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.
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.
【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 study the effects of recombinant human growth hormone (rhGH) on proliferation of human rectal cancer cell in vitro. MethodsThe experiment was divided into control group,rhGH group,Oxaliplatin (LOHP) group and rhGH+LOHP group. The double proliferation time of cells,cell inhibition rate,cell cycle, proliferation index (PI) and DNA inhibition rate of human rectal cancer line,HR8348,were studied by cell culture, MTT assay and flow cytometry on different concentration of rhGH. ResultsIn vitro the markedly accelerated effects of rhGH on multiplication of HR8348 cell line were not found: there was no statistical significance as compared rhGH group with control group or compared rhGH+LOHP group and LOHP group （Pgt;0.05）. The double proliferation time of cells was markedly lengthened, cell inhibition rate and the cells arrested in G0-G1 phase were obviously increased, meanwhile, the cells in S phase （P＜0.05） and G2-M phase and PI were markedly decreased and DNA inhibition rate was obviously risen as compared rhGH+LOHP group with control group or rhGH+LOHP group and rhGH group （P＜0.01）.ConclusionIn vitro rhGH does not accelerate the multiplication of human rectal cancer cells.
The effects of pentagastrin (PG) on the viable cell count (Α value) and the synthesis of DNA (CPM value) of primary cultured large bowel carcinoma cells in 25 patients were evaluated in vitro by MTT assay,3H-TdR incorporation. The results showed that Α value and CPM value in well， moderately and poorly-differentiated carcinoma cells were higher than normal control (Ｐlt;0.01，Ｐ＜0.05). The proliferative effect was significant at a dose of 0.3907 μg/ml in well-differentiated carcinoma cells, and at a dose of 6.2500μg/ml in moderately and poorly-differentiated carcinoma cells. These indicat that PG has the proliferative effect on large bowel carcinoma cells. These results provide an experimental foundation for the endocrine therapy for patients with large intestine carcinoma, especially by using gastrin receptor antagonists for well-differentiated carcinoma.
Objective To investigate the value of bronchial mucosa biopsy and quantitative culture in the differential diagnosis of lower airway bacterial colonization and infection. Methods A prospective observational cohort survey onMDR Pseudomonas aeruginosa and Acinetobacter baumannii was carried out in intubed or tracheotomized patients with invasive ventilation in respiratory intensive care unite ( RICU) . A total of 50 ICU patients were followed for the detection of MDR pathogen colonization or infection from June 2008 to October 2009. All subjects were divided into an infection group and a colonization group according to the outcome of patients discharged fromthe RICU. Baseline information, APACHEⅡ scores, and CPIS scores were recorded on individual forms for each patient untill discharge or death. Bronchial mucosa biopsy was conducted on appropriate time to identify whether the patient was comfirmed as infection. Microbiological diagnosis was performed with quantitative culture. Results Fifty patients were enrolled in this study, of which infected in 23 cases and colonized in 27 cases. The time of invasive mechanical ventilation, length ofICU stay, catheter indwelling time, and the kinds of disease were significantly different between the two groups( P lt; 0. 05) . The kinds of using antibiotics before onset of multi-drug resistance of bacteria showed that cefoxitin/ cefmetazole and mezlocillin also had significant difference between the infection group and the colonization group. The results of dynamic CPIS score of the infection group showed that scores at each timepoint were higher than those in the colonization group. However, the results of t-test showed that there was higher score in the infection group than that in the colonization group on 14 days after intubation ( P lt;0. 05) . The bronchial mucosa biopsy showed that airway inflammation was detected in 19 cases in the infection group and 9 cases in colonization group. The positive rate in the infection and the colonization group were 55. 6% and 25. 0% , respectively assessed by traditional threshold of 103 cfu/mL for PSB in quantitative bacterial culture. In addition, there was more inflammatory cells in the patients with drug-resistant pathogens infection than that in the patients without nosocomial infection. The combination of bronchial mucosa biopsy and microorganism quantitative cultures had the highest sensitivity and specificity and the highest diagnostic accuracy. Conclusions Bronchial mucosa biopsy combining microorganism quantitative culture is feasible in identifying colonized or infected bacteria. Invasive mechanical ventilation time, length of ICU stay and the catheter indwelling time extending are risk factors for bacterial colonization.
Objective To explore the application value of Mycoplasma pneumoniae （MP） rapid culture technique for diagnosis of lower respiratory tract infections （LRTIs ） inpatients. Methods 120 LRTIs inpatients in respiratory ward，Anzhen hospital from January 1，2010 to December 31，2010，were recruited in this study. Their pharynx swabs were obtained for rapid MP culture and the serum antibody detection of MP was performed by Gelatin particle agglutination method. Results There were 33 positive yields in 120 LRTIs patients by rapid culture method and 24 positive yields by serological assay. The positive rates were 27.5% and 20.0% respectively. There was no significant difference in the two detecting methods （Pgt;0.05）. Conclusions MP rapid culture method is a better early diagnostic method at the present. MP rapid culture method combined with serological detection can improve the positive yield and avoid missed diagnosis.
Objective To investigate the species distribution and antibiotic resistance of pathogens fromcatheter-related bloodstream infections ( CRBSI) in intensive care unit( ICU) , to provide evidence for the guidance of clinical rational administration.Methods A retrospective analysis was performed to review the microbiological and susceptibility test data of all CRBSI patients in ICU from January 2009 to December 2011. The patterns of antibiotic resistance among the top seven bacteria were compared. Results 67 cases of CRBSI were detected with 81 strains, including 40 Gram-positive ( G+ ) bacteria( 49.4% ) , 38 Gram-negative( G- ) bacteria ( 46.9% ) , and 3 fungi ( 3.7% ) . The main pathogens causing CRBSI were coagulase negative Staphylococci ( 27 strains, 33.3%) , Acinetobacter baumannii ( 12 strains, 14.8% ) , Klebsiella pneumoniae( 9 strains, 11. 1% ) , Staphylococcus aureus ( 8 strains, 9. 9% ) , Pseudomonas aeruginosa ( 7 strains, 8. 6% ) , Escherichia coli ( 6 strains, 7.4% ) , suggesting that Staphylococcus epidermidis was predominant pathogenic G+ bacteria, and Acinetobacter baumannii was predominant G- bacteria. The antibiotic resistance tests demonstrated that isolated G- bacillus was highly sensitive to carbopenem, while vancomycin-resistant G+ bacteria were not found. Conclusions Within the latest 3 years, the predominant pathogens of CRBSI in ICU are Staphylococcus epidermidis and Acinetobacter baumannii. Acinetobacter baumannii exhibited high drug resistance to all antibiotics.
Increasing evidence suggests that many types of cancers contain a population of cells that display stem cell properties. These cells are called cancer stem cells （CSCs），which are closely related to tumor initiation，growth，metastasis and chemoresistance. CSCs are also found in esophageal squamous cell carcinoma （ESCC）. These cells are characterized by potential of self-renewal and differentiation，tumor formation in nude mice and chemotherapy resistance，and thus may play an important role in targeted cancer therapies. Current methods for culturing and sorting CSCs in ESCC mainly include fluorescence activated cell sorting （FACS），magnetic activated cell sorting （MACS），suspension culture，and side population （SP） cell sorting. In this review，we focus on current research methods for CSCs in ESCC，their biological characteristics and areas for improvement. We believe that a combination of multiple cell-surface makers is needed for research of CSCs in ESCC.