OBJECTIVETo explore the effects of cyclopamine on the proliferation and apoptosis of LNCaP cells and the expression of the PCA3 gene in human prostate cancer in vitro.

METHODSLNCaP cells were treated with cyclopamine at the concentrations of 1, 5, 10 and 15 micromol/L for 24, 48 and 72 hours. The inhibitory effects of cyclopamine on the proliferation and apoptosis of the LNCaP cells were detected by MTT and flow cytometry respectively, the morphological changes of the cells observed by Hoechst 33258 staining, and the expression of the PCA3 gene determined by real-time fluorescence quantitative reverse transcriptase polymerase chain reaction (FQ-RT-PCR).

RESULTSCompared with the blank control group, cyclopamine significantly inhibited the proliferation of the LNCaP cells at 5, 10 and 15 micromol/L (P <0.01), reaching IC50 at 10 micro mol/L at 48 hours. The apoptosis rates of the LNCaP cells at 24, 48 and 72 hours were 37.21%, 57.38% and 57.98% in the 10 micromol/L group and 21. 16% , 71.31% and 72.90% in the 15 micro.mol/L group, significantly different from those in the control (P <0. 01). The cell apoptosis showed a rising trend with the increase of cyclopamine concentration and acting-time, while the expression of the PCA3 gene was decreasing with the increased concentration of cyclopamine, significantly lower than that of the blank control group (P <0.01) , and extremely low in the 10 micromo/L group

CONCLUSIONCyclopamine intervention at 10 and 15 micromol/L for 48 and 72 hours could significantly inhibit the at all time points. Proliferation and induce the apoptosis of LNCaP cells and reduce the expression level of PCA3.

Antigens, Neoplasm ; genetics ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Male ; Prostatic Neoplasms ; genetics ; pathology ; Veratrum Alkaloids ; pharmacology

The goal of this work was to explore the prospect of standardized application of an in-vitro bioactivity assay for recombinant human follicle-stimulating hormone based on a reporter gene. The relative accuracy, intermediate precision, linearity and applicable range of the method were validated according to the General Rules of Chinese Pharmacopoeia 2020 edition Volume IV (9401). Three laboratories used this method to determine the in-vitro biological activities of six batches of drug product and three batches of drug substance manufactured by two different companies. The consistency of the potency determined by three laboratories, the intra-laboratory precision and inter-laboratory precision were analyzed. The method was optimized during the collaborative validation. The results of method validation meet the requirements of the General Rules of Chinese Pharmacopoeia 2020 edition Volume IV (9401). Aiming to resolve the problems found in the collaborative validation, the medium for cell seeding, the pre-diluted buffer solution of standard and sample, and the means of removing and discarding supernatant after stimulation were optimized. After optimization, there was no significant difference in the bioactivity among the different laboratories (P > 0.05), indicating statistical equivalency. Intra-laboratory and inter-laboratory precision were good and the geometric coefficient of variation (GCV%) were both less than 15%. In conclusion, the reporter gene assay has good intra-laboratory repeatability and inter-laboratory reproducibility and is suitable for analyzing recombinant human follicle-stimulating hormone drug product and drug substance by different manufacturers. It is expected to be used as a standardized method for the determination of the in-vitro bioactivity of such products.

10.3969/j.issn.2095-4344.2013.25.007

AIM: To evaluate the influences of triptolide on serum cytokines, symptoms and pulmonary function in patients with steroid - resistant asthma, so as to investigate if there is therapeutic effect of triptolide on these patients. METHODS: Sixteen patients with steroid - resistant asthma were randomly divided into two groups (A and B, n =8 for each group). All of the patients took procaterol (50 - 100 μg/d) and theophylline (400 mg/d) orally as baseline treatment. Additionally, triptolide was prescribed for group A (33 μg, orally, three times per day for 4 weeks). Asthmatic symptom score calculation, serum cytokines ( interferon - γ, IFN - γ; interleukin - 4, IL - 4; and interleukin - 5, IL - 5)determination and pulmonary function test (FVC%, FEV1%, PEF%, V50% and V25% ) were undertaken before and at the end of the study. RESULTS: At baseline, no significant difference was found between group A and group B with respect to the above mentioned indices. Following the administration of triptolide, group A had significantly increased serum IFN -γlevel, FVC%, FEV1%, PEF%, V50% and V25%, and significantly decreased asthmatic symptom score and serum IL-4, IL-5 levels (P＜0.01 compared with baseline in the same group, and P＜0.05 compared with group B at the end of the study). Compared with baseline, no significant change was observed for group B regarding all the indices at the end of the study. CONCLUSION: Triptolide in combination with procaterol and theophylline may be a novel and effective strategy for the treatment of steroid - resistant asthma.

BACKGROUND: Some studies suggest that pre-injection of tumor necrosis factor-α (TNF-α)can protect focal cerebral ischemia in mice. Cerebral ischemia tolerance is related to the increase of TNF-α level; on the other hand, TNF-α is an injurious cytokine associated with stroke. Circulating antibody against anti-TNF-α can protect reperfused injury.OBJECTIVE: To study the effects of TNF-α pretreatment and post-treatment on cerebral ischemia-reperfusion injury and explore possible mechanism.DESIGN: Randomized controlled study.SETTING: Neurological Department, the Second Hospital Affiliated to Harbin Medical University.MATERIALS: The experiment was conducted at the Animal Experiment Center of Harbin Medical University from January to April 2002. Totally 120 healthy adult male Wistar rats were randomly divided into the following 8 groups: TNF-α 0.05 μg, 0.5 μg and 1.0 μg pretreatment groups and PBS group, TNF-α 0.05 μg, 0.5 μg and 1.0 μg post-treatment groups and PBS group with 15 in each group.METHODS: The focal brain ischemia model of middle cerebral artery occlusion (MCAO) was made using inserting thread method. TNF-α of different doses (0.05 μg, 0.5 μg or 1.0 μg) or PBS was injected intracisternally and 22-hour reperfusion, 8 rats from each group were killed. Then the perhour reperfusion, 7 rats from each group were killed. Then pathological changes were observed, glial fibrillary acidic protein (GFAP) and intercellular adhesion molecule-1 (ICAM-1) expression were inspected by immunohistochemical method. Histopathological and immunohistochemical evaluation was made with the computer-assisted image analyzing system,and the number of GFAP positive cells and ICAM-1 positive vessels in each hemisphere was counted.riliary acidic protein and ICAM-1.infarct volume: TNF-α 0.5 μg and TNF-α 1.0 μg pretreatment groups showed reduced volume of lesion; infarct volume reduced by 70.9% in TNF-α 0.5 μg pretreatment rats and 66.5% in TNF-α 1.0 μg pretreatment rats. TNF-α 0.5 μg and TNF-α 1.0 μg post-treatment groups showed increased volume of lesion; infarct volume increased by 22.3% in TNF-α 0.5 μg post-treatment rats and 46.7% in TNF-α 1.0 μg post-treatment rats.TNF-α 0.05 μg and 1.0 μg pretreatment groups did not differ significantly (P ＞ 0.05), but there was an obvious difference between TNF-α 0.5 μg and pared with PBS pretreatment group, TNF-α 0.5 μg and 1.0 μg pretreatment groups showed lessened tissue damage and edema. Compared with PBS post-treatment group, TNF-α 0.5 μg and TNF-α 1.0 μg post-treatment fibriliary acidic protein and ICAM-1: TNF-α 0.5 μg and TNF-α 1.0 μg pretreatment groups showed reduced volume of glial fibriliary acidic protein and ICAM-1 (P ＜ 0.05); but TNF-α 0.5 μg and TNF-α 1.0 μg posttreatment groups showed increased volume of glial fibriliary acidic protein and ICAM-1 (P ＜ 0.05). TNF-α 0.05 μg and 1.0 μg pretreatment groups did not differ significantly (P ＞ 0.05); but there was an obvious difference between TNF-α 0.5 μg and 1.0 μg post-treatment groups (P ＜ 0.05).cerebral ischemia reperfusion injury. This effect is not related to the repair given after cerebral ischemia reperfusion, ischemia exacerbates, which is α are determined by whether TNF-αis given before or after cerebral ischemia in a dose-dependent manner.

ObjectiveTo investigate portal hemodynamic features and its changes following devascularization in cirrhotics. MethodsHemodynamics of portal trunk (PT), right anterior branch (RAB) and splenic vein (SV), including maximum cross-sectional velocity(CS-Vmax), flow volume and congestion index(CI), were assessed in 69 cirrhotics and 46 normal volunteers by using a recently-developed color Doppler velocity profile(CDVP). Of 28 patients undergone devascularization procedure, portal hemodynamics were studied and compared before and after operation. ResultsCSVmax of PT and RAB was significantly lower in cirrhotic group than normal group;PT and SV flow volume and the ratio of SV to PT flow volume(SV/PT)were significantly greater in cirrhotic group compared with those of normal group;CI of PT, RAB and SV was significantly higher in cirrhotic group than normal group. Postoperative PT flow volume was significantly reduced( P ＜0.01),and the reduction was closely related to preoperative SV flow volume( r = 0.65, P ＜0.001). CS-Vmax and flow volume in RAB were decreased significantly following operation( P ＜0.01), and the reduction of RAB flow volume was highly related to preoperative RAB flow volume( r =0.74, P ＜0.001). After operation, free portal pressure(FPP)was declined by (6 ± 5)cmH2O[ (0.59 ± 0.49)kPa] ( P ＜ 0.001 ). There were no significant changes in CS-Vmax, CI in PT and CI in RAB following operation. ConclusionIn cirrhotics with portal hypertension, portal venous system coexists the elevated vascular resistance and hyperdynamics, but with different redorainance at different portion. SV hyperdynamics is the main source of increased portal blood flow. Devascularization procedure could markedly relieve portal hyperdynamics by elimination of SV inflow, which is one of the main mechanisms in obmining therapeutic goal. But the operation has no favorable effects on the increased portal resistance,and portal perfusion to the liver would be further declined after relieving portal hyperdynamics, which is unfavorable to maintenance of liver function.

BACKGROUND: The most commonly used rod-screw posterior spinal fixation system has some biomechanical drawbacks. The screw-plate system is more preferred for patients.OBJECTIVE: To prepare a new posterior spinal fixation system based on imaging results of distance between two pedicles,radian of spine flexion, extension and the height of intervertebral space of Chinese population.METHODS: The thoracolumbar data of 129 normal people were measured. According to the imaging measurements and relative documents, a device of gear-distraction plate (GDP) for spine reduction and fixation was designed. Eighteen fresh calf lumbar specimens were randomly divided into 3 groups, the test group was fixed by GDP and the other groups were fixed by CD and Steffee plate, respectively. The results of the displacement, strain, strength, stiffness and ultimate strength were measured under the states of vertical compression, flexion, extension and lateral bending respectively, and the results were statistically analyzed.RESULTS AND CONCLUSION: GDP could meet the need of strength and stiffness of human bodies. It showed superior to the CD and Steffee plate groups in strength and stiffness (P ＜ 0.05), with 13% or 14% torsion intensity greater than that of the CD or Steffee plate groups. The ultimate mechanical performance test showed that, the load bearing of the GDP group was greater than that of the CD and Steffee plate groups (P ＜ 0.05). The findings demonstrated that GDP for Chinese human presented with good biomechanical stability, which can promote vertebrae fracture union and prevent kyphosis relapses or vertebral height loses.

Objective To explore the effect of recombinant pEGFP-N3-APC vectors carrying various APC functional domains on the expression of β-catenin in human colorectal cancer cells HT-29.Methods The recombinant plasmids were transfected into HT-29 cells mediated by lipofectamine~(TM) 2000, and detected by green fluorescence and RT-PCR. Western blot was applied to detect β-catenin expression level in HT-29 cells after transfection, and gray scales of electrophoresis strips were analyzed by SPSS 13.0.Results Green fluorescence and RT-PCR made clear that all 5 recombinant plasmids were successfully expressed in HT-29 cells. Western blot showed that β-catenin expression level in HT-29 cells was not affected after being transfected with pEGFP-N3-APC1, pEGFP-N3-APC2 and pEGFP-N3-APC3, and was distinctly affected after being transfected with pEGFP-N3-APC4 and pEGFP-N3-APC5, especially the later one. Conclusion The selected APC5 gene fragment with 15-amino acid repeats and SAMP repeats, which is relatively short, can degrade β-catenin level in HT-29 cells and may be applied in the gene therapy.

Purpose To investigate the expression of gelsolin-like actin-capping protein (CapG) in gastric cancer and adjacent normal gastric tissues and its relationship with clinicopathological features and prognosis.Methods The immunohistochemical (IHC) assay was performed to detect the expression of CapG protein in 125 specimens of gastric cancer (study group) and 30 specimens of matched adjacent normal gastric tissues (control group).No patients had received chemotherapy or radiotherapy before surgery.Immunostaining was scored semiquantitatively by two independent observers who were blinded to the patients' outcome and other clinicopathological parameters.The correlation of the expression of CapG with clinicopathological characteristics and prognosis was analyzed.Results Cytoplasmic and nuclear CapG protein staining was detected in all gastric cancer tissues.CapG protein expression was absent or low in matched adjacent normal gastric tissues but significantly higher in gastric cancer tissues.Expression of CapG in the tumor was not significantly associated with gender,age,tumor size,tumor location (P ＞ 0.05).However,elevated CapG expression was strongly correlated with the depth of invasion (P =0.044),lymph node metastasis (P =0.026),distant metastasis (P =0.001),and AJCC stage (P =0.012).Significant poorer overall survival was observed in the high CapG expression group compared with that of the low CapG expression group (P ＜0.001).Multivariate analysis showed overall survival correlated with the patients' tumor location,lymph node metastasis,distant metastasis,AJCC stage and the expression of CapG protein.Conclusion CapG is highly expressed in gastric cancer.Tumors with CapG up-regulation tend to have poorer prognosis,suggesting overexpression of CapG may contribute to the prediction of poorer prognosis and may be a potential therapeutic target for gastric cancer.

Objective:Analysis of two ethnic Uygur and Han investigate ischemic stroke adiponectin (Adiponectin) gene single nucleotide rs182052 , rs6444175 , rs1501296 allele polymorphism point whether the differences.Methods:Gene sequencing methods were used to detect 210 was used cases of acute ischemic stroke patients ( case group ) and 104 healthy people ( control group ) Adiponectin gene ,using case-control association analysis of genotype and allele frequencies compared explore the two ethnic differences in different gene polymorphism loci.Results: When the case group and the control group were compared , it was found that the Adiponectin gene rs64441759G/A was significantly higher in the case group.The risk rate increased significantly to 1.481 times (OR=1.481;95%CI:1.219-1.910; P=0.000).Conclusion: Adiponectin gene rs6444175A allele may be susceptible to pathogenic gene.No significant differences show on the 3 SNP loci of Adiponectin genes between Uygur and Han patients.