[In vitro studies on the growth and proliferation characteristics of rat bone mesenchymal stem cells].

OBJECTIVE: This study was to investigate the growth and proliferation characteristics of rat bone mesenchymal stem cells (BMSCs) isolated by the method of whole bone marrow culture and to explore the effect of cell inoculation density and incubation period on cell proliferation, with an aim to provide multipotential seed cells for preventing from degenerative disease. METHODS: Bone mesenchymal stem cells were isolated by the method of whole bone marrow culture and then cultured in vitro. The cell morphologic features were observed by inverted microscope. The cell surface antigens were identified by flow cytometry. The effect of cell inoculation density and culture period on cell growth and proliferation was explored by analyzing the characteristics of a ten-day cell growth curve in 96-well plates. RESULTS: Flow cytometry results showed the detection rates for CD29, CD34 and CD45 were 97.68% (7607/7788), 7.93% (661/8340) and 2.76% (215/7788) respectively, which was consistent with the expression characteristics of BMSCs surface antigens. BMSCs became uniform after three cell passages, existing in a typical shape of whirlpool or radial colony. The senescent cells started to appear at 7(th) passage, and more senescent cells were found at 10(th) passage. The growth curve for moderate inoculation density was typically S-shaped. Lag phase was found during the first two days, and logarithm growth phase was in the following three days. Plateau phase started from the 6(th) day and cell numbers decreased slightly from the 8(th) day. CONCLUSION: The whole bone marrow culture is an effective way to obtain BMSCs. A moderate inoculation density was more advantageous to cell proliferation, by which more seed cells could be obtained.

[Effects of drynaria total flavonoid on osteogenic differentiation of bone marrow mesenchymal stem cells at different glucose concentrations: experiment with rats].

OBJECTIVE: To study the effects of drynaria total flavonoid on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at different glucose concentrations. METHODS: BMSCs of SD rats were isolated, cultivated in vitro, and divided into 6 groups to be induced to differentiate into osteoblasts under different conditions: (1) low glucose control group, (2) high glucose control group, (3) low glucose classical induction group (sodium glycerophosphate+vitamin C+dexamethasone), (4) high glucose classical induction group (sodium glycerophosphate+vitamin C+dexamethasone), (5) low glucose+drynaria total flavonoid group, and (6) high glucose with drynaria total flavonoid group. Alkaline phosphate (ALP) test kit was used to examine the level of ALP. The ALP staining positive rate was examined with modified calcium cobalt method. Alizarin red staining was adopted to observe the number of calcium nodes. Immunohistochemistry was used to detect type I collagen level. Advanced glycosylation end products (AGEs) were tested by ELISA. RESULTS: The A value indicating the ALP activity, ALP staining positive rate, calcium node number, and type I collagen expression score of the low glucose+drynaria total flavonoid group were (0.439±0.024), 48.7%, (9.75±1.71) nodes/HP, and (2.21±0.07) respectively, all significantly higher than those of the sodium glycerophosphate+vitamin C+dexamethasone [(0.385±0.029), 35.0%, (6.25±0.96) nodes/HP, and (1.93±0.13) respectively, all P<0.05]. The A value, ALP staining positive rate, calcium node number, and type I collagen expression score of the high glucose with drynaria total flavonoid group were (0.352±0.022), 25.3%, (4.50±1.29)/HP, and (1.70±0.03) respectively, all significantly higher than those of the sodium glycerophosphate+vitamin C+dexamethasone [(0.139±0.013), 22.7%, (3.25±1.50)/HP, and (1.28±0.27) respectively, all P<0.05]. The AGE expression levels of the high glucose classical induction group and high glucose+drynaria total flavonoid group were both significantly higher than those of the low glucose classical induction group and low glucose+drynaria total flavonoid group (both P<0.05). There were no significant differences in the AGE level among the low glucose control, low glucose classical induction, and low glucose+drynaria total flavonoid groups (all P<0.05); and among the high glucose control, high glucose classical induction, and high glucose+drynaria total flavonoid groups (all P<0.05). However, the AGE levels of the high glucose groups were all significantly higher than those of the corresponding low glucose groups (all P<0.05). Glucose increased the AGE levels dose- and time-dependently. The concentrations of AGEs were significantly negatively correlated with the expression of type I collagen (r=-0.410, P<0.05). CONCLUSIONS: Drynaria total flavonoid promotes the osteogenic differentiation of BMSCs and relieves the inhibitory effect of osteogenic differentiation by glucose at high concentration. Thus drynaria total flavonoid may provide a potential therapy for diabetic osteoporosis.

[Effect of TIEG-siRNA on Smad2, p-smad2 and collagen IV in diabetic nephropathy].

OBJECTIVE: To investigate the effect of TIEG-siRNA on Smad2, p-smad2 and collagen IV in diabetic nephropathy rats induced by streptozotocin (STZ). METHODS: Ten Sprague-Dawley rats injected with STZ were randomly divided into TIEG-siRNA and Control groups. Other five normal rats were used as control. Each of the TIEG-siRNA and Control groups were injected with TIEG-siRNA and Control 0.5 ml via tail vein at 0 and 72 hours respectively. All rats were sacrificed at week 4 after a successful modeling. To confirmed the efficacy of TIEG-siRNA in rat kidney, the TIEG levels were determined by fluorescence quantitative PCR. The expressions of Smad2, p-smad2 and collagen IV protein were detected by immunohistochemical method while Smad2 and p-smad2 examined by Western blot. RESULTS: The TIEG levels were greatly down-regulated in the TIEG-siRNA treated group (0.0636 ± 0.0066) versus the empty vector treated group (0.1054 ± 0.0111) (P < 0.05). The immunohistochemical semi-quantitative method indicated that there was a decrease in the TIEG-siRNA treated group versus the empty vector treated group: (2.13 ± 0.19)% vs (2.53 ± 0.34)% in Smad2, (21.77 ± 2.00)% vs (27.03 ± 2.51)% in p-smad2 and (3.67 ± 0.42)% vs (4.85 ± 0.43)% in collagen IV. Western blot also showed that smad2 in the TIEG-siRNA treated group (0.32 ± 0.09) was much lower than that in the empty vector treated group (0.50 ± 0.04). And p-smad2 in the TIEG-siRNA treated group (0.16 ± 0.01) was much lower than that in the empty vector treated group (0.32 ± 0.02) (P < 0.05). CONCLUSION: TIEG-siRNA may be useful in preventing the progression of diabetic nephropathy through influencing the expression of Smad2 and its activation and down-regulating collagen IV.