BACKGROUND: An ideal marked molecule has not been found to detarmine bone marrow mesenchymal stem cells (MSCs) so as to make sure the homogenicity.OBJECTIVE: To verify the in vitro differentiation from BrdU-treced MSCs into osteoblasts.DESIGN, TIME AND SETTING: A cytological observation/n vitro was performed in Shanxi Medical University from January to October 2008.MATERIALS: Wistar rats aging 4 weeks old were provided by Experimental Animal Center of Shanxi Medical University.METHODS: MSCs were isolated and cultured by using density gradient cantrifugation combined with attachment culture method.At about 80% confluence, trypsin was used for passage and amplification. MSCs at density of 5×1010/L were inoculated in a 25-mm culture dish with L-DMDM culture medium containing dexamethasone, β -phosphoglycarol, vitamin C, and 10% fetal bovine serum. The third-passaged MSCs were labeled in vitro with 10 μmol/L BrdU tracer. Thereafter, 10 visual fields were randomly selected to calculate numbers of positive and negative ceils so as to obtain BrdU tracing rate under a fluorescence microscope (×200).MAIN OUTCOME MEASURES: Inverted microscope was used to observe cell morphology; flow cytometry was used to detect cell surface antigen, differentiation into osteoblasts, and BrdU tracing rate in vitro.RESULTS: The purified MSCs which were like fibroblasts were adherent and fusiform. The third-passaged cells were changed equidirectionally and whirlpool-arranged, and the survival rate was more than 95%. The seventh-passaged cells still grew rapidly.CD44, CD71, and CD105 expressions were positive, but CD45 expression was negative. Black particles were visualized in MSCs after Von kossa staining. BrdU tracing rate was more than 90%.CONCLUSION: Density gradient centrifugation combined with attachment culture method can effectively isolate and purify rat MSCs which are cultured in vitro for a long period and differentiated into osteoblasts. BrdU tracer is safe, effective, and convenient to successfully label MSCs.

Objective:To observe the effect of metformin on the polarization state and photoreceptor cell activity of microglia (BV2 cells) in a high glucose environment.Methods:An experimental study. BV2 cells were divided into a control group, a high glucose group, and a metformin+high glucose group. The cells in the high glucose group were cultured with 75 mmol/L glucose in the medium; the cells in the metformin+high glucose group were pretreated with 2 mmol/L metformin for 12 h and then placed in 75 mmo/L glucose concentration medium. The relative expression of M1 marker inducible nitric oxide synthase (iNOS), CD86 and M2 markers arginase 1 (Arg-1), and CD206 protein were detected by Western blot. Interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-4 were detected by enzyme-linked immunosorbent assay (ELISA). BV2 cells were co-cultured with mouse retinal photoreceptor cells (661W cells) for 24 h. The proliferation rate of 661W cells in each group was measured by methyl thiazolyl tetrazolium (MTT) colorimetric assay; the apoptosis rate of 661W cells in each group was measured by flow cytometry and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL). An independent sample t-test was used for comparison between groups. Results:Western blot assay showed that the relative expression of iNOS and CD86 protein was increased and the relative expression of Arg-1 and CD206 protein was decreased in BV2 cells in the high glucose group compared with the control group, and the differences were all statistically significant ( t=-16.783, -11.605, 4.325, 4.649; P<0.05); compared with the high glucose group, the relative expression of iNOS and CD86 protein was decreased and the relative expression of Arg-1 and CD206 protein was increased in BV2 cells in the metformin + high glucose group compared with the high glucose group, and the differences were all statistically significant ( t=7.231, 5.560, -8.035, -8.824; P<0.01). ELISA results showed that compared with the control group, the BV2 cells in the high glucose group had increased IL-6, TNF-α content and IL-4 content was decreased in BV2 cells in the high glucose group compared with the control group, and the differences were all statistically significant ( t=-64.312, -127.147, 71.547; P<0.001); compared with the high glucose group, IL-6 and TNF-α content was significantly decreased and IL-4 content was significantly increased in BV2 cells in the metformin+high glucose group, and the differences were all statistically significant ( t=44.426, 83.232,-143.115; P<0.001). After co-culture of BV2 cells with 661W cells for 24 h, the results of MTT colorimetric assay showed that compared with the control group, the activity of 661W cells in the high glucose group was significantly reduced, and the difference was statistically significant ( t=7.456, P<0.01); compared with the high glucose group, the activity of 661W cells in the metformin+high glucose group was increased ( t=-3.076, P<0.05). TUNEL method and flow cytometry showed that the apoptosis rate of 661W cells in the high glucose group was significantly higher compared with the control group, and the differences were both statistically significant ( t=-22.248, -22.628; P<0.001); compared with the high glucose group, the apoptosis rate of 661W cells in the metformin+high glucose group was significantly decreased, and the difference was statistically significant ( t=11.767, 6.906; P<0.001, 0.01). Conclusion:In the high glucose environment, metformin inhibited the inflammatory response and attenuated the apoptosis of photoreceptor cells by regulating the polarization of microglia toward the M2 type.