Objective To investigate the biological properties of human amniotic mesenchymal stem cells (hAMSCs) which were preconditioned with phosphodiesterase-5 inhibitor (Vardenfil). Methods hAMSCs were in vitro isolated and cultured, hAMSCs were pre-treated with vardenfil in final concentration of 10 μmol/L. The morphology of Vard-hAMSCs was observed, and the immunological characteristics, proliferative capacity, and ability of anti-oxidative damage of hAMSCs and Vard-hAMSCs were analyzed by flow cytometry. Double labeling immunofluorescent staining was used to count the differences of differential potential between neural cells of hAMSCs and Vard-hAMSCs. Results (1)Flow cytometry revealed that both hAMSCs and Vard-hAMSCs positively expressed CD90、CD105 and CD73, and negatively expressed CD34、CD45、CD11b and HLA-DR. The SPF and PI in Vard-hAMSCs group were (0.57 ± 0.40)% and (2.20 ± 1.60)% respectively, there was no statistical significance compared with hAMSCs group; (2)After 4 hours treated by H2O2, the apoptosis rate in Vard-hAMSCs group were (7.67 ± 0.82)%,which were markedly lower than that in the hAMSCs group and specific blocker group; (3)Under the same induction condition, positive rates of MAP-2 and GFAP in Vard-hAMSCs group were (49.8 ± 6.42)%and (55.2 ± 6.10)% respectively detected by double labeling immunofluorescent staining, which were significantly higher than the control group. Conclusion The strategy that hAMSCs are treated with vandenfil can enrich the ability of anti-oxidative damage and the differential potential for neural cells in a certain time, and the morphology, immunological characteristics, proliferative capacity of Vard-hAMSCs have no significant change. It suggests that pre-treatment with vandenfil may provide a optimized experimental strategey for hAMSCs which were used to treat nervous system disease.

ObjectiveTo investigate the potential of human amnion-derived mesenchymal stem cells to differentiate into insulin secreting cells in vitro.MethodsThe hAD-MSCs were isolated from human amnion by trypsin-collagenase digestion.The phenotype of the isolated cells was identified by flow cytometry and immunocytochemical staining.The 3rd generation cells were inoculated at density of 2.5 × 106 unit/ml or 5 × 105 unit/ml in 6 well plates or preset coverslip 24 well plates.Induced groups were treated in serum-free HG-DMEM with 10 mmol/L nicotinamide and N2 supplement.The cells in the non-induced groups were incubated in LG-DMEM supplemented with 10％ fetal bovine serum.At days 7,14 and 21 after induction,insulin and β2 microglobulin was determined by immunocytochemical stain,the content of insulin in the culture supernatant was assayed by radioimmunoassay,and insulin mRNA and PDX-1 mRNA were detected by reverse transcriptase-polymerase chain reaction.Results( 1 ) The hAD-MSCs highly expressed CD29,CD44,CD73,CD166,and vimentin.( 2 ) At 7,14,and 21 days,the percentages of insulin-positive cells in hAD-MSCs induced groups were 74.67％ ± 1.53％,75.00％:1.00％,and 74.33％ ±1.53％,respectively.Contents of insulin in the supematant of hAD-MSCs induced groups were ( 331.62 ± 1.76 ),( 330.50 ± 1.22 ),and ( 331.65 ± 0.48 ) μIU/ml,respectively,but non-induced groups were negative.(3) PDX-1 mRNA and PDX-1 protein were expressed before and after the induction of hADMSCs,but insulin mRNA was expressed only in the induced groups.( 4 ) Both hAD-MSCs induced groups and non-induced groups expressed β2 microglobulin ( all P ＞ 0.05 ).ConclusionThe hAD-MSCs have a potential of differentiating into ISCs and thus may become a new cell source of therapy for type 1 diabetes.

This experimental research was aimed to establish an optimum system of enucleation, purification and identification for preparing the cytoplasts of suspension culture cells in order to undertake cell recombination. Human leukemia HL-60 cells in suspension culture were purified by 42% Percoll density gradient centrifugation and low-speed centrifugation at 1 500r/min, respectively. The purified HL-60 cells were treated with cytochalasin B (CB) alone or combined with colcchicine and enucleated by isopycnic gradient centrifugation on 50% Percoll at 25 degrees C and 34 degrees C, respectively. Cytoplasts made from HL-60 cells were purified through gradient centrifugation by 37%, 38% and 40% Percoll, respectively. The final cytoplasts were identified by Wright-Giemsa staining and 4,6-diamidino-2-phenylidole dihydrochloride (DAPI)/5, 6-carboxyflu-orescein diacetate succinimidyl ester (CFSE) double-staining. The phenotype and mitochondrial membrane potential of HL-60 cytoplasts were analyzed by flow cytometry. The results indicated that the enucleation ratio of HL-60 cells induced by CB combined with colcchicine was up to 91. 98% +/-4. 29%, which was significantly higher than that in CB alone group (74. 95% +/- 3. 02%)(P<0. 01). The rates of enucleation and cytoplast with diameter over 5min in 34 degrees C group were higher than those in 25 degrees C group (all P<0. 01). The cytoplast purities were (95.43 +/- 0. 59)% in 38% Percoll groups,which were higher than those of 40% Percoll (P<0. 05). Nucleus and caryoplasm could be clearly distinguished by DAPI and CFSE double labeling. The results further showed that the phenotype of HL-60 cytoplasts had no significant change, and the activity of the cytoplasts was above 80% within 12h. It is concluded that enucleation throuth density gradient centrifugation on 50% Percoll mediated by CB combined with colcchicine, 38%Percoll of purification followed by DAPI/CFSE double labeling and MMP detection is an optimum scheme for preparation and identification of cytoplast from suspension culture cells.
Cell Compartmentation ; Cell Nucleus ; Cell Separation ; Centrifugation, Density Gradient ; Colchicine ; analogs & derivatives ; pharmacology ; Cytochalasin B ; pharmacology ; Cytoplasm ; HL-60 Cells ; Humans

Objective To compare the therapeutic effects of orthotopic injection and tail vein injection of human amniotic mesenchymal stem cells(hAMSCs)on histological restoration and neurological functions of rats with spinal cord injury. Methods Transected spinal cord injury model in rats was established by transplanting DAPI prelabelled hAMSCs one week after injury.BBB scores were used to evaluate the hindlimb movement of rats. The histological patterns.and morphology of medullary sheath of spinal cord were observed. Results BBB scores in the orthotopic injection group and tail vein injection group were increased gradually from one to six week after hAM-SCs transplantation and reached 6.5 ± 0.5 and 7.12 ± 1.61 respectively 6 weeks after cell transplantation,higher than that of the control group(both P < 0.01). However,there was no statistical significance between the two groups.Histological results indicated that the repair of injured tissue in the orthotopic injection group and tail vein injection group were both better than that in the control group,and there were more vesica and loosened layers forming in the injured spinal cord of rats in the PBS control group as compared with the orthotopic and tail vein transplantation group. Conclusion hAMSCs transplantation through tail vein injection could promote histological restoration and neurological regeneration of rats with spinal cord injury,which has the similar therapeutic effects with hAMSCs orthotopic transplantation.

Objective To investigate the feasibility of human amniotic mesenchymal stem cells (hAMSCs) transplantation to improve the survival of ischemic ultra-long random skin flap vascularization,so as to promote the survival of skin flap.Methods The hAMSCs were isolated from human amnion,cultured in vitro,and identified by immunocytochemistry.The phenotype of hAMSCs was analyzed by flow cytometry.CellTrackerTM-CM-Dil was used to label before hAMSCs transplantation into skin flap.Twenty SD adult rats were selected and the 2 cm × 8 cm ischemic ultra-long random skin flap models were constructed in the left and right sides of the rat back.The pedicles of flaps were on the lliac crest level.The flaps were divided into left group (injection with 0.5 ml LG-DMEM) and right group (0.5 ml 1 × 106/ml hAMSCs) after the flap was lifted.The survival rate of flap was observed 7 d after surgery.The blood perfusion values,namely blood perfusion unit at pedicle and in the middle,were monitored by laser Doppler flow monitor at the immediate time,24 h,48 h,4 d and 7 d of the skin flap after surgery.The capillary density of the skin flap was observed through histological observation of the tissue (0.5 cm from adult and necrotic junction).The distribution and survival rate of CM-Dil labeled hAMSCs were observed by fluorescent microscope.Results In term of survival rate of the flap,left group was (50.6 ± 2.2) ％,and right group was (70.9 ± 2.1) ％.The survival rate of the flap in right group was greater than that of left group (P ＜ 0.05).Blood perfusion unit detected in the pedicle of left group at days 4 and 7 after surgery was higher than that of the right group (P ＜ 0.05).Blood perfusion unit in middle of flap of left group at 24 h,48 h,4 d and 7 d were lower than that of right group (P ＜ 0.05).The flap capillary density at 7 d after surgery were (8.8 ± 1.2)/mm2 in left group and (23.5 ± 1.6)/mm2 in right group (P ＜ 0.05).The tissue of flap was made frozen section,and the fluorescence microscope showed there were CM-Dil labeled hAMSCs in skip flap in right group,which could manifest the survival and distribution of hAMSCs in skip flap.Conclusion The application of hAMSCs in the middle and distal parts of ultralong random skin flap can significantly improve the survival rate of skin flap,and increase the density of microvascular reconstruction in the flap.

Objective To assess the viability and efficacy of employing automated segmentation for whole breast radiotherapy with simultaneous integrated boost to the medial tumor beds,a comparative analysis was conducted on the disparities in geometry,dosimetry,and working time between the auto-segmentation(AS)and manual segmentation(MS)groups.Methods A total of 30 patients with early breast cancer,who had undergone conserving surgery and received hypofractionated radiotherapy with a boost to the medial tumor bed,were enrolled from the First People's Hospital of Zunyi.AccuContour software was used in the AS group to obtain the whole breast planning target volume and cardiopulmonary structure.Geometric differences between AS and MS groups were assessed using Dice similarity coefficient(DSC)and 95%Hausdorff distance(95HD).Subsequently,a comparison was made between the two groups regarding target and cardiopulmonary dosimetry for PlanA and PlanM.Additionally,the time spent by each group was also compared.Results The DSC of PGTV,PTV,lung,and heart were 0.94(0.91,0.96),0.88(0.86,0.91),0.98(0.97,0.98)and 0.94(0.93,0.95),respectively.And the 95 HD(cm)were 0.25(0.20,0.33),0.99(0.56,1.20),0.29(0.25,0.35)and 0.50(0.50,0.59)respectively.The dosimetric results showed that the V95,D95,and Dmean of PGTV and PTV in the AS group were significantly lower than those in the MS group(P<0.05);while the V20 and MLD of the left lung were significantly higher(P<0.05).No significant difference was observed in cardiac dose between the two groups.The mean absolute differences of PGTV and cardiopulmonary dose parameters between the two groups were less than 1 Gy/1%,respec-tively.In terms of work efficiency,the AS approach substantially reduced contouring and planning time with over 70%of cases approved within two days.Conclusions The differences in geometric and dosimetric parameters between the auto-segmentation and manual segmentation groups were found to be negligible for whole breast radiotherapy with medial tumor bed boost patients.It is recommended that the PTV be manually modified prior to plan optimiza-tion,leading to a significant improvement in work efficiency.