prostate cancer cell line DU145 and its possible mechanisms,providing theoretical basis for the clinical application of salino-mycin. Methods (1)DU145 cells were treated with salinomycin. The percentage of ALDH+cells,which was used as the marker of cancer stem cells,was detected by flow cytometry.(2)After treated with salmonin,DU145 cells were subjected to Western-Blot analysis for the expression of mTORsignal pathway-related proteins such as p-70s6k, p-p70s6,p-s6 and so on. 3)DU145 cells were treated with salinomycin combined with mTOR signal pathway inhibi-tor rapamycin,and the ALDH+cancer stem cells were detected using flow cytometer. Results (1)Salmonomycin significantly inhibited ALDH-positive cancer stem cells in DU145cell line(inhibition rate in 77.8%),which was twice as high as that of traditional anticancer drug paclitaxel(which has a inhibition rate of 38.64%). This results suggesting that salinomycin would have the effect of inhibiting cancer stem cells. (2)The expression ofm-TOR p-70s6k,p-p70s6 and p-s6 in mTOR signaling pathway was inhibited by salinomycin in a time-dependent and dose-dependent manner,suggesting that salinomycin would inhibite mTOR signaling pathway.(3)Salinomycin combined with rapamycin can decrease the proportion of ALDH-positive DU145 cancer stem cells(inhibition rate in 77.95%), suggesting that salinomycin may inhibit ALDH-positive DU145 stem cells through the mTOR signaling pathway. Conclusion Salinomycin may play an important role in inhibiting cancer stem cells by inhibiting mTOR pathway signaling.

Objective To investigate the safe and effective method for laryngeal microsurgery in difficult la‐ryngeal exposure cases .Methods We selected 62 patients’ clinical data who had received laryngeal microsurgery with difficult laryngeal exposure and could not exposure by normal self -retaining laryngoscope between July 2012 and June 2015 .There were 42 cases of vocal cord polyp ,9 cases of the vocal cyst ,5 cases of the vocal amyloidosis , 4 cases of severe atypical hyperplasia of vocal cords and 2 cases of vocal cord high differentiated squamous carcino‐ma .We completed all kinds of laryngeal microsurgery to expose the glottis by adjusting the postures of patients ,in‐creasing the anesthesia depth ,using self -retaining laryngoscope with endoscopy which can be adjusted and pressing the throat .Results In 62 patients ,58 patients were successfully operated with adjustable self -retaining laryngo‐scope with endoscopy ,the success rate was 93 .55% .And 25 cases was exposed the glottis completely by increasing the anesthesia depth ,however ,when we increased the anesthesia depth ,there were 10 cases needed to combined with pressing the throat to expose .Five patients had retropharyngeal injure with different levels .One case with small jaw deformity of the vocal cord polyp surgery was not successful ,the success of electronic endoscopic under surface anesthesia surgery .The other one case with teeth unkempt and porcelain teeth and two cases of intraoperative frozen tip vocal cord cancer completed the operation of the open throat under the non trachea incision .Conclusion Most of difficult exposed laryngeal can be safely and effectively exposed through using the adjustable self -retaining laryngo‐scope with endoscopy while normal self -retaining laryngoscope can not .When necessary ,we can put 30°endoscope into the side channel of self -retaining laryngoscope to complete all kinds of laryngeal microsurgery .