Problem-based learning (PBL) teaching method combined with formative evalua-tion was used in the teaching practice of pediatrics education. This method was implemented by four phases: courses designing, group-preparing, problems-organizing and teaching practice. The method was evaluated by students' feedback and survey results of patients, teachers and teaching councilors. It was showed that the teaching effects of PBL combined with formative evaluation was better than tra-ditional teaching method in pediatrics teaching.

OBJECTIVESynthesis and identification of complete antigen of rutin, the traditional Chinese medicine active ingredient, and develop rapid detection of rutin using enzyme-linked immunoassay method (ELISA). Immunogenicity of the complete antigen was also studied.

METHODPrepare the complete antigen by sodium periodate solution and identified by UV scanning and SDS-PAGE test. Male New Zealand white rabbits were immunized by the antigen to obtain the antiserum.

RESULTThe results of UV analysis showed that the coupling ratio of complete antigen is 13: 1. SDS-PAGE display of the artificial antigen was delayed compared with bovine serum protein. The titer of rutin antibody is 1:4 000. The sensitivity of IC50 was 5.37 mg x L(-1), the lowest detection limit was 1 mg x L(-1), the average recovery was 102%, the intra and interspecific RSD were less than 10%, cross-reactivity rate of antibodies and other analogs were less than 1%.

CONCLUSIONRutin complete antigen was synthesized successfully, and the rapid detection of rutin by ELISA method was successfully established.

Animals ; Antibody Specificity ; immunology ; Antigens ; immunology ; Cattle ; Cross Reactions ; immunology ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Immune Sera ; immunology ; Immunization ; Male ; Periodic Acid ; chemistry ; Rabbits ; Rutin ; chemical synthesis ; immunology ; Serum Albumin, Bovine ; immunology ; Solutions ; chemistry

OBJECTIVETo evaluate the transfect results of recombinant adenovirus vector carrying tyrosinase gene (Ad-tyr) in vitro by magnetic resonance imaging (MRI) after the Ad-tyr was transfected into HepG2 cell.

METHODSThe Ad-tyr which carried the full-length cDNA of tyrosinase gene was transfected into HepG2 cell. The transfected cells were scan by MRI sequences of T1 weighted image (T1WI) , T2 weighted image (T2WI) , and short time inversion recovery (STIR) to observe the MRI signals of expressed melanin. Masson-Fontana staining was performed to search for melanin granules in transfected cells. Real-time PCR method was used to search for cDNA of tyrosinase gene.

RESULTSAd-tyr was transfected into HepG2 cells and synthesized a large amount of melanin inside. The synthesized melanin of 1 x 10(6) cells which had been transfected by Ad-tyr with the 50, 150, and 300 multiplicity of infection separately were all sufficient to be detected by MRI and showed high signals in MRI T1WI, T2WI, and STIR sequences. The signal intensities of MRI were positively correlated to the amounts of transfected Ad-tyr. The melanin granules were found in HepG2 cells in Masson-Fontana staining. The cDNA amount of tyrosinase gene in transfected HepG2 cells, which was detected by real-time PCR, was remarkably higher than that in nontransfected cells.

CONCLUSIONThe synthesized melanin of HepG2 cells, which controlled by expression of exogenous gene, can be detected by MRI, indicating that the adenovirus vector can efficiently carry the tyrosinase gene into HepG2 cells.

Adenoviridae ; genetics ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Hep G2 Cells ; Humans ; Magnetic Resonance Imaging ; methods ; Melanins ; analysis ; genetics ; Monophenol Monooxygenase ; biosynthesis ; genetics ; Transfection

SUMMARY Thehumanembryonicstemcells(hESCs)serveasaself-renewable,genetically-healthy, pluripotent and single source of all body cells,tissues and organs.Therefore,it is considered as the good standard for all human stem cells by US,Europe and international authorities.In this study,the standard and healthy human mesenchymal progenitors,ligament tissues,cardiomyocytes,keratinocytes,primary neurons,fibroblasts,and salivary serous cells were differentiated from hESCs.The human cellular health-safety of NaF,retinoic acid,5-fluorouracil,dexamethasone,penicillin G,adriamycin,lead ace-tate PbAc,bisphenol A-biglycidyl methacrylate (Bis-GMA)were evaluated selectively on the standar-dized platforms of hESCs,hESCs-derived cardiomyocytes,keratinocytes,primary neurons,and fibro-blasts.The evaluations were compared with those on the currently most adopted cellular platforms.Parti-cularly,the sensitivity difference of PM2.5 toxicity on standardized and healthy hESCs derived fibroblasts, currently adopted immortalized human bronchial epithelial cells Beas-2B and human umbilical vein endo-thelial cells (HUVECs)were evaluated.The results showed that the standardized hESCs cellular plat-forms provided more sensitivity and accuracy for human cellular health-safety evaluation.

Protopanaxadiol (PPD) has inhibitory effects on many tumors and receives much attention. However,it has poor water solubility and low utilization, which limits its clinical application. Considering these issues,in this study,we used hollow gold nanoparticles as transport carriers of PPD and synthesized PPD hollow gold nanoparticles (HAuNs). We conducted a number of experiments to investigate the in vitro anti-laryngeal cancer Hep-2 effect of a PPD HAuNs carrier. High performance liquid chromatography(HPLC) was used to detect the sustained release effect of PPD HAuNs. MTT assay was used to detect the inhibitory effect of PPD HAuNs on the proliferation of Hep-2 cells. Effect of PPD HAuNs on Hep-2 cell apoptosis was investigated by flow cytometry. The results of in vitro release showed that PPD HAuNs had sustained release effect. Compared with blank control group,HAuNs group and PPD group,the survival rate of Hep-2 cells in HAuNs-PEG-PPD group decreased more significantly and the apoptosis rate increased more significantly (p<0.01). PPD HAuNs could significantly enhance anti-laryngeal cancer effect of PPD in vitro and promote the apoptosis of tumor cells. It promotes tumor cell apoptosis, and is expected to be a new PPD drug delivery system, further promoting the application of PPD in clinical anti-laryngeal cancer.

Starting from the characteristics of nervous system,breaking the traditional subject-centered teaching model,combining the nervous system-related basic and clinical courses,Chongqing Medical University has established a nervous-module organ-system integration curriculum since 2010.This new teaching model is introduced and practiced in 5-year outstanding medical class of Chongqing Medical University.The new integrated curriculum guides students to learn knowledge from points to surface,helps them to combine knowledge vertically and horizontally,and simplifies the duplicate teaching content,so as to promote students to develop coherent and innovative thinking mode.Integrated curriculum pattern is an inevitable trend in medical education reform.

Objective:To evaluate the diagnostic value of 1, 3-β-D glucan (BDG), mannan IgM antibody (Mn-IgM) and mannan IgG antibody (Mn-IgG) in invasive candidiasis and to compare the differences in the diagnostic capability of serological markers used alone or in combination.Methods:Serum samples of 126 patients with invasive candidiasis and 104 healthy people who took physical examination during the same period were collected. BDG was detected by dynamic chromogenic method, and Mn-IgM and Mn-IgG were detected by ELISA. The sensitivity, specificity, positive predictive value, negative predictive value, Youden index, coincidence rate and Kappa value of the three serological markers used alone or in combination in the diagnosis of invasive candidiasis were analyzed and compared. The receiver operating characteristic (ROC) curves were drawn and the areas under the curves (AUCs) were calculated.Results:The levels of BDG, Mn-IgM and Mn-IgG in patients with invasive candidiasis were significantly higher than those in healthy people ( P<0.01). The sensitivity, specificity, Kappa value and AUC of BDG were 48.41%, 92.31%, 0.389 and 0.842. The sensitivity, specificity, Kappa value and AUC of Mn-IgM were 64.29%, 91.35%, 0.540 and 0.829. The sensitivity, specificity, Kappa value and AUC of Mn-IgG were 27.78%, 95.19%, 0.214 and 0.737. The sensitivity, specificity, Kappa value and AUC of BDG+ Mn-IgM were 76.19%, 88.46%, 0.637 and 0.921. The sensitivity, specificity, Kappa value and AUC of BDG+ Mn-IgG were 59.52%, 91.35%, 0.491 and 0.856. The sensitivity, specificity, Kappa value and AUC of Mn-IgM+ Mn-IgG were 69.84%, 90.38%, 0.588 and 0.891. The sensitivity, specificity, Kappa value and AUC of BDG+ Mn-IgM+ Mn-IgG were 80.16%, 88.46%, 0.679 and 0.922. Conclusions:The sensitivity of Mn-IgM was higher than that of BDG and Mn-IgG in the diagnosis of invasive candidiasis. When the serological biomarkers were used in combination, BDG+ Mn-IgM and BDG+ Mn-IgM+ Mn-IgG had relatively high Kappa value and AUC, showing high accuracy. The clinical diagnostic value of multiple serological biomarkers used in combination was significantly higher than that of any serological biomarkers used alone. Early combined detection and continuous monitoring of multiple serological biomarkers in patients with high risk of invasive candidiasis could be used clinically to adjust antifungal treatment strategies timely.

ObjectiveTo investigate the intervention effect of Danggui Buxuetang on oxidative stress and inflammatory response in diabetic kidney disease （DKD） rats from its improvement of podocyte mitochondrial dysfunction. MethodSD rats were randomly divided into the control group and modeling group， and the ones in the latter group rats were fed a high-glucose and high-fat diet and then intraperitoneally injected with a small dose of streptozotocin （STZ） for inducing type 2 diabetes. The successfully modeled rats were randomized into the model group， high- and low-dose （1.44 and 0.72 g·kg^-1） Danggui Buxuetang groups， and irbesartan （0.017 g·kg^-1）group and gavaged with the corresponding drugs， while those in the normal and model groups with an equal volume of normal saline. After 20 weeks of drug intervention， the urinary microalbumin-to-urine creatinine ratio （UACR） and serum malondialdehyde （MDA） content and manganese superoxide dismutase （MnSOD） activity in each group were measured. The pathological changes in renal tissue were observed by Masson trichrome staining， and periodic acid-silver metheramine （PASM） staining， followed by the observation of ultrastructural changes in podocytes under the transmission electron microscope （TEM）. The expression level of reactive oxygen species （ROS） in rat kidney tissue was detected using a fluorescent probe dihydroethidium （DHE）. The protein expression levels of peroxisome proliferator-activated receptor γ -coactivator -1α （PGC-1α）， nucleotide-binding domain like receptor protein 3 （NLRP3）， and Wilms tumor protein-1 （WT-1） were measured by immunohistochemistry （IHC）， and the expression levels of NLRP3， interleukin-1β （IL-1β），and WT-1 in podocytes by immunofluorescence （IF） assay. The mRNA expression levels of PGC-1α and NLRP3 in the renal tissues were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the protein expression levels of PGC-1α， MnSOD， NLRP3， and IL-1β were assayed by Western blot. ResultCompared with the normal group， the model group exhibited elevated UACR and MDA content， weakened MnSOD activity （P<0.01）， glomerular hypertrophy， thickened basement membrane， mesangial hyperplasia， increased extracellular matrix， K-W nodules， podocyte mitochondrial swelling， disordered mitochondrial cristae， foot process fusion or loss， vacuolization， increased ROS （P<0.01）， enhanced NLRP3 and IL-1β but diminished WT-1 expression in podocytes， down-regulated PGC-1α mRNA expression （P<0.01） and PGC-1α and MnSOD protein expression （P<0.01）， and up-regulated NLRP3 mRNA expression and NLRP3 and IL-1β protein expression （P<0.01）. Compared with the model group， Danggui Buxuetang high-dose group significantly decreased UACR and MDA， enhanced MnSOD activity （P<0.05， P<0.01）， improved renal histopathology and podocyte mitochondrial ultrastructure， decreased ROS （P<0.05， P<0.01） and NLRP3 and IL-1β expression in podocytes， enhanced WT-1 expression in podocytes， up-regulated the mRNA and protein levels of PGC-1α and MnSOD， and down-regulated the mRNA and protein levels of NLRP3 and IL-1β （P<0.05， P<0.01）. ConclusionDanggui Buxuetang alleviates oxidative stress， reduces inflammatory response， protects kidney， and delays the progression of DKD possibly by improving the mitochondrial dysfunction in podocytes of DKD rats.

ObjectiveTo observe the effect of Danggui Buxuetang on the podocyte injury and receptor-interacting protein kinase 1/receptor-interacting protein kinase3/mixed lineage kinase domain-like protein （RIPK1/RIPK3/MLKL） signaling pathway in diabetic kidney disease （DKD） ratsand to explore its possible mechanism against DKD. MethodEight of the 50 SD rats were randomly classified intoa normal group， and the remaining were fed a high-glucose and high-fat diet for six weeks and then intraperitoneally injected with 0.035 g·kg^-1streptozotocin （STZ） for inducing type 2 diabetes. After successful modeling，they were randomized into the model group，high- and low-dose （1.44，0.72 g·kg^-1） Danggui Buxuetang groups， and irbesartan （0.017 g·kg^-1）group. After 20 weeks of drug intervention， the fasting blood glucose （FBG）， kidney index （KI），and urinary microalbumin-to-urine creatinine ratio （UACR）were detected in each group. The pathological changes in renal tissue were observed by hematoxylin-eosin （HE） staining， followed by the observation of ultrastructural changes in podocytes under the transmission electron microscope. The levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in renal tissue of rats were determined by enzyme-linked immunosorbent assay （ELISA）， and the protein expression levels of RIPK1， RIPK3， and MLKL in rat kidney tissue by immunohistochemistry. The apoptosis rate of podocytes was detected by in situ nick end-labeling （TUNEL） assay. The mRNA expression levels of RIPK1， RIPK3， and MLKL in kidney tissue of rats were measured by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the protein expression levels of RIPK， RIPK3， and MLKL and podocyte marker Wilms tumor protein-1 （WT-1） in rat kidney tissue were assayed by Western blot. ResultCompared with the normal group， the model group exhibited elevated FBG， UACR， and KI （P<0.01）， glomerular hypertrophy， thickened basement membrane， increased extracellular matrix， mesangial hyperplasia， foot process fusion or loss， enhanced apoptosis in renal tissue， up-regulated TNF-α and IL-6 levels （P<0.01） and RIPK1/RIPK3/MLKL mRNA and protein expression （P<0.01）， and down-regulated WT-1 protein expression. Compared with the model group， Danggui Buxuetang high-dose group significantly reduced the levels of FBG， UACR， and KI， improved renal histopathology， podocyte loss， and apoptosis in renal tissue， down-regulated TNF-α and IL-6 levels and RIPK1/RIPK3/MLKL mRNA and protein expression （P<0.05， P<0.01）， and up-regulated WT-1 protein expression. ConclusionDanggui Buxuetang alleviates podocyte injury and delays the development of DKD possibly by regulating the RIPK1/RIPK3/MLKL signaling pathway.

ObjectiveTo observe the effect of Danggui Buxuetang on podocyte pyroptosis in diabetic kidney disease （DKD） rats and to explore the possible mechanism of its prevention and treatment of DKD and podocyte pyroptosis. MethodEight of the 50 male SD rats were randomly classified into a normal group， and the remaining 42 were fed a high-glucose and high-fat diet for six weeks and then intraperitoneally injected with 35 mg·kg^-1 streptozotocin （STZ） for inducing type 2 diabetes. After successful modeling， they were randomized into the model group， low- （0.72 g·kg^-1） and high-dose （1.44 g·kg^-1） Danggui Buxuetang group， and irbesartan （0.017 g·kg^-1） group and gavaged with the corresponding drugs， while those in the normal group and model group with an equal volume of normal saline， once per day， for 20 weeks. During the medication， the fasting blood glucose （FBG） and 24 h urine protein （24 h-UTP） were measured regularly. After administration， the pathological changes in renal tissues were observed by periodic acid-silver metheramine （PASM） staining， followed by the observation of ultrastructural changes in podocytes under the transmission electron microscope （TEM）. Serum levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） were determined by enzyme-linked immunosorbent assay （ELISA）. The DNA damage in renal tissue cells of rats was detected by in situ nick end-labeling （TUNEL） assay. The protein expression levels of thioredoxin interacting protein （TXNIP）， cysteine-dependent aspartate-directed protease-1 （Caspase-1）， and gasdermin D （GSDMD） in renal tissues of rats were detected by immunohistochemistry （IHC）， the expression levels of nucleotide binding domain like receptor protein 3 （NLRP3） and Wilms tumor protein-1 （WT-1） in podocytes by immunofluorescent （IF） staining， and the expression levels of TXNIP/NLRP3/Caspase-1/GSDMD pathway proteins and Synaptopodin in renal podocytes by Western blot. ResultCompared with the normal group， the model group exhibited increased FBG and 24 h UTP， glomerular hypertrophy， mesangial hyperplasia， increased extracellular matrix， thickened basement membrane， K-W nodules， vacuolar degeneration in renal tubular epithelial cells， foot process fusion or loss， elevated serum IL-1β and IL-18 levels and TUNEL-positive cells in renal tissue， enhanced NLRP3 but diminished WT-1 expression in podocytes， down-regulated Synaptopodin protein expression， and up-regulated TXNIP/NLRP3/Caspase-1/GSDMD protein expression （P<0.01）. Compared with the model group， Danggui Buxuetang high-dose group remarkably lowered FBG， 24-h UTP， and TUNEL-positive cells in renal tissue， improved renal histopathology and podocyte injury and loss， down-regulated NLRP3 expression in podocytes and TXNIP/NLRP3/Caspase-1/GSDMD protein expression levels， and up-regulated WT-1 expression in podocytes and Synaptopodin protein expression （P<0.05， P<0.01）. ConclusionDanggui Buxuetang inhibits podocyte pyroptosis to reduce proteinuria and delays the development of DKD possibly by regulating the TXNIP/NLRP3/GSDMD signaling pathway.