OBJECTIVE: To investigate the selection and identification of human keratinocyte stem cells(KSC) in vitro. METHODS: According to the characteristics of KSC which can adhere to extracellular matrix very fast, we selected 3 groups of different time(5 minutes, 20 minutes and 60 minutes) and unselected as control group. And the cells were identified by monoclone antibody of beta 1-integrin and cytokeratin 19 (Ck19), then the image analysis was done. Furthermore we analyzed the cultured cells with flow cytometer(FCM) and observed the ultrastructure of the cell by transmission electron microscope(TEM). RESULTS: The cell clones formed in all groups after 10 to 14 days, while the cells of 5 minute group grew more slowly than those of the other groups, however, the clones of this group were bigger. The expression of beta 1-integrin and Ck19 were found in all groups. The positive rate of beta 1-integrin was significant difference between 5 minute group and the other groups (P lt; 0.05). And the expression of Ck19 was no significant difference between 5 minute group and 20 minute group(P gt; 0.05), and between 60 minute group and control group. But significant difference was observed between the former and the later groups(P lt; 0.05). The result of FCM showed that most cells of the 5 minute group lied in G1 period of cell cycle, which was different from those of the other groups. At the same time, the cells of 5 minute group were smaller and contained fewer organelles than those of the other groups. CONCLUSION: The above results demonstrate that the cells of 5 minute group have a slow cell cycle, characteristics of immaturity, and behaving like clonogenic cells in vitro. The cells have the general anticipated properties for KSC. So the KSC can be selected by rapid attachment to extracellular matrix and identified by monoclone antibody of beta 1-integrin and Ck19.
Objective To identify glial cell line-derived neurotrophic factor (GDNF) recombinant retroviral vector and to establish its packaging cell line PA317. Methods PA317 cells were transfected with recombinant retroviral vector pLXSN-GDNF using liposomes. The recombinant retroviral particles were then harvested from culture media of G418 resistant transfected cells and analyzed using RT-PCR. Virus titers in supernatants were investigated. Results Sequencing date indicated that GDNF gene was exactly identical to the sequence in the GeneBank. PA317 cells were transfected with recombinant retroviral vector pLXSN-GDNF using liposomes, and virus titers insupernatants harvested from culture media of G418 resistant transfected cells were 104-105 CFU/ml. Conclusion Packaging cell line PA317/pLXSN-GDNF was established.
ObjectiveTo compare the biological features of early and late endothelial progenitor cells (EPCs) by isolating and culturing early and late EPCs from the human peripheral blood so as to find some unique properties of EPCs and to propose a suitable strategy for EPCs identification. MethodsMononuclear cells were isolated from the human peripheral blood using density gradient centrifugation. Then, the cells were inoculated in human fibronectin-coated culture flasks and cultured in endothelial cell basal medium 2. After 4-7 days and 2-3 weeks culture, early and late EPCs were obtained respectively. The morphology, proliferation potential, surface markers, cytokine secretion, angiogenic ability, and nitric oxide (NO) release were compared between 2 types of EPCs. Meanwhile, the human aortic endothelial cells (HAECs) were used as positive control. ResultsThe morphology of early and late EPCs was different:early EPCs formed a cell cluster with a spindle shape after 4-7 days of culture, and late EPCs showed a cobblestone appearance. Late EPCs were characterized by high proliferation potential and were able to form capillary tubes on Matrigel, but early EPCs did not have this feature. Both types EPCs could ingest acetylated low density lipoprotein and combine with ulex europaeus Ⅰ. Flow cytometry analysis showed that early EPCs did not express CD34 and CD133, but expressed the CD14 and CD45 of the hematopoietic stem cell markers;however, late EPCs expressed CD31 and CD34 of the endothelial cell markers, but did not express CD14, CD45, and CD133. By RT-PCR analysis, the expressions of vascular endothelial growth receptor 2 and vascular endothelial cadherin in early EPCs were significantly lower than those in the late EPCs and HAECs (P<0.05), but no significant difference was found in the expression of von Willebrand factor and endothelial nitric oxide synthase (eNOS) between 2 type EPCs (P>0.05). The concentrations of vascular endothelial growth factor, granulocyte colony-stimulating factor, and interleukin 8 were significantly higher in the supernatant of early EPCs than late EPCs (P<0.05). Western blot assay indicated eNOS expressed in both types EPCs, while the expression of eNOS in late EPCs was significantly higher than early EPCs at 5 weeks (P<0.05). Both cell types could produce similar amount of NO (P>0.05). ConclusionThe expression of eNOS and the production of NO could be used as common biological features to identify EPCs, and the strategy of a combination of multiple methods for EPCs identification is more feasible.
Objective To establish a simple and efficient method to isolate and culture the umbilical vein vascular endothelial cells in canine. Methods Twelve umbilical cords [(13.0 ± 1.5) cm in length] were taken from 12 newborn pups of Beagles. And then the vascular endothelial cells were isolated from these umbilical cords digested by 1% collagenase type I for 5, 7, and 10 minutes respectively (4 umbilical cords in each group). After cultured, the vascular endothelial cells were identified by morphology, immunofluorescence, and flow cytometry. And the growth curvature of umbilical vein vascular endothelial cells was detected by MTT assay. Results Few vascular endothelial cells were collected at 5 and 10 minutes after digestion; many vascular endothelial cells were seen at 7 minutes, and became cobblestone with culture time, with a large nucleus; after passage, cell morphology had no obvious change. Fluorescence microscope results showed that positive von Willebrand factor (vWF) and CD31 cells were observed in most of cells. The flow cytometry test displayed that the positive cell rates of vWF and CD31 were 99.0% ± 0.7% and 98.0% ± 1.2%, respectively. The above results indicated that cultured cells were vascular endothelial cells. MTT assay showed that vascular endothelial cells proliferation increased significantly with culture time. Conclusion Enzyme digestion is a convenient method to isolate vascular endothelial cells from canine umbilical vein, and a large number of cells and high purity of cells can be obtained by the method.
Objective To observe the morphology and growing status of mesenchymal stem cells(MSCs) of human bone marrow in vitro, in order to confirm that MSCs of human bone marrow are ideal seed cells and provide basic theory for further MSCs research. Methods The methods of density gradient centrifugation with lymphocyte separation medium for human and adherent filtration were used to isolate and purify MSCs of human bone marrow. We observed the cellular growth status and morphology of the primary MSCs and the surface antigens of second passage MSCs were tested. Results The primary culture cells fused into monolayer after 14-16 d. The passage cells kept the same morphological characteristics of primary culture cells. Ultrastructure of the second passage MSCs showed that the shape of nuclei was irregular, there were multiple nucleoli in some of the nuclei, and morphological differentiation of intracytoplasm organelles was immature. The growth curve of the first, fifth and tenth passage cells showed a logarithmic growth at day 3, a peak growth at day 5, and no clones occurred after tenth passage. Cloning efficiency of first passage, fifth passage and tenth passage was respectively 25.83%±2.93%, 14.67%±1.63% and 4.67%±0.52%. Test of MSCs phenotypic characteristics showed a high homogeneity among the cells and surface antigen profiles were positive for CD29, CD44 and negative for CD34, CD45. Conclusion The methods of density gradient centrifugation with lymphocyte separation medium for human and adherent filtration are simple, economic and efficient to isolate and purify MSCs from human bone marrow. With a high proliferating ability in vitro, MSCs from human bone marrow are ideal seed cells for tissue engineers.
ObjectiveTo explore value of ultrasound real-time elastography (RTE) technology for identification of benign and malignant solid thyroid nodules.MethodsA retrospective analysis was performed on 125 patients with thyroid nodules who underwent ultrasound RTE in this hospital from February 2018 to August 2019. All patients underwent RTE on the basis of conventional ultrasound. The ultrasound elasticity contrast index (ECI) was used as the evaluation index and the pathological examination result was used as the gold standard. The receiver operating characteristic (ROC) curve analysis was used to evaluate the value of ECI in the identification of benign and malignant solid thyroid nodules. Logistic regression analysis was used to analyze the influencing factors of ECI.ResultsAmong the 125 patients with solid thyroid nodules, 51 were malignant nodules, 74 were benign nodules. The ECI value of patients with benign thyroid nodules was lower than that of patients with malignant nodules (2.71±0.83 versus 3.42±1.14, t=–4.030, P<0.001). The result of ROC analysis showed that the cutoff value of ECI to distinguish benign and malignant solid thyroid nodules was 3.07, area under curve of ROC was 0.806 [95%CI (0.717, 0.894), P<0.001], sensitivity was 80.3%, specificity was 70.4%. The multivariate logistic regression analysis showed that the thyroid nodules with diffuse lesions, calcification, and maximum nodule diameter ≥1 cm were the risk factors for elevated ECI values (P<0.05). For the solid thyroid nodules without diffuse lesions, without calcification, and maximum nodule diameter <1 cm, ECI had the higher sensitivity, specificity, accuracy, and positive predictive value for the differential diagnosis of benign and malignant thyroid nodules (all exceed 80%), but these indexes were lower (under 60%) for the differential diagnosis of solid thyroid nodules with diffuse diseases, with calcification, and maximum nodule diameter ≥1 cm.ConclusionsECI obtained by ultrasound RTE can be used to differentiate solid thyroid nodules from benign ones. The presence or absence of diffuse lesions, calcification, and maximum nodule diameter are the influencing factors for ECI to differentiate solid thyroid nodules. In clinical diagnosis, it should be paid attention to the comprehensive analysis of the above factors.
In this paper, the research has been conducted by the Microsoft kinect for windows v2 for obtaining the walking trajectory data from hemiplegic patients, based on which we achieved automatic identification of the hemiplegic gait and sorted the significance of identified features. First of all, the experimental group and two control groups were set up in the study. The three groups of subjects respectively completed the prescribed standard movements according to the requirements. The walking track data of the subjects were obtained straightaway by Kinect, from which the gait identification features were extracted: the moving range of pace, stride and center of mass (up and down/left and right). Then, the bayesian classification algorithm was utilized to classify the sample set of these features so as to automatically recognize the hemiplegia gait. Finally, the random forest algorithm was used to identify the significance of each feature, providing references for the diagnose of disease by ranking the importance of each feature. This thesis states that the accuracy of classification approach based on bayesian algorithm reaches 96%; the sequence of significance based on the random forest algorithm is step speed, stride, left-right moving distance of the center of mass, and up-down moving distance of the center of mass. The combination of step speed and stride, and the combination of step speed and center of mass moving distance are important reference for analyzing and diagnosing of the hemiplegia gait. The results may provide creative mind and new references for the intelligent diagnosis of hemiplegia gait.
In order to develop safe training intensity and training methods for the passive balance rehabilitation training system, we propose in this paper a mathematical model for human standing balance adjustment based on T-S fuzzy identification method. This model takes the acceleration of a multidimensional motion platform as its inputs, and human joint angles as its outputs. We used the artificial bee colony optimization algorithm to improve fuzzy C-means clustering algorithm, which enhanced the efficiency of the identification for antecedent parameters. Through some experiments, the data of 9 testees were collected, which were used for model training and model results validation. With the mean square error and cross-correlation between the simulation data and measured data, we concluded that the model was accurate and reasonable.
Objective To investigate the effect of monocyte chemoattractant protein 1 (MCP-1) on the migration of the induced and differentiated mouse bone marrow mesenchymal stem cells (BMSCs) for raising the efficacy of intravenous transplantation of BMSCs. Methods The BMSCs were cultured with the method of differential adhesion and density gradient centrifugation of C57/BL10 mice, and were identified by alkal ine phosphatase Gomori modified staining after osteogenic inducing. At the 3rd passage, the BMSCs were induced to the myoblasts with 5-azacytidine (5-Aza). The chemotaxis of MCP-1 in the induced and differentiated BMSCs in vitro at concentrations of 25, 50, 100, 200, and 400 ng/mL was observed through the migration test, by counting the number of the migrated cells. The expression of the chemokine receptor 2 (CKR-2) in the induced and differentiated BMSCs was detected with the flow cytometry. Results The cells could be cultured with the methods of differential adhesion and density gradient centrifugation and still had higher prol iferative and differentiative potency; the induced cells at the 3rd passage could differenciate to the osteoblasts, confirming that the cells were BMSCs; the myogenic induced BMSCs possesed the sarcotubule structure. The number of the migrating BMSCs at MCP-1 concentrations of 25-400 ng/ mL were respectively 35.066 7 ± 6.584 2, 43.200 0 ± 6.460 8, 44.466 7 ± 4.823 5, 45.600 0 ± 8.650 3, and 50.733 3 ± 7.582 5; showing significant difference when compared with control group (28.333 3 ± 8.917 6, P lt; 0.05), and presenting significant difference among 25, 50, 400 ng/mL groups compared with each other (P lt; 0.05). The expression of CKR-2 in the mouse BMSCs (48.0%) was significantly higher (P lt; 0.001) than those of blank control (0.6%) and negative control (17.0%). Conclusion The results indicate that the MCP-1 can induce the migration of mouse BMSCs by MCP-1/CKR-2 pathway.
Objective To investigate the clinical significance of visual identification and intraoperative neuromonitoring of recurrent laryngeal nerve (RLN) during thyroidectomy. Methods Totally 1 664 patients underwent thyroidectomy with RLN protection from January 2009 to December 2009 were included in this study, in which 1 447 cases were protected by visual identification only, and 217 complex thyroidectomy cases were protected by visual identification and intraoperative monitoring. Results By the “multisites, three steps” RLN exposure method, 1 417 cases （85.16%） were successfully recognized and the recognition time was (3.57±1.26) min. The recognition time in the rest 30 complex cases (2.07%) without intraoperative neuromonitoring was (17.02±5.48) min. By this method, the temporary RLN injury occurred in 23 cases (1.54%) and 15 cases (65.22%) recovered within 2 weeks. In patients undewent intraoperative neuromonitoring, the recognition rate was 100% (217/217) and recognition time was (2.18±0.67) min. The temporary RLN injury occurred in 4 cases (1.84%) and 3 cases (75.00%) recovered within 2 weeks. All temporary RLN injuries recovered within 1 month and no persistent RLN injury occurred. Conclusions Conventional visual identification can reduce the RLN injury, but not meet the needs of the RLN protection during complex thyroidectomy. The combination of visual identification and intraoperative neuromonitoring can further improve the recognition rate and shorten the recovery time of vocal cord dyskinesia.