| Objective: Osteogenic Growth Peptide (OGP) is a polypeptide consisting of fourteen amino acids. It exists in mammalian as the same form. Former researches have shown that OGP can promote osteogenesis both in vivo and in vitro. We have synthesized OGP (sOGP) in vitro. Bone Marrow Mesenchymal Stem Cells (BMSCs) belong to adult stem cells and have a multilineage potential. In the first and second parts of this experiment, we examined the effect of sOGP on the proliferation and differentiation of the cultured BMSCs obtained from rodents (Rat) and human to prove if the osteogenesis effect of OGP in vivo is based on its effect on BMSCs. In the third part of the experiment, we examined the change of expression levels of three members of the MAPK family: ERK, JNK and p38 to investigate if this signaling pathway is involved in the action of sOGP.Methods: In the first part of the experiment, we examined the effect sOGP on the proliferation and differentiation of the cultured BMSCs of rats. We obtained BMSCs from the bone marrow in femur of rats and cultured in vitro. After adding three different concentrations of sOGP to the cultured BMSCs, we observed the morphological changes using inverted microscope and electromicroscope, measured the proliferation rate using MTT method and compared the proliferation curve of each group, and detected the osteogenesis markers(including alkaline phosphatase, type I collagen and osteocalcin) at the level of both mRNA and protein with biochemical methods , semi-quantitive RT-PCR and histochemical staining with image analysing, and compared with the results of the classic osteogenic inducer dexmethsone.In the second part of the experiment, we examined the effect sOGP on the proliferation and differentiation of the cultured BMSCs of human. We obtained BMSCs from the bone marrow in the iliac crest of the volunteers and cultured in vitro. The technique used is same as the first part. Then we compared the results of human to those of rats.In the third part of the experiment, we try to find the signaling pathway of the effect of sOGP. After adding sOGP to the human BMSCs, at 1, 2, 4, 6, 8,10, 12, 14, 16 days, we examined the change of expression levels of three members of theMAPK family: ERK, JNK and p38 and their activated form using Western-blot technique. After that, we used U0126, a specific blocker of the ERK pathway, to pretreat the BMSCs, and observed the influence on the effect of sOGP. Results: After treated with sOGP, BMSCs of rats had obvious changes in morphology. The cellular nodule and the ossicle structure appeared in the cultured cells. The electromicroscope shows the activated status of the cells. The influence of sOGP on proliferation depends on its concentrations. sOGP at the concentration of 10-11 mol/L slightly promoted the proliferation of BMSCs, while at the concentration of 10-7and 10 -9mol/L, it showed a slight suppression effect. RT-PCR testify the expression of all three marks in the sOGP group, and the measurement of ALP and cell staining showed the expression of ALP and COL I in the protein level, and proved the exist of calcium nodule. Quantitative measurement shows at the concentration of 10-9mol/L, all three osteogenic markers had the highest level of expression, and higher than that of the dexmethsone, i.e. sOGP had the greatest ability of promoting osteogenesis at this concentration.The results of human BMSCs are not the same as that of the rat BMSCs. After treated with sOGP, they also had an obvious change in morphology and cellular nodule appeared, but the ossicle structure did not appear. The influence on proliferation also depends on the concentration of sOGP, basicly it showed a slight promotion effect, while at the concentration of 10-9mol/L, the promotion effect is maximal. RT-PCR, ALP measurement and cell staining also proved the expression of the three osteogenic markers at the level of both mRNA and protein. And quantitative measurement also shows at the concentration of 10-9mol/L, sOGP has the greatest ability of promoting osteogenesis.After the action of sOGP, the expression of cellular ERK 1/2 maintained at almost the same level, while the expression of phospho-ERK1/2 elevated at the beginning of day 2 and maintained about 4days, and reached the highest level at day 4(about 5-6 times higher than the base level). This period is also the key period for the transformation of BMSCs into osteogenesis. The level of p38 also maintained during the period, while the expression of phospho-p38 elevated at the beginning of day 8 and reached the highest level at day 10(about 3 times higher than the base level). JNK and phospho-JNK did not change during the period. After the pretreatment of U0126, the elevation of ALP caused by sOGP almost disappeared, and the cells had a positive reaction to the oil red O staining, which shows BMSCs transformed into adipogenic lineage. Conclusion:1. sOGP can promote BMSCs of both rats and human into osteogenic lineage, this effect is related to its concentrations. For both rats and human BMSCs the strongest effect appear at the concentration of 10-9mol/L.2. The influence of sOGP on the proliferation of BMSCs depends on its concentrations. And it shows a slight promotion or suppression effect.3. ERK1/2 signaling pathway is essential for the effect of sOGP. After the blockage of ERK1/2 pathway, BMSCs cannot transform into osteogenic lineage but transform into adipogenic lineage.
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