Objective To establish human heart valve interstitial cells calcification culture model in vitro,and observe the effect of bone morphogenetic protein-2 (BMP-2) on calcification of human heart valve interstitial cells.Methods Human heart valve interstitial cells were cultured in vitro,and divided into control group:cells were cultured in conventional media plus recombinant human BMP-2 treatmnent and experimental group:besides above treaments,calcification inducers (recombinant hunan BMP-2,β-glycerophosphate,L-ascorbic acid,dexamethasone) were added to the culture media.The two group of cells were cultured for 14 days and were stained by Von Kossa,then the cell calcification was observed in this valvular interstitial cells calcification culture model in vitro.Protein expression of intercellular adhesion molecule 1 (ICAM-1),interleukin 8,BMP-2 and BMP-4 was determined by Western blot and BMP-2 secretion was measured by ELISA.Results In the control group,the structure of human heart valve interstitial cells was clear,and the spindle and radial growth shaped cellular morphology was visible,and Von Kossa staining was negative.In the experimental group,the nuclei become darker in color,and granular sediment distribution was seen surrounding cells,and Von Kossa staining was positive,the cells were forming nodules of calcification.The protein expression of ICAM-1,interleukin 8,BMP-2 and BMP-4 in the experimental was significantly higher than that of the control group (all P ＜ 0.05).The expression of BMP-2 in the experimental group was also significantly higher than that in control group ((92.5 ±4.9) pg/ml vs.(22.2 ± 1.9) pg/ml,P ＜ 0.05).Conclusion Human BMP-2,β-glycerophosphate,L-ascorbic acid,and dexamethasone can induce human heart valve interstitial cells calcification and enhance inflammation in vitro by stimulating the secretion of BMP-2.

Objective To observe the protein expression of Notch 1 in the cultured calcified human heart valve interstitial cells (hVICs) in vitro and related mechanisms.Methods hVICs were divided into two groups:control hVICs were cultured in conventional media for 14 days and calcified hVICs were cultured with calcification inducers:[3-glycerophosphate (500 μl),ascorbic acid (200 μl),dexamethasone (100 μl) for 7 days.The calcified hVICs were further divided into calcified hVICs group and inhibited calcified hVICs by adding specific Notch1 inhibitor DAPT (50 μmol/L(4 μl/hole))groups and cultured for another 7 days.Inflammatory response of all groups were induced by lipopolysaccharide (LPS) for 8 to 12 hours.Western blot was used to detect the protein expression of Notch1,phosphorylation nuclear transcription factor κB (p-NF-κB),bone morphogenetic protein-2/4(BMP-2/4).ELISA was applied to detect the content of BMP-2 secretion of the groups.Von Kossa staining was used to observe of cellular calcification.Results (1)Von Kossa staining is positive in the induced calcification group,the expression of Notch1,p-NF-κB,BMP-2 and BMP-4 is significantly higher in the induced calcification group than in the control group (all P ＜ 0.05).The expression of BMP-2 is significantly higher in the induced calcification group than in control group ((88.23 ± 3.28) pg/ml vs.(25.41 ± 3.68) pg/ml,P =0.02).(2) After treatment with DAPT,the calcification and the expression of Notch1,p-NF-κB,BMP-2 and BMP-4 were significantly decreased compared to calcification group (all P ＜ 0.05).The expression of BMP-2 is (26.74 ± 4.62) pg/ml in the calcification inhibition group and (80.41 ± 2.96) pg/ml in calcified control group (P =0.02).Conclusions Upregulated Notch 1 expression promotes BMP-2/4 secretion in LPS stimulated hVICs,and contributes to osteogenic changes in hVICs.Inhibiting Notch1 can decrease the BMP-2/4 secretion and calcification in hVICs,which may serve as a novel therapeutic option for treating calcific valve disease.

We reported a case of monochorionic monoamniotic twins discordant for anencephaly diagnosed by second-trimester ultrasonography at the First Affiliated Hospital of Fujian Medical University.Ultrasound at seven weeks of gestation showed only one gestational sac with an embryo inside.Another 12 gestational weeks' ultrasound scan performed at another hospital found one gestational sac and one fetus (crown-rump length was 6.11 cm and nuchal translucency was 0.11 cm) in the upper-middle uterine cavity.The ultrasound examination at 22+6 gestational weeks identified one placenta and two fetuses without obvious diaphragm echo in between.Although no structural abnormality was observed in one fetus,frog-like eyes,absence of skull image and brain tissue echo were presented in the other fetus.The patient was transferred to a higher level hospital and was successfully performed radiofrequency ablation for selective reduction at 23+4 weeks of gestation.At 35 weeks,a premature live boy and an anencephalic stillbirth fetus were born vaginally after premature rupture of membranes.The baby boy was healthy at follow-up at four months old.