BACKGROUND:Kunming mouse embryonic fibroblasts are the most common feeder layers at present, and there are rare reports addressing C57BL/6 mouse embryonic fibroblasts as feeder layers.
OBJECTIVE:To separate and culture C57BL/6 mouse embryonic fibroblasts in vitro, and produce feeder layers to enlarge the resources of mouse embryonic fibroblasts.
METHODS:C57BL/6 mouse embryonic fibroblasts were isolated and cultured by trypsin digestion method in vitro. The biological characteristics and growth rule of the fibroblasts were investigated, then the feeder layers for the cel culture were produced. The growth of cel colonies on the prepared feeder layer was tested.
RESULTS AND CONCLUSION:C57BL/6 mouse embryonic fibroblasts grew wel with a large amount, by trypsin digestion method at different concentrations. There was no significance in the survival rate after cryopreservation for 1 week, 2 weeks, 1 month, 3 months and 6 months. The cel s were proliferative from the second to fifth passage and declined sharply after the sixth passage. The planted mouse embryonic fibroblasts feeder layers had a high activity within 3 days, but got a sharp decline after 4 days. So it is best to use C57BL/6 mouse embryonic fibroblast feeder layers within 3 days after they’re inactivated. C57BL/6 mouse embryonic fibroblast feeder layer can support embryonic stem cel s and induce pluripotent stem cel s to grow as Kunming mouse embryonic fibroblasts.

Objective To investigate the effect of oxaliplatin combined with capecitabine on the rate of local control and anal sphincter preservation of rectal cancer.Methods Eighty patients with stage Ⅱ/Ⅲ colorectal adenocarcinoma were randomly divided into control group and observation group,each group in 40cases.The patients in the observation group were treated with oxaliplatin and capecitabine.The control group was treated with oxaliplatin combined with leucovorin and 5-fluorouracil.The patients were followed up for 2~4 weeks.The local curative effect, perioperative biochemical index,R0 (excision margin without cancer cell) resection rate and sphincter preserving rate were compared between the two groups.Results In the control group, the short-term curative effect was 47.50%, which in the observation group was 60.00%, the difference was statistically significant (x2=4.22,P<0.05).There were no statistically significant differences in the biochemical indicators between the two groups (all P>0.05).The R0 resection rates of the control group and the observation group were 62.50%,57.50% (x2=0.67,P>0.05).The anal sphincter preservation rates of the control group and observation group were 32.50%,37.50% respectively (x2=0.67,P>0.05).The local control rates of the control group and observation group were 90.00%,92.50%,which of the observation group was higher than the control group, but the difference was not statistically significant(x2 =0.32,P>0.05).Conclusion The combination of oxaliplatin and capecitabine can improve the local curative effect in patients with rectal cancer without affecting the biochemical indicators of perioperative patients, but the effect of R0 resection rate and preserving anus rate in patients with rectal cancer compared with the chemotherapy regimen of FOLFOX4 has no significant difference, it is worthy of clinical promotion.

Objective To report the diagnosis and treatment of a case of renal inflammatory myofibroblastoma. Methods A 30-year-old man presented with space-occupying lesion of the kidney with no symptoms in physical examination.No abnormality was found in the laboratory tests.B-ultrasound showed a clear-edged,solid mass which was 4.5 cm?3.7 cm in size and had heterogeneous echo in the upper,middle part of the left kidney.CT showed a similar value of the mass to that of the normal tissue.Enhanced MRI showed heterogeneous intensification of the mass whose signal was slightly lower than that of the normal tissue.Preliminary diagnosis of renal cancer was made. Results Nephrectomy was performed.On pathological examination the tumor was mainly composed of spindle-shaped,fibrous cells, and positive staining for Vimentin,SMA and HHF35 were observed immunohistochemically.Inflammatory myofibroblastoma of the kidney was diagnosed.Follow-up of 54 months showed no recurrence and metastasis of the tumor. Conclusions Inflammatory myofibroblastoma is a kind of rare,benign or low-grade malignant tumor of the kidney, and the clinical diagnosis is often difficult.Definite diagnosis relies on pathology.For the sake of avoidance of resecting the kidney by mistake, pathological examination is the preferred choice during operation.

Brain-derived neurotropic factor (BDNF) contents in blood and cerebrospinal fluid (CSF) were detected by ELISA method in 30 cases of hypertensive cerebral hemorrhage (HCH group) and 30 healthy subjects (control group).The performance and neurological functions of patients were evaluated by National Institutes of Health Stroke Scale( NIHSS),Hamilton Depression Scale ( HAMD),Barthel Index of Daily Living Skills.The result showed that the contents of BDNF in blood and CSF of HCH patients in recovery stage were significantly higher than those in acute stage and those of control group ( P ＜ 0.05 ) ; the contents of BDNF in blood and CSF were negatively correlated with neurological impairment degree ( P ＜0.05).The results suggest that BDNF in the blood or the CSF may promote the recovery of neurological function in patients with hypertensive cerebral hemorrhage.

3T3-L1 adipocytes were cultured and differentiated into mature adipocytes in vitro. The adipocytes were intervened by ACAT inhibitor( 2 μg/ml) and ox-LDL with various concentrations (0,25,50,75,and 100 μg/ml)for 48 h,ACAT inhibitor( 2 μg/ml) and ox-LDL( 50 μg/ml) at the 0,6,18,36,and 48 h,or ACAT inhibitor( 2 μg/ml),ox-LDL( 50 μg/ml),and TUDCA with various concentrations(0,100,200,and 400 μ mol/L)for 48 h,respectively.The levels of visfatin in supernatant were examined by ELISA and the expressions of protein GRP78 and CHOP in adipocytes were detected by Western blot.After the adipocytes were treated with ACAT inhibitor and ox-LDL at different concentrations for 48 h,the cholesterol concentration and the expressions of GRP78 and CHOP protein in adipocytes and the visfatin levels in the supernatant fluid were increased with the increase of the ox-LDL concentration.The differences had statistical significance in the experimental groups compared with blank control group( all P＜0.05 ).After the intervention with ACAT inhibitor and ox-LDL for different durations,the expressions of GRP78 and CHOP protein in adipocytes and the visfatin levels in the supernatant fluid were up-regulated in a timedependent manner.The differences between experimental groups and blank control had statistical significance( all P＜0.05 ).After the intervention with ACAT inhibitor,ox-LDL,and different concentrations of TUDCA for 48 hours,the expressions of GRP78 and CHOP protein in adipocytes and visfatin levels in the supernatant fluid were down-regulated in a dose-dependent manner and as compared with blank control group the difference were statistically significant( all P＜ 0.05 ).The increase of cholesterol load in adipocyte may promote the visfatin secretion,denoting that the mechanism might be due to the enhancement of endoplasmic reticulum stress in aidpocytes.

Objective To screen the proteins interact with cytoplasmic tail of NECL1.Methods The cytoplasmic tail sequence of human NECL1 gene was inserted into the "bait" vector pGBKT7 in frame.The recombinant pGBKT7-NECL1C was then transformed into yeast cells and followed by screening of human fetal brain cDNA library.Then the GST pull down assay was used to confirm the interactions between the proteins.Results Nine different sequences by sequencing the positive clones and five alternative proteins were obtained by the amino acid sequence blast.Using the GST pull down analysis,we confirmed that two of them can interact with the cytoplasmic tail of NECL1 in vitro.Conclusion The alternative proteins obtained with the yeast dual-system may interact with the cytoplasmic tail of NECL1.

Objective To compare clinical effect and safety of left-double-lumen tube and bron-chial blocker in vedio-assisted atrial fibrillation ablation.Methods Forty-eight patients,26 males and 22 females,aged 45-65 years,ASA physical status Ⅰ or Ⅱ,who underwent vedio-assisted atrial fi-brillation ablation were randomly divided into double-lumen tube (group A)and bronchial blocker (group B),with 24 patients in each group.The tube type of group A was left-double-lumen tube. Bronchofiberscope was used for location in every patient.The mean artery pressure (MAP)and heart rate (HR)before intubation,intubation positioning time,peak airway pressure (Ppeak)after 5 min of one-lung ventilation,lung collapse,incidences of hoarseness,pharyngalgia and choke were ob-served.Results Intubation positioning time between two groups was not statistically significant. MAP and HR were significantly increased at intubation positioning time in both groups,to be specif-ic,they were significantly in group A than in group B (P <0.05 ).When left lungs blocked,Ppeak and qualities of lung collapse were not statistically different between the two groups.When right lungs blocked,group A was higher than that in group B (P < 0.05 ).Cough,hoarseness and sore throat were more frequently seen in group A than in group B.Conclusion Both double-lumen tube and bron-chial blocker can be used in video-assisted atrial fibrillation ablation with satisfying effects.As for the quality of lung isolation,double-lumen tube was better than bronchial blocker.However,compared with bronchial blocker,double-lumen tube results in more unstable hemodynamics and higher occur-rence of hoarseness,pharyngalgia and choke.

Objective To explore the expression and significance of vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1) in hepatic fibrosis.Methods Fifty-six cases of patients with hepatic fibrosis were selected as observation group and 50 healthy persons as control group.Immunohistochemistry were performed to detect VEGF and TGF-β1 in two groups.Results Serum VEGF and TGF-β1 in observation group were significantly higher than those in control group(VEGF:(110.87 ±32.64) μg/L vs (15.98 ±6.75) μg/L,t =20.166,P ＜0.001;TGF-β1:(15.08 ±4.27) ng/L vs (7.17 ±2.86) ng/L,t =11.066,P ＜ 0.001) ;There were significant differences on VEGF and TGF-β1 level among S1,S2,S3 and S4 subgroups(VEGF:(84.25 ±16.86) μg/L vs (101.87 ±36.70) μg/L vs (118.04 ±40.75)μg/L vs (134.65 ± 45.73) μg/L,F =15.689,P =0.015 ; TGF-β1:(10.87 ± 2.64) ng/L vs (13.06 ± 2.74)ng/L vs (17.87 ± 3.28) ng/L vs (22.76 ± 4.75) ng/L,F =12.438,P =0.026).VEGF had positive correlation with TGF-β1 (r =0.532,P =0.013).Conclusion VEGF and TGF-β1 level have close relationship with the occurrence and development of hepatic fibrosis.Combined detection of VEGF and TGF-β1 can be serum index for diagnosis and evaluation disease condition.

Objective　To investigate the change of epithelium growth factor(EGF), tumor necrosis factor (TNF), superoxide dismutese(SOD) and hemorrheology (HL) in patients with diabetic retinopathy.Methods　EGF、TNF、SOD levels and HL changes have been measured in 30 NIDDM patients with retinopathy (DR group), 29 NIDDM patients without retinopathy (NDR group), and 29 patients with senile cataract and without diabetes as control (C group). Results　The mean levels of EGF、TNF and blood viscosity were significantly higher in both DR and NDR group than those in C group; the mean levels of SOD was significantly lower in both DR and NDR group than that in C group. The EGF、TNF and blood viscosity was significantly higher in proliferative type DR than in background DR; The SOD was significantly lower in proliferative type DR than in background DR.Conclusion　The development of diabetic retinopathy is related to the increase of EGF、TNF、blood viscosity and decrease of serum SOD.

Objective To explore the applicability of the childhood autism spectrum test (CAST) of children in Mainland China according to autism screening,in order to provide the effective reference for the identification and diagnosis of autism spectrum disorders of children.Methods Two hundreds and fifty-two typical developing children,and 61 autistic children who were confirmed by psychological clinic of Child Healthcare Clinic,Shenzhen Luohu District Maternal and Child Health Care Hospital who were conformed with diagnosis standards of Diagnostic and Statistical Manual of Mental Disorders Fifth Edition(DSM-5) by using CAST Chinese version were assessed,and the reliability and validity analysis were made on the CAST scale.The receiver operating characteristic curve was drew to determine the optimal threshold value,and the sensitivity,specificity and positive predictive value were calculated.Results CAST Chinese version had good reliability,Cronbach's α was 0.880.Split-half reliability coefficient r was 0.821,and the whole scale calibration reliability coefficient was 0.902.There was statistically significant difference between items and the total score(P＜0.05).The consistency between the CAST ≥4 years old children and diagnosis standards of DSM-5 reached 0.884(P=0.000).The consistency 3-＜4 years old children and diagnosis standards of DSM-5 reached 0.867 (P=0.000).Among the screening of the children who were ≥4 years old,the cut off was 15 scores that the sensitivity and specificity were best.With the children of 3-＜4 years old,a designated cut-point should be 17 scores.Conclusions There are good reliability and validity of CAST,which can be used to mainland China,as well as to screen autistic children more than 3 years old.