Hepatocellular carcinoma (HCC) is a serious threat for human health, the incidence of HCC in China accounts for more than 50% worldwide. There is an urgent need to develop novel anticancer agents for the treatment of HCC patients. Here we characterized the inhibitory effect and the molecular mechanism of protopine on HCC cancer cells. The results of a CCK-8 assay indicated that protopine displays anticancer activities on HCC cells. Flow cytometry and JC-1 staining confirmed that treatment with protopine decreased the mitochondrial membrane potential and induced apoptosis in HCC cells. Western blot analysis showed that protopine was able to increase protein expression in the mitochondrial apoptotic pathway; the level of cytochrome C, apoptotic protease activating factor-1 (Apaf-1), Bax, cleaved-poly ADP-ribose polymerase (cleaved-PARP), cleaved-caspase-3, and cleaved-caspase-9 were increased while the expression of Bcl-2 was suppressed significantly. An in vivo study revealed that protopine significantly suppressed the growth of tumors in nude mice bearing HepG2 cells. Administration of protopine intraperitoneally at a concentration of 50 mg·kg^-1 inhibited tumor growth by 72.46%. Animal experiments were carried out according to the Regulation of the Animal Ethics Committee of Southwest Medical University. This study provides preliminary evidence that there is potential to develop protopine as a lead compound for the treatment of HCC.

Objective To explore the diagnostic sensitivity and specificity of echocardiography parameters ,and the diagnostic value of multiple parameters score . Methods Forty‐nine cases of the fetus checked by fetal echocardiography and were indicated aortic coarctation . According to the results of follow‐up ,the cases were divided into the positive and negative groups . Comprehensive analysis were performed in fetal echocardiography parameters ,such as diameter ratio and velocity ratio of ventricle ,aorta ,pulmonary artery ,ductus artery ,middle cerebral artery and umbilical artery . Results Diameter ratio of left ventricle/right ventricle ( LV/RV ) ,pulmonary artery/aorta ( PA/AO ) ,aortic isthmus/ductus artery ( AI/DA ) ,and velocity ratioof middle cerebral artery/inner umbilical artery ( MCA/inUA ) were calculated ,ROC curve showed their area under ROC were>0 .5 with a significant statistical significance ( P < 0 .05) . The Chi‐square test showed that the consistency of any single ratio was relatively low . When the number of the ratiois increased to 3-4 ,the Kappa value was 0 .687( P = 0 .000) and 0 .649( P = 0 .000) ,respectively , which had relatively high diagnostic value . Conclusions Ultrasonic diagnosis of fetal coarctation of aorta has certain difficulty ,and the value of single hemodynamic parameter of fetal echocardiography in diagnosis of fetal aortic coarctation is lower ,so that the comprehensive evaluation and combined multiple ultrasonic parameters can improve the sensitivity and specificity of ultrasound diagnosis .

BACKGROUND:Stem cel therapy has been used for prevention and treatment of degenerative disc disease. Considering the special microenvironment in the intervertebral disc, the survival rate and differentiation ability of transplanted cels are decreased, which may lead to the poor efficacy of stem cel therapy. How to improve the survival ability and therapeutic effect of the transplanted cels is the focus of stem cel therapy for degenerative disc disease.
OBJECTIVE: To investigate the effects of cobalt chloride combined with hypertonic solution pretreatment on bone marrow mesenchymal stem cels that wil be transplanted for treatment of degenerative disc disease.
METHODS:(1)In vitro cel experiment: bone marrow mesenchymal stem cels were divided into three groups and subjected to normal culture medium (normal control group), 1% hypertonic mother solution (hypertonic group), 100 μmol/L cobalt chloride (hypoxia group), or 1% hypertonic mother solution plus 100 μmol/L cobalt chloride (combined group) for 1 week. Then, 2% hypertonic solution and 200 μmol/L cobalt chloride cobalt chloride were used to simulate the anaerobic and hypertonic environment intervenes in pretreated and untreated bone marrow mesenchymal stem cels for 24 hours. After that, RT-PCR was used to detect the expression of caspase-3 for apoptosis evaluation. (2)In vivo animal experiment: Sprague-Dawley rats were divided into model, cel transplantation and hypertonic plus hypoxic groups. Rat models of intervertebral disc degeneration were made in these three groups. After modeling, rats in these three groups were given no treatment, bone marrow mesenchymal stem cel transplantation or transplantation of bone marrow mesenchymal stem cels which were subjected to hypertonic and hypoxia pretreatments into the intervertebral disc. Two weeks later, immunohistochemistry and RT-PCR methods were used to detect cel distribution and related gene expression, respectively, thereby to evaluate the therapeutic effect of stem cels.
RESULTS AND CONCLUSION: (1)In vitro cel experiment: caspase-3 mRNA expression was significantly reduced in pretreated bone marrow mesenchymal stem cels compared with the untreated cels (P < 0.05). (2)In vivo animal experiment: compared with the control group, the caspase-3 and interleukin-1β in the intervertebral disc and a number of degenerative indexes were decreased in the cel transplantation. Compared with the cel transplantation group, these indicators had better outcomes in the hypertonic plus hypoxic group (P < 0.05). These findings indicate that bone marrow mesenchymal stem cels have therapeutic potential for degenerative disc disease, and have better adaptability and transplantation effects by hypertonic and hypoxia pretreatments.

Objective To study local and systemic immune response in an animal model treated with recombinant hIFN-α-2b-BCG instillation. Methods The MB49 orthotopic bladder cancer model in C57BL/6 mice was established and treated separately with rBCG, wild BCG, wild BCG combined with IFN-α-2b and PBS as the control. The changes of lymphocyte subgroups in peripheral blood were analyzed with FCM, and mTNF-α and mIL-12 in peripheral blood of mice were detected with ELISA.Immunohistochemistry was carried out to detect the local immune reaction, T cell subsets and FAS, in bladder cancer after being treated with rBCG or wBCG. Results The content of CD4+ T lymphocyte was up-regulated in the rBCG group. The CD4+/CD8+ ratio of 2. 63 was up-regulated than pretreatment, significantly different than that of wBCG group(P＜0.05). ELISA assay showed that BCG significantly up-regulated the level of mTNF-α and mIL-12 in serum of orthotopie murine bladder cancer mice. The mTNF-α 806 pg/ml, mIL-12 860 pg/ml in rBCG group, was not significantly higher than those in wBCG group and combination group. The immunocompetent cell numbers with CD3, CD4,CD8 phenotype increased significantly in the tumor tissue of BCG treated group than the control(P＜0.05). The results of CD4+ in rBCG group and the combination group, and CD8+ in rBCG group were significantly higher than that of the wBCG(P＜0.05). The expression of Fas in tumor tissues treated with intravesical BCG was increased(P＜0. 05). Conclusions The recombinant IFN-α-2b-BCG can retrieve the disproportion of systemic lymphocyte subgroups, and increases Th1-type factors and local Fas expression in orthotopic murine bladder cancer. The recombinant IFN-α-2b-BCG is effective in regulating local and systemic immune reaction in orthotopic murine bladder cancer model.

Objective To explore genes expression of adiponectin receptors during differentiation of SW872 preadipocytes. Met-(hods) SW872 preadipocytes were cultured in vitro and induced to differentiate by 0.6 mmol/L oleic acid. During the progress of diffe-(rentiation), the morphological changes of SW872 cells were observed and the differentiation rate was assayed by oil-red O staining. Adiponectin receptors mRNA was measured by reverse transcription-polymerase chain reaction during differentiation of SW872 preadipocytes. Results 1.After stimulated by 0.6 mmol/L oleic acid for 72 hours, almost all SW872 cells were differentiated,and there were lots of fat droplets in the cells.2.There were adiponectin receptors genes expressions in SW872 preadipocytes.After 72 hours,and the levels of adiponectin receptor(AdipoR) 1 mRNA and AdipoR 2 mRNA were markedly increased up to 2.54 and 4.09 times,respectively. Conclusion There are AdipoR1 and AdipoR2 genes expressions in fat cells and the expressions are differentiation-dependent.

Objective To study the effect of oleic acid on the differentiation of SW872 preadipocytes.Methods SW872 prea-(dipocytes) were cultured and induced to differentiate by 0.6 mmol/L oleic acid in vitro.After 24 h,48 h and 72 h of differentiation,the morphological changes of SW872 preadipocytes were observed and the differentiation rate was assayed by oil-red O staining.In addition,triglyceride(TG) mass was detected by chemical colorimetry methods.During the differentiation of SW872 preadipocytes,transcription factors including peroxisome proliferator activated receptor-?_2(PPAR-?_2) and CAAT/enhancer binding protein-?(C/EBP-?)(mRNA) were also measured by reverse transcription-polymerase chain reaction(RT-PCR).Results 1.SW872 preadipocytes were fibroblastic and had no obvious fat droplet in cytoplasm.However,when stimulated for 72 hby 0.6 mmol/L oleic acid,SW872 preadipocytes became more bigger and rounder and differentiated into mature adipocytes with lots of fat droplets in the cells.2.Compared with that of predifferentiation,the concentration of TG mass increased by 14 folds after 72-hour differentiation(P

ObjectiveTo explore the effect of different nutritional support mdoes on humoral immunity and outcomes after esophagectomy in patients with esophageal carcinoma.MethodsForty-six patients with middle or low thoracic esophageal carcinoma underwent Ivor Lewis esophagectomy.The patients were randomized into enteral nutrition group ( EN,n =23 ) and enteral combined parenteral nutrition group ( EN + PN,n =23 ) based on the nutrition support modes.Serum levels of immunoglobulin (IgG,IgA,IgM,IgE,κ/λ light chain) and comphments (C3/C4) were assayed and compared on the 1st pre-operative day and at 18 hours as well as 3rd and 7th day after operation.The clinical outcomes including infection-related complications and hospital stay were compared between two group s.ResultsThere was no significant difference in all humoral immunity indicators between two groups at the eachpost-operative time point.In both two groups,the levels ofIgG [ (8.90 ± 1.75),(7.53 ±1.41) g/Land (8.64±2.44),(7.48±2.16) g/L],κ [ (2.14±0.46),(1.78±0.41) g/L,and (2.15 ±0.63),( 1.86 ± 0.62) g/L] and λ light chain [ ( 1.34 ± 0.45 ),( 1.11 ± 0.31 ) g/L and ( 1.20 ± 0.32),( 1.08 ± 0.35 ) g/L] were significantly lower 18 hours and 3rd day after operation than the pre-operative levels [ (12.15±2.86)and (11.11±2.96) g/L,(2.90±0.77) and (2.77±0.79) g/L,(1.79±0.57) and (1.56±0.41) g/L] (P=0.000,P=0.000,and P=0.004,P=0.000,and P=0.000,P=0.000,and P=0.011,P=0.000,and P=0.004,P=0.000,and P =0.008,P =0.000),and returned to the preoperative levels by the postoperative 7th day (P＞0.05),except for the level of κ light chain 7th day after operation in EN group [ ( 2.42 ± 0.69) g/L] ( P =0.027 ).The levels of IgA,IgE,and C3 were not significantly different during the perioperative period ( P ＞ 0.05 ).The level of IgM was not significantly different during the perioperative period in EN group (P ＞0.05),and was significantly lower on the 3rd post-operative day [ ( 1.00 ±0.53) g/L] than the pre-operative level [ ( 1.47 ±0.76) g/L] in the EN + PN group (P =0.031 ),and were not significantly different on the other time points (P ＞ 0.05 ).In the EN group,the C4 level was significantly lower at the postoperative 18 hours [ (0.24 ±0.08) g/L] than the pre-operative level [ (0.37 ±0.36) g/L] (P =0.030),and were not significantly different at the other time points ( P ＞ 0.05 ).In the EN + PN group,the C4 level was not significantly different during the perioperative period ( P ＞ 0.05 ).There was no significant difference in the infection-related complications and hospital stay between these two groups ( P =0.300,P =0.371 ).ConclusionsThe effects of EN or EN + PN on humoral immunity and outcomes after esophagectomy in patients with esophageal carcinoma are not different.Both these two nutritional support modes can not completely alleviate the harm to the humoral immunity.The EN is more cost-effective.

ObjectiveTo explore the relationship of trefoil factor family 3 (TFF3),vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1α in gastric cancer SGC-7901 cells under hypoxic condition and try to investigate the mechanism of TFF3 in the genesis and development of gastric cancer. Methods The hypoxic model of gastric cancer SGC-7901 cell was induced by CoCl2 Gastric cancer cell line SGC-7901 cells were transfccted with pU6-siTFF3 plasmid which carrying RNAi targeted to human TFF3 and pU6-mock.Puromycin was selected as screening medicine.The stable and specific TFF3 inhibited gastric cancer cell line was established. Gastric cancer cell line SGC-7901 and TFF3 RNAi targeted gastric cancer cell line SGC 7901 were cultured under hypoxic condition and normoxic condition. The expression of TFF3,VEGF and HIF-1a at protein and mRNA level were detected by RT-PCR,Western blot and ELISA assay.The distribution and expression of TFF3 and HIF-1α in gastric cancer cell line SGC-7901 cells uuder normoxia and hypoxic condition were determined with immunofluorescence staining.Results The expressions of HIF-1a,TFF3 and VEGF in gastric cancer SGC-7901 cell increased under CoCl2 induced hypoxic condition (33.4 =1.8,14.8 ± 1.1 and 15.1 ± 1.2,respectively). Under hypoxie condition,the expression of VEGF and HIF-1α protein reduced in stable TFF3 RNAi SGC-7901cells.Conclusion TFF3 mediated the regulation of VEGF and HIF-1α expression under hypoxic condition.TFF3might be a potential anti-angiogenic target in gastric cancer treatment.

Objective To analyze gene mutations of a Niemann-Pick disease type C (NPC) proband,and carry out prenatal diagnosis for the family.Methods The coding regions of NPC1 gene in the proband (late-infantile form) and white blood cell (WBC) in peripheral blood of its parents were amplified by polymerase chain reaction and direct DNA sequencing in both directions was performed.The sequencing results were compared with human NPC1 gene sequence (NM_000271) in GenBank,and sequences of mutated exons were determined.Direct sequencing was used on 50 normal Chinese individuals' DNA samples (control) to exclude mutation's single nucleotide polymorphism (SNP).An inter-species alignment of homologous NPC1 proteins was performed using ClustalX 1.81 software.During the second pregnancy of the proband's mother,the amniotic fluid was obtained at 18 weeks of gestation and the amniocytes were cultured for gene mutation analysis.Neonate's DNA of WBC in peripheral blood was also extracted for NPC1 gene analysis.Results Mutation analysis of NPC1 gene revealed two novel heterozygous mutations (c.2284-2287 delCTCT and p.V959G) in the proband,which originated from her father and mother,respectively.These two mutations were absent in the control,suggesting that these mutations were not SNP.While comparing with the amino acid in NPC1 protein of human,mouse,rat,rabbit,cat and pig,it revealed that p.V959 belonged to a conservative amino acid region and the missense mutation of p.V959G may perturb the function of NPC protein.Neither mutation was found in DNA from amniotic fluid or from the cultivated amniocytes in the second pregnancy,suggesting a normal fetus.c.2284-2287 delCTCT and p.V959G mutation were not found in NPC1 gene analysis of WBC in peripheral blood of the neonate,which was consistent with the prenatal diagnosis.Conclusions PCR-direct sequencing could be used as genetic diagnosis for NPC proband and prenatal diagnosis for its family.The mutation p.V959G may be correlated to late infantile form of NPC.

Objective Establish a method for measuring the activities of Galactocerebrosidase (GALC), α-Glucosidase(GAA), α-Galactosidase (GLA) and α-L-Iduronidase (IDUA) in dried blood spots specimen by tandem mass spectrometry ( MS/MS ).Methods A total of 2175 dried blood spot samples forinborn errors of metabolism in neonatalscreening center of Shanghai Xinhua hospital were collected in July and November, 2013.And twenty dried blood spot samples from patients withlysosomalstorage disorders( LSDs) of Shanghai Xin Hua Hospital were collected from September 2012 to January 2014.The extraction of DBS was incubated with enzyme substrates and internal standards.After liquid-liquid and solid-phase extraction, the extraction solution was dried under nitrogen and reconstituted.Then enzyme reaction products and internal standards were analyzed by MS/MS.Linearity, precision, accuracy and the limit of detection were evaluated.2175 dried blood spot samples were detected to establish the normal reference range for the activities of four enzymes according to 0.5th to 99.5th percentiles.20 specimens from patients withLSDs were detected to verify the reference range inclinical judgment.Results The intraassay and interassay precisions ranged from 1.7%to 11.8%, and the intraassay and interassay accuracies ranged from 85%to 115%.The linear coefficients for measured concentration of enzyme products/internal standards and theoretical concentration were 0.997-0.999.The limits of detection forGALC, GAA, GLA and GLA were 0.03 μmol/(L· h), 0.09 μmol/(L· h), 0.12 μmol/(L· h) and 0.16 μmol/(L· h) .The normal reference values for GALC, GAA, GLA and GLAwere 0.51-8.51μmol( L· h) ,1.99-22.22μmol/( L· h),1.68-41.59 μmol/(L· h) and 2.36-19.21 μmol/(L· h).The enzymes of 20 patients with LSDs were remarkably decreased compared to the normal range.The Krabbe, Pompe, Fabry, MPSⅠpatients can be effectively detected by this MS/MS method.Conclusions A MS/MS method for measuring GALC, GAA, GLA and IDUA enzyme activities in DBShas been established.