Objective To study the double restriction fluorescent labeling (DRFL) method for fluorescent labeling of trace DNA samples and its effect in enhancing the pathogen detection sensitivity of microarray assays. Method SARS-CoV RNA samples were reversely transcribed and then further amplified with the restriction display (RD)-PCR and fluorescently labeled by conventional restriction labeling directly with Cy-universal primer and the novel double labeling with Cy-universal primer and CydNTP. The labeled samples were applied to the microarray with the viral probes, processed and analyzed. Results Compared with the conventional method, DRFL labeling resulted in 3. 5835 times higher fluorescent intensity of all the SARS probes on average, even though increased fluorescent intensities for different probes varied considerably. Conclusion Signal to noise ratio can be enhanced by the DRFL method which improves the sensitivity of microarray technology in trace pathogen detections.

Objective To investigate the effect of ischemia and ischemia/reperfusion(I/R)in rat heart on matrix metalloproteinase-1(MMP-1).Methods The I/R animal models were established by shutting down and reopening the anterior interventricular branch with a silver clamp,then the distribution and amount of MMP-1 of the normal and I/R rat hearts were observed by immunohistochemical staining and Western blotting and analyzed by computer image analysis.Results 1.Immunohistochemical staining showed MMP-1 existed mainly in the cardiac matrix.There were strong positive reactions in fibrocytes,smooth muscle cells of the blood vessel and endotheliaI cells of capillaries.MMP-1 didn't show distinct changes 30 minutes after ischemia,while its concentration increased dramatically 60 minutes after ischemia.The positive reaction of MMP-1 increased 30 minutes after I/R,and 60 minutes after I/R there was large fusion areas in MMP-1 existing reglons.2.Quantitative analysis showed no dramatic changes of MMP-1 after ischemia for 30 minutes(P＞0.05),while dramatic changes were seen 60 minutes after ischemia(P＜0.05).MMP-1 changed dramatically 30 minutes and 60 minutes after I/R.3.Western blotting showed that there were no distinct naked-eye-observable changes.The bands of MMP-1 became widened 30 minutes after I/R,and became obviously widened 60 minutes after I/R.Conclusion 1.MMP-1 is secreted by fibrocytes,smooth muscle cells and endothelial cells of cardiac tissue under physiological conditions,and cardiomyocytes has the potential to secrete MMP-1 under ischemia or I/R.2.The longer time the heart ischemia lasts,the greater MMP-1 concentration will increase.Reperfusion can increase MMP-1 concentration to an even higher level,which may be the main cause of the collagen destruction after heart I/R.

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 When cumulus-enclosed oocytes are isolated from ovarian follicles and cultured in vitro,they undergo germinal vesicle breakdown(GVBD) and extrude the first polar body,then remain in meiotic arrest.In this study,we examined the timing of progression of human oocytes through meiosis using in vitro maturation conditions.Measurements made include nuclear changes,oocyte diameter and the perivitelline space(PVS).Methods Oocytes were collected from polycystic ovary patients by transvaginal immature follicle puncture(TV-IMFP) and matured in vitro.At 4,8,12,16,20,24,and 28?h from the onset of culture,oocytes were put into culture droplets and analyzed under light microscope to measure their diameter and the PVS width,then fixed and stained to evaluate the chromosomal status.Results About 50% of the oocytes underwent GVBD before 10?h culture,maximum chromatin condensation peaked at 8?h to 12?h,the majority of oocytes reached metaphase Ⅰ at 20?h,and soon anaphase and telophase Ⅰ progression occurred from 20?h to 24?h.Finally about 70% of oocytes extruded the first polar body by 28?h.With the maturation progressing,oocyte diameter did not change significantly;however,the perivitelline space width enlarged from 5.27+/-0.88??m to 17.18+/-1.26??m during this time.Conclusion The results presented here delineate the timing of nuclear events in the human oocyte during maturation in vitro with this culture system. The size of PVS is related to human oocyte meiotic progression.Thus,the size of the perivitelline may be a useful indication of the maturation state of the ooeyte.

Objective To study the changes of biological characteristics of CD34~+/CD133~+/VEGFR-3~+ lymphatic endothelial progenitor cells(EPCs) isolated from umbilical cord blood after induction with VEGF-C and investigate the mechanisms of EPCs differentiation toward lymphatic endothelial cells. Methods Mononuclear cells were isolated from umbilical cord blood using density centrifugation with Percoll solution.CD34~+/CD133~+/VEGFR-3~+ cells were sorted with FACS.Differentiation of the cells was induced with VEGF-C.The ultrastructural changes of the cells were viewed under scanning and transmission electron microscopes.Changes in expression of markers of the cells were viewed under confocal laser scanning microscope. Results Lymphatic EPCs isolated from umbilical cord blood expressed CD34,CD133 and VEGFR-3.At 7 day after induction with VEGF-C,CD34~+/CD133~+/VEGFR-3~+ cells were shuttle-like,there were a lamellipodium,numerous filopodia and many short microvilli.Caveolae were observed.Mitochondrion and rough endoplasmic reticulum were rich in cytoplasm.At 14 day after induction,the cells demonstrated appearance of the endothelial cell and expressed lymphatic endothelial specific makers LYVE-1 and 5'-nucleotidase,the expression of CD133 disappeared.Weibel-Palade body was observed in the cytoplasm of the cell.Conclusion There are CD34~+/CD133~+/VEGFR-3~+ lymphatic EPCs in human umbilical cord blood.The cells may differentiate into lymphatic endothelial cells through VEGF-C/VEGFR-3 signaling pathway after induction with VEGF-C.

Objective To study isolation and identification and culture of rat type A spermatogonial cells in vitro.Methods Percoll discontinue density gradient centrifugation combined with different speeds of different cells adhering to dish was used to purify the type A spermatogonial cells.The c-kit and TERT special antibodies were used to identify the type A spermatogonial cells.The purified cells were cultured in vitro. Results 0.614?10~6 cells per testis finally were obtained and the percentage of viable cells was 92.1% by trypan blue dye exclusion test.The percentage of type A spermatogonial cells expressing c-kit and TERT were 91.7?1.2% and 90.8?1.0% respectively.Type A spermatogonial cells could proliferate and self-renew in the DMEM containing 10% NBS.Conclusion Percoll discontinue density gradient centrifugation combined with different speeds of different cells adhering to dish is an efficiency method for isolation of rat type A spermatogonial cells.The purified cells are type A spermatogonial cells by identification of the immunohistochemistry of c-kit and TERT antibodies.Type A spermatogonial cells can proliferate and self-renew in vitro.

Objective To investigate the effect of steroid hormones on lymphatic stomata of ovarian bursa and fluid adsorption. Methods The lymphatic adsorption was traced by trypan blue,and the ultrastructure of the lymphatic stomata and estrogen receptor level of mesothelial cells on ovarian bursa was observed by scanning electron microscopy,transmission electron microscopy and immuno-colloid golden technology. Results The absorption and opening area of lymphatic stomata were enhanced in estrogen group mice,and decreased in androgen group as compared to control group mice.Estrogen receptor was first identified in the nuclei of mesothelial cells on the inner layer of mice ovarian bursa and the level was down-regulated by estrogen and slightly up-regulated by androgen.Conclusion The absorption and the opening area of lymphatic stomata can be regulated by steroid hormones in mice,and the machanism may be related to the expression levels of estrogen receptor in the nuclei of mesothelial cells.

Objective To study the effect of FSH on the secretion of gastrin in the rat stomach,offering experimental evidence for reproductive endocrine hormone regulating digestive function. Methods The FSH was directly injected into the stomach of rats to observe the change of density of gastrin immunoreactive positive cells in the stomach by immunohistochemical SABC method and the gastrin level in circulating blood and gastric liquid by ELISA. Results Compared with the control group,which was injected with saline into the stomach,the density of immunoreactive positive cells was significantly increased in the stomach with FSH treatment group(P

Objective To investigate the distribution of somatostatin in mammary gland of the later lactating rats.Methods The timely immunohistochemical method was used to detect the somatostatin in mammary gland of the later lactating Sprague-Dawley rats. Results The timely immunohistochemical method showed that intense staining for somatostatin in the whole epithelial cell cytoplasm and the secretory material.Conclusion There are the location of somatostatin in the epithelial cell cytoplasm and the secretory material of the rat mammary gland.

Objective To investigate the whole differentiating process of myogenic satellite cells in vitro,and the expression of N-ACh receptors.Methods Skeletal muscle satellite cells were isolated by trypsin digestion in vitro and purified by preferential attachment method.The cells were observed morphologically and identified by ?-sarcomeric actin,desmin and skeletal myosin antibodies.Contraction of myotubes was observed after adding ACh in the culture medium, N-AChRs in myotube were also be identified by bungarotoxin.Results Cultured satellite cells in vitro differentiated into myoblasts and fused to form myotubes later.These cells were positive by ?-sarcomeric actin, skeletal myosin and desmin antibodies' staining.Adding ACh directly in the medium stimulated the contraction of myotubes.No obvious expression of ACh receptors was observed on the surface of satellite cells.Cluster of receptors aggregated on myotubes from neonatal rats' satellite cells cultured in differentiation media.Conclusion The myotubes differentiated from myogenic satellite cells have the function of contraction.N-ACh receptors were synthesized gradually during the process of differentiation.