地黄多糖对MSCs向神经细胞分化过程中脑区相关基因和BMP4的影响
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摘要
目的：探讨地黄多糖对骨髓间充质干细胞(MSCs)向神经细胞分化过程中脑区相关基因和骨形态发生蛋白4(BMP4)表达的影响,从MSCs跨胚层分化角度阐述中医”肾生髓,通于脑”的理论。
方法
1.采用体外贴壁培养法筛选分离小鼠MSCs,于不同时间首次换液,用细胞计数和免疫细胞化学法检测MSCs的纯度和生长特性。
2.传3代后的细胞分为完全培养基组、地黄多糖组、BMP4组和地黄多糖合BMP4组,培养24小时后,完全培养基组、地黄多糖组用逆转录-聚合酶链反应(RT-PCR)法检测MSCs分化细胞的BMP4 mRNA表达水平。同时用免疫细胞化学法检测四组的MSCs分化细胞的NF200和GFAP表达。
3.传3代后的细胞分为完全培养基组、地黄多糖组和地黄多糖合BMP4组。培养24小时后,用RT-PCR半定量分析三组中脑区相关基因Emx2 mRNA, Otx2 mRNA和Hoxbl mRNA的表达。
结果
1.24h首次换液组,细胞生长较缓慢,对数生长期晚,CD44阳性表达率最高,为(88.11±1.67)%,CD45阳性表达率最低,为(26.37±2.60)%；3d首次换液组,细胞增殖速度快,对数生长期早；5d首次换液组,前5d生长速度较快,6d后生长渐缓慢,CD44阳性表达率为(79.63±1.53)
%,CD45阳性表达率最高,为(36.55±1.22)%。三组间比较CD44、CD45阳性表达率均有极显著性差异(P＜0.01)。
2.在完全培养基组有较低水平的BMP4 mRNA表达,为(0.25±0.3),而地黄多糖能促进BMP4 mRNA的表达,为(0.58±0.7)。两者相比有极显著性差异(P＜0.01)。
3.完全培养基组仅有少量NF200和GFAP阳性细胞分化,分别为(8.47±1.65)%和(5.97±1.48)%,与地黄多糖组、BMP4组和地黄多糖合BMP4组相比均有极显著性差异(P＜0.01)。地黄多糖、BMP4、地黄多糖合BMP4均能促进上述NF200和GFAP阳性细胞的分化水平,其阳性表达率分别为(63.43±4.68)%和(14.05±2.88)%、(48.93±5.23)%和(16.55±1.96)%、(77.25±3.27)%和(23.36±2.96)%,均以NF200为主。在NF200、GFAP阳性表达率两方面,地黄多糖联合BMP4组与地黄多糖组和BMP4组相比都有极显著差异(P＜0.01)。而地黄多糖组NF200阳性表达率较BMP4组增多(P＜0.01),GFAP表达率较BMP4组少(P＜0.05)。
4.完全培养基组有较低水平的Emx2 mRNA、Otx2 mRNA、Hoxb1 mRNA的表达,分别为(0.49±0.028)、(0.28±0.045)和(0.077±0.016),地黄多糖、地黄多糖合BMP4能促进上述表达.分别为(0.77±0.045)、(0.50±0.043)、(0.311±0.0 49)和(0.83±0.042)、(0.62±0.047)、(0.405±0.0 35)。与完全培养基组比较,地黄多糖组和地黄多糖合BMP4组表达增高显著(P＜0.01)；其中地黄多糖合BMP4组表达最高,与地黄多糖组比Emx2mRNA、Otx2mRNA表达显著增多(P<0.01), Hoxb1 mRNA的表达也较多(P＜0.05)。
结论
1.不同时间首次换液对贴壁法MSCs的生长有影响,单从细胞纯度来考虑,24h首次换液较为理想,从细胞生长情况和纯度综合考虑,3d首次换液较为理想。
2.BMP4能促进MSCs向神经细胞分化,地黄多糖通过影响BMP4诱导MSCs向神经细胞分化。
3. MSCs向神经分化过程中有脑区相关基因Emx2 mRNA,Otx2 mRNA和Hoxbl mRNA的表达,地黄多糖能增强这些基因的表达。
4.从MSCs跨胚层分化角度阐述了中医“肾生髓,通于脑”的理论,进一步丰富了中医脏象学说。
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.
方法
1.采用体外贴壁培养法筛选分离小鼠MSCs,于不同时间首次换液,用细胞计数和免疫细胞化学法检测MSCs的纯度和生长特性。
2.传3代后的细胞分为完全培养基组、地黄多糖组、BMP4组和地黄多糖合BMP4组,培养24小时后,完全培养基组、地黄多糖组用逆转录-聚合酶链反应(RT-PCR)法检测MSCs分化细胞的BMP4 mRNA表达水平。同时用免疫细胞化学法检测四组的MSCs分化细胞的NF200和GFAP表达。
3.传3代后的细胞分为完全培养基组、地黄多糖组和地黄多糖合BMP4组。培养24小时后,用RT-PCR半定量分析三组中脑区相关基因Emx2 mRNA, Otx2 mRNA和Hoxbl mRNA的表达。
结果
1.24h首次换液组,细胞生长较缓慢,对数生长期晚,CD44阳性表达率最高,为(88.11±1.67)%,CD45阳性表达率最低,为(26.37±2.60)%；3d首次换液组,细胞增殖速度快,对数生长期早；5d首次换液组,前5d生长速度较快,6d后生长渐缓慢,CD44阳性表达率为(79.63±1.53)
%,CD45阳性表达率最高,为(36.55±1.22)%。三组间比较CD44、CD45阳性表达率均有极显著性差异(P＜0.01)。
2.在完全培养基组有较低水平的BMP4 mRNA表达,为(0.25±0.3),而地黄多糖能促进BMP4 mRNA的表达,为(0.58±0.7)。两者相比有极显著性差异(P＜0.01)。
3.完全培养基组仅有少量NF200和GFAP阳性细胞分化,分别为(8.47±1.65)%和(5.97±1.48)%,与地黄多糖组、BMP4组和地黄多糖合BMP4组相比均有极显著性差异(P＜0.01)。地黄多糖、BMP4、地黄多糖合BMP4均能促进上述NF200和GFAP阳性细胞的分化水平,其阳性表达率分别为(63.43±4.68)%和(14.05±2.88)%、(48.93±5.23)%和(16.55±1.96)%、(77.25±3.27)%和(23.36±2.96)%,均以NF200为主。在NF200、GFAP阳性表达率两方面,地黄多糖联合BMP4组与地黄多糖组和BMP4组相比都有极显著差异(P＜0.01)。而地黄多糖组NF200阳性表达率较BMP4组增多(P＜0.01),GFAP表达率较BMP4组少(P＜0.05)。
4.完全培养基组有较低水平的Emx2 mRNA、Otx2 mRNA、Hoxb1 mRNA的表达,分别为(0.49±0.028)、(0.28±0.045)和(0.077±0.016),地黄多糖、地黄多糖合BMP4能促进上述表达.分别为(0.77±0.045)、(0.50±0.043)、(0.311±0.0 49)和(0.83±0.042)、(0.62±0.047)、(0.405±0.0 35)。与完全培养基组比较,地黄多糖组和地黄多糖合BMP4组表达增高显著(P＜0.01)；其中地黄多糖合BMP4组表达最高,与地黄多糖组比Emx2mRNA、Otx2mRNA表达显著增多(P<0.01), Hoxb1 mRNA的表达也较多(P＜0.05)。
结论
1.不同时间首次换液对贴壁法MSCs的生长有影响,单从细胞纯度来考虑,24h首次换液较为理想,从细胞生长情况和纯度综合考虑,3d首次换液较为理想。
2.BMP4能促进MSCs向神经细胞分化,地黄多糖通过影响BMP4诱导MSCs向神经细胞分化。
3. MSCs向神经分化过程中有脑区相关基因Emx2 mRNA,Otx2 mRNA和Hoxbl mRNA的表达,地黄多糖能增强这些基因的表达。
4.从MSCs跨胚层分化角度阐述了中医“肾生髓,通于脑”的理论,进一步丰富了中医脏象学说。
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.
引文
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