Abstract:The deepening of our understanding on the cardiac electrophysiology mechanism and the progress on cell transplantation technique have expanded our view in treating various kinds of cardiac diseases. Over the past few years literatures have reported many kinds of cell transplantation techniques for treating various types of bradyarrhythmias, such as complete heart block and sinoatrial node dysfunction, and impressive achievements have been made by far. This review discusses the advantages and flaws of the existing strategies and the future of biological pacemakers

Abstract:Objeetive：To investigate the survival of rabbits receiving autologous left auricle cardiomyocytes transplantation into the infarcted myocardium and to assess the effect of transplantation on the peripheral blood supply, heart rate, and cardiac function. Methods： Healthy adult rabbits were randomly divided into transplantation group （n = 8） and control group （n = 8）. The myocardial infarction （MI） models were established by ligating the left anterior descending arteries in all rabbits. Four weeks later the left auricles were harvested and the auricle cardiomyocytes were isolated and labelled with DAPI ex vivo. Rabbits in the transplantation group were injected with DAPI-labelled cell suspension into the infarcted areas and those in control group received culture medium. All the rabbits were examined by electrocardiogram （ECG） and 2-D ultrasonic cardiogram （UCG） 4 weeks after transplantation. Specimens were harvested from the transplanted areas and observed histologically. Resalts：All rabbits survived 4 weeks after transplantation. ECG showed that the heart rate in transplantation group was faster than that in control group （P〈0.05）, with no ectopic rhythm. UCG showed that the left ventricle function of the transplant group were better than that of the control group. Besides, the infarcted areas in control group showed typical histological changes of myocardial infarction and in the transplantation group there were ＂cell islands＂. Fluorescence detection certified the survival of implanted cells. Conclusion： Isolated autologous left auricle cardiomyocytes can survive after grafted into rabbit ventricular myocardium following myocardial infarction and may improve the left ventricular function

Abstract:Objective： To compare the histological, immunological, and biomechanical characteristics of decellularized porcine aortic valve scaffold created by 3 different decellularization protocols and to search for a more suitable technique for creating aeellular tissue-engineered cardiac valve conduit. Methods： Porcine aortic valve leaflets and whole aortic roots were deeellularized by 3 different protocols. Decellularization procedure in group Ⅰ involved treatment with 0.01% trypsin, 1% Triton, and nuclease for 24 h; that in group Ⅱ involved treatment with 0.01% trypsin （8 h）, 1% DCA, and nuclease for 24 h; and that in group Ⅲ involved treatment with 1% DCA and nuclease for 32 h. All the treatments were conducted during continuous shaking at 37℃. Porcine aortic valve leaflets and whole aortic roots treated with PBS were taken as control. The deeellularization efficiencies of each protocol were assessed by H-E staining, scanning electron microscopy, and transmission electron microscopy. The biomechanical features of the acellular valve matrices were examined by stress-strain tests and tensile strength tests. The immunogenicity and inflarfimatory responses of the decellularized matrices, valve leaflets, and aortic wall were investigated by subcutaneous implantation of them in rats. Results： The native ceils in porcine aortic valve leaflets and aortic roots were completely removed in group Ⅱ , which was superior to group Ⅰ and Ⅲ. The values of elasticity modulus and ultimate tensile strength （UTS） of groupⅡ were greater than those in group Ⅰ （[5.77±0. 951 MPa vs [4.15±1.13] MPa and [7.82± 1.51] MPa vs [4.65±0.85] MPa, respectively; P〈0.05）. The extension ratios at 1.5 MPa and at rupture in group Ⅱ were less than those in group Ⅰ （[0.33±0.04] vs [-0.41±0. 091 and [-0.45±0. 021 vs [-0.60±0. 06]; P〈0.05）, but the extension ratio at rupture was similar to that of fresh porcine aortic valves （ [0.45±0.02] vs （[-0.46±0.03]）. Histological analysis showed only slight inflammatory responses in grouplland the host cells grew into the matrix, rebuilding the acellular matrices gradually. Conclusion： Decellularization using 8-hour pretreatment with 0. 01% trypsin, followed by 24 hours incubation with 1% DCA plus nuclease is effective and convenient; it not only removes the cells but also decreases the immunogenicity of the aortic valve matrices, making the product an excellent material for tissue-engineered cardiac valve conduit.

Abstract:Objective： To modify the pulsatile bioreactor we constructed previously for simulating the high-flow, highpressure hemodynamics of heart valve in vivo, and to experimentally cultivate the tissue-engineered heart valves （TEHV） in the modified bioreactor. Methods： T-PLS system （NewheartBio Co. ,Ltd Korea） was used to generate pulsatile flow in the modified bioreactor and we designed a new air-exchange pathway to avoid contamination. The TEHV were made by seeding human bone mesenchymal stem cells （BMSCs） on deellularized porcine heart valve. After cultured under static condition for 4 d, the TEHVs were moved to the modified bioreactor and exposed to low-flow （0-600 ml/min） or high-flow（0-4 800 ml/min） pulsatile hydrodynamics for 7d, then the cells on TEHVs were evaluated. Results： After modification, the flow range expanded from （0- 1 200） ml/min to （0-6 000） ml/min and the pressure range expanded from （0-40） mmHg to （0-180） mmHg. In culture experiments, 26.3 % of the seeded cells remained under low-flow environment and cells were completely lost under the high-flow dynamics. Conclusion： The modified bioreactor can basically simulate the dynamics of heart valve in vivo and can be used in TEHV cultivation research. However, the current TEHV can not tolerate the high-flow pulsatile hydrodynamics

Abstract:Objective： To assess the effect of auto bone marrow mononuclear cells （BMMNCs） transplantation on reendothelialization and neointima formation in vein grafts. Methods： BMMNCs were extracted from the bone marrows of adult rabbit under sterile environment and were labelled with DAPI before transplanted into vein grafts. Twenty adult rabbits were randomly divided into 2 groups： BMMNCs transplantation group（group Ⅰ ,n= 10） and PBS transplantation group （group Ⅱ , n= 10）. The left external jugular vein of 20 rabbits were harvested and transplanted between ipsolateral common carotid artery （AVG）. Three days later, animals in group Ⅰ were transplanted with BMMNCs（6×10^8 cells）/100μl via periotic veins and those in group Ⅱ were injected with 100 μl PBS. Animals were killed 4 weeks later and graft veins were harvested to observe the reendothelialization and the thickness of vein grafts. Results： We found that the transplanted cells survived and were incorporated into the endothelium of vein grafts in group Ⅰ. The endothelium integrity of the vein grafts in group Ⅰ was significantly better than that of group Ⅱ. The intima thickness of vein grafts in group Ⅰ was significantly thicker than that of groupU. Condusion.. BMMCs transplantation therapy may improve reendothelialization of the vein graft and inhibits intimal hyperplasi the vein graft

Abstract:Objective：To investigate the effects of chitosan （CHI） and PC-chitosan （PC-CHI）-coated membranes on the proliferation, migration, and hemocompatibility of vascular endothelial cells. Methods： CHI and PC-CHI were separately sprayed on the bottom of culture dish evenly to form the polymer membrane; the cylinder was made of 316 L stainless steel flake. Pig iliac endothelial cells （PIEC） were cultured on the polymer membranes, the stainless steel bottom of the cylinder, and the untreated bottom of culture dish （as blank control）. Then the cell morphology was observed by light microscope and scan electron microscope; cell proliferation was measured by Cell Counting Kit-8 （CCK-8）; and cell migration was also assayed. Whole blood activated partial thromboplastin time （APTT）, prothrombin time （PT）, fibrinogen （FIB）, and thrombin time （TT） were examined by clotting method. Results： PIEC grew well on the membranes of CHI and PC-CHI, with normal morphology. After 24 h culture, the proliferation rates of PIEC were 88.8% and 77.8% on CHI and PC-CHI membranes, respectively. The survived PIEC on CHI and PC-CHI membranes were significantly more than those on 316 L stainless steel flake（P〈0.01）, and those on CHI membrane were significantly more than those on PC-CHI membrane（P〈0.01）. After 72 h culture, the numbers of migration cells on CHI and PC-CHI membranes were significantly more than those on 316 L stainless steel flake（P〈0. 01）, and those on PC-CHI membrane were significantly more than those on CHI membrane（P〈0.01 ）. APTT in PCCHI, 316 L stainless steel flake, and blank control groups were significantly longer than that of C HI group（P〈0.01, P〈0.05）, and FiB levels in the former 3 groups were higher than that of CHI group（P〈0.01 ）. Conclusion.. Coating with CHI and PC-CHI can improve the proliferation and migration of vascular endothelial cells on the culture dishes, indicating an excellent biocompatibility of CHI and PC-CHI membranes. PC-PCH membrane has more potent antithrombotic effect than CHI does.

Abstract:Objective： To investigate the application of a novel degradable biomaterial-short chitin fiber reinforced polycaprolactone （PCL） as a chest wall prosthesis, so as to assess its feasibility in clinical chest wall recomstruction. Methods： Two kinds of chest wall defects models （ 10 cm× 10 cm） were established in the present study, namely, group I with simple rib resection （n=2） and group II with full-thickness resection （the ribs, rib periosteum, intercostal muscle, parietal pleura, n=8）. The defects in both groups were repaired with short chitin fiber reinforced PCL plates. The implanted chest wall prosthesis and the regeneration of the chest wall tissue were dynamically observed postoperatively by X-ray, CT scanning, and histological examinations. Results： No operative/peri-operative death was observed in both groups; flail chest and paradoxical movement were not found in dogs. In group Ⅰ neogenetic bone tissues were found growing along the surface of chest wall prosthesis and new ribs appeared between prosthesis and parietal pleura. In group Ⅱ artificial chest wall prosthesis integrated tightly with chest wall ribs and muscle tissues around. Conclusion： Degradable chitin fiber reinforced PCL has fine biocompatibility and can provide effective support for chest walls, making it a promising biomaterial for chest wall reconstruction

Abstract:Objeetive ： To improve the traditional in vitro culture method for umbilical cord blood mesenchymal stem cells （UCB-MSCs） in an effort to increase the successful culture rate and to study the biological characteristics of the cultured cells. Methods. Full-term UCB samples were obtained with the mothers＇ consent and processed within 12 h of collection （n= 28）. Mononuclear cells （MNCs） were isolated from UCB by centrifugation at 2 500 r/min for 20 rain and were suspended in presence of a-minimum essential medium（α-MEM） containing 10% fetal bovine serum （FBS） and 100 U/ml penicillin. The medium was half changed after 5-7 days＇ primary culture and then was totally changed at a .3-4 days＇ interval. After the cells adhered to the culture wall, the following procedures were carried out according to our improved methods. In group Ⅰ , the suspension containing detached mesenchymal-like cells were removed onto a new culture dish when osteoclast-like cells reached over 80% confluence. In group Ⅱ , 15% calf serum instead of 10% FBS was used in the culture medium when osteoclast-like cells were growing confluence, and α-MEM medium containing 10% PBS was used again until most of the osteoclast-like cells were detached. The morphology of UCB-MSCs was observed under microscope and the phenotypes of them were examined by flow cytometry. The 5＇h passage cells were cultured with osteogenic medium and adipogenic medium separately and cytochemical staining was carried out subsequently. Results： Adhered cells were cultured from 20 of the 28 samples. UCB-MSCs were cultured from 13 of the 20 samples, which could grow to confluence and could be stably passaged to P22. The cultured UCB-MSCs were all positive for MSC-related antigens, such as CD29 and CD105, but negative for CD34, CD45, and CD106. We also found that the cultured UCB-MSCs could differentiate into osteoblasts and fat cells under specified conditions. Conclusion： MSCs do exist in UCB, and improvement of culture method can increase the yielding of UCB-MSCs. The cultured cells, with the similar phenotype and differentiation potential as those derived from other resources, can be amplified easily and passaged stably in vitro, making them promising seed cells for future bone tissue engineering

Abstract:Objective：To investigate the effect of sodium butytate （different concentrations） on the growth and proliferation of rat liver oval cell line WB-F344, and to discuss the conditions and rules for sodium butyrate-inducd WB-F344 cells differentiation into biliary lineage in vitro. Methods： WB-F344 cells were treated with sodium butyrate （0.75, 2.25, 3.75, 4.5 mmol/L） and the cell growth and morphological changes were observed; routinely cultured WB-F344 cells were taken as control. The changes of CK19 protein expression were examined immunohistochemically after WB-F244 cells were treated with 3.75% sodium butyrate; and the expression of phenotypic markers, such as 7-glutamyltransferase （GGT） ,β4-integrin, CK19, AFP and ALB at mRNA level were determined by RT-PCR. Untreated WB-F344 cells were used as blank control. Results： We found that sodium butyrate inhibited the growth of WB-F344 cells. The optical densities were significantly decreased in 3.75 and 4. 5 mmol/L groups compared with that in control group（P〈0.01）; but no significant difference was found between 0. 75, 2.25 mmol/L groups with control group. WB-F344 cells treated with 3. 75, 4. 5 mmol/L sodium butyrate became larger and round, with increased nuclei and decreased nucleus to cytoplasm ratio; those treated with 0.75, 2.25 mmol/L had no obvious changes. Immunohistochemical results showed that sodium butyrate significantly increased CK19 expression compared with control group（92.3±1.1]% vs [1. 3±0. 2]% , P〈0.01）. RT-PCR showed increased expression of β4-integrin in sodium butyrate treated groups, but not in control group; the expression of GGT and CK19 was higher than that of control group. Alpha-fetoprotein （AFP） expression was observed in blank control group, but not in sodium butyrate treated cells. Albumin expression was not detected in the 2 groups. Conclusion.. Sodium butyrate at 3. 75 mmol/L is suitable for inducing WB-F344 cells differentiate into the biliary lineage in vitro

Abstract:Objective： To investigate the effects of secreted protein acidic and rich in cysteine （SPARC） peptide on ultramicrostructure and extracellular matrix secretion of human mesangial cells cultured in vitro. Methods： Human mesangial cells （HMC） were incubated at the presence of SPARC peptide （50μg/ml） for 48 h and 96 h separately; HMC cultured without SPARC peptide was taken as control. The cell ultramicrostructure was observed by transmission electron microscope. Real-time fluorescent quantitative PCR （RT-PCR） was used to detect mRNA levels of collagen typeⅠ （ColⅠ ）, collagen type Ⅳ （ColⅣ）, fibronectin （FN）, and laminin （LN） 96 hours after culture, and the results were compared between the 2 groups. The secreted levels of ColⅠ , ColⅣ, FN, and LN protein were measured and compared by enzyme-linked immunosorbent assay （ELISA） in the 2 groups. Results： There was no obvious change in HMC in the control group. After cultured for 48 hours, a few HMC in experimental group showed initial appearance of apoptosis; after cultured for 96 h, a great number of HMC had a typical apoptotic appearance and there were typical apoptotic bodies under electron microscope. RT-PCR showed that, compared with the control group, the experimental group had significantly decreased levels of ColⅠ , ColⅣ and FN mRNA （P〈0.05, P〈0. 01） ; ELISA results showed that the secretions of ColⅠ , ColⅣ, and FN protein in the experimental group were greatly lower than those in the control garoup （P〈0.05, P〈0.01）. Conclusion： SPARC peptide can effectively induce apoptosis of human mesangial cells and down regulate the secretion of extracellular matrix in vitro

Abstract:Objectire：To construct a recombinant lentivirus harboring RNAi sequence targeting rat nogo receptor gene and to observe its infection efficiency of 293T cells. Methods： Lentivirus shuttle plasmid containing siNgR199 cDNA was constructed by gene engineering and was used to transfect 293T cells in the presence of packaging plasmids. Forty-eight hours later the supernatant was collected and the titer of virus was determined. The recombinant lentivirus and the standard lentivirus were used to transfect 293T cells at 1 MOI,3 MOI,5 MOI, 10 MOI and 20 MOI. Polymerase chain reaction （PCR） was used to detect the recombinant vectort enhanced green fluorescent protein （EGFP） expression was used to determine the titer and the infection rate of the recombinant lentivirus under fluorescent microscope. Results： Restriction endonuclease and PCR analysis confirmed that the siNgR199 cDNA was successfully inserted into the lentivirus vector. The titer of the recombinant lentivirus harboring siNgR199 was 1 ×10^8 ifu/ml and the best MOI was 3. Conclusion- The recombinant lentivirus containing siNgR199 gene has been successfully constructed, which lays a foundation for future axon regeneration in treatment of spinal cord injury

Abstract:Objective：To investigate the effect of Na^＋,K^＋-ATPase on the increase of cytosolic Ca^2＋ level induced by hypoxia in rat cortical neurons. Methods： Using confocal laser scanning microscope and video based motion edge detection system, we measured the intraeellular Ca^2＋ concentration （[Ca2＋]i ） and Na^＋ concentration （[Na^-]i ） in cortical neurons exposed to hypoxia for different time periods and at presence of different concentrations of dihydroouabain （DHO, an inhibitor of Na^＋,K^＋-ATPase ）. We also observed the effect ofNa^＋,K^＋-ATPase on the increase of cytosolic Ca^2＋ level induced by hypoxia in rat cortical neurons. Results： DHO （10^-9-10^-3mol/L） dose-dependently and hypoxia（4-20 min） time-dependently increased the levels of [Ca^2＋] and [Na^＋] in cultured cortical neurons. [Ca^2＋]i and [Na^＋]i increased significantly 4 min after cortical neurons were exposed to hypoxia, and addition of DHO （ 10^-3mol/L） further increased their concentrations. While 15 min after hypoxia exposure, different concentrations of DHO did not further increase [Ca^2＋] and [Na^＋]i We also found that hypoxia exposure did not enhance DHO pretreatment-induced [Ca^2＋]i increase. Couclusion： Inhibition of Na^＋,K^＋-ATPase activity is one of the mechanisms responsible for hypoxia-induced elevation of [Ca2＋]i in cortical neurons.

Abstract:Objective： To quantitatively study the damage of hepatocyte mtDNA in rats with obstructive jaundice and to understand its relationship with the damage of mitochondrial function. Methods： Forty-eight Wistar rats were randomly divided into 2 groups, namely, sham-operation group and obstructive jaundice group （model group）. The obstructive jaundice model was induced by double ligation and section of the common bile duct. The changes of mitochondrial function were routinely assessed; the whole mtDNA and deleted mtDNA in hepatocytes were determined by real-time fluorescent quantitative PCR. Results： Compared with sham-operation group, model group had reduced mitochondrial function and mtDNA copies （P〈0.01）. The deleted mtDNA was found in the model group but not in the sham operation group. With the continuation of the obstruction time, mtDNA copies decreased in the model group and the percentage of deleted mtDNA in the whole mtDNA copies increased （P〈0.01）, which was consistant to the deterioration of mitochondrial function. Conclusion： The damage of hepatocyte mtDNA is an important factor for the damage of mitochondrial function in rats with obstructive jaundice

Abstract:Objective：To investigate CDH1 methylation of epithelial-cadherin（E-cadherin） gene in intrahepatic cholangiocarcinomas （ICCs）. Methods： Forty-two liver samples were obtained from ICC patients （32 males and 10 females） during surgical resection in Eastern Hepatobiliary Surgery Hospital. The ICC tissue samples and the adjacent tissue samples were paraffinembedded and fresh ice-frozen. A methylation-specific polymerase chain reaction （MSP） was used for analyzing the methylation of CDH1 gene; E-cadherin protein and mRNA expression was detected by immunohistochemical method and RT-PCR analysis, respectively. Results： The methylation rate of CDH1 was 28.6% in ICC patients. The expression of E-cadherin mRNA and protein was decreased in 64.3% and 69.1% of the samples, respectively. The methylation of CDH1 gene was correlated with the expression of E-cadherin protein and mRNA and metastasis of ICCs （P=0. 008, P=0. 031, and P=0. 020, respectively） ,but not with the prognosis of ICC. The abnormal expression of E-cadherin was significantly correlated with the survival of patients （P=0. 002）. Conclusion; The methylation of CDH1 gene and down-regulation of E-cadherin are frequently seen in ICC patients, indicating that they may be closely related to the development and progression of ICCs

Abstract:Objective：To investigate the inhibitory effects of RNA silencing via adenovirus-mediated vascular endothelial growth factor（VEGF） shRNA on proliferation of lung adenocarcinoma ceils in vitro and in vivo. Methods： PCR method was used to construct a pAd-Easy/VEGF adenovirus vector containing enhanced green fluorescent protein （EGFP） gene and expressing VEGF shRNA. The 293 ceils were transfected with the linearized pAd-Easy/VEGF using Lipofectamine2000. Then lung adenocarcinoma cells A459 were transfected with the constructed vector. The EGFP expression was detected by fluorescent microscopy and flow cytometry. VEGF mRNA expression was examined by RT-PCR and Western blotting. The cell growth was observed with MTT method and the growth curve was plotted. Meanwhile, nude mice were transplanted with A549 cells to establish tumor models and the growth of tumors were observed. Results： The recombinant pAd-Easy carrying shRNA targeting VEGF had been constructed and the aim sequence had been obtained. The transfection efficiencies in pAd-Easy/VEGF and blank vector transfected A549 cells were 100% and 99.7%, respectively. RT-PCR and Western blotting showed a remarkable decrease of VEGF expression in the pAd-Easy/VEGF group compared with normal saline group. The tumor growth in pad-Easy/VEGF group was obviously slowed down and the weight and volume of tumors were both significantly lower than those of the control group （all P〈0. 01）. Conclusion： The shRNA targeting VEGF constructed in the present study can efficiently decrease the VEGF expression in A549 cells in vitro and suppress the growth of A549 ceils in vivo.

Abstract:Objective： To investigate the protein expression of HPV16 and its early gene E6, E7 in human laryngeal squamous cell carcinoma tissues, and to analyze their relationship with the clinical stage and pathological classification of laryngeal squamous cell carcinoma. Methods： The expression of HPV16, HPV16 E6, and HPV16 E7 protein was detected in 147 specimens of different laryngeal lesions immunohistochemically. The specimens included 82 laryngeal carcinoma, 39 noncarcinoma tissues （including 27 specimens of vocal cord polyp and 12 specimens of normal laryngeal tissues taken from more than 1.0 cm adjacent to the carcinoma）, and 26 precancerous lesions （leukoplakia） of the larynx. The relationship between the protein expression with the clinical stage and histopathological classification of laryngeal squamous cell carcinoma was analyzed. Results： The positive rates of HPV16, HPV16 E6, and HPV16 E7 protein in precancerous tissues （30. 77%, 26. 92%, and 26.92%, respectively） were significantly lower than those in laryngeal carcinoma lesions （45. 12%, 39.02%, and 42. 68%, respectively; P〈0. 05 or 0. 01）, but were significantly higher than those in non-carcinoma tissues （23. 08G, 5. 13%, and 2. 56%, respectively; P〈0.05 or 0.01）. In non-carcinoma tissues, the positive rate of HPV16 protein was significantly higher than that of E6 or E7 （P〈0.05 or 0.01）, while there was no difference between their positive rates in laryngeal carcinoma or precancerous lesions. We found that human laryngeal carcinoma tissues with different clinical stages and different pathological classifications also had different positive rate of HPV16 protein （P〈0.05）, but they had a similar positive rate of HPV16 E6 and E7. Conclusion： The expression of HPV16 E6, E7 proteins after the HPV16 infection might be one of the reasons for development of human laryngeal Carcinoma. Inhibition of HPV16E7 expression by immunologic strategy may have a potential for treatment of laryngeal carcinoma

Abstract:Objective： To investigate the effect of hyperlipemia on endothelial function and histomorphology of venous conduit in rabbits before grafting. Methods：Fifty adult male rabbits were randomly divided into 2 groups： one group was fed with normal diet （control group, n=25） and the other with high-cholesterol diet （hyperlipemia group, n= 25）. The blood samples and cervical vein specimens were harvested before and 2, 4, 8 and 12 weeks after feeding. The serum levels of total cholesterol （TC）, low-density lipoproteins （LDL）, triglycerides （TG）, and high-density lipoproteins（HDL） were determined. The expression of endothelial NO synthase （eNOS） protein, the production of NO, and the histopathological changes （including the thickness of intima and media, the diameters of the veins, and the presence of lipid or plaque） in the vein specimens were examined. Results： Eight weeks after feeding, the serum levels of TC, LDL, TG, and HDL in hyperlipemia group were significantly higher than those in the control group （P〈0.01 ）. Obvious lipid plaques were formed in the carotid artery of rabbits in hyperlipemia group. The jugular veins of hyperlipemia rabbits had significantly lower eNOS expression and NO production （P〈0.05）. Endothelial denudation was noticed and the elastic fibers almost disappeared in hyperlipemia group; there were no foam cells and lipid plaques. Conclusion： Hyperlipemia may result in endothelium dysfunction and histomorphological change of venous conduit

Abstract:Objective：To investigate the incidence of BK virus infection in renal transplant recipients and to analyze the risk factors of BKV infection. Methods： The urine samples and peripheral blood samples of 63 renal transplant recipients were collected at 1, 2, 3, 4, 6 and 8 months after transplantation. A real-time fluorescent PCR procedure was used to detect BK virus DNA in the samples and the samples were divided into the following 3 groups according to the detection outcomes： UV^＋PV^＋ group （BKV DNA positive in both urine and blood samples）, UV^-PV^- group （BKV DNA positive in urine but negative in blood samples）, and UV^-PV^- group （BKV DNA negative in both urine and blood samples）. Urinary sediment smears of patients were checked for decoy cells and the decoy cells were subjected to cytological immunostaining. Renal graft biopsy was performed when a sample was highly suspected of BKVAN by its clinical manifestations. The age, cold ischemia time, hematodialysis duration, immunosuppressive agents, panel reactive antibody, and other clinical parameters were compared between the 3 groups and a Logistic regression was performed to analyze the risk factors of BK virus infection. Results： There were 19 （30.1 %） patients in UV^＋PV^- group, 9 （14.3%）in UV^＋PV^＋ group, and 35 in UV^-PV^- group. The median time for the first detection of BK virus was 4 months in UV^＋ PV^- group and 3 months in UV^＋ PV^＋ group. Decoy cells were detected in 39. 7% of the 63 patients and the positive rate of decoy cell immunostaining was 54. 3%. One patient showed no BDVAN manifestation in renal biopsy. Logistic regression found that the cold ischemia time was significantly related to the BKV DNA infection （P= 0. 048, OR = 1. 151 ）, but not to other parameters （ P = 0. 069 ）. Conclusion ： Real-time fluorescent quantitative PCR is a good way for detection of BKV infection after renal transplantation. The peak time for BKV shedding is 3-4 months after transplantation. Cold ischemia time may be one of the risk factors of BKV infection, and immunosupressive regiment may has no obvious influence on BK virus infection

Abstract:Objective：To elucidate the major reasons for unexpected death nude mice when they were used for establishing human metastatic cancer models. Methods： The fresh specimens of human renal cell carcinoma （RCC）, prostate cancer （PC）, and DU-145 cells were transplanted / injected into nude mice ecotopically and orthotopically. Tumorigenesis and pathological changes （including the symptoms, pathological sections, survival time, etc. ） of mouse liver were investigated subsequently. Results： The tumorigenesis and metastasis rates were respectively 21.7% （35/161） and 1.2% （2/161） after implantation of RCC sample, and were respectively 100V0 （20/20） and 25M （5/20） after implantation of DU-145 cell line, while there was no tumorigenesis or metastasis after implantation of PC specimens. Liver pathological changes were found in 58. 4%（94/161） of mice implanted with RCC samples and in 43.4% （46/ 106） of mice implanted with PC samples. No pathological lesion was found in mice implanted with DU-145 cells. The death peak of mice with pathological changes after implanting RCC and PC samples was consistent with that of the total mice used in this study, all oceurring in the winter and spring of the year. Conclusion： The pathological changes of liver appear to be the major reason of unexpected death of the nude mice when they were used for establishing human metastatic cancer models. A specified pathogen-free environment is very important for establishment of the models

Abstract:Objective：To investigate the effects of various degrees of rapid eye movement （REM） sleep deprivation （RSD） and sleep recovery on cognitive function （learning and memory） and expression of brain-derived neurotrophic factor （BDNF） in the rat hippocampus. Methods, Male SD rats were divided into 8 groups （n= 12）： blank control group （with normal sleep）, environmental control, RSD 1 d, RSD 3 d, RSD 5 d, RSD 7 d, recover sleep 6 h after 7 d RSD （RS 6 h）, and recover sleep 12 h after 7 d RSD （RS 12 h）. The modified multiple platform method （MMPM） was used to establish sleep deprivation model in rats. The cognitive functions of rats were tested by Y-type maze. The hippocampal BDNF mRNA and protein levels were detected by real-time PCR and immunohistochemical method. Results： The failure reaction times of all RSD groups and the 2 RS groups were significantly more than those in control group and environmental control group （P~0.05）. Compared with the control group, the total reaction time in RSD 1 d, 3 d groups was significantly lower （P〈0.05）, but that of RSD7 d group was significantly higher （P〈0.05）. Compared with the blank control group, expression of BDNF mRNA was significantly increased in RSD 1 d group （P〈0.05）, and reached the peak in RSD 3 d group. The protein expression of BDNF in CA1, CA3, and denatae gyrus areas of RSD1 d, 3 d groups and in the denatae gyrus area of RSD5 d and RS6 h groups was significantly higher than that of blank control group （P〈0.05）. Conclusion： Short-time RSD can lead to decrease of learning and memory ability, and recovery of sleep can partially improve their ability. The increase of BDNF expression may compensate the result of sleep deprivation and protect the cognitive function. However, as the prolongation of deprivation time, the compensation may become invalid

Abstract:Objective：To prepare a pulmonary targeting DepoFoam loaded MR contrast media： gadopentetate dimeglumine （Gd-DTPA）, and to observe its physi-chemical and biologic characteristics. Methods： The routine magnetic resonance contrast media Gd-DTPA was encapsulated with multivesicular liposome （DepoFoam, Depo） labelled with red fluorescence to obtain a new particle Depo-Gd. The morphology, diameter, and the encapsulation rate of the new particle were examined. The targeting efficiency of the particle was assessed by animal test with mice. Ten mice were evenly divided into 2 groups： one group received caudal vein-injection of Depo-Gd and the other group received vena mesenterica-injection of Depo-Gd. Twenty minutes after the injection the slices of the mouse liver, lung, and kidney were obtained from both group and the fluorescence labeling were observed. Results： Under microscope, the produced Depo-Gd particles were round in shape and stable in status, with good mobility and well-distributed diameter （18μm in average）. The encapsulation rate of the particles was 50% and the concentration of Gd was 0. 0417 mmol/ml. The animal fluorescence test showed that mice in the 2 groups were all active and with normal respiratory and conscious status after the injection. In the Depo-Gd caudal vein-injection group, large amount of red dyeing was found in the lung while little red dyeing was found in the liver and kidney; in vena mesenterica-injection group, satisfactory dyeing was found in the liver but not in the lung. Conclusion： The produced Depo-Gd particles have a stable physicchemical property and with satisfactory encapsulation rate. There is no obvious biological toxicity and allergic reaction. Besides, the particles have a good pulmonary targeting ability when injected into peripheral veins, making them promising in future clinical application

Abstract:Objective：To investigate the priorities of each factor influencing the casualties in refugees living in border areas, so as to provide evidence for casualty forecasting in the refugees. Methods： We summarized the factors affecting the refugee casualty in the border area through searching and reviewing the related literatures. The identified factors were classified into 3 levels and the quantitative index system was established by using Delphi method, i.e. expert consulting method. The names and the meanings of each index were revised according to experts＇ suggestions after 3 rounds of consulting. The weights of each index were determined by analytic hierarchy process （AHP） and comparing-reordering method. Results.. A 3-level quantitative system was successfully constructed, which consisted of 4 first level indices （including natural factors, social factors, medical factors, and war factors）, 12 second level indices, and 37 third level indices; the weights of all indices were determined. Conclusion：The result of our study can be used in predicting refugee casualty and provide a reference for the medical service of refugees living in the border areas

Abstract:Objective：To analyze the construct validity of performance evaluation system of clinical centers by exploratory factor analysis strategy, so as to provide evidence for optimizing the parameters of the evaluation system. Methods： Using the exploratory factor analysis method, we evaluated the construct validity of the constructed performance index system. Results： Factor analysis showed that the factor components （listed in an increasing importance order） were： discipline superiority, cultivation of talented persons, medical service, scientific research capability, and infrastructure construction, etc. （the cumulative contribution of the first 6 factors was 90.17 %）. Most of index factor loadings matched with the meaning of their corresponding factors, indicating that the construct validity of the evaluation system was consistent with the expectation of its designers. Conclusion： The constructed clinical center performance evaluation system has a reasonable factor structure

Abstract:临床资料 患者，男，32岁，发现左侧腰部一生长缓慢的无痛性肿块近6年，体检见肿瘤区皮色正常，但有小静脉轻度怒张，触诊肿瘤大小约50mm×40mm，类似椭圆形，质韧偏硬，境界清楚，活动度极好，触痛不明显。声像图表现：二维图像见肿块位于皮下组织层与腹外斜肌筋膜之间，椭圆形，大小约45mm×25mm.[第一段]

Abstract:Hedgehog （HH） signaling pathway was firstly discovered in the regulation of embryonic segments development in Drosophila; later, the biochemical and functional homologs of Drosophila HH signaling genes were also isolated in vertebrates （including human）. Researchers found that HH signaling not only controls the embryonic development, but also plays an important role in tumorigenesis, Gli, a zinc finger transcription factor in the vertebral HH signaling pathway, combines to the special sequences of distal HH targeted genes and directly controls the transcription of targeted genes, playing a key role in HH signaling pathway. In this article, we summarize the role of Gli in HH signaling pathway and its prospect in cancer therapy

Abstract:Objective：To search for a new method for synthesis of 9-fluorenylmethoxycarbonyl amino acid tert-butyl ester. Methods： Glycine, L-Proline, and L-Phenylanaline were separately allowed to react with 9-fluorenylmethylchloroformate to obtain the corresponding 9-fluorenylmethoxycarbonyl amino acids. With 4-（dimethylamine） pyridine （DMAP） used as catalyst, the 9-fluorenylmethoxycarbonyl amino acids were allowed to react with Tert-butyl dicarbonate for the corresponding 9-fluorenyl- methoxycarbonyl amino acid tert-butyl esters. Results： Three 9-fluorenylmethoxycarbonyl amino acid tert-butyl esters were successfully synthesized by this method and their structures were confirmed by 1^HNMR. Conclusion： Ours is a simple method with mild condition and high yielding rate for synthesis of 9-fluorenylmethoxycarbonyl amino acid tert-butyl esters.

Abstract:在肝移植领域中，缺血再灌注损伤是一个亟待解决的重大课题。加氧冷灌注是防治肝移植缺血再灌注损伤的一种新方法，它能够有效恢复缺血供肝的氧供，提高供肝的ATP水平，改善供肝在缺血期间的能量代谢，从而最终提高肝移植的成功率。预计加氧冷灌在肝移植领域将有广阔的应用前景

Abstract:目的:观察及评价超选择性动脉溶栓治疗急性脑梗死的临床疗效和安全性.方法:对43例发病6 h内的急性脑梗死患者行动脉内超选择性重组组织型纤溶酶原激活物(rt-PA)溶栓治疗.结果:发现闭塞30例(69.8﹪),溶栓后完全再通17例(56.7﹪),部分再通9例(30.0﹪),未再通4例(13.3﹪),总体血管再通率86.7﹪.颅内出血3例(7.0﹪),死亡4例(9.3﹪).NIHSS评分:术前(12.8±2.9),术后24 h(12.1±2.6),术后14 d(8.1±2.4),术后3个月(5.5±2.0),术前与术后24 h评分差异无统计学意义;术前与术后14 d及3个月比较评分差异非常显著(P＜0.01).结论:超选择性动脉溶栓能提高闭塞血管再通率,明显改善预后,是治疗急性脑梗死的一种有效和较安全的方法.

Abstract:目的:观察前列腺增生症(BPH)患者手术前后的尿动力学指标的变化,探讨尿动力学检查(UDS)对BPH诊断、术前评估、术后疗效评价的价值.方法:357例BPH患者术前均有严重的排尿梗阻症状,国际前列腺症状评分(I-PSS)为(27.8±3.5)分,UDS检查均提示BPH诊断.其中17例患者UDS检查发现伴有膀胱尿道功能性疾患(膀胱逼尿肌收缩力异常与不稳定收缩、尿道外括约肌收缩强度过高等),包括低顺应性膀胱5例、逼尿肌收缩无力6例、逼尿肌反射亢进1例、逼尿肌外括约肌协同失调8例、不稳定膀胱7例、逼尿肌排尿后持续低幅度收缩1例、尿道外括约肌痉挛2例.术后对所有患者进行随访及UDS检查.结果:术后1个月随访,340例术前UDS检查无膀胱尿道功能性疾患患者排尿通畅,无梗阻症状,I-PSS (2.3±1.5)分,UDS指标基本恢复正常(最大尿流率、平均尿流率、最大尿流率时间、总尿流时间、尿量及尿流曲线改善等);17例术前存在膀胱尿道功能疾患患者,术后排尿仍不满意,I-PSS(26.8±2.1)分,UDS示膀胱尿道功能疾患仍存在,膀胱与尿道外括约肌的异常收缩无显著改变,但尿道膀胱镜检查未发现机械性梗阻.结论:手术治疗能解除大部分BPH患者的机械性梗阻症状,但对功能性梗阻患者效果可能不明显,手术前后有必要进行UDS检查,以进一步明确诊断、选择手术时机并对术后疗效进行正确的评估.

Abstract:目的:探讨乳腺黏液癌的临床特点及预后情况及相应治疗方案.方法:对39例乳腺黏液癌患者进行回顾性分析,单纯型27例,混合型12例,均接受手术、化疗等综合治疗.分析临床特点及治疗与患者生存率的关系.结果:女性乳腺黏液癌患者中位年龄48岁,绝经前69﹪,病灶大小1～15 cm,1/3病例误诊为良性肿瘤.单纯型黏液癌无腋淋巴结转移(0/27),混合型转移率50﹪(6/12).总5年生存率89﹪(单纯型96﹪,混合型75﹪).结论:国内35～50岁女性良性肿瘤外观的乳房肿块需排除乳腺黏液癌.乳腺黏液癌预后较好,混合型即便有淋巴结转移综合治疗效果也优于最常见的浸润性导管癌.乳腺单纯型黏液癌无腋窝淋巴结转移,更无需行腋窝淋巴结清扫.

Abstract:目的:观察松弛素样因子(relaxin-like factor, RLF)mRNA在离体培养的大鼠卵巢颗粒细胞和黄体细胞中的表达,初步探讨影响其表达调控的可能机制.方法:离体培养来自健康Wistar大鼠卵巢的颗粒细胞和黄体细胞,分别加入血小板活化因子(PAF)、促性腺激素(FSH或hCG)、腺苷酸环化酶激动剂(Forscolin)、蛋白激酶C(PKC)激动剂佛波酯(PMA),以培养液作为对照.采用RT-PCR方法观察各组RLF mRNA的表达,每组设5个复孔.结果:离体培养的大鼠卵巢颗粒细胞和黄体细胞均有RLF mRNA的表达,颗粒细胞中加入PAF、FSH、Forscolin、PMA组RLF mRNA的表达均较对照组增强(P＜0.05或P＜0.01);黄体细胞中加入PAF、Forscolin、PMA组RLF mRNA的表达均较对照组增强(P＜0.05),但加入hCG组无明显变化.结论:大鼠卵巢颗粒细胞和黄体细胞RLF mRNA的表达受PAF的正性调节,蛋白激酶A(PKA)和PKC信号通路的激活可能促进RLF mRNA的表达.

Abstract:目的：建立高效液相色谱法，测定不同产地首乌藤中大黄素含量的方法。方法：以YMC-PackODS-A（150mm×4．6mm，5μm）为色谱柱，流动相为甲醇-0．2mol／L磷酸二氢钠溶液（80：20,pH=3．0），检测波长256nm，流速1．0ml／min；柱温30℃，以外标法测定不同产地首乌藤中大黄素含量。结果：大黄素在1．52～7．58μg／ml范围内与峰面积呈良好线性关系，r=0．9997，大黄素平均加样回收率为100．97％，RSD为1．48％（n=6）。不同产地药材中大黄素含量分别为江苏0．500mg／g、贵州1．137mg／g、湖南0．333mg／g、河南0．121mg／g和湖北0．363mg／g。结论：本法可用于首乌藤中大黄素的含量测定；5个产地的首乌藤中大黄素含量有显著差异，贵州产首乌藤中大黄素的含量最高

Abstract:临床资料，患者，男，49岁，健康体检时超声扫查意外发现右侧肾上腺占位性病灶，入我院手术治疗。术前再次超声检查发现右侧肾上腺有3枚明确的圆形低回声结节，大小相近，均在10-15mm（图1A），左侧肾上腺也可见1枚椭圆形的类似低回声结节，略大。[第一段]