Influence of varying intensities of water-jet force on autologous fat draft viability: an in-vitro study / 中华医学美学美容杂志

Objective To observe the effect of varying intensities of water-jet force on autologous fat graft viability.Methods Lipoaspirate was taken from 12 female patients undergoing waterjet assisted abdominal liposuction at our department.According to the intensity of water-jet force,the experimental group was divided into four subgroups:R1 (pressure,30 bar),R2 (pressure,50 bar),R3 (pressure,70 bar) and R4 (pressure,90 bar).Hand-held suction was taken as the control group C.Adipose tissue was filtered with cotton cushion and centrifuged at low speed,and the composition ratio of water and fat tissue from each group was observed.Calcein-AM/Hoechst 33342 staining was used to detect the viability of adipocytes.Results Fat aspirates was divided into four layers:oil layer,pure fat tissue,liquid and bottom sediment.Oil ratios of R1,R2,R3,R4 and C were (8.9 ± 2.3) ％,(9.6±2.1)％,(10.3±1.3)％,(14.2±1.6)％ and (9.5±1.8)％,respectively.There was no statistically significant difference between R1,R2,R3 and C (P＞0.05).Statistically significant difference was found between R4 and other groups (P＜0.001).Viability of adipocytes from R1,R2,R3,R4 and C groups were (88.1±2.8)％,(89.9±1.9)％,(84.8±2.3)％,(78.0±1.7)％ and (91.1±2.9)％ respectively.There was no statistically significant difference between R1,R2 and C (P＞ 0.05).Statistically significant difference was found between R3,R4 and C (P ＜ 0.05).Conclusions Viability of fat graft harvested under lower intensity of water-jet force (R1,R2) is higher than that harvested under higher intensity of water-jet force (R3,R4).

Optimization of extracting technology of total triterpenoids in Antrodia camphorata by central composite design-response surface methodology and study on antitumor activity of total triterpenoids / 中草药

Objective: To study the extracting technology of Antrodiacamphorata total triterpenoids (ACTT) and their anti-tumor activity. Methods: To optimize the ACTT extraction process by central composite design of response surface methodology (CCD-RSM).To compare the inhibition of ACTT extract at different concentration and different time on in vitroproliferation of cultured HepG2 cell using MTT assay;To observe the HepG2 cell morphology by fluorescence microscopy and flow cytometry andto detect the cell apoptosis rate by AnnexinV apoptosis assay kit. Results:The optimized process was plus 20 times amount of 90% ethanol, ultrasonic extraction twice, 40min each time, ultrasonic power of 400W.ACTT extracting rate was 11.87%, deviation between the actual and predicted values of ACTT extraction rate was 1.56%.MTT assay showed that in 12.5-200μg/mL, ACTT extract at different concentration (12.5-200μg/mL) on the proliferation of HepG2 cells showed significant inhibition(P<0.01).Microscopic observation and direct form Hoechst33342 staining were used to observethenuclear enrichment and nuclear fragmentation and other typical features of apoptosis and apoptotic bodies;Flow cytometry showed that there was significant differenceof apoptosis rate in the administration group (P<0.01) compared with the control group. Conclusion: UsingCCD-RSM to optimize antrodia ultrasonic alcohol extraction process, theactual and predicted values are consistent with high and good predictability which is feasible.ACTT extract can significantly inhibit the proliferation and induce apoptosis on HepG2 cells in vitro.

Evaluation of a side population of canine lymphoma cells using Hoechst 33342 dye

Cancer stem cell (CSC) research has increased exponentially to gain further insight into the mechanisms underlying both carcinogenesis and chemotherapy resistance. The present study was performed to explore the potential value of a side population (SP) assay for identifying and characterizing putative CSCs among canine lymphoma cells. Canine lymphoma cells from cell lines and clinical samples were subjected to the SP assay consisting of Hoechst 33342 staining and subsequent flow cytometric analysis. The SP assay revealed various amounts of a SP fraction among the canine lymphoma cells. The percentages of SP were not affected by inhibitors of membrane transporters, verapamil hydrochloride, or fumitremorgin C. Most of the canine lymphoma cells expressed high levels of Bmi-1 and membrane transporter proteins such as ABCG2 and phosphorylated (p)-glycoprotein. This investigation lays the groundwork for further studies of the biological behaviors and molecular characteristics of CSCs in cases of canine lymphoma.

Isolation of Putative Corneal Epithelial Stem Cells from Cultured Limbal Tissue

PURPOSE: To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue. METHODS: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37 degrees C or 4 degrees C for 1 or 16 hours, respectively, with 1.2U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37 degrees C in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation. RESULTS: The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3x10(4) cell/ml and 8.06x10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21x10(6) cell/ml with a trypsin/EDTA treatment (p<0.05). CFE was 9.67+/-2.13% and 6.63+/-2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61+/-0.42% and 5.21+/-4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67+/-2.24% and 1.17+/-6.13%, respectively (p<0.05). CONCLUSIONS: Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.

Study of Survival and Migration of Schwann Cells Transplanted to Central Nervous System for A Long-term / 中国康复理论与实践

@#ObjectiveTo investigate Schwann cells whether survival and migration after transplanted to central nervous system for a long-term.MethodsThe Schwann cells of rat were expended in vitro, the part of them were labeled with 5'-bromodeoxyuridine (BrdU) and transplanted to rat's middle brain injured by electric needle stimulus, the others were labeled with Hoechst 33342, seeded to PLGA scaffold, and transplanted to rat's transected spinal cord. 8 and 11 months later, rat brain and spinal cord were taken out respectively, examined by BrdU immunohistochemistry and fluorescence microscope.ResultsBrdU positive cells could be seen after 8 months and migrated toward cerebral cortex. Hoechst 33342 positive cells could be identified in scaffold and transected spinal cord after 11 months under fluorescence microscope.ConclusionGrafted Schwann cells can survive in central nervous system for a long-term and migrate toward distance.

OBSERVATION OF RAT TRACHEAL STEM CELLS DURING THE INJURE AND REGENERATION INDUCED BY 5-FU / 解剖学报

Objective To observe the rat tracheal stem cells in situ.Methods Tracheal rings of Wistar rats treated with 5-FU to make an injure model in vitro then observing the process of regeneration in sequence time from 3*!hours to 48*!hours. Use light microscope to detect the development of tracheal epithelium,assay the level of proliferating cell nuclear antigen (PCNA) immunohistochemistry and perform Hoechst33342 staining to search the negative staining cells. Results Twelve hours after 5-FU it could be seen that tracheal mucosa exfoliated with nuke-nucleus like cells nailed just above the basement membrane,6*!hours after removing 5-FU we could see that the tracheal rings were covered with squamous epithelium. It could be seen that PCNA immunohistochemistry negative staining cells located bordering on the positive one. Meanwhile under fluorescent contrast microscope negative cells with no Hoechst dye combinations were among most of the positive cells. 12*!hours after removing 5-FU mucous granular cells and cilia cells could be seen. After 48*!hours the whole tracheal rings were covered with pseudostratified columnar ciliated epithelium.Conclusion 5-FU is a cell-cycle specific antimetabolite cytotoxicant.It can make the cycling cells degeneration and necrosis but has little effect on the G 0 cells. Few G 0 cells which look like nuke nucleus cells randomly on the basement have the ability to efflux the dye Hoechst33342.The tracheal rings are regenerated through these cells differentiation and proliferation.