To establish a gene regulation system compatible with biopharmaceutical industry and gene therapy, we constructed a fusion protein of biotin ligase from Bacillus subtilis (BS-BirA) and the trans-activation domain, and used its expression vector as the regulatory vector. Meanwhile, BS-BirA-specific operators were ligated upstream of attenuated CMV promoter to obtain the response vector. In this way, a novel eukaryotic gene regulation system responsive to biotin was established and named BS-Biotin-On system. BS-Biotin-On system was further investigated with the enhancing green fluorescent protein (EGFP) as the reporter gene. The results showed that our system was superior to the current similar regulation system in its higher induction ratio, and that the expression of interest gene could be tuned in a rapid and efficient manner by changing the biotin concentrations in the cultures, Our results show that the established system may provide a new alternative for the exogenous gene modulation.

The blastoderm cells isolated from the stage X embryos of chicken were cultured on feeder layer of mouse embryonic fibroblasts(MEF)with high glucose DMEM supplemented with 10 ng/mL bFGF,20 ng/mL hIGF-1,2 ng/mL mSCF,2 ng/mL hIL-11 and 1 000 U/mL LIF.Typical chicken embryonic stem cells(cESCs)colonies with high endogenous AKP activity were observed in the culture system.The cESCs derived from blastoderm cells expressed pluripotent markers SSEA-1 and Oct-4 as determined by immunocytological analysis.RT-PCR analysis further confirmed the expression of cENS-1,a gene specifically expressed in cESCs and early embryo.These results demonstrate the feasibility for using the culture system in isolating cESCs from the stage X chicken embryos and supporting the growth of cESCs with undifferentiating state in vitro.

BACKGROUND: The aim of myocardial tissue engineering is to repair or regenerate damaged myocardium with engineered cardiac tissue constructed by a combination of cells and scaffolds in vitro. However, this strategy has been hampered by the lack of cardiomyocytes and the significant cell death after transplantation in vivo.METHODS: In this study we explored the feasibility of in vitro construction of vascularized cardiac muscle using genetically modified mouse embryonic stem cells (ESCs) transfected by pMHC-neo/SV40-hygro. A stirred bioreactor was used to facilitate the formation of a large number of ESC-derived cardiomyocytes, which were then mixed with human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblasts (MEFs) in a liquid collagen scaffold to construct highly vascularized cardiac tissue in vitro.RESULTS: The resulting tissue constructs were transplanted into dorsal subcutaneous sites of nude mice. Tumor formation was not detected in all samples and vascularized cardiac tissue could survive after transplantation. Vascularization of the implanted cardiac muscle was significantly enhanced by the addition of HUVECs and MEFs, which resulted in a thicker myocardium. The combination of genetically modified ESCs and stirred bioreactor cultivation not only benefited the large-scale production of pure ESC-derived cardiomyocytes, but also effectively controlled the potential risk of undifferentiated ESCs.CONCLUSIONS: Using liquid collagen as scaffold, the enriched cardiomyocytes derived from genetically modified ESCs mixed with HUVECs and MEFs in 3-dimensional culture resulted in highly vascularized cardiac tissues. J Heart Lung Transplant 2012;31:204-12 (C) 2012 International Society for Heart and Lung Transplantation. All rights reserved.

Embryonic stem cells (ESCs) can propagate unlimitedly in vitro and differentiate into cardiomyocytes, which have been proposed as unlimited cell sources for cardiac cell therapy. This was limited by difficulties in large-scale generation of pure cardiomyocytes. In this study, we used stirred bioreactors to optimize the differentiation condition for mass production of embryoid bodies (EBs) derived from genetically modified mouse ESCs. Stirred suspension culture could more efficiently produce EBs and have a more uniform EB population without large necrotic centers, compared with the conventional static culture. Importantly, the cardiac-specific gene expressions (GATA binding protein 4, α-cardiac myosin heavy chain and myosin light chain-2v) were increased within EBs cultured in stirred bioreactor. Stirred suspension culture significantly increased the proportion of spontaneously contracting EBs, yielded a greater percentage of α-sarcomeric actinin-positive cells detected via flow cytometry, and harvested relatively more cardiomyocytes after G418 selection. Stirred suspension culture provided a more ideal culture condition facilitating the growth of EBs and enhancing the cardiogenic differentiation of genetically modified ESCs, which may be valuable in large-scale generation of pure cardiomyocytes.

Objective:To investigate the growth and metabolism of Vero cells immobilized cultured on various microcarriers.Methods:Cell viability and cell morphology of the Vero cells cultured in 1%(v/v) newborn bovine serum DMEM/F12,the growth of the Vero cells cultured on 2D MicroHex,Biosilon,Cytodex 1 and Cytopore 1 was evaluated on the viable cell density;The specific consumption rate of glucose(qglc),the specific production rate of lactate(qlac),the specific consumption rate of glutamine(qgln),and the specific consumption rate of glutamate(qglu) were used as the evaluation indexes,the metabolism of the Vero cells cultured on various microcarriers was determined.Results:After 7 d in culture the viable cell density of the Vero cells cultured on 2D MicroHex,Biosilon,Cytodex 1 and Cytopore 1 was 18.4×105 cells/ml,21.9×105 cells/ml,23.9×105 cells/ml and 16.2×105 cells/ml,respectively.And,the Vero cells growth on Cytodex 1 formed compact cell layers with distinct cell morphology;Metabolic indexes of the Vero cells cultured on various microcarriers were basically the same.Conclusion:Among these microcarriers,Cytodex 1 is comparatively good for the growth of Vero cells,and could be used as the favor choice of microcarrier for the large-scale cultivation of Vero cells for viral vaccine production.

Beta-cell transplantation is considered to be the most effective approach to cure type 1 diabetes (T1D). Unfortunately, the scarce availability of donor tissue limits the applicability of this therapy. Recent stem cell research progress shows stem cell therapy may be a potential means to solve this problem. Bone marrow-derived mesenchymal stem cells (MSCs) are self-renewable and multipotent adult stem cells which can differentiate into the three germ layers. Here we aimed to investigate whether MSCs could be reprogrammed into insulin-producing cells (IPCs). We isolated and characterized MSCs obtained from rat bone marrow. Then MSCs were induced to transdifferentiate into IPCs under specific conditions containing high concentrations of glucose, activin A, all-trans retinoic acid, and other maturation factors. The induced cells expressed multiple genes related to pancreatic beta-cell development and function, such as insulin1, glucagon, Pdx1, Pax6, and Glut-2. Insulin1 and C-peptide production were identified by immunocytochemistry. In vitro glucose challenge studies showed the induced cells secreted insulin in a glucose-dependent manner, as do normal pancreatic beta-cells. Transplantation of these MSC-derived insulin-positive cells could reverse the hyperglycemia of streptozotcin (STZ)-induced diabetic rats. These results demonstrated that MSCs could be reprogrammed into IPCs and might be a potential autologous cell source for transplantation therapy of T1D.

Apoptosis plays an important role in limiting viable cell density and production performance in large-scale animal cell culture.Overexpression of anti-apoptotic genes is one of the most common strategies used to improve apoptosis resistance of industrial cells.HEK293 cells with adenoviral anti-apoptotic E1B-19K gene were transfected and picked out several representative clones.These clones were further investigated in the apoptotic percentages under various inducing culture conditions and cell growth and metabolism under normal culture condition.The results showed that the overexpression of E1B-19K in HEK293 cells,endowed cells anti-apoptotic ability by decreasing cell death by 60%~80% under inducing culture conditions with reduced glucose,low serum or without glutamine,delayed cells' entry into decay phase by 2 d under normal culture conditions;and had no significant affect on cell metabolisms of glucose,lactate and glutamine.The above results indicate that the overexpression of E1B-19K is a promising strategy to reduce cell death in mammalian cell culture.

Objective:To investigate the effect of sunitinib on platelet-derived growth factor(PDGF)induced human airway smooth muscle cells(HASMCs)proliferation,migration and phosphorylation of ERK(p-ERK).Methods:HASMCs derived from human bronchus were divided into six groups:normal group,PDGF-BB group,PDGF-BB and sunitinib intervention group,sunitinib interven-tion group,U0126 intervention group,PDGF-BB and U0126 intervention group.Cell cycle progression was analyzed by flow cytometry.Migration was examined by Transwell test.The protein expressions of p-ERK were determined by Western blot analysis.Results:PDGF-BB(20 ng/ml)increased the proliferation and migration of HASMCs significantly(P 0.01);sunitinib(3 nmol/L)decreased the proliferation and migration of HASMCs stimulated by PDGF-BB significantly(P 0.05),which was no difference compared with U0126.The expression of p-ERK in PDGF-BB group was higher than that of the normal group(P 0.01),which can be decreased by sunitinib.There is no difference between the effect of sunitinib and U0126 on p-ERK expression induced by PDGF-BB.Conclusion:S unitinib can directly inhibit HASMCs proliferation and migration induced by PDGF-BB through the modulation of the ERK pathway.

BACKGROUND: Extract of injured pancreatic gland contains specific growth factor, which can promote β cell proliferation and insulin secretion, resulting in insulin regeneration and cell differentiation. OBJECTIVE: To investigate effects of the injured rat pancreatic extract on in vitro trans-differentiation of bone marrow mesenchymal stem cells (BMSCs) into insulin-like cells. DESIGN, TIME AND SETTING: A cytology in vitro experiment was performed at the Institute of Biotechnology, Academy of Military Medical Sciences of Chinese PLA from June 2005 to March 2007. MATERIALS: Six clean male Sprague-Dawley rats were obtained from the Animal Experimental Center of Academy of Military Medical Sciences. Activin A, Retinoic Acid and trypsin inhibitor were bought from Sigma, USA. Streptozotocin was purchased from Sigma company. METHODS: Streptozotocin was intraperitoneally infused into rats. Two days later, pancreatic tissues were obtained when blood glucose exceeded 10 mmol/L. Homogenate was prepared in phosphate buffer saline containing pancreatic protein inhibitor. Supernatant was obtained after twice centrifuge, which was injured pancreatic extract. BMSCs were isolated from rats by density gradient centrifugation. The passage cells were randomly divided into 4 groups when 70%-80% passage cells were confluent. BMSCs in the control group were incubated in low-glucose DMEM containing 2% fetal bovine serum for 11 days. BMSCs in the Activin A and Retinoic Acid (AR) group were separately treated with Activin A and Retinoic Acid each for 24 hours, and with basic fibroblast growth factor, nicotinic amide, nicotinic amide+exendin each for 3 days. BMSCs in the Activin A, Retinoic Acid and injured pancreatic extract (AR+E) group were treated with injured pancreatic extract on the basis of former groups. BMSCs in the injured pancreatic extract (E) group were only incubated with injured pancreatic extract for 11 days. MAIN OUTCOME MEASURES: Phenotypes of cells were examined by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Insulin secretion levels were determined by enzyme labeled immunosorbent assay (ELISA). RESULTS: More insulin-positive cells existed in AR and AR+E groups, and insulin-like structure was bigger and more in the AR+E group compared with the AR group. There were a few insulin-positive cells and insulin-like structure in the E group. Insulin-1 mRNA expression existed in the experimental groups. Insulin secretion was detected in supernatant. Insulin secretion levels were greater in the AR+E group compared with the AR group, and secretion levels were the lowest in the E group (P < 0.05). Each index was negative in the control group. CONCLUSION: On the basis of previous study of Activin A and Retinoic Acid and other maturation factors, the injured pancreatic extract can increase the efficiency of BMSCs differentiation into insulin-like cells, resulting in elevating differentiation efficiency.

By using the cell density, cell viability, size distribution of cell aggregates,specific consumption rate of glucose (q_ glc ), specific production rate of lactate (q_ lac ), lactate transform rate (Y_ lac/glc ) and amino acids utilization as the evaluation indexes, the growth and metabolism of HEK293 cells under carrier-free immobilization culture mode were examined and compared with those of HEK293 cells cultured in static tissue flasks. It was found that HEK293 cells grown as suspended cell aggregates in spinner flasks maintained the basic growth and metabolism characteristics of HEK293 cells in stationary anchored culture, and HEK293 cells under carrier-free immobilization culture mode as suspended aggregates in stirred bioreactor facilitate perfusion performance and increase unit productivity. Cultivation of HEK293 cells in carrier-free immobilization culture mode has potential for further improving mammalian cells culture technique.

利用HEK293细胞在悬浮培养中具有聚集成团的体外培养特性,在250ml的Bellco的搅拌培养体系中,以细胞团粒径、细胞粒径、细胞数、细胞活力、葡萄糖比消耗速率 (qglc)、乳酸比生产速率 (qlac) 和乳酸对葡萄糖得率 (Ylac/glc) 为观察指标,考察了HEK293细胞在Ca2+浓度设置为0μmol/L、250μmol/L、500μmol/L、750μmol/L和1000μmol/L的搅拌培养体系中的细胞团形成、细胞生长和代谢.实验发现:培养基中的Ca2+浓度决定着HEK293细胞在悬浮培养中能否形成细胞团并影响着细胞团的粒径分布和细胞团内细胞相互连接的紧密程度;在250～1000μmol/L的Ca2+浓度范围内,HEK293细胞团的平均粒径与培养基中的Ca2+浓度成正比;Ca2+浓度对以细胞团的形式悬浮培养的HEK293细胞的生长和代谢无明显的影响.实验结果提示,Ca2+浓度是调节HEK293细胞团粒径分布、维持悬浮细胞团中HEK293细胞正常生长和代谢的有效控制参数.

经不同浓度staurosporine处理诱导凋亡的G7细胞样品,分别用YO-PRO-1/PI和AV/PI进行荧光染色,借助流式细胞仪检测凋亡情况,将两种检测方法得到的结果进行统计学分析显示,二者有显著的相关性（r=0.9659,P＜0.01）,且没有显著性差异（P＜0.05）;另外,上述凋亡细胞样品经YO-PRO-1/PI染色后在荧光显微镜下计数凋亡细胞比例的结果与AV/PI流式细胞仪的检测结果也有显著的相关性（r=0.9903,P＜0.01）,且没有显著性差异（P＜0.05）。以上这些结果表明,用YO-PRO-1/PI对细胞进行染色、借助流式细胞仪和荧光显微镜均能准确地检测细胞凋亡,可替代AV/PI流式细胞仪方法用于细胞凋亡的检测。

Aim: To setup a convenient method for the measurement of free arachidonic acid (AA) in neurons and to analyze the effects of heat treatment on the AA levels in neurons so as to give a reference to the study on heat injury in neural system. Methods: Primary cultured striatum neurons of rats were treated at a temperature of 43°C for 1 h. Cells of the heat treated group and control group were homogenized, and the fatty acids in cell lysate and cell membrane were extracted respectively. After heptadecanoic acid (C17:0) was added as an internal standard, the levels of fatty acid methanol esters were detected with Gas/Mass Chromatography. Results: The relative abundant fatty acid, including saturated (palmitic acid and stearic acid) and unsaturated (oleic acid, linoleic acid and arachidonic acid) were detectable, and did not change significantly during heat treatment, either in cell lysate or in cell membrane. There were no significant difference between saturated acid and unsaturated acid. While the level of AA in cytoplasm increased 100% from 0.53 ± 0.13 to 1.00 ± 0.56 after heat treatment (F = 5.71, P < 0.05), but the level of AA in cell membrane was undetectable, even after heat treatment. Conclusion: More amount of AA is released from neurons in striatum induced by heat treatment, and the increased AA may be crucial in the injury signal transduction.