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 observe the effects of transplantation of autologous adipose-derived stem cells (ASCs) on osteoporosis (OP) in a rabbit ovariectomy (OVX) model.Methods A total of fifteen 6-month-old female New Zealand white rabbits were randomly divided into two groups:ovariectomy group (group A,n =12) and sham operation group (group B,n =3).All rabbits were subjected to bilateral ovariectomy in the group A.Six months later,bone mineral density (BMD) of group A and group B were measured by dual energy X-ray absorptiometry (DXA) to check the result of OVX-OP.ASCs harvested from adipose of OP rabbits were cultured to be expanded and differentiated in osteogenic medium in vitro.Osteogenesis was evaluated by alizarin red staining,alkaline phosphatase (ALP) staining and quantitative assays of osteocalcin (OCN).Autologous osteo-induced ASCs were mixed in calcium alginate hydrogel (CAH) and then transplanted in the left distal femurs,while CAH was transplanted in the right distal femurs of OP rabbits.At 12 weeks after implantation,BMD,micro-CT and histomorphological analyses were performed on these rabbits.Results The BMD of femurs in group A rabbits were obviously lower than that of group B rabbits (P ＜ 0.05) at 6 months after OVX.Compared with control group,ASCs cultured in osteo-induction medium had similar proliferation rate as the non-induced cells,but displayed positive ALP and alizarin red staining and OCN contents.At 12 weeks after implantation,the cell-treated femurs displayed higher BMD,bone trabecula number,trabecula thickness and separation than those of control group,while the structure model index and porosity were lower (P ＜ 0.05).Histological examination indicated that the trabecular thickness increased with complete CAH resorption in cell-treated group,while CAH remained in control group.Conclusions Transplantation of autologous ASCs can help strengthen osteoporotic bone in OVX-OP rabbits,providing a novel approach to OP treatment.

Objective To study the expressions of the tumor suppressor gene TSH receptor( TSHR),P16, and RAS in papillary thyroid carcinoma ( PTC ) , and the correlation between the occurrence of tumor and the aberrant promoter hypermethylation of three tumor suppressor genes. Methods RT-PCR was used to detect the mRNA expression of three tumor suppressor genes in tissues of 50 cases of PTC ,20 cases of nodular goiter,and 12 cases of thyroid adenoma. The promoter methylation status of three tumor suppressor genes was examined by methylation-specific PCR technique( MSP). Gene sequencing was used to test if the hypermethylation existed in the promoter of three tumor suppressor genes. Results In 68.0% (34/50) TSHR gene, 54.0% (27/50) P16 gene, and 60.0% ( 30/50 ) RAS gene in PTCs, hypermethylation in promoter region was detected, the respective results 21.9% (7/32) , 15. 6% (5/32) ,and 31. 3% (10/32) were found in control tissues. The rates of the three genes with promoter hypermethylation in PTC were significantly higher than those in control tissues ( all P<0. 05). The mRNA expressions of TSHR,P16,and RAS were significantly lower in PTC than those in control tissues (0. 41 ± 0.11 vs 0.63±0. 08,0. 51±0. 17 vs 0. 72±0. 22,0. 56±0. 10 vs 0. 67±0. 16, all P<0. 05). The sequencing confirmed that there was CC to TC transmission in the promoters of three tumor suppressor genes. Conclusions The methylation of three tumor suppressor genes in promoter region is a common molecule event and may be involved in the genesis and development of human PTC.

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

Objective To explore whether the use of spatiotemporal image correlation (STIC) can supply additional information with respect to conventional fetal echocardiography in the prenatal diagnosis of total anomalous pulmonary venous connection(TAPVC).Methods Twenty-five cases diagnosed as TAPVC received fetal echocardiography examination.Four-dimensional volumes from 19 cases of suspected TAPVC were compared to conventional echocardiography.Echocardiographic characteristics were compared with the results of postnatal work-up and pathology.Results TAPVC was found in 25 cases by fetal echocardiography,four cases were isolated TAPVC,21 TAPVC had associated cardiac anomalies.Among them,nine cases were supracardiac types,six cases were infracardiac types,nine cases were intracardiac types and one was indefinite.Three true positivecases of TAPVC were confirmed after birth.Twenty-two choose to terminate pregnancy,9 of which refused to autopsy,and 10 true positive cases of TAPVC were confirmed at autopsy while 3 were considered false-positive cases.4D ultrasound with STIC clearly visualized the anomalous PV confluence and/or the draining vertical vein in all nineteen cases examined.Conclusions STIC may be proposed to identify abnormal venous drainage at the screening level,thus supplying additional information over that provided by 2D fetal echocardiography.

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.

Objective To investigate the changes and correlation of mitral valve coaptation length index CLI and coaptation area index CAI after mitral valvuloplasty MVP Methods A total of 30 subjects undergoing MVP for mitral regurgitation MR were studied Coaptation length CL CLI coaptation area CA and CAI were determined before and after surgery by 2-dimensional transoesophageal echocardiography 2D-TEE and 3-dimensional transoesophageal echocardiography 3D-TEE Results Compared with preoperative measurements CL CLI CA and CAI were significantly increased in postoperative studies CL 4 7±0 7 mm vs 9 4± 1 1 mm CLI 9 1 ±3 3 vs 38 5 ±4 1 CA 148 9 ± 65 3 mm 2 vs 371 9 ± 144 3 mm 2 CAI 9 3 ±3 1 vs 35 9 ± 7 5 all P < 0 05 CLI was significantly correlated with CAI both preoperatively r = 0 770 P < 0 01 and postoperatively r = 0 771 P <0 01 Furthermore CLI and CAI were significantly negative correlated with the degree of MR r =-0 897 P <0 01 r =-0 886 P <0 01 Conclusions Coaptation variables increased significantly in subjects after MVP CLI by 2D-TEE was related to CAI by 3D-TEE and both were useful for the assessment of mitral valve coaptation But CLI by 2D-TEE was more simple and feasible in clinic.