BACKGROUND:Constructing a three-dimensional tissue-like structure in vitro plays a critical role in modern tissue engineering and regenerative medicine. Several advances have been made in the past decade. However, it is stil a chal enge to promote microvascular-like structure formation and improve limited nutritional transportation, thereby promoting cel viability.
OBJECTIVE:To explore the feasibility of constructing a three-dimensional microvascular-like structure through the co-culture technique.
METHODS:Human bone marrow mesenchymal stem cel s and human endothelial cel s were co-cultured on a three-dimensional porous silk scaffold. Cel proliferation was analyzed by Pico-green DNA assay. Their growth profiles were evaluated by scanning electron microscope and laser scanning confocal microscopy, respectively. The mRNA levels of von Wil ebrand factor and CD31, two key functional markers of endothelial cel s, in the co-cultured endothelial cel s was assayed by real-time quantitative reverse transcription-PCR.
RESULTS AND CONCLUSION:The three-dimensional culture system constructed by the silk scaffold and bone marrow mesenchymal stem cel s provided an ideal microenvironment for cel growth and proliferation in vitro. Moreover, this microenvironment was capable of promoting endothelial cel differentiation evidenced by their significantly improved mRNA levels of von Wil ebrand factor and CD31. Premicrovascular-like structure was also observed in the co-cultures under the confocal microscope. Thus, al the data supported that the unique co-culture system could promote endothelial cel differentiation and self-assembling in vitro. This culture system provides a robust tool for the studies addressing microvessel-based tissue engineering.

BACKGROUND:The quantity and quality of seed cel s is a critical bottleneck of the development of vascular tissue engineering. To address this issue, stem cel-derived endothelial cel s have been a hot spot in this field due to their potential in providing the ideal seed cel s.
OBJECTIVE:To elucidate the effect of vascular endothelial growth factor (VEGF) supplementation combined with hypoxic culture condition on the lineage-specific differentiation of embryonic stem cel s into endothelial cel s.
METHODS:Serum-free medium mTeSR?1 was applied to cultivate H9 cel s in vitro. A conditioned medium containing 50μg/L vascular endothelial growth factor was utilized to induce H9 cel s to differentiate into endothelial cel s under the hypoxic culture condition (5%O2). The cel under normal condition (5%CO2) with or without vascular endothelial growth factor served as controls. The phenotype and function of human embryonic stem cel s-derived endothelial cel s were assayed by immunofluorescence staining, quantitative RT-PCR, and low-density lipoprotein uptake experiment.
RESULTS AND CONCLUSION:Compared with the control group, the H9 cel s were induced to be differentiated into endothelial-like cel s more efficiently when they were cultivated under a conditioned medium with vascular endothelial growth factor supplementation under the hypoxic condition. These differentiated cel s not only expressed some important surface markers of endothelia cel s, including kdr, pecam, but also took in low-density lipoprotein to form microvessle-like structures. This culture system supports a synergy effect of vascular endothelial growth factor and hypoxic environment that can efficiently promotes the lineage-specific differentiation
of embryonic stem cel s into endothelial cel s with good phenotype and functionality.

Objective To explore the gene expression in the neural stem cells as well as the cells after the clone was differentiated. Methods Neural stem cell culture, Immunocytochemistry and RT-PCR technique were used. Results There was Aromatase expression in the neural stem cells, after the stem cells were differentiated, the Aromatase strongly expressed in the neruons and weakly expressed in the astrocytes. The aromatase gene in these cells was expressed by means of the brain-specific promoter 1. 4.Conclusion It may provid a new clue for the source of estrogen in the central nerve system.

Objective:To evaluate the efficacy and safety of talc poudrage pleurodesis via semi-rigid medical thoracoscopy in the treatment of malignant pleural effusions,as well as the factors that may influence the outcomes.Methods:A series of 27 patients with malignant pleural effusion underwent medical thoracoscopic talc poudrage pleurodesis between July 2005 and September 2007 in Peking University First Hospital.Results:There were 16 male and 11 female patients in the series,the average age being 65.2 years.All the patients had documented malignant pleural effusions,including 16 cases of adenocarcinoma,6 of malignant mesothelioma,2 of squamous cell carcinoma,1 of lymphoepithelioma-like carcinoma,1of small cell carcinoma and 1 of undifferentiated lung cancer.Thirty days after the procedures,complete successful pleurodesis was achieved in 22 cases,and partial successful in 4 cases.Pleurodesis was not successful in one case.Overall successful rate was 96.3%(26/27).The average duration of thoracic tubing was 6.85 days.Chest pain,fever and an increase in peripheral WBC after the procedure occurred in 19(70.4%,19/27),21(77.8%,21/27),and 12(44.4%,12/27)cases respectively.No respiratory failure occurred.Conclusion:Medical thoracoscopic talc poudrage pleurodesis is a safe and effective method for the treatment of malignant pleural effusion.

Objective To investigate the relationship between the resting heart rate (RHR) and target organ damage (TOD) in elderly patients with metabolic syndrome(MS). Methods 264 elderly patients with MS were divided into four groups according to the level of RHR: RHR1 group, RHR<65 beats/minute (bpm) (46 cases) ;RHR2 group, 65≤RHR<75 bpm (77 cases);RHR3 group, 75 bpm≤RHR<85 bpm (89 cases);RHR4 group, RHR≥85 bpm (52 cases).Electrocardiography, echocardiography, carotid uhrasonography, crcatinine clearance rate (Ccr) and quantitative assay of 24 hours' albuminuria were performed. Results (1) Compared with RHR1, RHR2 and RHR3 groups, RHR4 group showed higher levels of carotid intima-medial thickness (IMT), carotid arterial diameter (CAD), left ventricular mass index (LVMI) and albuminuria(P< 0.05 or P<0.01), and lower levels of left ventricular ejection fraction (LVEF) and Ccr (all P< 0.01). (2) The IMT, CAD, LVMI and albuminuria were positively correlated with RHR (r=0.33, 0.23, 0.61, 0.58, respectively, all P<0.01). However, the LVEF and Ccr were negatively correlated with RHR (r=-0.59, -0.51, all P<0.01). (3) Logistic multivariate analysis showed that RHR and pulse pressure (PP) had effects on myocardial hypertrophy, coronary heart disease, heart failure, cerebral stroke and renal dysfunction(P<0.05 or P<0.01). Except heart failure, PP played a more important role than RHR. Coneinsions RHR may be an independent risk factors for TOD in elderly patients with MS,and RHR regulation is important for the development of MS in the elderly.

AIM:To construct nine novel L-asparaginase mutants and study their enzyme-activity.METHODS:The mutants were constructed using overlap extension PCR according to the principle of alanine-scanning mutagenesis. The enzyme-activity was detected by Nessler's method. RESULTS:The DNA sequencing showed that the mutagenesis was consistent with the theoretical prediction. The enzyme-activity assay demonstrated that each mutant possessed enzyme activity equal to the original enzyme. CONCLUSION:Through gene modification,epitop of L-asparaginase was changed without activity loss.These results provide foundation for further study of the structure-function relationship of L-asparaginase.

Objective To simulate the three-dimensional(3-D) growth pattern of stem cells in vivo by a 3-D culture system in vitro constructed by rat tail collagen scaffold.Methods Circular strands were prepared by mixing suspended murine embryonic stem cells(mESCs) with rat tail collagen.Growth profile of mESCs within the collagen strand was observed with phase contrast microscopy.Their metabolic activity was evaluated by glucose/lactic acid contents.To evaluate the effect of 3D culture system on ESCs differentiation,ES-derived cardiomyocytes were detected by immunohistochemistry,RT-PCR and transmission electron microscopy respectively.Results ESCs grew well in the 3-D culture system constructed by rat tail collagen.Cell connections can be found in those cell clusters formed within collagen stands,which indicated that tissue-like cultures should be produced during the process of 3-D culture in vitro.ESCs cultured by 3-D collagen strand differentiated into cardiomyocytes spontaneously.Conclusion Collagen provides an ideal growth matrix for ESCs proliferation in vitro and promotes ESCs differentiation towards tissue-like structures.Thus,the three dimensional culture system constructed by rat tail collagen can be applied to study ESCs differentiation in vivo.

Objective To get a better medical image segmentation result by studing a new image segmentation method. Methods Medical images were segmented using image segmentation method based on texture character and generalized radial basis function neural networks. The texture character parameters were obtained according to gray level co-occurrence matrix. The parameters were input to the generalized radial basis function neural networks to train the network. Results Comparatively, perfect binary images were obtained by using this new image segmentation method. Conclusion The emulational results show that the method is an effective medical image segmentation method.

Aim To investigate the effect of metformin (Met) on connexin43(Cx43) expression in H9c2 cells cultured with high glucose and the relevant mecha-nisms.Methods Rat myocardial cells H9c2 were cul-tured with high glucose , then combined with 3μmol· L-1 and 5 μmol · L-1 of Met for 24 h respectively . The viability and toxicity of H9c2 cells were detected by MTT and LDH , respectively .The expression and distribution of Cx43 were detected by immunofluores-cence staining .The intracellular oxygen species reac-tive ( ROS) level was detected by fluorescencemicrosco-py.And the expression of Cx43, P-AMPK, AMPK and GAPDH was determined by Western blot .Results The viability of H9c2 cells was increased and the level of intracellular ROS was decreased in Met group , while there existed no significant difference in LDH release among groups.Met significantly increased the phos-phorylation of AMPK and the expression of Cx 43, and improved the distribution of Cx 43 also.Conclusion The beneficial effect of Met on cardiovascular system in diabetic patients may via up-regulating the expression of Cx43 and down-regulating of intracellular ROS through activation of AMPK .

AIM:To investigate the effect of all-trans retinoic acid ( ATRA) on the viability of gastric cancer cell SGC-7901 and the sensitivity to radiotherapy .METHODS:MTT assay was used to examine the cell viability .Radio-sensitivity and cell cycle were determined by colony formation assay and flow cytometry , respectively .The mRNA levels of Bax, Bcl-2, survivin and NF-κB in the cells were measured by RT-qPCR.RESULTS: ATRA inhibited the viability of SGC-7901 cells in a concentration-dependent manner .The maximal inhibition was at concentration of 8 μmol/L.Colony formation assay revealed that the combination of ATRA with X-ray treatment significantly reduced the values of D 0 and Dq, and shifted down the fitting survival curve , as compared with radiotherapy alone .Moreover , ATAR markedly decreased the percentage of G2/M phase in the SGC-7901 cells (P<0.05).In addition, following ATRA treatment, the mRNA levels of Bcl-2 and survivin were decreased (P<0.05), whereas the mRNA levels of Bax and NF-κB were increased (P<0.05). CONCLUSION:ATRA enhances the sensitivity of SGC-7901 cells to radiotherapy , inhibits G2/M arrest and regulates the mRNA expression of Bax , Bcl-2, survivin and NF-κB.