BACKGROUND:There is still controversy whether human bone marrow mesenchymal stem cells can maintain their biological characteristics,energy metabolism patterns,and multidirectional differentiation potential after long-term expression in vitro.Further comprehensive and systematic research is needed. OBJECTIVE:To evaluate the effects of long-term expansion in vitro on the biological characteristics of mesenchymal stem cells. METHODS:Human bone marrow mesenchymal stem cells cultured to passage 5,10,and 15 in vitro.MTT assay was used to detect cell proliferation ability.Flow cytometry was used to detect cell cycle.The multi-differentiation potential of mesenchymal stem cells was detected by inducing to adipogenic,osteogenic and chondrogenic differentiation.Cell migration and invasion abilities were detected by scratch test and Transwell assay.The mitochondrial oxidative phosphorylation and glycolysis function were analyzed using energy metabolism analyzer.The cell senescence was detected by senescence-associated β-galactosidase staining.The expression levels of p21,p16,and p53 proteins were detected by western blot assay. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells at passages 5,10,and 15 grew adherently;the volume of passage 15 mesenchymal stem cells increased and its proliferation ability decreased;the percentage of S-phase cells decreased(P<0.05).With the increase of culture passages,the migration and invasion abilities decreased gradually(P<0.05).There was no significant difference in the differentiation potential,demonstrated by adipogenic,osteogenic and chondrogenesis induction.The ability of oxidative phosphorylation of mitochondria and glycolysis decreased gradually(P<0.05).The number of senescence-associated β-galactosidase-positive cells increased with the increase of passages(P<0.05),and the expression of senescence protein p21,p16,and p53 increased gradually(P<0.05).The results indicated that the biological characterization of mesenchymal stem cells changed after long-term in vitro expansion.Mesenchymal stem cells cultured over 10 passages may have a reduced activity due to increasing senescence.Therefore,bone marrow mesenchymal stem cells cultured less than 10 passages are suitable for clinical research/therapy.

ObjectiveTo investigate the chemosensitization effect of gallic acid （GA） combined with sorafenib （Sora） on hepatocellular carcinoma HepG2 cells and related mechanisms. MethodsHepG2 cells were randomly divided into control group， GA group， Sora group， and GA+Sora group. CCK8 assay was used to measure cell viability； CompuSyn software was used to analyze combination index （CI）； colony formation assay was used to evaluate the colony formation ability of cells； flow cytometry was used to measure cell apoptosis； wound healing assay and Transwell chamber assay were used to observe the migration and invasion abilities of cells； Western Blot was used to measure the expression matrix metalloproteinase 2 （MMP-2）， matrix metalloproteinase 9 （MMP-9）， and apoptosis-related proteins. HepG2 cells were subcutaneously inoculated into the lower right back of mice， and 6 days later， the mice were divided into control group， GA group， Sora group， and GA+Sora group. Tumor size and body weight were measured once a week， and drug intervention was performed for 21 days. Then the nude mice were sacrificed， and tumor weight was measured. A one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsThe mean IC₅₀ values of GA and Sora for the treatment of HepG2 cells for 48 hours were 123.47±5.16 μmol/L and 9.87±0.98 μmol/L， respectively， and when Sora was combined with 70 μmol/L GA （IC₃₀）， IC₅₀ decreased to 2.06±0.35 μmol/L； the CI value was<1 for Sora at different concentrations combined with 70 μmol/L GA. The number of cell colonies was 234.0±20.4， 147.0±12.1， 129.3±13.3， and 73.0±7.6， respectively， in the four groups， and the GA+Sora group had a significantly lower number of cell colonies than the control group， the GA group， and the Sora group （all P<0.05）. After 48 hours of treatment， the cell apoptosis rate was 1.98%±0.29%， 15.17%± 1.56%， 18.65%±1.48%， and 34.60%±5.36%， respectively， in the four groups， and the GA+Sora group had a significantly higher cell apoptosis rate than the control group， the GA group， and the Sora group （all P<0.05）. After 24 hours of treatment， the cell migration rate was 55.59%±5.08%， 29.34%±4.36%， 21.80%±5.16%， and 6.47%±2.75%， respectively， in the four groups， and the GA+Sora group had a significantly lower cell migration rate than the control group， the GA group， and the Sora group （all P<0.05）. After 48 hours of treatment， the number of transmembrane cells was 223.7±13.0， 168.3±10.9， 155.3±29.1， and 62.7±19.7， respectively， in the four groups， and the GA+Sora group had a significantly lower number of transmembrane cells than the control group， the GA group， and the Sora group （all P<0.05）. Compared with the control group， the GA group， the Sora group， and the GA+Sora group had significant reductions in the protein expression levels of MMP-2， MMP-9， and Bcl-2 （all P<0.05） and significant increases in the protein expression levels of Bax and cleaved caspase-3 （all P<0.05）. Compared with the control group， the GA， Sora， and GA+Sora groups had significant reductions in tumor volume and weight （all P<0.05）， and compared with the Sora group， the GA+Sora group had significant reductions in tumor volume and weight in nude mice （both P<0.05）. ConclusionGA can increase the sensitivity of HepG2 cells to Sora chemotherapy， possibly by promoting cell apoptosis and inhibiting cell migration and invasion after combination with Sora.