BACKGROUND:Currently, the enzymatic digestion combined with magnetic activated cel sorting for isolating microvascular endothelial cel s are cumbersome and do harm to cel s. Therefore, how to simplify the isolation and culture of human dermal microvascular endothelial cel s to obtain highly purified endothelial cel s in vitro becomes a hotspot.
OBJECTIVE:To explore a simple and effective cultivation method of microvascular endothelial cel s from diabetic patient skins in vitro, and to detect the cel growth.
METHODS:Diabetic patients with chronic foot wounds after amputation were enrol ed to col ect the limb proximal skin and topical skin around the wound superficial dermal tissue. Human dermal microvascular endothelial cel s were obtained using adherent method and trypsin method, fol oewd by purified utilizing trypsin digestion and repeated attachment method when passage culture.
RESULTS AND CONCLUSION:Human dermal microvascular endothelial cells were obtained successfully, Primary cultured endothelial cells completely adhered to the wall at 24 hours, entered the logarithmic phase at the 10th day, and the cell concentration reached 80%at the 12th-13th day. While the passage cells grew more actively than primary cells, and fully covered the bottom in a“cobblestone”arrangement after 5-7 days of culture. Immunohistochemical staining showed that cultured cells were positive for FVIII and CD31-associated antigens with 100%positive rate. MTT assay showed that cell growth curves of 2, 4, and 5 generations of dermal microvascular endothelial presented the invertedSshape. These results suggest that abundant highly purified human dermal microvascular endothelial cells can be obtained through the adherent method and a small amount of short-term trypsin method.

Education, Dental ; Oral Medicine ; education

Limit test of flavones in diterpene ginkgolides meglumine injection materials by UV-Vis and HPLC-DAD method was studied in this essay. The HPLC-DAD method has lower LOD (about 1% of the UV-Vis), that is, the sensitivity is higher than UV-Vis method. Through the analysis of the kinds of flavonoids ingredients in the samples by LC-MS, the three compounds with highest contents are kaempferol, quercetin and isorhamnetin. Kaempferol, quercetin and isorhamnetin were chosen as reference compounds for HPLC analysis, and the HPLC separation analysis was carried on an Agilent Eclipse plus C18 column (4.6 mm x 250 mm, 5 μm) with methanol and water containing 0.4% phosphoric acid (50: 50) as mobile phase, and the flow rate was 1.0 mL x min(-1). The detection wavelength was set at 360 nm. This method has good specificity, precision and reproducibility. The LODs of quercetin, kaempferide and isorhamnetin were 27.6, 22.3, 29.5 μg x L(-1). The average recovery was 87.9% (RSD 3.3%), 91.7% (RSD 3.1%), 88.3 (RSD 1.3%) for quercetin, kaempferide and isorhamnetin, respectively. Based on the 10 batches of sample results and sensitivity of different HPLC, the content of total flavonoids ingredients of diterpene ginkgolides meglumine injection materials was limited no more than 2 x 10(-5). This method is simple, quick and has good maneuverability, and could be used to the limit test of flavonoids in the diterpene ginkgolides meglumine injection materials.
Chromatography, High Pressure Liquid ; methods ; Diterpenes ; analysis ; Drugs, Chinese Herbal ; analysis ; Flavones ; analysis ; Ginkgolides ; analysis ; Limit of Detection ; Mass Spectrometry ; methods

Objective To explore the feasibility and effectiveness of the revised OSCE stations.Methods 106 sstudents of grade 2008 and 2009 from Xinhua clinical medical school of Shanghai Jiaotong University School of Medicine were involved,32 of whom (experimental group) tool modified OSCE examination and 74 of whom (control group) tool traditional OSCE examination.The revised OSCE exam was the one which on the basis of original OSCE test station setup,improved the operating part of the test stand,adopted clinical cases as stem,asked the students to make possible diagnosis,and perform the corresponding operation.We evaluated the students based on the OSCE scores from the school exam and the overall performances in the national clinical skills competition.Comparison between the two groups was performed by using t test.Results All students who have received the new training methods agreed that it stood closer to the clinical setting.Revised-OSCE trained group of students test scores than traditional OSCE trained group of students,though it didn't have statistical significance.Students from the revised-OSCE group had the score of (80.50 ± 15.45),while the traditional group had the score of (78.30 ± 17.68).By taking the revised-OSCE trainings,all the students have improved their clinical skills and two years in a row to win the Shanghai Jiaotong University School of Medicine Clinical Skills Competition.Conclusions Case-based OSCE stations are well accepted.The new OSCE stations are much closer to clinical teaching and can make better objective assessment of students,so it plays a good role in improving medical students' overall performances.

Objective To determine the normal range of minimal erythema dose (MED) for ultraviolet A (UVA) and B (UVB) in volunteers from Urumqi region.Methods One hundred and twenty-seven volunteers including healthy subjects and patients with noninflammatory skin disorders were enrolled in this study.SUV-1000 type UV simulator was used as the light source to determine MED of UVA and UVB in these subjects.Results These subjects included 48 persons with Fitzpatrick skin type Ⅲ,79 with Fitzpatrick skin type Ⅳ,51 males and 76 females.The median MED value for UVA and UVB was 38.1 J/cm2 and 31.8 mJ/cm2 respectively in subjects with skin type Ⅲ,59.16 J/cm2 and 48.00 mJ/cm2 respectively in subjects with skin type Ⅳ.Significantly lower median MED values of UVA (both P ＜ 0.01) and UVB (both P ＜ 0.05) were observed in the male and female subjects with skin type Ⅲ compared with those with skin type Ⅳ.The male subjects showed a significantly higher median UVA-MED value (59.16 J/cm2 vs.41.10 J/cm2,P ＜ 0.05),but a similar UVB-MED value (39.60 mJ/cm2 vs.35.55 mJ/cm2,P ＞ 0.05) compared with the female subjects.No significant difference was observed in the median value of UVA-or UVB-MED in subjects with skin type Ⅲ or Ⅳ between Han and Uygur nationality (all P ＞ 0.05).Also,no correlation was found in the median value of UVA-or UVB-MED with age or duration of outdoor exposure in the male or female subjects (all P ＞ 0.05).The lower reference limit was 33.38 J/cm2 for UVA-MED and 27.90 mJ/cm2 for UVB-MED in the population in Urumqi region.Conclusion Skin phototype may be an important determinant of MED.

ObjectiveTo investigate the relationship between Dicer expression and clinicopathological characteristics and prognosis by detecting the expression of Dicer in hilar cholangiocarcinoma tissues and cells.MethodsThe expression of Dicer in tissues was detected using immunohistochemistry.Western blotting and RT-PCR were used to investigate Dicer expression in QBC939 and HIBEpic cells.The relationship between Dicer expression and clinicopathological characteristics was analyzed.A Kaplan-Maier analysis was performed to analyze the disease-free survival (DFS) and overall survival (OS) after radical surgical resection of hilar cholangiocarcinoma.ResultsWhen compared to control,Dicer was significantly down-regulated in hilar cholangiocarcinoma tissues (P＜0.05) and in QBC939 (P＜0.05).The expression of Dicer was higher in well differentiated adenocarcinoma than poorly and moderately differentiated tumours. Univariate analysis showed low expression of Dicer protein was significantly correlated with short disease-free survival and overall survival of patients with hilar cholangiocarcinoma after radical surgical resection (P＜0.01). Multivariate analysis revealed that the expression of Dicer was the most important factor for predicting prognosis after radical surgical resection of hilar cholangiocarcinoma (P＜0.05).ConclusionsDicer could be used as a prognostic marker for hilar cholangiocarcinoma.

Objective To evaluate the value of preoperative ultrasonographic parameters in predicting the outcome of TURP.Methods A total of 202 patients with symptomatic benign prostatic hyperplasia (sBPH) entering our department for surgical therapy were prospectively recruited,with mean age of (65.5 ± 8.1) years,international prostate symptom score (IPSS) of 16.6 ± 8.1 and quality of life (QOL) score of 5 (3,6).Preoperative combined test of ultrasonography and urodynamics has found total prostate volume (TPV),transitional zone volume (TZV),transitional zone index (TZI),intravesical prostatic protrusion (IPP),resistive index (RI),postvoiding residue (PVR),detrusor wall index (DWT),ultrasonic estimation of bladder weight (UEBW) and maximum flow rate (Qmax) to be (75.0 ±38.5) ml,(49.9 ± 32.4) ml,0.59 ±0.14,(17.2 ±5.0) mm,0.63 ±0.12,(132.7 ±97.8)ml,(16.3 ±7.9)mm,(44.8 ± 7.1)g and (6.1 ± 6.0)ml/s respectively.A 6-monthsfollow-up after standard TURP were applied including re-measurement of IPSS,QOL score and Qmax.The patients were classified into 2 groups of effective and ineffective after the recovery being stratified into 4 levels of none,fair,good and excellent.The influence of preoperative ultrasonographic parameters on surgical outcome was analyzed by logistic regression and receiver operating characteristic (ROC) curve.Results The group of effective has 149 patients,with the preoperative TZI,IPP,RI,DWT and UEBW of 0.65 ± 0.27,(18.3 ± 3.1) mm,0.77 ± 0.18,(19.0 ± 5.0) mm and (46.6 ± 7.1) g,which were significantly higher than that of the group of ineffective (P ＜ 0.05) Lower RI,DWT and UEBW were found to be risk factors of unfavorable surgical efficacy (P ＜ 0.05) from multivariable analysis.The area under curve (AUC) of RI,DWT and UEBW in outcome prediction was 0.816,0.732 and 0.723 respectively from ROC curve,indicating the good predictive value of the 3 parameters with combined positive predictive value (PPV) of 96.3％.Conclusion RI,DWT and UEBW have favorable value in predicting TURP outcome.Measuring these parameters by preoperative ultrasonography might aid in determining the need for surgical intervention in sBPH patients.

OBJECTIVETo investigate the effect of allogene hepatic nonparenchymal cell (NPC) on the survival of grafted skin in mice and its underlying mechanism.

METHODSSixty-five C(3)H and fifty-eight C(57)BL/6 mice were employed in the study. Twenty C(3)H mice were used as skin donor and forty as the source of hepatic NPC. The rest five served as the stimulators of mixed lymphocyte culture (MLC) before and on the 7th, 18th, 30th, and 60th day after NPC infusion with 1 at each time point. MLC was determined and expressed as count per minute (CPM). Fifty-eight C(57)BL/6 mice were further divided into experimental (E, n = 50) and control groups (C, n = 8). The mice in C group only underwent skin grafting without NPC infusion. The mice in E group received with 2 x 10(7) NPC via caudal vein, followed by peritoneal injection of cytoxan (200 mg/kg) 48 hours later; They were grafted with skin donated from C(3)H mice 18 days after injections. The survival time of the mice in the two groups was observed. The serum levels of interleukin-4, chimera and MLC in the two groups were determined before and on 7th, 18th, 30th, 60th days after NPC infusion, and micro-chimera were aslo assessed on the 1st and 3rd day after NPC infusion. Five mice were sacrificed at each time point.

RESULTSThe survival time of skin graft in E group (70.0 +/- 17.2 day) was obviously longer than that in C group. The serum levels of IL-4, chimera in E group were increased gradually, while MLC response decreased gradually. The serum IL-4 level reached 251.5 +/- 11.0 ng/L and splenic chimera level to 26.30 +/- 1.04% on the 60th day after NPC infusion.

CONCLUSIONThe high levels of IL-4 and chimera might play important roles in inducing and maintaining immune tolerance.

Animals ; Female ; Graft Survival ; immunology ; Hepatocytes ; immunology ; Immune Tolerance ; Interleukin-4 ; metabolism ; Male ; Mice ; Mice, Inbred C3H ; Mice, Inbred C57BL ; Skin Transplantation ; immunology ; Surgical Flaps ; Transplantation Chimera ; Transplantation, Homologous ; immunology

Objective To observe the effect of Tangmaikang particles on insulin sensitivity in the treatment of type 2 diabetes.Methods A total of 120 patients with type 2 diabetes were selected, randomly divided into control group ( n=60) and treatment group ( n=60).Patients in control group received routine hypoglycemic, hypotensive and lipid adjusting treatment, while patients in treatment group were given Tangmaikang par-ticles 15 g? d-1 on the basis of conventional treatment.The treatment lasted for 3 months.The data of postprandial plasma sugar ( PPG) , gly-cosylated hemoglobin ( HbA1 c ) , fasting insulin ( FINS ) , insulin sensi-tivity ( HOMA-IS ) , low density lipoprotein ( LDL -C ) , triglyceride (TG), endothelin -1( ET-1) and the change of clinical symptoms in two groups before and after treatment were observed as well as the inci-dence of adverse reactions in 2 groups.Results The total effective rate was 88.33% in treatment group, better than control group (75.00%) .After treatment, the data of FINS, HOMA -IS, PPG, HbA1c were all significantly improved in two groups( P<0.05 ) , and treatment group improved more obviously than control group ( P<0.05 ) .Symptoms in two groups were both improved compared with before treatment( P<0.05) , and the treatment group was better than control group( P<0.05).But the TG level wasn′t significantly decreased in two groups( P>0.05).After treatment, LDL-C and ET-1 level in treatment group were significantly decreased ( P<0.05 ) , and the control group was not obvious (P>0.05).After the treatment for early diabetic nephropathy, ET -1 in treatment group was significantly lower (P<0.05), and the control group was not obvious (P>0.05).There was no adverse reactions found during treat-ment.Conclusion Tangmaikang particles are safe and effective for type 2 diabetes, which also can improve insulin sensibility, reduce insulin resistance and ET-1 level, especially can reduce the ET-1 level of patients with diabetic nephropathy.

OBJECTIVETo investigate the methylation status of LRP15 gene in acute leukemia (AL) patients and its role in the tumorigenesis.

METHODSThe methylation of LRP15 promoter and first exon of bone marrow mononuclear cells in 73 patients with AL, 10 with chronic leukemia (CL), 9 with hematological benign diseases, and 20 healthy transplantation donors was analyzed by using methylation specific polymerase chain reaction. The methylation of LRP15 gene promoter and first exon in COS7, K562, and HL60 cell lines was also assayed.

RESULTSNo LRP15 gene promoter methylation was detected in COS7 cell line. LRP15 gene promoter was methylated in K562 and HL60 cell lines. No deletion of LRP15 gene was detected in all samples. In nearly all French-American-British leukemia subtypes, we found that frequency of LRP15 methylation in adult patients with AL was 71.23% (52/73). There was no detectable methylation in any of the 20 healthy donors and 8 chronic myeloid leukemia patients. The difference in frequency of LRP15 methylation between AL patients and healthy donors or CL patients (10.00%, 1/10) was significant (P < 0.01). Hypermethylation of LRP15 gene was found in 57.14% (16/28) of newly diagnosed AL patients, 83.33% of relapsed AL patients respectively, which was significantly different (P < 0.05). We also demonstrated LRP15 methylation in 55.56% (5/9) adults with benign hematological diseases.

CONCLUSIONSLRP15 methylation changes are common abnormalities in leukemia. LRP15 is postulated to be a tumor suppressor gene.

Acute Disease ; Animals ; Base Sequence ; COS Cells ; Cell Line, Tumor ; Cercopithecus aethiops ; DNA Methylation ; DNA Primers ; Humans ; Leukemia ; genetics ; Neoplasm Proteins ; genetics ; Promoter Regions, Genetic