Objective To investigate the possibility of creation of tissue engineered heart valve leaflets in vitro . Methods Aorta were obtained from 9 hybrid young pigs. The endothelial cell, fibroblast and smooth muscle cells were isolated and cultured to get enough cell. The expanded fibroblast, smooth muscle cell,and endothelial cells were seeded on the polymers sequentially. The cell polymer constructs were sent for scanning electron microscopy(SEM) examination after cultured for 7, 14, and 28 days. Histological examination were performed after the cell polymer constructs cultured for 28 days. Results SEM showed that the number of cells on the polymers increased as the culture time prolonged, with the formation of matrix. After 28 days, there were a great number of cells and large amount of matrix on the scaffolds. The confluent cell had covered a large area of the polymers. Hematoxylin and eosin(HE) stain showed large amount of cells attached to the polymers. Conclusion With the viability of the cultured cellular scaffolds,it is possible to create tissue engineered heart valve leaflets in vitro.
ObjectiveTo observe the effects of A549 cells under hypoxicconditions on the migration of human umbilical vein endothelial cells (HUVECs) and microvascular formation. MethodsAfter cultured for 24 h in normoxia condition(21% O2),hypoxia condition (2% O2),and anaerobic condition (0% O2),respectively,morphology of A549 cells was observed with inverted phase contrast microscope,proliferation was detected by MTT assay,and intracellular hypoxia-inducible factor-1α (HIF-1α) protein was detected by immunocyto-chemical technique,for determining whether the hypoxia model is successful. Then A549 cells' supernatant in the normoxic group,the hypoxia group and HUVECs culture medium were taken to intervene HUVECs. The migration of HUVECs was observed with cell scratch test,pseudopodia formation of HUVECs was observed with microfilament green fluorescent staining method,and blood vessel formation was observed with three-dimensional culture techniques in vitro. ResultsCompared with the normoxic group,the growth of A549 cells was better in the hypoxia group with more proliferation,and was poor in the anaerobic group with decreased number of cells. A549 cells in the hypoxia group and the anaerobic group both expressed HIF-1α protein,which was more obvious in the anaerobic group. Compared with the HUVECs supernatant intervention group,the hypoxia supernatant intervention group and the normoxic supernatant intervention group both had varying degrees of migration,pseudopodia structure formation and vascular lumen sample structure formation,which were more obvious in the former group. ConclusionA549 cells in hypoxic environment grow very well,proliferated significantly,but anaerobic environment is not conducive to the growth of A549 cells which found to be apoptosis. A549 cells in hypoxic environment can promote HUVECs migration,pseudopodia formation and angiogenesis.
【摘要】 目的 建立ICU专科护士培养模式，并对其培养模式的实施效果进行评价。 方法 课题研究采用德尔菲法，根据函询结果构建ICU专科护士培养模式，并将模式应用于29名护理人员的ICU专科培训。 结果 学员的理论水平、技术水平、综合能力均有明显提高。 结论 建立的培养模式符合ICU专科护士培养需求。【Abstract】 Objective To establish the training mode for ICU nurses and evaluate the effect of the training mode. Methods The training mode for ICU nursing was established via the Delphi method. According to the result of consultation, we put the training mode into practice; 29 qualified ICU nurses were trained. Results The level of theory and skills of the trained nurses improved, and the all-round ability was better than before. Conclusion The training mode is suitable to train ICU nurses.
护士角色的多元化发展以及新的医改政策与我国大多数医学院校现行的涉外护理专业人才培养模式形成了巨大的反差。如何突破传统观念,锐意改革,建立新型涉外护理专业人才培养模式,已成为一个既重要又急迫的课题。本文重点讨论根据国际护士角色的多元化发展趋势,明确建立新型涉外护理专业人才培养模式的要点。
目的：机械分离、培养小鼠耳蜗螺旋神经元，并进行免疫荧光细胞学鉴定，为后期进一步的实验研究提供实验材料。方法：采用初出生1～5天以内的昆明小鼠进行解剖、机械分离以获得螺旋神经节组织，进行原代培养后，应用神经微丝蛋白（Neurofilament protein,NFP－H）单克隆抗体进行免疫荧光细胞学鉴定。结果：机械分离后获得的螺旋神经节组织中的螺旋神经元，在体外培养条件下可以存活并进行正常分化。典型的螺旋神经元，其细胞形态呈椭圆形,胞体透明光滑、接近生理形态。荧光染色标记后，胞体和神经突起均显色好，Schwann细胞和成纤维细胞未着色。结论：应用机械分离的方法获得小鼠耳蜗螺旋神经节组织并进行培养，耳蜗螺旋神经元在体外可以稳定地存活生长。培养获得的细胞形态和生存状态接近生理状态，满足电生理、免疫细胞化学、药理学等研究。应用特异性的神经微丝蛋白对培养获得的螺旋神经元进行免疫荧光细胞学鉴定，特异性好，荧光显色好。
ObjectiveTo investigate the growth characteristics of pancreatic cancer cells in the twodimensional culture system (monolayer) and threedimensional culture system (type Ⅰ collagen and extracellular matrix gel). MethodsThree pancreatic cancer cell lines (SW1990, PCT, and ASPC1) were cultured in monolayer, type Ⅰ collagen, and extracellular matrix gel, respectively. The growth patterns were observed, growth curves were detected by CCK8 test, and the cell cycle distributions were analyzed by propidium iodide staining. Results In the twodimensional culture system, cells grew in monolayer. In the type Ⅰ collagen and the ECM gel threedimensional culture system, cells formed multicellular spheroids (MCS), of which the growth rates were slower than those of the cells in monolayer. The proportions of S phase of SW1990, PCT, and ASPC1 cells in twodimensional culture system were significantly more than those in the type Ⅰ collagen on 4 d and 8 d 〔(29.6±3.0)% vs. (18.2±5.1)%, (33.6±2.1)% vs. (14.5±3.2)%, (33.1±1.8)% vs. (24.7±2.6)%; Plt;0.05〕, while the difference of proportion of three cell lines in G2/M phase was not different between twodimensional culture system and type Ⅰ collagen (Pgt;0.05). The proportions of G0/G1 phase of SW1990 and PCT cells cultured in the type Ⅰ collagen on 4 d and 8 d and ASPC1 cells cultured in the type Ⅰ collagen on 4 d were significant more than those cultured in twodimensional culture system (Plt;0.05). The proportions of S phase of ASPC1 cells and SW1990 cells cultured in the type Ⅰ collagen on 4 d were significant more than those cultured in the type Ⅰ collagen on 8 d (Plt;0.05). ConclusionsThe characteristics of pancreatic cancer cells in twodimensional and threedimensional culture systems are different. MCS culture system can better mimic the in vivo growth environment of cells in tumors.
Objective To develop a reliable method for primary culture of normal human peritoneal mesothelial cells. Methods Human peritoneal mesothelial cells were dissociated by a mixture of pancreatin and ethylene diamine tetraacetic acid with a magnetic puddler. Inverted phase contrast microscope was used to observe the morphological structures of cells, approximate process of growth. Calretinin was used to identify the mesothelial cells. Results On the 4th d of culture, mesothelial cells adhered to the culture dish. After day 14, mesothelial cells confluenced gradually and grew well like the slabstone. Calretinin was positively expressed by mesothelial cells after 5 d of cultivation. The mesothelial cell population of subculture was less than that of the primary culture. Conclusion A reliable method for primary culture of normal human peritoneal mesothelial cells has been successfully developed, by which sufficient amount of highly purified normal human peritoneal mesothelial cells can be obtained.
Objective To study a simple and practical method of isolation, culture and identification of hepatic oval cells from adult rat. Methods Wistar adult rats were fed by 2-acetaminofluorere （AAF） and were stimulated by partial hepatectomy to activate the proliferation of hepatic oval cells. After operation 12 days, the livers were resected for isolating oval cells. Hepatic tissue was digested by 0.10% collagenase Ⅳ and the obtained heterogeneous liver cells were then isolated and purified by density gradient centrifugation. The expressions of albumin and CK19 mRNA in hepatic oval cells were analyzed by immuno-fluorescence and RT-PCR. Results The survival rate of the newly isolated oval cells was more than 90%. The hepatic stem cells were shown by immuno-fluorescence of stem cell’s antigen c-kit. The expressions of mRNA CK19 and albumin of the oval cell were also detected by PCR. The proliferation activity of the newly isolated oval cells was significantly high and they could be induced to differentiate into both hepatic and bile ductal cells by some growth factors. Conclusion The successful development of the simple and feasible isolation and purification procedure as well as the identification method for hepatic oval cells may provide a fundamental for further studies about bionomics of the hepatic stem cell and the relation between stem cells and hepatic carcinoma.
【Abstract】Objective To study the influence of transplantation of cultured parathyroid cells on the survival of the allografts in rats. Methods Parathyroid cells digested with collagenase and trypsin were cultured and transplanted under the left renal capsule. The survival time of the allografts was recorded and the allografts were examined by transmission electron microscopy.Results In fresh parathyroid cells group, the mean survival time was (9.25±3.45) days. While in cultured parathyroid cells group, the survival time was (46.25±7.44) days (P＜0.01). During the 50 days of observation, serum calcium and PTH remained normal in 6 of 8 rats. There were intact parathyroid cells in the allografts which had abundant rough endoplasmic reticula,mitochondria and secretory granules. Conclusion Transplantation of cultured parathyroid cells in rats can prolong the survival time of allografts and is a potent way to cure hypoparathyroidism.
ObjectiveTo explore the suitable method for isolation and maintenance of primary cultures of human gallbladder epithelial cells (GECs) for establishing the basis of research works in physiological function of gallbladder and its related diseases.MethodsGECs were isolated with collagenase type Ⅳ and blunt separation.The dishes were coated with fibronectin, laminin and polyDlysine respectively.Additional 10 ng/ml epidermal growth factor was added to DMEM medium containing 20% fetal calf serum.The cells were studied under light and electron microscope to determine their shape and distribution.ResultsEach gallbladder yielded approximately (1-5)×107columnar epithelial cells,greater than 95% of which were viable by trypan blue exclusion.The cells grew vigorously within one week which was flat and multangular in shape. CK19 expressed positive.Electron microscope showed typical gallbladder epithelia with microvilli,tight junctions and mucus droplets.ConclusionCombination of collagenase type Ⅳ，mechanical blunt separation and twostep attachment is of great benefit for separating and harvesting GEC.Fibronectin coated culture dish and DMEM medium containing 20% calf serum and 10 ng/ml hEGF is of great benefit for culturing gallbladder epithelial cells.