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OBJECTIVE： To study the effects of aspirin on the growth and autoghagy of human gastric cancer cells SGC-7901 and BGC-823. METHODS： SGC-7901 and BGC-823 cells were selected as research objects， with phosphate buffer （PBS） as negative control treated for 48 h， MTT assay was used to detect the effects of 1， 2， 4， 6， 8， 10 mmol/L aspirin， 5 mmol/L aspirin alone or combined with 2.5 μmol/L chloroquine， 2.5 μmol/L 3-methyladenine （3-MA） on survival rate of gastric cancer cells. Flow cytometry was used to detect the effects of 2 and 5 mmol/L aspirin， 5 mmol/L aspirin alone or combined with 2.5 μmol/L chloroquine and 2.5 μmol/L 3-MA on the apoptosis rate and cell cycle distribution of gastric cancer cells. Hoechst33258 staining was used to observe the effects of 5 mmol/L aspirin on morphology of gastric cancer cell nucleus； Transwell chamber test was adopted to detect the effects of 5 mmol/L aspirin on the migration of gastric cancer cell. Laser confocal scanning microscopy was used to observe the effects of 5 mmol/L aspirin on autophagy formation in gastric cancer cells. Western blot method was used to detect the effects of 2 and 5 mmol/L aspirin on the protein expression of autophagy markers LC3-Ⅱin gastric cancer cells. RESULTS： Compared with negative control group， aspirin could inhibit the survival rates of SGC-7901 and BGC-823 cells in dose-dependent manner， but had no significant effects on apoptosis rate of SGC-7901 and BGC-823 cells； SGC-7901 and BGC-823 cells were blocked in G1 phase. Compared with aspirin alone group， the survival rates of SGC-7901 and BGC-823 were increased significantly after treated with aspirin+chloroquine and aspirin+3-MA， while the distribution rate of SGC-7901 and BGC-823 cells at G1 phase were decreased significantly， with statistical significance （P＜0.05 or P＜0.01）. Compared with negative control group， there were no obvious DNA fragmentation fragments， apoptotic bodies and fragments of dense bright blue， while the number of migration cells were decreased significantly in SGC-7901 and BGC-823 cells after treated with aspirin （P＜0.001）； the number of autophagosome was increased significantly and the protein expression of LC3-Ⅱ was enhanced significantly （P＜0.05）. CONCLUSIONS： Aspirin can significantly inhibit the growth of SGC-7901 and BGC-823 cells， and arrest cell cycle in G1 phase， the mechanism of which may be associated with the activation of autophagy.
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Objective To explore the biocompatibility of nano-hydroxyapatite/polyamide 66 (nHA/PA66) with human bone mesenchymal stem cells (hBMSCs) after osteogenic induction.Methods After hBMSCs were isolated and cultured in vitro,the experiment was conducted in 3 groups.Group A were hBMSCs subjected to no osteogenic induction,group B hBMSCs subjected to osteogenic induction,and group C was the composite of nHA/PA66 with hBMSCs subjected to osteogenic induction.Adhesion of the cells onto the nHA/PA66 in group C was observed by electron microscope scanning.Growth and proliferation of the cells in groups B and C were detected by MTI test at 1,2 and 3 weeks.The ability of osteogenic differentiation of hBMSCs in vitro was analyzed by alkaline phosphatase (ALP) activity and alizarin red staining.The ability of osteogenic differentiation of hBMSCs on nHA/PA66 was tested by ALP activity.Results Electron microscope scanning showed that the cells spread and attached well on the surface of the composite scaffold in group C;the proliferation of the cells in groups B and C showed no significant difference (P ＞ O.05).These suggested that the proliferation of hBMSCs was not affected by nHA/PA66.The number of mineralized nodules in group B was significantly larger than in group A (P ＜ O.05);the ALP activity of the cells in group A was significantly lower than in group B at 6 and 12 days (P ＜ 0.05);no significant differences were observed between groups B and C (P ＞ 0.05).These indicated that the hBMSCs were capable of osteogenic differentiation which was not affected by nHA/PA66.In groups B and C,the ALP activity of the cells at 12 days was significantly higher than at 6 days,indicating the ALP activity increased with increased induction time (P ＜ 0.05).Conclusion nHA/PA66 can be used as a carrier of hBMSCs in bone tissue engineering because hBMSCs can well adhere to,proliferate,and differentiate into bone on nHA/PA66 scaffolds.
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BACKGROUND:Orthopedists should pay more attentions to nonunion prevention in view of nonunion treatment, that is, active interventions should be taken to avoid bone delayed union and nonunion. OBJECTIVE:To explore the effect of composite tissue-engineered scaffold constructed by nano-hydroxyapatite/polyamide 66 (nHA/PA 66) combined with bone marrow mesenchymal stem cels to repair a femoral fracture with severe nonunion. METHODS:Rat bone marrow mesenchymal stem cels were isolated and culturedin vitro, and then they were divided into three groups: bone marrow mesenchymal stem cels without osteogenic induction, with osteogenic induction or combined with nHA/PA 66 folowed by osteogenic induction as control group, test group or composite group, respectively. Then osteogenic differentiation of bone marrow mesenchymal stem celsin vitrowas analyzed by measuring alkaline phosphatase activity and alizarin red staining, cel adhesion on the nHA/PA 66 was observed using scanning electron microscopy, and the celgrowth and proliferation were detected by MTT assay. In the meanwhile, established Sprague-Dawley rat femur nonunion models were randomly divided into three groups: the areas of nonunion were implanted with nothing as blank control group,those were with nHA/PA 66 as simple scaffold group, and the others were with nHA/PA 66 combined with bone marrow mesenchymal stem cels as composite scaffold group. Afterwards, X-ray examination, micro-CT and Masson staining were used to evaluate the femoral healing. RESULTS AND CONCLUSION:At 6 and 12 days after osteogenic induction, alkaline phosphatase activity in the test group was significantly higher than that in the control group; at 14 days, compared with the control group, the amount of mineralized nodules in the test group was significantly higher, which indicated that bone marrow mesenchymal stem cels after osteogenic induction could differentiate into osteoblasts. Attached cels spread wel on the scaffold with good proliferation activity, suggesting that nHA/PA 66 is suitable for cel adherence, proliferation and osteogenic differentiation. Besides, at 12 weeks after modeling, in the blank control group, no calus appeared in the nonunion region. In the simple scaffold group, the broken femur did not heal at 8 and 12 weeks after surgery. In the composite scaffold group, the broken femur did not heal at 8 weeks, but a lot of calus appeared; at 12 weeks, bone healing achieved and the scaffold was encased and absorbed.These findings demonstrate that the tissue-engineered bone scaffolds constructed by bone marrow mesenchymal stem cels and nHA/PA 66 effectively prevent bone nonunion by accelerating femoral healing in a rat femur nonunion model.