ABSTRACT
AIM:To evaluate the effect of α2-macroglobulin(α2M) against X-ray induced obstacle on osteo-genic differentiation of human bone marrow mesenchymal stem cells(hBMMSCs). METHODS:hBMMSCs were cultured in vitro. The 4th generation of hBMMSCs was irradiated with 8 Gy X-ray,then induced osteogenic differentiation and trea-ted with different concentrations of α2M(0.5 and 1.0 g/L). The alkaline phosphatase(ALP) activity and the mRNA ex-pression of runt-related transcription factor-2 (RUNX2) were detected on day 7 after osteogenic induction. The protein ex-pression of osteoglycin (OGN) was evaluated by Western blot on day 14 after osteogenic induction. The formation of calci-um nodules was detected by alizarin red staining on day 21 after osteogenic induction. The activity of superoxide dismutase (SOD) and the protein expression of MnSOD of irradiated hBMMSCs with 8 Gy X-ray were determined at 24 h after α2M treatment. RESULTS:Compared with 8 Gy X-ray group,the activity of ALP,the mRNA expression of RUNX2,the pro-tein expression of OGN and MnSOD,as well as SOD activity were higher than those in the hBMMSCs treated with α2M at 0.5 and 1.0 g/L after 8 Gy X-ray irradiation,and the calcium nodules were also increased. CONCLUSION:α2M signifi-cantly improves the osteogenic differentiation ability,the SOD activity and MnSOD protein expression of hBMMSCs after ra-diation injury.
ABSTRACT
Objective To establish an assay for detecting α2-macroglobulin activity in Cohn fraction Ⅳ.Methodsα2-M reacted with trypsin to form α2-M-trypsin complex.After the chromogenic substrate Na-benzoyl-DL-arginine 4-nitroanilide hydrochloride ( BAPNA) was added, absorption at 410 nm was detected with the microplate reader .α2-M activity in Cohn fractionⅣwas quantitatively detected according to the established standard curve of plasma α2-M activity. Result Several critical parameters in this assay were optimized .A standard curve of plasma α2-M activity was established . According to this standard curve ,α2-M activity in Cohn fraction Ⅳsample was detected to be 1.578 PU/ml.Conclusion Using normal human plasma as the reference material , theα2-M activity in Cohn fractionⅣcan be detected through chro-mogenic substrate assay.This study provides a simple method to detect α2-M activity during the purification process of α2-M from Cohn fraction Ⅳ.
ABSTRACT
Normal human blood plasma showed hydrolytic activities on several synthetic substrates for proteases, the most effective being H-D-Ile-Pro-Arg-p-nitroanilide, H-D-Pro- Phe-Arg-p-nitroanilide and H-D-Val-Leu-Arg-p-nitroanilide. When plasma was preincubated for 12 h at 37°C, there was no significant alteration of the hydrolytic activities. On incubation for 12 h with king cobra venom (2 μg for 0·1 ml plasma), there was considerable decrease in the activities and complete abolition of the protease binding capacity of α2-macroglobulin. On chromatography on Sephadex G-200, α2-macroglobulin activity and bulk of the protease activity of normal plasma were eluted in the void volume region. A minor protease peak was eluted with a Ve/Vo value of 2·5. With venom treated plasma, there was no decrease with this peak. The major protease peak and α2-macroglobulin activity were drastically reduced. Chromatography on red Sepharose showed that all the a2- macroglobulin activity and bulk of the protease activity in normal plasma were bound to the column. In venom treated plasma there was marked reduction in the bound fraction. The data suggest that cobra venom proteases directly or through proteases generated in plasma in situ causes limited cleavage of α2-macroglobulin as well as α2– macroglobulin bound proteases, inactivating them.