BACKGROUND:As a new kind of adult stem cells, adipose-derived stem cells get more and more attention, because of rich source, drawing materials easily and powerful proliferation. OBJECTIVE:To isolate and culture adipose-derived stem cells from the epididymal adipose tissue in mice, and to identify their biological characteristics. METHODS:Adipose tissue was obtained from epididymis in mice by aseptical y cutting. The tissue was digested using col agenase. Adipose-derived stem cells were separated and purified by using one digestion, multiple col ection method and differential adhesion method. The morphology of adipose-derived stem cells was observed using inverted microscopy and transmission electron microscopy. Growth curve of adipose-derived stem cells was drawn. Immunophenotype of adipose-derived stem cells was identified by flow cytometry. Adipose-derived stem cells were induced to differentiate into adipocytes and osteocytes using cellinductors. Compatibility of adipose-derived stem cells and col agen scaffold material was observed using scanning electron microscope. RESULTS AND CONCLUSION:Adipose-derived stem cells exhibited long spindle-like or fibroblast-like appearance, grew intensively and arranged in scrol and fascicular shape. In vitro, adipose-derived stem cells could be passaged to passage 9 under the inverted microscope. Under the transmission electron microscope, adipose-derived stem cells showed abundant microvil i on the cellsurface. The nuclei were big in size. Some organel es were seen in cytoplasma, such as mitochondria and rough endoplasmic reticulum. Adipose-derived stem cells expressed CD44 and CD29, did not express CD34. After inducing by inductor, many smal lipid droplets were seen in the cytoplasm of adipose-derived stem cells. The smal lipid droplets were dyed red with oil red O. After induction of osteogenic inductor, the boundary line among adipose-derived stem cells was not clear and the structure of cells was fuzzy in the growth-intensive areas. There were many strong refractive granular material deposits at that field after dyeing with alizarin red. Scanning electron microscope revealed that adipose-derived stem cells were spread on the col agen scaffold. Results suggested that adipose-derived stem cells isolated by this method could amplify in vitro and stably subcultured. Under a certain inducing condition, adipose-derived stem cells could differentiate into osteoblasts and adipocytes, which showed a good compatibility with col agen scaffold.

Objective BRAFV600E mutation, RET/PTC rearrangement, and the concomitant of Hashimoto's thyroiditis(HT) could influence clinicopathological features of papillary thyroid carcinoma (PTC).This study is to investigate the distribution of three factors in PTC and to analyze their associations with clinicopathological characteristics.Methods Fine-needle aspiration samples were collected in a total of 122 conventional PTC patients, who were confirmed by surgery.The clinicopathological features were collected to analyze its association with different factors.BRAFV600E mutation and RET/PTC rearrangement were detected by pyrosequencing and Taqman-qPCR, respectively.Results BRAFV600E mutation was significantly correlated with an older age and a less coexistence with HT(P＜0.05).In contrast, RET/PTC rearrangement was more prevalent in young patients and was associated with the concomitant of HT(P＜0.05).In the age of ≥20 year and＜45 year groups, BRAFV600E mutation was significantly associated with extrathyroidal invasiveness.RET/PTC rearrangement was significantly associated with bilateral lymph node metastasis and the number of metastatic lymph node.Conclusions The distribution of three factors were different in PTC patients.In addition to the age at diagnosis, all of three factors should also be considered together to analyze the association of clinicopathological features of PTC.

Objective To investigate the dynamic expression of nitric oxide synthase(NOS) in the apoptosis of primary cultured rat cortical neruons following hypoxia/reoxygenation(H/R) and the protective role of extract of ginkgo biloba(EGB). Methods The cortical neurons of E16-17 days fetal rat were primarily cultured.The apoptosis model of primary cultured cortical nurons following H/R was established by using W-G staning,electromicroscopy,TUNEL staining.The dynamic expression of NOS different H/R times was investigated with NADPH-diaphorase histochemical method. Results H/R can cause apoptosis of primary cultured rat cortical neurons.In the experiment of H-2R-0,H-4R-0, H-6R-0,H-8R-0 and H-2R 18,H-4R 18,H-6R 18 H-8R 18,the apoptosis cells occurred after 4 hour hypoxia.The increasing of apoptosis cell acted as time-dependence and the peak value was at H-8R 18.The expression of NOS increased both after 2 hour hypoxia and reoxygenation 18 hour after 8 hour hypoxia compared with the normal control group.EGB could inhibit the increasing and decrease the percentage of apoptosis.Conclusion The apoptosis of primary cultured rat cortical neurons could be induced by H/R.The increasing of NO might be one of the mechannisms of apoptosis.EGB could singnificantly inhibit the apoptosis by means of inhibiting the expression of NOS and reducing the production of NO.;

Tendon and ligament (T/L) have been known to be obviously different from each other in tissue level. However, due to the overlapping gene markers, distinction in cellular level has not been clearly verified yet. Recently, the use of nuclear magnetic resonance (NMR) spectroscopy has shown the potential to detect biological markers in cellular level. Therefore, in this study we applied a non-invasive technique based on NMR spectroscopy to establish biomarkers to distinguish between T/L fibroblasts. In addition the cellular morphologies and gene expression patterns were also investigated for comparison through optical microscopy and real-time polymerase chain reaction (PCR). No difference was observed from morphology and real-time PCR results, either as expected. However, we found clear differences in their metabolomic spectra using ¹H NMR spectroscopy. The calculated integral values of fatty acids (with chemical shifts at ~0.9, 1.26, 1.59, 2.05, 2.25, and 2.81 ppm), lactate (~1.33 ppm), and leucine (~2.72 ppm) were significantly different between the two types of fibroblasts. To be specific tendon group exhibited higher level of the metabolite than ligament group. In conclusion, in-cell metabolomic evaluation by NMR technique used in this study is believed to provide a promising tool in distinguishing cell types, especially T/L cells, which cannot be classified by conventional biological assays.
Biological Assay ; Biomarkers ; Fatty Acids ; Fibroblasts* ; Gene Expression ; Genes, Overlapping ; Lactic Acid ; Leucine ; Ligaments* ; Magnetic Resonance Spectroscopy* ; Metabolomics ; Microscopy ; Real-Time Polymerase Chain Reaction ; Spectrum Analysis* ; Tendons*

This study aims to investigate the roles and effects of EGCG (epigallocatechin-3-gallate) during the osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro. Recent studies have shown that proper mechanical stimuli can induce osteogenic differentiation of hMSCs apart from biochemical factors. In this study, the hMSC cultures were subjected to: (1) 25 uM EGCG alone or (2) 3% mechanical stretching (0.2 Hz for 4 h/day for 4 days) or (3) in combination with 3% mechanical stretching (0.2 Hz for 4 h/day for 4 days). The two factors were applied to the cell cultures separately and in combination to investigate the individual and synergistic effect of both mechanical stimulation and ECGC in the osteogenic differentiation of hMSCs. Utilizing real time PCR, we measured various osteogenic markers and even those related to intracellular signalings. Further investigation of mitochondria was performed that mitochondria biogenesis, antioxidant capacity, and morphological related markers were measured. hMSCs were to be osteogenic or myogenic differentiated when they were under 3% stretching only. However, when EGCG was applied along with stretching they were to be osteogenic differentiated rather than to be myogenic differentiated. This was supported by evaluating intracellular signalings: BMP-2 and VEGF. Therefore, the synergistical effects of simultaneous employment of stretching and EGCG on osteogenic differentiation were confirmed. Moreover, simultaneous employment was found positive in mitochondria biogenesis, antioxidant capacity, and morphological changes. Through this study, we came into the conclusion that the combination of proper mechanical stretching, 3% in this study, and EGCG promote osteogenic differentiation. Reflecting that EGCG can be obtained from plants not from the chemical syntheses, it is worth to be studied further either by animal tests or long-term experiments for clinical applications.
Animals ; Cell Culture Techniques ; Employment ; Humans* ; In Vitro Techniques ; Mesenchymal Stromal Cells* ; Mitochondria ; Osteogenesis ; Real-Time Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A

Aiming at the teaching characteristics of Medical Organic Chemistry in medical colleges and universities and the learning features of students, this article takes information technology as the medium and fully integrates autonomous learning mode of "teaching" and "learning" into the teaching process. In the teaching of Medical Organic Chemistry for students in grade 2017of Air Force Medical University, we adopted flipped classroom that is autonomous learning based on information resources-, student-centered micro-class assisted teaching, and self-learning mode such as self-designed experimental teaching based on network. The comprehensive scores of students of grade 2017 were compared with those of students of grade 2016 who adopted traditional teaching mode. The results showed that this mode of "teaching" and "learning" combined with "dominant-subject" can fully mobilize students' learning enthusiasm and initiative in Medical Organic Chemistry. It is more conducive for students to mastering learning methods, integrating knowledge and improving their ability to analyze and solve practical problems. The application of autonomous "teaching" and "learning" mode has effectively addressed the problems of insufficient interest of medical students in chemistry learning and problems of only one teaching mode for them. To a certain extent, it improves the quality and efficiency of Medical Organic Chemistry teaching.

Objectives: . Using tissue-engineered materials for esophageal reconstruction is a technically challenging task in animals that requires bioreactor training to enhance cellular reactivity. There have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in initial epithelialization in the special environment of peristalsis. The purpose of this study was to evaluate the potential of an artificial esophagus that can enhance the regeneration of esophageal mucosa and muscle through the optimal combination of a double-layered polymeric scaffold and a custom-designed mesenchymal stem cell-based bioreactor system in a canine model. Methods: . We fabricated a novel double-layered scaffold as a tissue-engineered esophagus using an electrospinning technique. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. After 3 days of cultivation using the bioreactor system, tissue-engineered artificial esophagus was transplanted into a partial esophageal defect (5×3 cm-long resection) in a canine model. Results: . Scanning electron microscopy (SEM) showed that the electrospun fibers in a tubular scaffold were randomly and circumferentially located toward the inner and outer surfaces. Complete recovery of the esophageal mucosa was confirmed by endoscopic analysis and SEM. Esophagogastroduodenoscopy and computed tomography also showed that there were no signs of leakage or stricture and that there was a normal lumen with complete epithelialization. Significant regeneration of the mucosal layer was observed by keratin-5 immunostaining. Alpha-smooth muscle actin immunostaining showed significantly greater esophageal muscle regeneration at 12 months than at 6 months. Conclusion . Custom-designed bioreactor cultured electrospun polyurethane scaffolds can be a promising approach for esophageal tissue engineering.

OBJECTIVE： To screen and characterize effective components of immunopotentiating activity in Senecionis cannabifolii Herba. METHODS： The polysaccharide components were obtained by water extraction and alcohol precipitation method to yield 50％ alcohol precipitation sample （SCHE-1） and 80％ alcohol precipitation sample （SCHE-2）. The cells from mice mononuclear macrophage line RAW264.7 were divided into blank group （medium without serum）， negative control group （medium with serum）， lipopolysaccharide group （LPS， positive control drug， 1 μg/mL）， SCHE-1 and SCHE-2 low-dose and high-dose groups （0.5， 1 mg/mL）. The cell viability of RAW264.7 cells was detected by MTT assay. The levels of IL-1β， IL-6 and TNF-α in RAW264.7 were detected by ELISA. These were used to investigate the effects of SCHE-1 and SCHE-2 on the immunological enhancing activity of RAW264.7 cells. The molecular weight and distribution of SCHE-1 were determined by size exclusion chromatography； the monosaccharide composition of SCHE-1 was determined by HPLC pre-column derivatization. Methylation analysis of SCHE-1 was conducted by NaOH method. RESULTS： Compared with negative control group， the activity of RAW264.7 cells was enhanced significantly in SCHE-1 groups and LPS group， which also significantly increased the levels of IL-1β， IL-6 and TNF-α in cell culture fluids （P＜0.01）. SCHE-1 was an effective component with immunopotentiating activity. The neutral sugar content of SCHE-1 was 40.05％， the uronic acid was 35.62％， and the protein was 8.89％. SCHE-1 was a mixture， molecular weight of which was 62-6 119 Da； monosaccharide was mainly composed of galacturonic acid， arabinose （Ara） and galactose （Gal）. The results of methylation analysis showed that the backbone was composed of 1→3， 1→4 and 1→6 linked Gal， and branches were on the O-6 position of the 1→3 linked Gal， and the non-reducing terminals were Ara. CONCLUSIONS： SCHE-1 may be the effective component of immuno potentiating activity， and main component of SCHE-1 is polysaccharide. SCHE-1 may regulate the immune function by activating macrophages to release IL-1β， IL-6 and TNF-α.

The binding of talin-F0 domain to ras-related protein 1b (Rap1b) plays an important role in the formation of thrombosis. However, since talin is a force-sensitive protein, it remains unclear whether and how force regulates the talin-F0/Rap1b interaction. To explore the effect of force on the binding affinity and the dynamics mechanisms of talin-F0/Rap1b, molecular dynamics simulation was used to observe and compare the changes in functional and conformational information of the complex under different forces. Our results showed that when the complex was subjected to tensile forces, there were at least two dissociation pathways with significantly different mechanical strengths. The key event determining the mechanical strength difference between the two pathways was whether the β4 sheet of the F0 domain was pulled away from the original β1-β4 parallel structure. As the force increased, the talin-F0/Rap1b interaction first strengthened and then weakened, exhibiting the signature of a transition from catch bonds to slip bonds. The mechanical load of 20 pN increased the interaction index of two residue pairs, ASP ⁵⁴-ARG ⁴¹ and GLN ¹⁸-THR ⁶⁵, which resulted in a significant increase in the affinity of the complex. This study predicts the regulatory mechanism of the talin-F0/Rap1b interaction by forces in the intracellular environment and provides novel ideas for the treatment of related diseases and drug development.
Molecular Dynamics Simulation ; Talin