Objective To investigate the mechanism of protective effects of resveratrol on tissueengineered cartilage.Methods The chondrogenesis of alginate-encapsulated bone marrow mesenchymal stem cells (BMSCs) were evaluated by toluidine blue staining and immunostain.The morphology of BMSCs-derived chondrocytes cultured on chitosan-gelatin scaffolds (CGS) was evaluated by scanning electron microscope and laser confocal microscope.When these cells on CGS were pre-stimulated with interleukin-1β (IL-1β) or cotreated with IL-1β and resveratrol in the absence and presence of specific β1-integrin blocking antibody,collagen type Ⅱ,aggrecan,matrix metalloproteinase-13 (MMP-13) expression,and the translocation of nuclear factor kappaB (NF-κB) were analyzed by Western blotting.ANOVA was used for statistical analysis.Results Alginate bead culture plus conditional medium together could induce the cartilage-specific collagen type Ⅱ,aggrecan expression and extracellular matrix accumulation in differentiated chondrocytes.CGS supported differentiated cell attachment,proliferation,and migration.When those cells cultured on CGS were stimulated with IL-1β alone,collagen type Ⅱ and aggrecan expression was inhibited.However,MMP-13 expression increased.By Western blotting semi-quantitative analysis,the expression level of cartilage-specific collagen type Ⅱ of the control group was 0.484±0.006; the expression level of resveratrol intervention group was 0.474±0.014.The difference between these two groups was not statistically significant (P＞0.05).The expression level of the IL-1β intervention group reduced to 0.155±0.009,which was statistically significant different from the above two groups(P＜0.05).Resveratrol could antagonist the negative effect of IL-1β,and increase collagen type Ⅱ to 0.468±0.014,the difference between these two was statistically significant (P＜0.05),and no significant difference when compared to the control group (P＞0.05).Specific β1-integrin blocking antibody could abrogate these effects of resveratrol,decrease collagen Ⅱ expression to 0.169±0.011,the difference was significant (P＜0.05),but there was no difference when compared to the IL-1β group (P＞0.05).Aggrecan semi-quantitative expression has the same trend in the expression of type Ⅱ collagen while the expression of MMP-13,NF-κB had the reversal trend.These indicated that the resveratrol reversed the catabolic effects by reducing the nuclear translocation of NF-κB.Specific β1-integrin blocking antibody abrogated these effects of resveratrol.Conclusion Resveratrol,by regulating β1-integrin,acts as a NF-κB nuclear trans-location inhibitor to protect tissue-engineered cartilage.

Objective To explore the method of culture of rat adipose-derived stem cells(ADSCs) and differentiation induction into myoblasts. Methods Adipose tissues were obtained from SD rats,and were isolated by enzyme digestion and cultured into ADSCs.The expression of surface antigen CD90,CD105 and CD34 was detected by immunofluorescence and flow cytometry.ADSCs of the second passage with logarithmic growth were obtained,and culture media containing 5-azacytidine(5-aza) and basic culture media were employed for cells in induction group and control group,respectively.The induction lasted for 7 d,14 d,21 d,28 d and 35 d,respectively.Cell growth and cell morphology were observed by inverted phase contrast microscope,and immunofluorescence and flow cytometry were utilized to detect the expression of myoblast specific antigens desmin and myosin. Results ADSCs were successfully isolated and cultured,and were identified to be stem cells.On the 28th day of induction,cells in induction group displayed "swirl" morpholgy,and multinucleation was observed.It was revealed by immunofluorescence and flow cytometry that the highest expression rates of desmin and myosin were 52.57% and 50.04%,respectively on the 28th day of induction,while there was no expression before induction and in control group. Conclusion ADSCs can be isolated and cultured from rat adipose tissues,and can further differentiate into myoblasts after induction by culture media with 5-aza.The expression of myoblast specific antigen is the highest on the 28th day of induction.

Objective To explore the application value of two-dimensional echocardiography with spatio-temporal im-age correlation (STIC) in fetal congenital heart disease (CHD) prenatal ultrasonography. Methods 11 036 hearts of fetus were inspected by severalviews order scanning method and for STIC volume database acquisition and off-line a-nalysis congenital heart disease fetus for autopsy after induced labor or contrast the follow-up results after birth. Re-sults 97 cases with spatio-temp-oral image correlation in 176 cases congenital heart and great vessels exception (dysrhythmias not including) with simple two-dimensional echocardiography,92 cases were accordant (one case with incorporative intracardiac malformation missed diagnosis);in screened congenital heart disease fetus,STIC (n=79) and routine ultrasonography (n=87) took (7.76±2.42) min and (9.68±2.13) min per case,respectively;in dif-ferent gestational weeks,the quality of the images derived from volume datasets were comparable to that directly ob-tained from 2D echocardiography. Conclusion STIC technology can be used as effective supplementary means of 2D echocardiography, and the combination can further improve the prenatal diagnosis of fetal congenital heart disease.

Objective To explore the rare primary mt-DNA mutation in LHON patients in China.Design Clinical study.Partici- pants Clinically diagnosed LHON patients whose common primary mt-DNA mutations were negative and normal persons.Methods Polymerase chain reaction(PCR)and DNA sequencing were used to detect the mutations of G11778A,G3460A and T14484C in mt-DNA of clinically diagnosed LHON patients.Fragments of mt-DNA 3962～4356 and 11320～11789 were PCR amplified and se- quenced if all the three mutations above were negative and also in the control group.The sequence results were analyzed by the BLAST service provided by NCBI and compared with the 2001 Revised Cambridge Reference Sequence for the mt-DNA mutations C4171A and G11696A.Main Outcome Measures The sequence results of mt-DNA.Results There were totally 56 eases of clinical diagnosed LHON patients included.The mutation of G11778A was found in 36 cases,G3460A was found in 3 cases,and T14484C was found in 5 eases.Twelve cases of clinical diagnosed LHON patients didn't harbor any of the three common primary mutations,one case harbored the mutation C4171A,the mutation of G11696A was not found.There were totally 25 cases in the control group,the mutations of G11696A and C4171A were not found.Conclusion C4171A may be rare primary mt-DNA mutation in LHON patients in China.(Oph- thalmol CHN,2007,16:382-385)

Objective To investigate the effect of glucocorticoid and bisphosphonate on Hedgehog signaling pathway in human bone mesenchymal stem cells (hBMSCs) and bone tissue.Methods ① Bone biopsy test:forty cases of systemic lupus erythematosus (SLE) patients were divided into 2 groups:20 cases in newly diagnosis group,20 cases in the GCs group (the dosage of glucocorticoids was higher than 1 mg·kg-1·d-1).Patients in the GCs group was randomly divided into 2 groups:10 cases in the control group were without anti-osteoporosis treatment,10 cases in treatment group received alendronate 70 mg once a week.All of the patients had bone marrow puncture after24 weeks,and the value of average optical density of Gli1 was tested with immunohistochemistry.② Cell culture:hBMSCs cultured in normal medium were intervened with rh-SHHN and methylprednisolone (10-3 mol/L,10-5 mol/L) after successfully identified.Quantitative polymerase chain reaction (PCR) was used to detectthe mRNA expression of Gli1.The final calcium nodules was detected by Alizarin red staining.hBMSCs cultured in osteogenesis medium were intervened with bisphosphonate and methylprednisolone (10-3 mol/L) after successfully identified.Quantitative PCR was used to detect the expression of Gli1 mRNA.Alizarin red staining was used to detect the final calcium nodules.Comparisons between the two groups were carried out using t-test while the difference analysis of multi-groups were tested by factorial analysis.Results The average optical density of Gli1 in the GCs group (47±7) was less than the newly diagnosed group (61±12) (t=4.442,P＜0.01),and it was less in the control group (51±6) than in the treatment group (42±6) (t=3.701,P=0.002).When normally cultured,high and moderate concentration of methylprednisolone suppressed the mRNA (0.38±0.04; 0.68±0.24) (F=8.748,P＜0.01) expression of Gli1 which was initially stimulated by rh-SHHN (2.01 ±0.38).And the final calcium nodules in groups which contained methylprednisolone were much less than rh-SHHN only group.When hBMSCs were cultured in osteogenesis medium,compared with the methylprednisolone group (0.024±0.011),the expression of Gli1mRNA(0.034-0.006) (t=7.62,P＜0.01) and the final calcium nodules were significantly improved by bisphosphonate.Conclusion High and moderate doses of glucocorticoids inhibit hBMSC osteogenesis by inhibiting Gli1.Low concentration of alendronate can not only stimulate hBMSC osteogenesis differentiation but also can remit the inhibition effects of GCs through the way of Hedgehog.

Objective To compare the perioperative outcomes of laparoscopic liver resection (LLR) versus open liver resection (OLR) for hepatocellular carcinoma (HCC).Methods A total of 89 HCC patients undergoing liver resection between January 2012 and November 2016 were enrolled.Nonparametric tests were employed to compare the clinicalpathological characters and preoperative outcomes.Results No significant difference was observed in clinicalpathological features and postoperative morbidity.LLR group had shorter hospital stay (Z =4.642,P ＜0.01),lower serum ALT level in 1st,3rd and 5 day (Z =2.157,3.089,2.384,all P ＜0.05) and AST level in 1st-and 3rd-day postoperatively (Z =2.688,2.566,all P ＜0.05).The growth rate in serum total protein (TP) and albumin (ALB) postoperatively is higher for LLR group (y =2.348 4x + 51.696 vs.y =0.902 9 + 35.532),(y =1.539 9x + 29.68 vs.y =0.732 9x + 30.406).Conclusion LLR allows quicker liver function recovery and shortens patients' postoperative hospital stay.

Objective To investigate the efficacy and safety of vasopressin receptor antagonist tolvaptan for treating dilutional hyponatremia casused by decompensated liver cirrhosis.Methods Ninety-six subjects with decompensated liver cirrhosis complicated by dilutional hyponatremia were divided into test group (n =56) and control group (n =40) by double blind method.Test group were treated with a single dose of tolvaptan 15 mg orally in addition to routine therapy,while control group were treated with routine therapy plus placebo.The changes in serum sodium concentration,liver functions,ascites,and edema of lower extremities between the two groups were observed.Measurement data were compared by t test,and categorical data were compared by chisquare test.Results On day 7,36 (64.3％) patients in test group and 9 (22.5％) patients in control group reached normal serum sodium concentrations (x2 =5.241,P＜0.01).All the patients in test group and only 3 patients in control group had 24 hours' total urine volume above 3500 mL.Difference of 24 hours' total urine volume during 7-day treatment between the two groups was statistically significant (t=20.899,P＜0.01).Thirty-nine patients in test group and 15 patients in control group had reduced ascites (x2 =4.260,P=0.039),but improvement of edema in lower extremities of both groups was comparable (12 cases in test group and 7 cases in control group; x2 =0.227,P=0.634).Serum alanine aminotransferase (ALT),total bilirubin (TBil),potassium and creatinine levels of test group were improved after treatment,but were not significantly different from either baseline levels (t=1.509,0.783,1.107,1.237; both P＞0.05) or those of the control group (t=1.712,1.635,1.121,0.873; both P＞0.05).Conclusions Single dose of tolvaptan (15 mg) exerts a strong diuretic effect and is able to greatly increase serum sodium concentration,thus has distinct therapeutic effect for patients with decompensated liver cirrhosis complicated by dilutional hyponatremia.

Objective To evaluate the application of liver ultrasound hemodynamic parameters in diagnosing moderate to severe esophageal varices(EV) among patients suffering from portal hypertension.Methods The control group consisted of twenty nine patients without EV.Sixty patients with EV diagnosed by endoscopy were divided into two groups equal in number according to grade of EV.One being mild,the other was moderate to severe.All patients underwent color Doppler flow imaging and contrastenhanced ultrasound,and the dynamic angiography data were collected.Time intensity curves were drawn by software to acquire the arrive time of hepatic artery (HAAT),hepatic vein (HVAT) and portal vein (PVAT).Then HV-HA interval time,PV HA interval time and PV-HV interval time were calculated.The quantitative parameters including HV-HA,PV-HA,PV-HV,damping index (DI) of hepatic vein and portal vein velocity(PVV) were compared.ROC curve was used to assess the value of the parameters above in predicting moderate to severe esophageal varices.Results The difference of PV-HV,PV HA,HV-HA,PVV,DI between the control and the moderate to severe group were statistically significant (P =0.000).PV-HV,PV-HA,HV-HA,DI in moderate to severe group showed statistically significant compared with mild group (P =0.000,P =0.002,P =0.000,P =0.002),however,PVV had no significant (P ＞0.05) difference between the two groups.Mild group contrasted to the control group,the differences of PV-HA and HV-HA were statistically significant (P =0.000),whereas PV-HV,DI,PVV displayed no significant (P ＞0.05) difference.The areas under ROC curve of PV-HV,PV-HA,HV-HA,PVV,DI were 0.952,0.775,0.847,0.699,0.808 respectively.The best cutoff value of PV-HV for diagnosing moderate to severe EV was 0.5 second,with sensitivity and specificity of 86.2％ and 94.8％ respectively.Conclusions The liver ultrasound hemodynamic parameters is helpful in the diagnosis and evaluation of moderate to severe esophageal varices,which is expected to become a new noninvasive method.

OBJECTIVEThis study aimed to evaluate the diagnostic value of a 24-hour esophageal combined multichannel intraluminal impedance pH (24 h our MII-pH) monitoring in children with gastroesophageal reflux (GER) disease and recurrent pneumonia.

METHODSA total of 17 cases with a suspected diagnosis of GER disease children with unexplained recurrent pneumonia underwent a 24-hour MII-pH monitoring to analysis of the nature and characteristics of reflux.

RESULTSIn the 17 cases of GER disease with recurrent pneumonia, 11 (65%) were confirmed positively by a 24-hours of MII-pH monitoring. A total of 853 reflux cycle were detected, of which 65.3% were acid refluxes predominantly occurring within 2 hours after meal, and 71.6% were mixed refluxes. Refluxes occurred most frequently in the supine position, followed by the lateral position and the prone position. Distal reflux was the predominant form of reflux. According to the Biox-Ochoa classification, 73% of cases (8/11) were severe GER.

CONCLUSIONSMII-pH monitoring may effectively characterize refluxate properties and thereby increase the detection rate of GER disease.