BACKGROUND:Construction of tissue engineering adult cardiac myocytes has been a new research hotspot in cardiovascular fields.
OBJECTIVE:To explore a simple, fast method for the separation of adult rat cardiomyocytes, and preliminarily explore the construction methods of tissue engineering adult cardiac myocytes.
METHODS:Segmented enzymatic digestion method was used to isolate cardiac myocytes from adult rats. Subsequently, cardiac myocytes were transfected with adenovirus and liposome-mediated red fluorescent protein gene. Construction efficiency of tissue engineering cells was qualified using inverted fluorescence microscopy and flow cytometry. Final y, cardiac myocytes were transfected with adenovirus-mediated hypoxia-inducible factor-1a, and the expression of hypoxia-inducible factor-1a was examined by western blot analysis.
RESULTS AND CONCLUSION:A lot of cardiac myocytes were col ected using the segmented enzyme digestion method. Flow cytometric analysis showed that the survival of adult cardiomyocytes was (87.03±0.70)%. Compared with liposomal transfection (transfection efficiency was 0), adenovirus infection efficiency was (70.31± 1.39)%, and the cells expressed red fluorescence under fluorescence microscope. After 4 days of adenovirus infection, transfected cells expressed hypoxia-inducible factor-1a protein. These results showed that segmented enzyme digestion is a fast way to isolate adult cardiac myocytes, and recombinant adenovirus vector is a good vector to transfect cardiac myocytes.

Objective The experiment aims at probing the best condition of the isolation and purification of rat islets.Methods The islets were isolated from rat pancreas by hepatopancreatic duct perfusing with collagenase and purified with Ficoll 400 discontinuous density gradient centrifugation.Then the purified islets were subjected to histological staining,electron microscopy and radioimmunoassay for identification of specificity and viability.Results The histological staining revealed that the viability and the purity of the purified islets were above 95％and 85％respectively.Electron microscopy showed that the purified islets were morphologically intact with clear membrane and plenty of secreting granules.Radioimmunoassay demonstrated the secreted insulin concerntration between low-glucose groups and high-glucose groups varied significantly,which verified the good function of the islets.Conclusion Hepatopancreatic duct perfusing with collagenase is a good method for digestion.There aye many factors that influence the quantity and quality of the acquired islets,such as the completed expansion of pancreas,the concentration and viability of collagenase and the digested time,etc.

Objective To explore the effect of psychotherapy in patients with methadone maintenance treatment.Methods 60 patients accepted drug and 69 patients accepted drug with psychotherapy treatment were evaluated with Self-Rating Anxiety Scale(SAS) and Self-rating Depression Scale(SDS) in the beginning of psychotherapy therapy and 3 months later.All of them were assessed for heroin use at 3 months after the end of a 3-month treatment period.Results SAS、SDS did not show any significant differences between the two groups in the beginning(P ＞0.05).After 3 months of psychotherapy,the differences in SDS and SAS between the two groups showed statistically significant(t =- 2.858,- 2.465,all P ＜0.05).After 2 months of psychotherapy,the number of relapsing again patients of group psychotherapy treatment was significantly lower than control group(x2 =6.906,5.054,5.100,4.247,all P ＜0.05).Conclusion The mental healthy status of patients with heroin dependence is poor.Psychological treatment plays an important role in short-term prevent hernoin addicts from relapse again.

Objective To observe the pulmonary X-ray changes of malignant malaria.Methods The chest routine X-ray examination in 86 cases with malaria in company with respiratory tract symptoms such as fever was performed,the pulmonary changes were obsenved.Results Of 86 cases, 58 cases were abnormal on chest filmes,including lung framework marked in 33 cases,miliary,small or large patchy shadow in 24 cases and multiple round opaque mass lesion in one case.Conclusion The pulmonary X-ray changes of malignant malaria are varied,the common characteristic is that most of them are situated at middle and lower portion of the lungs.This may attribute to blood dynamic abnormality resulting from malaria protozoan and the pathological changes of involved tissues.

To study the clinicopathological and immunohistochemical characteristics of omental and mesenteric stromal tumor, 8 cases of stromal tumor were investigated, and followed up. The results showed that these tumors were solitary nodule, most were large. Histologically, cytoplasmic eosinophilia and perinuclear vacuolization were often seen in omental and mesenteric stromal tumor of spindle cell type. 3 cases showed epithelioid cytologic features. An immunohistochemical study showed that all the patients were C kit positive (100%),7 were CD34 positive, whereas 6 were SMA positive, positive expression of p53 and PCNA was found in 2 cases of malignant stromal tumor, Lower expression of PCNA was observed in 1 case of benign one.The results suggested that omental and mesenteric stromal tumors have distinct clinicopathological features, the expression of C kit and CD34 plays an important role in its diagnosis.

Objective The experiment aims at probing the best condition of the isolation and purification of rat islets. Methods The islets were isolated from rat pancreas by hepatopancreatic duct perfusing with collagenase and purified with Ficoll 400 discontinuous density gradient centrifugation.Then the purified islets were subjected to histological staining,electron microscopy and radioimmunoassay for identification of specificity and viability. Results The histological staining revealed that the viability and the purity of the purified islets were above 95% and 85% respectively.Electron microscopy showed that the purified islets were morphologically intact with clear membrane and plenty of secreting granules.Radioimmunoassay demonstrated the secreted insulin concerntration between low-glucose groups and high-glucose groups varied significantly,which verified the good function of the islets.Conclusion Hepatopancreatic duct perfusing with collagenase is a good method for digestion.There are many factors that influence the quantity and quality of the acquired islets,such as the completed expansion of pancreas,the concentration and viability of collagenase and the digested time,etc.

Objective To investigate the clinical characteristics, the antenatal management, the outcome and prognosis of chronic myeloproliferative disorders (CMPD) complicating pregnancy. Methods Retrospectively analyze the clinical data of eleven patients with CMPD complicating pregnancy hospitalized in Peking University People' s Hospital from 2000 to 2009, including five patients with essential thrombocythemia, one with primary myelofibrosis and five with chronic myeloid leukemia. Results (1)Five pregnancies had periodic antenatal care and laboratory monitorings like full blood count. Reasonable anti-coagulation therapy was given to prevent the complications. One patient with PMF diagnosed before conception had her first pregnancy ended with mild pre-eclampsia and intrauterine death at the gestational age of 32 weeks. During the first trimester of her second pregnancy two years later, the test for anti-β2 glycoprotein antibody was positive. She received low-dose aspirin and low-molecular-weight heparin as anticoagulants. An uneventful course was obtained and she delivered a healthy term infant. (2) Five pregnancies had occasional antenatal examination, including two patients with ET and three patients with CML One patient with ET developed severe pre-eclampsia at the gestational age of 25 weeks. Umbilical artery Doppler showed reversed end-diastolic velocity. The management with anti-convulsants, antihypertensives and anti-coagulants showed no effect. An emergency cesarean section had to be performed because of the aggressive hypertension and placental abruption, with still birth as a result. Two pregnancies never had an antenatal care. Both of them were admitted on labor and the diagnoses of CML were made. (3)Four pregnancies developed oligohydramnios and three developed preelampsia(two severe pre-eclampsia and one mild pre-eclampsia). There was no other hemorrhage and thrombosis event. (4) Eight pregnancies reached full-term with four cesarean sections and four vaginal births. Two preterm cesarean sections were performed because of a progressive oligohydramnios. The ten live neonates weighed 1820 - 3600 g. All were appropriate for gestational age, except one fetal growth retardation (FGR) developed in one patient with severe pre-eclampsia. (5) As for the CMPD, the eleven patients were all in stable conditions. Three patients with CML received hydroxyurea in the third trimester, four with ET and one with CML had plateletpheresis before delivery with favorable effect. All patients were uneventful postpartum, except one with CML who died in 5 months after childbirth. Conclusions The pregnancy outcomes for patients with CMPD are mostly good. However, antenatal care should pay more attention to the complications such as thromboembolic accidents, pre-eclampsia, still birth and fetal growth retardation. Management including reasonable anticoagulation therapy should be considered, which may help improve the prognosis.

[ABSTRACT]OBJECTIVETo explore the method on reconstruction of long and special tracheal defects which can mostly match with natural airway: pedicle rib cartilage with cilia endothelial wall.METHODS8 experimental model of rabbits were trained with cervical double belt blood supply fascia embedding and autologous costal cartilage and nasal septum mucos in the first period, then followed by transplantation in the second period. After the operation, we would assess the physiology, breathing and histopathology index of the rabbits, etc. After the animal experiment, we tried to apply the method to an appropriate clinical case.RESULTS8 cases of experimental rabbits dead after the second period operation with the average survival time of 21.9 days and caused by asphyxia. Histopathological results: rib cartilage and trachea ring up of cartilage cells and fibers have high similarity in histology; cartilage of all cases under the cultivation of the pedicle fascial package has not been absorbed; all cases' nasal septum mucosa in the body and blood supply to differentiation under fascia nutrition. Then we applied the method on a clinical case.CONCLUSIONTrachea ring rib cartilage had a higher similarity to the tracheal cartilage on the histology and biological characteristics that can be used as the preparation of artificial trachea shaping material or cell culture to tissue engineering materials. Package of rib cartilage pedicle fascial can provide adequate blood supply to make up for a free training rib cartilage defect to its absorption. Nasal septum mucosa of pressure in the body and blood supply of the fascia nutrition can simulate the trachea ciliated epithelium, which can play its biological characteristics similar to the inner wall of the trachea.

Objective: To explore the sensitivity of Kit or PDGFRA mutants related to gastrointestinal stromal tumor (GIST) to Gleevec.Methods: The recombinant plasmids of KIT Del559-560, KIT Ins IPYD579, PDGFRA D842V and PDG-FRA L839P gene mutants were transiently transformed into the CHO cells by liposome methods.Western blot was used to detect the expression of the related protein and their phosphorylated forms after the cells were incubated with Gleevec for 90 min.At 72 hours after incubation with Gleevec, MTT was used to detect cell proliferation.Results: Western blot results showed that Gleevec at 0.1 μM can notably reduce phosphorylation of KIT Del559-560.Gleevec at 1μM completely blocked phosphorylation of KIT Ins IPYD579 and PDGFRA L839P, but did not affect PDGFRA D842V phosphorylation.MTT analy-sis indicated that growth of CHOPDGFRA L839P was inhibited by Gleevec at 1μM, however, CHOPDGFRA D842V was re-sistant to Gleevec at 5 μM.Conclusion: Gleevec can decrease the expression of phosphorylated protein CHOPDGFRA L839P and CHOKIT Ins IPYD579, and can remarkably inhibit the proliferation of cells containing PDGFRA L839P mutant.

A method was developed for the determination of furocoumarin compounds(psoralen, oxypeucedanin, imperatorin, isoimperatorin) in traditional Chinese medicine samples by dispersive liquid-liquid microextraction(DLLME) coupled with high performance liquid chromatography and the relation of sample solution volume, sediment phase volume with enrichment factors was deducted. Some important parameters that influenced the extraction efficiency were optimized: 50 μL carbon tetrachloride and 300 μL acetonitrile were rapidly injected into 1.5 mL sample solution containing 2.5%(w/V) NaCl. After centrifugation at 3500 r/min for 3 min, the sedimented CCl_4 phase was pumped and recorded the volume, then dissolved with methanol and injected into the HPLC for analysis. Under the optimum conditions, a good linear relationship was obtained in the range of 0.006 6.00 mg/L of both psoralen and oxypeucedanin and 0.006-12.0 mg/L of both imperatorin and isoimperatorin. The limits of detection were 1.0-3.0 μg/L(S/N=3). The relative standard deviations were 2.3%-5.4%(n=5). The enrichment factors ranged from 12.6 to 38.5 folds. The average recoveries ranged from 97.5% to 114.8%. The four kinds of furocoumarin compounds in Radix Angelicae dahuricae and Yuanhu Zhitong tablet were determined by the proposed method with satisfactory results.