| Objective:To study the effect of related gene of encephalic region and bone morhogenetic protein 4 (BMP4) in the processing of from mesenchyrnal stem cells (MSCs) into neurons induced by rchmannia glutnosa polysaccharides (RPS), and on the ground of that clarify the relation of kidney being in charge of formation of brain, marrow and bone MSCs straddle embryonic layer angle.Methods:1. MSCs were sepatrated from thighbones marrow of mousse, different time for the first exchange of medium, the purity and grew character were evaluated by cell counting and indirect immunocytochemisty staining.2. The 3rd generation of MSCs were induced by RPS, BMP4, RPS relates of BMP4. After 24h, We use reverse transcription-polymerase chain reaction (RT-PCR) to detect BMP4 mRNA expression and indirect immunocytochemisty staining to detect the neuronal specific markers neurofilement protein (NF) and glial fibriliary acidic protein (GAFP). 3. The 3rd generation of MSCs were induced by RPS, BMP4,RPS relates of BMP4. After 24h, We use reverse transcription-polymerase chain reaction (RT-PCR) to detect the related gene of encephalic region:Emx2 mRNA, Otx2 mRNA and Hoxbl mRNA expression.Results:1. The cells after first exchange of medium in 24 hours were slow growth, the positive rate of CD44 and CD45 is (88.11±1.67)%and (26.37±2.60)%. The cells after first exchange of medium in 3 days grew up well the positive rate of CD44 and CD45 is (85.18±1.86)%and (29.68±1.29)%.The cells after first exchange of medium in 5 days grew up well in the first 6 days,but decreased thereafter the positive rate of CD44 and CD45 is (79.63±1.53)and (36.55±1.22) %. Among in the 3 groups, Compared with each other's surface markes,the positive rate of CD44,CD45 had significantly difference(P<0.01).2. There is few BMP4 mRNA expression in the complete medium group, is (0.25±0.3), RPS can promote BMP4 mRNA expression, is (0.58±0.7), having significantlydifference between the two groups(P< 0.01).3. There are few NF200 and GFAP expression in the complete medium group, which is (8.47±1.65)%and (5.97±1.48)%, compared with RPS group,BMP4 group and RPS related BMP4 group(P< 0.01). RPS group,BMP4 group and RPS related BMP4 group can promote NF200 and GFAP expression, the positive rate is (63.43±4.68)%,(14.05±2.88)%,(48.93±5.23)%and(16.55±1.96) %,(77.25±3.27)%,(23.36±2.96)%. At the positive rate of NF200 and GFAP,the RPS related BMP4 group compared with RPS group and BMP4 group had significantly difference(P<0.01). In the RPS group the NF200 positive expression is more than in the BMP4 group(P<0.01),but GFAP positive expression is less than BMP4 group (P<0.05).4. There is few Emx2 mRNA. Otx2 mRNA, Hoxbl mRNA expression in the-complete medium group, which is (0.49±0.028),(0.28±0.045) and (0.077±0.016), RPS group,BMP4 group and RPS related BMP4 group can promote their Expression,is (0.77±0.045), (0.50±0.043), (0.311±0.049), (0.83±0.042), (0.62±0.047),(0.405±0.035). Compareing With the complete medium group, the RPS group and RPS related BMP4 group are Significantly increase (P<0.01) compared with RPS group, the Emx2 mRNA and Otx2 mRNA expression in RPS related BMP4 group are increase(P<0.01),the Hoxbl mRNA expression also increase (P<0.05).Conclusion:1. Different time for the first exchange of medium to bone mesenchymal stem cells with wall sticking method has influence.If it is only to consider the cells' purity, the cells first exchange of medium in 24 hours were better. If synthetical consideration of growth and purity, he cells first exchange of medium in 24 hours were better.2. BMP4 can promote MSCs differentiate into nerve cells, and RPS induce MSCs differentiate in to nerve cells by the effect of BMP4.3. There are related gene of encephalic region Emx2 mRNA,Otx2 mRNA,Hoxbl mRNA expression in the process of MSCs differentiate into nerve cells,and RPS can promote these genes expression.4. From MSCs straddle embryonic layer angle, we clarify the relation of kidney being in charge of formation of brain and marrow.
|
PDF Full Text Request