BACKGROUND: The long-term cryopreservation of embryonic stem cells is an important link in its researches and application. Traditional protectants of cryopreservation often contain dimethyl sulfoxide, glycerine, animal serum, etc., but they cannot effectively ensure the survival rate of some cells, and even damage some functions of the cells.OBJECTIVE: To develop the suitable protectant for the cropreservation of mouse embryonic stem cells. DESIGN: An observational study.SETTINGS: Department of Physiology, Conghua College of Guangzhou Medical College; Sinocells Bio Technologies Co.,Ltd.MATERIALS: The experiment was carried out in the laboratory of Sinocells Bio Technologies Co.,Ltd. from October 2004 to September 2005. Mouse cell line mES-1 was given as a present by the Research Room of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences of Chinese PLA.METHODS: The cells collected from cultured mES-1, a mouse embryonic stem cell line. Dimethyl sulfoxide was used as the main protectant for cryopreservation. There were four different groups according to different protectants. In the control group, the protectant for cryopreservation contained DMEM (high sugar), 10% dimethyl sulfoxide, 10% FBS,10 mg/mL ascorbicacid, 0.18 mg/mL inositol and 0.44 mg/mL folic acid; Besides, 7.56 mg/mL mannose in the mannose group, 34.23 mg/mL mycose in the mycose and 41.94 mg/mL saccharu in the saccharu group were supplemented respectively. The effects of different polyols on the survival rate and ability of multiple differentiation of mouse embryonic stem cells after cryopreservation were observed.MATN OUTCOME MEASURES: Formation rate of mouse embryonic stem cells colony; Formation rate of embryoid bodies.were all significantly decreased as compared with that before cryopreservation [control group: (24.0±8.8)%; mannose group: (42.0±10.1)%; mycose group: (84.0±8.2)%; saccharu group (70.0±14.2)%; before cryopreservation: (95.0±4.7)%,embryoid bodies in the mycose group was higher than those in the control group and mannose group [(90.0±5.2)%,(80.0±6.9)%, (82.0±9.6)%, P＜0.05], but had no significant difference as compared with that in the saccharu group.CONCLUSION: The protectant for cryopreservation by taking mycose as the main component can effectively maintain the abilities of self-renewing and multiple differentiation of mouse embryonic stem cells.

Objective To analyze the related influencing factors of progressive ischemic stroke, and to investigate the value of serum markers for prediction of progressive ischemic stroke. Methods Three hundred and six patients with acute cerebral infarction were divided into progressive ischemic stroke (PIS) group (n=91) and non-progressive ischemic stroke (NPIS) group (n=215). Data of gender, age, past medical history, personal history and serum markers were collected and compared in two groups. Binary Logistic regression was used to analyze the influencing factors of PIS. The receiver operating characteristic (ROC) curves of neutrophils to lymphocytes ratio (NLR), fibrinogen and fasting blood glucose were analyzed in two groups. Results Positive rates of hypertension history, diabetes history, hypercholesterolemia history, incidence of large artery atherosclerotic (LAA) stroke were significantly higher in PIS group than those in NPIS group ( P<0.05). The levels of neutrophils, NLR, fibrinogen and fasting blood glucose were significantly higher, but the level of lymphocytes was significantly lower in patients with PIS than those in patients with NPIS (P<0.05). Logistic regression analysis confirmed that diabetes, LAA stroke, NLR, fibrinogen and fasting blood glucose were independent risk factors for the PIS ( P<0.05). The areas under ROC curve by NLR, fibrinogen and fasting blood glucose were 0.777, 0.560 and 0.574, respectively. The sensitivities of NLR, fibrinogen and fasting blood glucose were 72.5%, 59.8%and 47.3%, respectively;and the specificities were 76.7%, 51.2% and 69.8%, respectively. Conclusion The occurrence of PIS is related with blood pressure, blood lipids, inflammatory cells in peripheral bolld, fibrinogen and fasting blood glucose. The level of NLR in peripheral blood can predict the occurrence of PIS, which can be used as an important reference index for early diagnosis of PIS.

Objective To study the relationship between serum uric acid (UA) level and cerebrovascular stenosis in acute ischemic stroke (AIS) patients.Methods Five hundred and thirteen patients with AIS or old IS admitted to our hospital from April 2014 to April 2016 were divided into primary IS group (n=236),recurrent IS group (n=136),and old IS group (n=141).Venous blood samples were taken on admission for biochemical testing.The patients were further divided into serum UA≤255 μmol/L group (n=128),serum UA=256-312μmol/L group (n=129),serum UA=313-371 μmol/L group (n=129),and serum UA＞371 μmol/L group (n=127).Their cerebrovascular stenosis was assessed by CT angiography or magnetic resonance angiography after admission.Results The severity of cerebrovascular stenosis,hypertension,diabetes and hyperlipidemia was significantly different in primary IS group,recurrent IS group and old IS group (27.5％ vs 33.8％ vs 12.8％、24.2％ vs 28.7％ vs 46.8％、61.9％ vs 49.3％ vs 40.4％、71.6％ vs 61.8％ vs 46.8％,P=0.000).Logistic regression analysis showed that serum UA≤ 255 μmol/L was an independent risk factor for cerebrovascular stenosis (OR =2.787,95 ％CI:2.209-3.365,P=0.001).The risk of cerebrovascular stenosis decreased gradually with the elevated serum UA level.Conclusion Serum UA level is not directly related with recurrent IS.Low serum UA level is a risk factor for cerebrovascular stenosis in AIS.

Objective To evaluate analytical performance of homocysteine (Hcy) by circulating enzymatic method .Methods Re‐ferring to CLSI evaluation project and pertinent literature ,and by combining our actual works .we designed a verification procedure and experimental method .By using these above ,the precision ,accuracy ,analytical measurement range ,clinical reportable range of Hcy by circulating enzymatic method were evaluated .Results would be compared with the declaration of the manufacturer (NingBo Medical System Biotechnology Co .,Ltd) or desirable specifications derived from biologic variation .Results The results showed that the within‐run CV were 1 .26% and 0 .84% ,and the total CV were 1 .36% and 1 .32% ,less than 10% of the manufacturer′s statement .The relative bias between the results measured for calibrator at tow levels and target value was 3 .69% and 0 .69% ,less 10% .AMR was 3 .38-51 .81 μmol/L ,and the most suitable dilution rate was 1∶3 ,so the CRR was 3 .38-155 .43 μmol/L .Con‐clusion The analytical performance of Hcy analyzed by circulating enzymatic method is consistent with the standards which manu‐facturers has proclaimed ,so it is conform to the requirements of clinical .

Objective To study expression and activation of p38 mitogen activated protein kinase (MAPK) in vascular endothelial cells dysfunction in preeclampsia. Methods From Sept. 2009 to Mar.2010, 54 pregnant women underwent deliveries in the First Affiliated Hospital of Chongqing Medical University were enrolled in this study, including 20 patients in mild preeclampsia group, 16 patients in severe preeclampsia group and 18 women with term cesarean section without perinatal complications as control group. Placental endothelial cells were labeled by CD34 to assay microvessel density (MVD) of each group. Immunohistochemical SP and western blot were used to detect localization and expression of p-p38 MAPK protein, respectively. The levels of sera soluble fms-like tyrosine kinase-1 (sFlt-1)and soluble endoglin(sEng) were measured by ELISA. Results ①The MVD of placenta were 103 ± 3 in control group, 81 ±5 in mild preeclampsia group and 63±4 in severe group, respectively, which showed statistical difference among each group (P＜0.05).②The expression of p38 MAPK protein were 0.84±0.05 in control group,0.90±0.14 in mild group and 0. 86 ±0.18 in severe group, which did not reach remarkable difference among each group (P＞0.05). The expression of p-p38 MAPK protein were 0.13±0.05 in control group,0.59±0.12 in mild group and 1.16±0.18 in severe group, which show statistical difference among each group(P＜0.05).(3) The localization of p-p38 was in trophoblast, endothelial cells and a few (5.2±0.3)and(10.9±0.4)μg/L in control group,(12.5±1.2) and (20.4±5.3)μg/L in mild group and (19.3±3.0) and (29. 5 ±3.7) μg/L in severe group. When drawing paired comparison in those p-p38 MAPK protein levels and the concentrations of serum sFlt-1, sEng in preeclampsia groups (r=0.68,P＜0.05;r=0.87,P＜0.05). Conclusions The remarkable activation of the p38 MAPK in the placenta of patients with preeclampsia induced the increased levels of sFlt-1 and sEng in maternal serum, which confer the injury of vascular endothelial cells that caused the significant decline of MVD in placentas. p38 MAPK signaling might be one of the key pathways in vascular endothelial cell dysfunction in preeclampsia.

Objective To investigate the features of gallbladder carcinoma in two-phase spiral CT, and to analysis the values of two-phase spiral CT for the differential diagnosis between gallbladder carcinoma and chronic cholecystitis. Methods The two-phase spiral CT manifestations of 30 cases of gallbladder carcinoma, proved by surgery and pathology, and 30 cases of chronic cholecystitis were analyzed. Results According to the CT findings, the gallbladder carcinoma was categorized into 3 types: intraluminal mass of gallbladder in 6 out of 30 (20.0%), thickening of the gallbladder wall in 11 (33.7%), and mass replacing the normal gallbladder in 13(43.4%). The most common enhancement patterns of the wall in gallbladder carcinoma were hyperattenuation during the arterial phase, while isoattenuation with the adjacent hepatic parenchyma during the venous phase; or hyperattenuation during both phases. The most common enhancement pattern of the wall in chronic cholecystitis was isoattenuation during both phases, with clear hypoattenuation linear shadow in the gallbladder fossa. Other ancillary features of gallbladder carcinomas included: infiltration of the adjacent parenchyma, local lymphadenopathy and intrahepatic metastasis. Conclusion Two-phase spiral CT scan can identify the features of the gallbladder carcinoma and is helpful for the differential diagnosis of these two different disease entities.

Objective:To analyze clinical characteristics of ketosis-associated prurigo pigmentosa after ketogenic diet and bariatric surgery.Methods:Clinical data were collected from patients with ketosis-associated prurigo pigmentosa, who were diagnosed and treated in Department of Dermatology, Peking University People′s Hospital from September 2018 to September 2020. The clinical characteristics, sequelae and therapeutic effect of dietary modification were analyzed and summarized.Results:A total of 6 patients with ketosis-associated prurigo pigmentosa were collected, including 5 females who developed prurigo pigmentosa after ketogenic diet, and 1 male who developed prurigo pigmentosa after bariatric surgery. The skin lesions mainly involved the chest, back, waist and abdomen, and rarely involved the eyelids, axillae, elbows and mons pubis. Common skin lesions included urticaria-like erythema, papules and pigmentation arranged in a reticular distribution, and rare skin lesions included mung bean- to soybean-sized blisters, whose walls were liable to break. Among 5 patients undergoing routine urine analysis, 4 were positive (from + to ++++) for ketone bodies in the urine, and 3 were positive for urinary protein (+) . Pathological examination in 2 patients showed epidermal spongiosis, scattered necrotic keratinocytes, basal cell liquefaction, lymphocyte infiltration in the superficial dermis, and erythrocyte extravasation. The 6 patients were advised to eat staple foods. After dietary modification, 5 patients were nearly cured within 1 week; 1 patient, who continued ketogenic diet for weight loss, still received marked improvement after the treatment with minocycline at a dose of 100 mg/d in spite of restriction of carbohydrate intake. The levels of urinary ketone bodies and urinary protein in the 6 patients all returned to normal within 1 week after treatment.Conclusions:Ketosis plays an important role in the occurrence of prurigo pigmentosa. Dietary modification alone or adjuvant medical treatment such as minocycline is effective for the treatment of ketosis-related prurigo pigmentosa.

BACKGROUND:Transient receptor potential channel C6 (TRPC6) is a new and important slit diaphragm-associated protein in podocytes involved in regulating glomerular filter function. Glomerular TRPC6 expression is closely associated with proteinuria in diabetic kidney disease. OBJECTIVE: To investigate the expression of canonical TRPC6 in mouse podocytes induced by high glucose, and to explore the possible mechanism of diabetic kidney disease. METHODS:Mouse podocyte cels were cultured and divided into normal glucose group (5.6 mmol/L D-glucose), normal control group (5.6 mmol/L D-glucose+25 mmol/L mannitol) and experimental groups which were in the environment of high glucose (30 mmol/L). The experimental groups included high glucose group, valsartan treatment groups (10-5 mol/L) and U73122 control group (10μmol/L U73122). After 48 hours, the expressions of mRNA and proteins of TRPC6, nephrin and angiotensin II (AngII) were detected respectively by real-time quantitative PCR and western blot analysis. RESULTS AND CONCLUSION:Compared with the normal control group, the expressions of mRNA and proteins of TRPC6 and angiotensin II were markedly elevated in the high glucose group (P < 0.01), while the expressions of mRNA and proteins of nephrin were decreased (P < 0.01). The mRNA and proteins of TRPC6 and angiotensin II expressions were significantly down-regulated by valsartan (P < 0.05,P < 0.01), while the mRNA and protein expressions of nephrin were effectively up-regulated (P < 0.05). Compared with the high glucose group, the expressions of mRNA and proteins of TRPC6 and angiotensin II were ameliorated in the U73122 control group. The expressions of mRNA and proteins of TRPC6, nephrin and angiotensin II had no statistical significance between the normal control group and normal glucose group (P > 0.05). Angiotensin II-TRPC6 signaling pathway may mediate high glucose-induced podocyte injury, meanwhile it provides a new theoretical basis for the treatment of diabetic kidney disease, by which the angiotensin receptor blockers can protect podocytes in diabetic kidney disease.

Objective To study the effects of matrine on proliferation, cell cycle and apoptosis of human hepatocarcinoma cell line HepG2 and probe into the mechanisms of its anti-hepatocarcinoma effects.Methods The HepG2 cells were treated with different concentration of matrine (0.0125, 0.02, 0.05, 0.1, 0.2,0.4, 0.8, 1.6 g/L) for different time (24, 48, 72 h), then investigate the effects of matrine on cell proliferation by MTT, and the effects on cell cycle and apoptosis by flow cytometry. Results Matrine can inhibit the HepG2 cells proliferation at the concentration of 0.1 g/L and above in a concentration-dependent and timedependent manner(P ＜0.01). The result of FCM showed that the cell cycle of HepG2 was retarded at G1 phase treated with matrine for 48 h at the concentration of 0.8 g/L [(75.3±6.5)% vs (64.1±6.3)%, P ＜0.05], whereas was retarded at G2 phase treated with matrine for 48 h at the concentration of 1.6 g/L [(29.1 ±9.1)% vs (11.6±2.1)%, P ＜0.01]. The apoptosis of HepG2 cells can be induced by matrine for 12, 24 h or 48 h at the concentration of 0.4, 0.8, 1.6 g/L. Conclusion Matrine can inhibit cell proliferation, interfere cell cycle and inducing apoptosis of hepatocarcinoma cells, which may be involved in the mechanisms of matrine's antihepatocarcinoma effects.

Objective:To evaluate the effect of semiconductor lasers irradiation after routine root canal preparation on root cannal seal.Methods:60 Single-rooted freshly extracted human teeth were randomly divided into 6 groups(n=10).The crowns were removed at the cementoenamel junction and the roots were endodontically prepared with conventional methods.The roots in groups A and B were irradiated with 1 W semiconductor laser for 20 s,in group C and D were ultrasonically washed for 1 min,in group E and F without any treatment were used as the controls.Then all the roots were filled by vertical condensation of warm gutta-percha.The root cannal seal was evaluated with microleakage measurement.The data was analyzed by ANOVA.The teeth of group B,D and F were sectioned and examined under scanning electron microscope(SEM).Results:The microleakage(mm) of group A,C and E was 1.70±0.82,2.02±0.40 and 4.56±2.72 respectively(A vs E,P<0.01;C vs E,P<0.05;A vs C,P>0.05).SEM observation showed the melting,narrowness or closure of most dentinal tubules in group B,past and/or gutta-percha in the most dentinal tubules of group D.Conclusion:Semiconductor laser irradiation prior to root cannal filling can promote the effects of cannal seal.