Objective To establish a method for culturing neonatal mice cardiomyocytes, and construct an in vitro model of cardiomyocytes for assessing the cardiac toxicity of chemicals. Methods Hearts of neonatal mice of 1 day old were digested with enzyme mixture of trypsin/collagenase type Ⅱ/dispase and the cell suspensions were pre-plated to flask for a short time and then seeded on coated dishes. The cultured cells were treated with 5-fiuorine-deoxy-uridine(15 μg/ml)and uridine(35 μg/ml)to enrich cardiomyocytes that were identified according to the morphology and immunocytochemistry of α-actin antigen. Cardiac myocytes were incubated with 0-64 μg/ml of a fusarium mycotoxin butenolide(BUT) for 12 h. Cell viability was then evaluated by MTT assay. Microscopic observation showed that BUT induced significant morphological changes including cellular swelling, vacuolation and breakage of muscle fibers. Results It was found that 96% of the cultured cells were cardiomyocytes and the myocytes kept beating after 90 days of culture. Concentration-dependent decreases in cell viability following exposure of cardiac myocytes to BUT were observed. Conclusion The results indicated that relatively pure primary culture of neonatal mice cardiomyocytes is successfully established. BUT possesses the potential to induce myocardial toxicity.

OBJECTIVETo investigate the pathogenicity of Leptospira interrogans fliR gene to J774A.1 cells.

METHODSfliR gene from L. interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 and kana gene from plasmid pET42a were amplified by PCR. Suicide plasmid of fliR gene was constructed; and specific siRNA for fliR gene was designed and synthesized. fliR gene mutants were constructed by gene knock-out with suicide plasmid (56601fliR-Kana) and gene silencing with siRNA (56601siRNA-R2). The mutants were identified by PCR, sequencing and semi-quantitative RT-PCR. Adhesion to mouse mononuclear-macrophage J774A.1 and induction of cell necrosis and apoptosis by 56601fliR-Kana and 56601siRNA-R2 were examined by adhesion test and flow cytometry, respectively.

RESULTThe nucleotide and putative amino acid sequences of cloned fliR gene had 99.9% and 100% similarities to those of reported sequences in GenBank. The nucleotide sequence of the cloned kana gene was identical to the corresponding sequence in pET42a map. The results of PCR and sequencing confirmed that kana gene was inserted in the sequence of 56601fliR-Kana fliR gene. The mRNA level of fliR gene in 56601fliR-Kana was remarkably decreased (P<0.01) while the mRNA level of fliR gene in 56601siRNA-R2 was much lower than that in wild strain 56601 (P<0.05). 56601fliR-Kana and 56601siRNA-R2 lost the ability to adhere J774A.1 cells; and their ability to induce cell necrosis and apoptosis was markedly weakened (P<0.01).

CONCLUSIONfliR is a virulence-associated gene of L. interrogans and the function of the gene is closely related to adhesion, induction of cell necrosis and apoptosis of the microbe.

Animals ; Apoptosis ; Bacterial Adhesion ; Bacterial Proteins ; genetics ; metabolism ; Cell Line ; Leptospira interrogans ; genetics ; pathogenicity ; Macrophages ; microbiology ; pathology ; Membrane Proteins ; genetics ; metabolism ; Mice ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics

Objective To develop and evaluate a porcine model for training the single needle running suture method of laparoscopie urethrovesical anastomosis(LUA). Methods Twenty minipigs with mean weight of 30kg were general anaesthetized with Sumianxin solution 0. 1 ml/kg intramuscularly. Pneumoperitoneum was created by insufflation of carbon dioxide by a veress needle inserted through the umbilicus. One 10mm port and two 5mm ports were positioned after the establishment of pneumoperitoneum. The intestine was used as "bladder". The procedures were completed with the single needle running suture method of laparoscopic urethrovesical anastomosis. Six trainees performed the LUA procedure based on the models during a laparoscopic training course, following the technique used in the operation room. The learning curve was analyzed by operative time. Results The porcine model for laparoscopic training was established successfully and 3 LUAs could be performed on each pig. Each trainee performed 10 LUAs based on the models during the training course of laparoscopic urology. The operative time declined from (55.3±10. 4)min initially to (22.4±4.8)min (P＜0. 01) after the training course. At the end of training, all trainees could accomplish a watertight LUR procedure on the model. Conclusions The establishment of the training model is feasible. The trainees could acquire the skills necessary to perform LUA in vivo based on this model. The model provides a platform for training the basic techniques of LUA procedures.

Choosing the right solvent and timely use is the basis of rational drug use and the most direct and efficient way to improve the safety of traditional Chinese medicine injections. This study aimed to evaluate the influence of solvent and drug preparation time on Shuanghuanglian injection inducing pseudo-allergic reactions with mouse mode. The two tests were carried out: (1) Comparative experiment between different solvent: Shuanghuanglian injection preparation to the appropriate concentration with 0.9% sodium chloride injection and 5% dextrose injection, mixed with Evans blue, at one time intravenous injected into mice, 30 minutes later, the mouse ears vascular permeability were observed and compared. (2) Comparative experiment among different preparation time: placed 10 min, 2.5 h, 6 h and 24 h after Shuanghuanglian injection were prepared and then to detect the pseudo-allergic reactions in mice using the same methods as in (1). The results showed that there was no significant difference in the pseudo-allergic reactions in mice which induced by the same dose of Shuanghuanglian injection, respectively with 0.9% sodium chloride injection and 5% dextrose injection preparation, and with the extension of preparation time, the degree of pseudo-allergic reactions of Shuanghuanglian injection was gradually severe.
Animals ; Drug Compounding ; Drug Hypersensitivity ; etiology ; Drugs, Chinese Herbal ; adverse effects ; Injections ; Male ; Mice ; Mice, Inbred ICR ; Solvents ; Time Factors

To evaluated the pseudo-allergic reactions of cordate houttuynia, pulse-activating and Qingkailing injection in mice, the ICR mouse were divided into different test groups, then were intravenously injected with three traditional Chinese medicine injections, positive control compound 48/80 and physiological saline as normal control. All test substances were mixed with 0.4% Evans blue. The reaction and vascular permeability of the ears were observed and measured 30 min after injection. At the same time, the mechanisms of the traditional Chinese injections' pseudo-allergic reactions was studyed. ICR mice were injected with the test substances as above without EB, blood in a part of mice were collected after 5 min, and the level of histamine in the plasma were measured. Blood in the other part of mice were collected after 30 min, and the level of VEGF, TNF-α and IL-10 in the serum were measured. The reasults showed that except the cordate houttuynia injection, pulse-activating injection in 1. 5 times clinical concentration and Qingkailing injection in 3.3 times clinical concentration caused mild pseudo-allergic reactions mainly for vascular permeability, no pseudo-allergic reactions occurred when the concentration of the two injections was below the concentration metioned above; 5 minutes after intravenous injection of the three TCM injections into ICR mice with the highest dose, the levels of histamine in plasma of pulse-activating injection and Qingkailing injection groups were increased significantly, 30 minutes later, the levels of VEGF, TNF-α and IL-10 in the serum of all groups were no significant change compared to normal group. The mouse of pulse-activating and Qingkailing injection groups showed inflammatory changes in ear and lung tissues. It can be conluded that when the dose or concentration increased to a certain extent, pulse-activating and Qingkailing injection could induce pseudo-allergic reactions on ICR mice, the increased histamine realease maybe the main reason for pseudo-allergic reactions of the two traditional Chinese medicine injections. In addition the author preliminary thought that inflammatory mechanisms leading to hyperpermeabilities were the main reason of the traditional Chinese medicine injection's pseudo-allergic reaction.
Animals ; Drug Hypersensitivity ; etiology ; Humans ; Injections ; Interleukin-10 ; blood ; Male ; Medicine, Chinese Traditional ; adverse effects ; Mice ; Mice, Inbred ICR

Epidemiology is a discipline characterized by complicated theory and practice.How to make the practice course function better is a topic worthy of exploring in educational reform for clinical students.The article explored the‘Student-Dominated’ Model based on ‘Problem-Based Learning ’ and ‘Team Based Learning ’ in teaching process and compared the model with the traditional one ( Teacher-Dominated Model) .Suggestions were given to further improve effectiveness of epidemiology practice courses.

Objective To evaluate the gender differences in dose-response curve with cisatra-curium in epileptics.Methods Eighty ASA grade Ⅰ or Ⅱ epileptics were enrolled in this study.All patients were divided into male and the female groups and received the method of single dose injection under midazolam-fentanyl intravenous anesthesia.Each patient received intravenous bolus of 20,30, 40,50μg/kg of cisatracurium respectively.The neuromuscular block was measured by Neuromuscular Transmission Monitor and the responses were defined in terms of the percentages of maximum sup-pression in T1 of TOF of the adductor pollicis muscle.According to log-probit transformation of the data of dose and response,the dose-response curve of cisatracurium was established through linear re-gression.The onset time of cisatracurium was also observed.Results The ED50 ,ED75 ,ED90 ,ED95 values of cisatracurium in male epilepsy patients were 37.2±9.7,48.1±11.3,60.4±12.8,69.3± 14.0 μg/kg and that of female epilepsy patients were 36.6±4.3,47.5±7.7,60.5±14.0,70.1± 19.4 μg/kg.There was no significant difference between the two gender groups.No significant change in onset time was observed among 4 dose groups.Conclusion No gender differences are ob-served in dose-response curve of cisatracurium in epileptics.

Objective To investigate the relationship of pathological response of breast cancer after neoadjuvant chemotherapy with the imaging findings in dynamic contrast-enhanced MRI.Methods Forty- five patients with pathologically confirmed breast carcinoma who finished courses of neoadjuvant chemotherapy had breast MRI prior to operation.Dynamic contrast-enhanced MRI scans were performed on a 1.5 T scanner using 3D SPGR sequence before and repeated 6 times after administration of Gd-DTPA. Pathological response was assessed by a pathologist according to Miller & Payne five points classification blinded to breast MRI results.Grade 5 was defined as pCR(pathological complete response).Grade 4 and 5 were defined as major histopathological response(MHR).The type of time signal intensity curve(TIC) (three types),pattern of residual enhancement of each breast cancer were recorded and correlated with pathological findings.Fisher exact test was used for statistical analysis.Results Grade 5 responses were achieved in seven patients;grade 4 in sixteen patients;grade 3 in sixteen patients and grade 1—2 in six patients.70.0%(14/20)of type Ⅰ time signal intensity curve correlated with MHR,while all 6 type Ⅲ curves showed non-MHR response.The type of time signal intensity curve and pathological response grades had statistically significant correlation(P=0.001).18 of the 23 cases with MHR exhibited residual enhancement,while the remaining 5 cases showed no enhancement.Of the 18 MHR cases with residual enhancement,11 showed non-mass-like enhancement and 7 showed mass-like enhancement.The mass(non- mass)morphological pattern in dynamic contrast enhanced-MRI had statistically significant differences in pathological response(P=0.012).Conclusions Pathological response of breast carcinoma after neoadjuvant chemotherapy could be characterized using dynamic contrast-enhanced MRI by identifying patterns of residual contrast enhancement and kinetic curve.Favorable pathological responses correlated with Type Ⅰ TIC,non-enhancement,and non-mass-like residual enhancement.

OBJECTIVETo investigate biological characteristics of rat bone marrow mesenchymal stem cells (MSC) cultured in vitro and to explore their potential applications.

METHODSMSC were isolated from rat bone marrow by density gradient centrifugation and were induced to differentiation. Flow cytometry was used to characterize their surface antigen expression, cell cycle status and cell growth parameters. Telomerase activity was determined by TRAP-ELISA assay.

RESULTSFusiform MSC became larger and flattener with increasing passages of culture. After the fourth passage, the MSC showed an immunophenotype of CD29 (94.75% +/- 3.68%), CD71 (95.43% +/- 2.23%), and CD90 (98.08% +/- 3.88%). After the seventh passage, MSC with such immunophenotype decreased with CD29: 50.00% +/- 3.35%, CD71: 50.70% +/- 2.43%, and CD90: 48.60% +/- 2.83%. Cells with such immunoprofile completely disappeared after passage 9. Overall, MSC grew faster during the first 5 passages. The number of MSC in S and G(2)/M phases were 38.36% +/- 2.01% and those in G(0)/G(1) phase were 61.64% +/- 2.13% after 3 passages. The cell growth decreased after passage 7. Percentage of MSCs in S and G(2)/M phases was 10.83% +/- 1.63% and that in G(0)/G(1) was 89.17% +/- 1.96% after passage 12, after which the cells failed to further divide. After passage 9, MSCs lost their ability to differentiate to Von Kossa and oil red O positive staining cells. In addition, telomerase activity of MSC also gradually decreased with the prolonged passages, from the original 52.7% +/- 0.78% to no telomerase activity.

CONCLUSIONThe biological and immunophenotypical characteristics of cultured MSC showed obvious alterations with increasing numbers of passage of culture.

Animals ; Antigens, CD ; metabolism ; Bone Marrow Cells ; cytology ; metabolism ; Cell Cycle ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Immunophenotyping ; Integrin beta1 ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Rats ; Receptors, Transferrin ; metabolism ; Thy-1 Antigens ; metabolism

BACKGROUNDRecent studies have shown that T helper type-2 (Th2) cells can induce the apoptosis of CD4+CD25+ Treg cells or resist the immunosuppressive effect of Treg cells. We hypothesize that an imbalance of Th2/Treg is present in patients with allergic asthma.

METHODSTwenty-two patients with mild asthma, 17 patients with moderate to severe asthma, and 20 healthy donors were enrolled. All patients were allergic to house dust mites. The proportion of peripheral blood CD4+CD25+ Treg cells and Th2 cells were determined by flow cytometry. The concentration of interleukin (IL)-10, transforming growth factor (TGF)-β and IL-4 in plasma was determined by enzyme linked immunosorbent assay. In these subjects, peripheral blood mononuclear cells from 17 mild asthmatic patients, 13 moderate to severe asthmatic patients and 14 healthy donors were acquired and expression of forkhead box P3 (Foxp3) and GATA-3 mRNA was detected by reverse-transcriptase polymerase chain reaction.

RESULTSCompared with healthy donors and patients with mild asthma, the percent of CD4+CD25+ Treg cells and plasma IL-10 levels were decreased in patients with moderate to severe asthma. There were no significant differences in Foxp3 mRNA expression among three groups, but a downward trend seen among patients with asthma. However, the percent of Th2 cells, IL-4 levels and expression of GATA-3 mRNA was markedly higher in patients with mild and moderate to severe asthma than in the control group. The ratio of Th2/Treg and their cytokines was increased in allergic asthma, especially for moderate to severe asthma. The ratio of GATA-3/Foxp3 mRNA was also increased in allergic asthma. In patients with moderate to severe asthma, the percentage of peripheral blood Treg cells was negatively correlated to the percentage of Th2 cells and IL-4 levels.

CONCLUSIONSThe decline of CD4+CD25+ Treg cells in patients with moderate to severe asthma may play an important role in progress of the disease. Furthermore, the deficiency of CD4+CD25+ Treg cells was associated with the over-expression of Th2 response.

Asthma ; etiology ; immunology ; Cytokines ; blood ; Forkhead Transcription Factors ; genetics ; GATA3 Transcription Factor ; genetics ; Humans ; RNA, Messenger ; analysis ; T-Lymphocytes, Regulatory ; immunology ; Th2 Cells ; immunology