BACKGROUND:At present, a lot of research about culture methods for umbilical cord mesenchymal stem cels, but not for the waste of primary system. OBJECTIVE:To explore the best culture method of human umbilical cord mesenchymal stem celsin vitro. METHODS:Human umbilical cord mesenchymal stem cels were prepared by tissue explants method, recorded as initial culture group. The centrifugal fluid and tissue of the primary culture flask were centrifuged and divided into three groups for secondary culture: tissue group, mixed group and pure liquid group. Cel morphology, time for cel acquisition, and yield of primary cels in the four groups were observed; the cel growth curve was analyzed by MTT assay; and cel cycle and phenotype were detected by flow cytometry. RESULTS AND CONCLUSION: The average time for cel acquisition in the initial culture group, tissue group, mixed group and pure liquid group were (15.00±0.45), (7.0±0.3), (8.00±0.25) and (8.00±0.25) days, respectively. The number of cels at first generation was (4.0±0.5)×105, (9.0±0.55)×105, (15.0±0.2)×105 and (7.0±0.33)×105 markers of the four groups had no significant differences. The human umbilical cord mesenchymal stem cels can be obtained rapidly and largely through the secondary culture to the primary culture system. T75 culture bottle, respectively. Under the inverted microscope, cels in the four groups were fusiform-like adherent cels, which were in paralel or circinate arrangement. Growth curve, proliferative activity, surface markers of the four groups had no significant differences. The human umbilical cord mesenchymal stem cells can be obtained rapidly and largely through the secondary culture to the primary culture system.

Objective To explore the role of apelin-13 in regulating stem cell differentiation into vascular net. Meth?ods Mesenchymal stem cells were isolated from human umbilical Wharton’s jelly using tissue adherence method.Their immunophenotypes were detected by flow cytometry . Passage 3 of WJ-MSCs (Wharton’s jelly-mesenchymal stem cells) were inoculated in 4 flasks, denoted as A1, A2, A3, A4 group. TwentyμL of apelin-13 at concentrations of 0, 1×10-6, 10 × 10-6 and 100 ×10-6 mol/L were added to A1, A2, A3 and A4 respectively each day. After being induced for 7 days, cell mor?phology and viability were observed under inverted microscope. Von Willebrand factor (vWF) was examined by immunofluo?rescence and CD31 was identified by flow cytometry. Upon incubating with three dimensional culture medium of hydrogel, those cultured A1, A2, A3 and A4 were renumbered as S1, S2, S3, S4. Again, twentyμL of apelin-13 at concentrations of 0, 1×10-6, 10 ×10-6and 100 ×10-6 mol/L were used to treat S1, S2, S3 and S4 respectively. After 7 days, cell morphology, via?bility and vas-like networks were observed with inverted microscope. Results Our study showed that WJ-MSCs can be in?duced by apelin 13 to differentiate into endothelial cells lineage indicated by positive of vWF staining. Moreover, CD31 expres?sion increases significantly upon apelin-13 addition in a dosage dependent manner. The endothelial cells line formed vas like networks when cultured with three-dimensional medium containing hydrogel. Conclusion This study demonstrated that ape?lin-13 could promote human umbilical cord-MSCs to differentiate into endothelium lineage then to form vascular networks.

Objective To explore the association of methyl-CpG-binding protein2 (MeCP2) polymorphisms with female schizophrenia in Chinese Han population.Methods By using the technology based on microarray Chip,gene polymorphism analysis of 4 locus located in the gene MeCP2 was performed in 126 schizophrenia patients fulfilled with DSM-Ⅳ-TR criteria for schizophrenia and in 144 healthy controls.Chi square test was used to compare the inter-group differences of genotypic and allelic distribution.Haplotype case-control association analysis based on linkage disequilibrium was conducted using SNP stats online software after the data was screened.The significance of results was corrected by permutation test.Results To the point of Locus rs1616369 which located in Gene MeCP2,the distribution of the genotype G/A between two groups(24.8％ vs 34.3％) reached significance (P＜0.05) ; rs3027933,genotype G/C(25.6％ vs 34.8％),P＜0.05 ; rs17435,genotype T/A(25.6％ vs 35.5％),P ＜ 0.05 ; rs2239464,genotype C/A (58.5％ vs 71.8％),P＜ 0.05.The distribution prevalence of Haplotype GCCA was 0.1581 in case group,and 0.2389 in control group,reached significant difference(P＜0.05).Conclusion As to female MeCP2 gene,the genotype on the four locations might associate with the onset of schizophrenia,MeCP2 might be susceptibility gene of female schizophrenia.The Haplotype GCCA maybe the protective factor.

Objective:To evaluate the value of growth differentiation factor 15(GDF-15)and hepatocyte growth factor(HGF)in mortality prediction for patients with chronic heart failure(CHF)during a 5-year follow-up.Methods:This prospective case-control study enrolled 141 CHF patients hospitalized at the First Affiliated Hospital of Anhui Medical University between August 2015 and September 2017, including 59 with preserved ejection fraction(HFpEF)and 82 with non-preserved ejection fraction(non-HFpEF). Using all-cause mortality as the endpoint during the 60-month follow-up, there were 93 cases in the survival group and 48 cases in the death group.Clinical baseline data of patients in the two groups were compared, and the prognostic value of GDF-15 and HGF for CHF was assessed using multivariate logistic regression analysis, receiver operating characteristic curves(ROC curves), the area under the ROC curve(AUC), and Kaplan-Meier survival curves.Results:The results of multivariate Logistic regression analysis showed that GDF-15, HGF, glomerular filtration rate, and body mass index were independent risk factors for CHF prognosis during the 60-month follow-up; The degrees of predictive ability on mortality in 60-months in patients with heart failure were estimated for GDF-15( AUC=0.769, 95% CI: 0.685-0.854), HGF( AUC=0.765, 95% CI: 0.676 to 0.854), body mass index( AUC=0.689, 95% CI: 0.594 to 0.783), and glomerular filtration rate( AUC=0.612, 95% CI: 0.518 to 0.705). The AUC values of GDF-15 and HGF were greater than those of the body mass index and the glomerular filtration rate.Using GDF-15=2 326 ng/L and HGF=1, 603 ng/L as the cut-off values, the Kaplan-Meier survival curves showed statistically significant differences in survival rates between the two groups( P<0.05)The mortality rate in the non-HFpEF group was higher when GDF15 ≥2, 326 ng/L and HGF ≥1, 603 ng/L(100%, 15/15)than that in the HFpEF group(50%, 2/4), and the difference was statistically significant( χ2=5.526, P<0.05). Conclusions:GDF-15, HGF, the estimated glomerular filtration rate and the body mass index are independent prognostic risk factors for CHF during a 60-month follow-up period.GDF-15 and HGF are independent predictors of all-cause death in patients with CHF, especially those with non-HFpEF during 5-years.

Objective:To investigate the value of counting microvessel density (MVD) and lymphatic vessel density (LVD) in predicting distant metastasis of pancreatic cancer within 1 year after surgery.Methods:The clinicopathological data of 47 patients with pancreatic cancer who underwent surgery in Laibin People's Hospital from January 2020 to December 2021 were retrospectively analyzed. The patients were divided into non-metastasis group( n=24) and metastasis group( n=23) according to whether distant metastasis occurred during 1-year follow-up. Immunohistochemistry was used to detect the CD 34 expression in microvascular epithelial cells and D2-40 level in lymphatic epithelial cells from pancreatic cancer tissues. MVD and LVD in cancer tissues and adjacent normal tissues were counted. The relationship between MVD and LVD in cancer tissues and clinicopathological characteristics such as gender, age, tumor diameter, tumor differentiation, lymph node metastasis, vascular invasion, nerve invasion and tumor stage were analyzed. The receiver operating characteristic curve (ROC) was drawn and the area under the curve (AUC) was calculated to evaluate the value of MVD and LVD in predicting distant metastasis of pancreatic cancer within 1 year after surgery. The effects of MVD and LVD on the distant metastasis rate of pancreatic cancer within one year after operation were evaluated. Univariate and multivariate logistic regression were used to analyze the independent influencing factors for distant metastasis of pancreatic cancer within 1 year after surgery. Results:MVD and LVD in metastatic cancer tissues were higher than those in adjacent normal tissues [(72.52±9.73) vs (51.73±7.95)/400 times field of view, (23.78±6.87) vs (14.00±5.66)/400 times field of view]. MVD and LVD in the non-metastasis group were also higher than those in the adjacent normal tissues [(63.20±6.52) vs (54.79±5.80)/400 times field of view, (16.25±5.15) vs (13.62±5.03)/400 times field of view], and all the differences were statistically significant ( P<0.05). MVD in cancer tissue was significantly increased in patients with tumor diameter ≥2 cm, lymph node metastasis, vascular invasion and high TNM stage ( P<0.05), and LVD was significantly increased in patients with tumor diameter ≥2 cm, lymph node metastasis, moderate and low differentiation, vascular invasion, nerve invasion and high TNM stage ( P<0.05). The AUC values of MVD and LVD in predicting distant metastasis of pancreatic cancer within 1 year after surgery were 0.799 (95% CI 0.659-0.939) and 0.803(95% CI 0.676-0.929), and the cut-off values were 70.5 and 20.5/400 times field of view, respectively. The sensitivity was 73.9% and 69.6%, and the specificity was 87.5% and 83.7%. The cumulative distant metastasis rate within 1 year after operation in high MVD and high LVD groups was significantly higher than that in low MVD and low LVD groups ( P<0.05). Multivariate logitic regression analysis showed that tumor diameter ≥2 cm ( OR=1.757, 95% CI 1.536-3.846, P<0.05), lymph node metastasis ( OR=2.364, 95% CI 1.036-4.175, P<0.05), high MVD ( OR=4.345, 95% CI 1.245-3.736, P<0.05) and high LVD ( OR=3.637, 95% CI 1.426-4.035, P<0.05) were independent risk factors for distant metastasis of pancreatic cancer within 1 year after surgery. Conclusions:Increased MVD and LVD in pancreatic cancer tissues are independent influencing factors for distant metastasis within 1 year after surgery, which can be used to predict whether patients have distant metastasis within 1 year after surgery.

OBJECTIVE：To study the effects of costunolide on the proliferation ，migration and apoptosis of breast cancer SK-BR-3 cells and its mechanism. METHODS ：SK-BR-3 cells in logarithmic growth period were collected and cultured with different concentrations （10，20，30，40，50 μmol/L）of costunolide for 24，48，72 h. Inhibitory rate of costunolide on cell proliferation was detected with CCK- 8. The cells were divided into blank control group and costunolide group （10，20，30 μmol/L）. Hoechst 33258 fluorescence was used to observe the morphology and apoptosis of cells ，and apoptotic rate of cells were calculated. Cell scratch test was used to detect the migration ability of cells and calculate the migration rate. Western blotting was used to detect the relative expression level of Bcl- 2，Bax，Caspase-3 and Cleaved Caspase- 3 in cells. RESULTS ：The proliferation of SK-BR-3 cells were significantly inhibited by costunolide （P＜0.05 or P＜0.01），and it shows a trend of concentration and time dependence. In the blank control group ，cells possessed clear contour ，regular shape and good adherence . Compared with blank control group，the number of cells were decreased significantly in 10，20，30 μmol/L costunolide groups，the cell structure was loose，the volume was reduced ，and the gap became larger ，and most of the cell contour disappeared and became round ，the cell adherence was poor ；cell migration rate and Bcl- 2 protein relative expression level were decreased significantly ，while apoptosis rate and the relative expression level of Bax ，Caspase-3 and Cleaved Caspase- 3 protein were significantly increased （P＜0.05 or P＜0.01）. CONCLUSIONS ： Costunolide can inhibit the proliferation and migration ，and induce apoptosis of human breast cancer SK-BR- 3 cells，mechanism of which may be through up-regulating the expression of Bax ，Caspase-3 and Cleaved Caspase- 3 while down-regulating the expression ofBcl-2.