损伤胰腺提取液在促进BMSCs分化为胰岛素分泌细胞的机制研究
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摘要
第一部分：损伤胰腺组织提取液对大鼠BMSCs转分化为胰岛素分泌细胞的影响
目的：
在大鼠损伤胰腺组织提取液促进BMSCs转分化为胰岛素分泌细胞过程中,动态检测转分化的胰岛素分泌细胞胰腺发育相关基因、蛋白、以及细胞形态的变化,探讨损伤胰腺组织提取液对BMSCs转分化为胰岛素分泌细胞的效果和机制。方法：
将SD大鼠胰腺切除90%,两天后处死大鼠取剩余胰腺组织匀浆,提取组织液。在大鼠BMSCs培养过程中加入损伤胰腺组织提取液,培养14天,动态检测大鼠BMSCs是否有胰腺发育相关基因和蛋白的表达,以及细胞形态学改变,对转分化的胰岛素分泌细胞检测胰岛素阳性细胞效率,葡萄糖刺激实验评价转分化的胰岛素分泌细胞分泌胰岛素的水平。
结果：
损伤胰腺组织提取液可以诱导大鼠BMSCs转分化为胰岛素分泌细胞,在转分化过程中依次表达与胰腺发育密切相关的基因,如PDX-1,NKX6.1,GLUT2,PC2,C肽等；也表达成熟胰腺表达的蛋白,如：C肽,Insulin,Nkx6.1等；分泌胰岛素的细胞占13.3±1.8%,胰岛素分泌水平为天然胰岛的1/20-1/50。
结论：
胰腺损伤后,损伤胰腺组织分泌富含胰腺修复和发育分化的相关蛋白,这些蛋白可以促进大鼠BMSCs向胰岛素分泌细胞分化。
第二部分应用飞行时间质谱技术筛选损伤胰腺组织提取液中促进转分化的相关蛋白
目的：
损伤胰腺组织提取液富含促进BMSCs转分化为胰岛素分泌细胞的相关蛋白,应用飞行时间质谱技术,对损伤胰腺蛋白提取液和正常胰腺蛋白提取液进行差异性蛋白筛选,寻找能高效定向诱导BMSCs分化为胰岛素分泌细胞的特异性可溶性蛋白。方法：
采用二维凝胶电泳(2-DE)分离和分析损伤胰腺组织提取液和正常胰腺组织提取液的蛋白差异,凝胶用硝酸银染色扫描,ImagemasterZDv5.0软件分析,筛选差异性蛋白位点,考马斯亮兰染色后切下差异蛋白位点,酶解消化后获得多肽,利用基质辅助激光解吸离子化飞行时间质谱(MALDI-TOF/TOF (MS/MS)对差异蛋白质进行鉴定,Western blot分析蛋白质cofilin-1, NDPKA, PRDX6和丝氨酸蛋白酶HTRA2在大鼠损伤胰腺组织提取液中的表达,以验证2-DE的结果。
结果：
2-DE显示损伤胰腺组织提取液与正常胰腺组织提取液平均蛋白质位点分别为1227±17个和1507±29个,其中共有50个蛋白质点出现2倍以上的表达差异。所有差异蛋白质点经MS鉴定出20种蛋白,其中表达上调者7个(编号为：7,15,16,23,34,35,42)；表达下调者13个。其中骨架蛋白的调节蛋白(cofilin-1),二磷酸核昔酸激酶A(NDPKA),组氨酸三聚体核苷结合蛋白1 (HINT1),肽基脯氨酰顺反异构酶(PPIB),过氧化物酶6 (PRDX6)和丝氨酸蛋白酶HTRA2 (serine protease HTRA2)与细胞分化和增殖有关,这些蛋白质经Western blot验证在损伤胰腺提取液中均有表达。
结论：
损伤胰腺组织提取液中存在促进胰腺发育分化的相关蛋白,应用飞行时间质谱技术筛选出cofilin-1, NDPKA, HINT1, PPIB, PRDX6和丝氨酸蛋白酶HTRA2,这些蛋白可能具有促进BMSCs转分化为胰岛素分泌细胞的作用,为干细胞高效定向分化为胰岛素分泌细胞提供了新的转分化因子。
Part I:
The effect of the injured rat pancreatic extract on transdifferentiation of BMSCs into insulin-secreting cells
Objective:To explore the effect and the mechanism of injured pancreatic extract from Sprague-Dawley rats on induction of pancreatic differentiation of BMSCs through continuous dynamic monitoring of gene, protein, cell morphological changes.
Methods:About 90% partial pancreatectomy was performed with six-week-old rats. injured pancreatic extract derived from the remnant pancreas after 48 h. After the BMSCs were induced with injured pancreatic extract for 14 days in vitro, morphological changes of the induced cells were observed. Immunocytochemical staining were used to examine pancreatic developmental protein. RT-PCR and real-time PCR were conducted to detect the expression of pancreatic endocrine gene, and Flow cytometry was used to detect the efficiency of transdifferentiation. ELISA were performed to evaluate the function of the IPCs.
Results:Insulin-secreting cells can be transdifferentiated from BMSCs by the inducement of injured pancreatic extract. during the process of induction, BMSCs can express a series of genes closely related to pancreatic development, such as PDX-1, NKX6.1, GLUT2, PC2, C-peptide, etc. and also express the protein expression of mature pancreas, such as:C-peptide, Insulin, Nkx6.1 etc. the differentiated BMSCs cells obtained nearly 13.3±1.8% insulin-positive cells by flow cytometry analysis and released 1/20-1/50 insulin in response to glucose stimuli comparable to that of natual islets.
Conclusion:affter pancreatic injured,injured pancreatic extract is rich in pancreatic development associated protein,which can induce transdifferentiation of BMSCs to insulin-secreting cells.
Part II:
Screening the related protein induced differentiation from injured pancreatic extract by mass spectrometry
Objective:Injured pancreatic extract contain associated protein which can induced transdifferentiation of BMSCs into insulin-secreting cells.by using mass spectrometry. To filter out soluble proteins from injured rats pancreatic extract and normal rats pancreatic extract that can highly efficiently induced BMSCs to differentiate into insulin-secreting cells in vitro.
Methods:differential protein profile betwent injured rats pancreatic extract and normal rats pancreatic extract were analyed by using two-dimensional gel electrophoresis (2-DE). The gels were visualized by silver staining and analysed with Image Master 2D Elite software.The protein spots with signifieant changes were visualized by Coomassie blue staining for further identifieation. Afterwards, the differential proteins were identified via in-gel digestion combined with MALDI-TOF-TOF-MS. Cofilin-1,NDPKA,PRDX6 and HTRA2 were analyzed in rat injured pancreatic extract with Western blot to confirm the resμlts of 2-DE.
Resμlts:The average spots in gels for injured rats pancreatic extract and normal rats pancreatic extract were 1227±17 and1507±29, respectively.50 spots displayed at least 2-fold of differential expression. and 20 proteins were successfμlly identified by MS,7 spots(7,15,16,23,34,35,42) were up-regμlated, and 13 spots were down-regμlated. cofilin-1,NDPKA,HINT1,PPIB,PRDX6 and HTRA2 are associated with cells differentiation and proliferation. These protein were detected from injured rats pancreatic extract by Western blot.
Conclusions:Injured rats pancreatic extract contain pancreatic development associated protein. By MS,we found injured rats pancreatic extract contains cofilin-1,NDPKA,HINT1,PPIB,PRDX6 and HTRA2 which can promote transdifferentiation of BMSCs to insulin-secreting cells. They can provide new factor to highly efficiently induced cell differentiation.
目的：
在大鼠损伤胰腺组织提取液促进BMSCs转分化为胰岛素分泌细胞过程中,动态检测转分化的胰岛素分泌细胞胰腺发育相关基因、蛋白、以及细胞形态的变化,探讨损伤胰腺组织提取液对BMSCs转分化为胰岛素分泌细胞的效果和机制。方法：
将SD大鼠胰腺切除90%,两天后处死大鼠取剩余胰腺组织匀浆,提取组织液。在大鼠BMSCs培养过程中加入损伤胰腺组织提取液,培养14天,动态检测大鼠BMSCs是否有胰腺发育相关基因和蛋白的表达,以及细胞形态学改变,对转分化的胰岛素分泌细胞检测胰岛素阳性细胞效率,葡萄糖刺激实验评价转分化的胰岛素分泌细胞分泌胰岛素的水平。
结果：
损伤胰腺组织提取液可以诱导大鼠BMSCs转分化为胰岛素分泌细胞,在转分化过程中依次表达与胰腺发育密切相关的基因,如PDX-1,NKX6.1,GLUT2,PC2,C肽等；也表达成熟胰腺表达的蛋白,如：C肽,Insulin,Nkx6.1等；分泌胰岛素的细胞占13.3±1.8%,胰岛素分泌水平为天然胰岛的1/20-1/50。
结论：
胰腺损伤后,损伤胰腺组织分泌富含胰腺修复和发育分化的相关蛋白,这些蛋白可以促进大鼠BMSCs向胰岛素分泌细胞分化。
第二部分应用飞行时间质谱技术筛选损伤胰腺组织提取液中促进转分化的相关蛋白
目的：
损伤胰腺组织提取液富含促进BMSCs转分化为胰岛素分泌细胞的相关蛋白,应用飞行时间质谱技术,对损伤胰腺蛋白提取液和正常胰腺蛋白提取液进行差异性蛋白筛选,寻找能高效定向诱导BMSCs分化为胰岛素分泌细胞的特异性可溶性蛋白。方法：
采用二维凝胶电泳(2-DE)分离和分析损伤胰腺组织提取液和正常胰腺组织提取液的蛋白差异,凝胶用硝酸银染色扫描,ImagemasterZDv5.0软件分析,筛选差异性蛋白位点,考马斯亮兰染色后切下差异蛋白位点,酶解消化后获得多肽,利用基质辅助激光解吸离子化飞行时间质谱(MALDI-TOF/TOF (MS/MS)对差异蛋白质进行鉴定,Western blot分析蛋白质cofilin-1, NDPKA, PRDX6和丝氨酸蛋白酶HTRA2在大鼠损伤胰腺组织提取液中的表达,以验证2-DE的结果。
结果：
2-DE显示损伤胰腺组织提取液与正常胰腺组织提取液平均蛋白质位点分别为1227±17个和1507±29个,其中共有50个蛋白质点出现2倍以上的表达差异。所有差异蛋白质点经MS鉴定出20种蛋白,其中表达上调者7个(编号为：7,15,16,23,34,35,42)；表达下调者13个。其中骨架蛋白的调节蛋白(cofilin-1),二磷酸核昔酸激酶A(NDPKA),组氨酸三聚体核苷结合蛋白1 (HINT1),肽基脯氨酰顺反异构酶(PPIB),过氧化物酶6 (PRDX6)和丝氨酸蛋白酶HTRA2 (serine protease HTRA2)与细胞分化和增殖有关,这些蛋白质经Western blot验证在损伤胰腺提取液中均有表达。
结论：
损伤胰腺组织提取液中存在促进胰腺发育分化的相关蛋白,应用飞行时间质谱技术筛选出cofilin-1, NDPKA, HINT1, PPIB, PRDX6和丝氨酸蛋白酶HTRA2,这些蛋白可能具有促进BMSCs转分化为胰岛素分泌细胞的作用,为干细胞高效定向分化为胰岛素分泌细胞提供了新的转分化因子。
Part I:
The effect of the injured rat pancreatic extract on transdifferentiation of BMSCs into insulin-secreting cells
Objective:To explore the effect and the mechanism of injured pancreatic extract from Sprague-Dawley rats on induction of pancreatic differentiation of BMSCs through continuous dynamic monitoring of gene, protein, cell morphological changes.
Methods:About 90% partial pancreatectomy was performed with six-week-old rats. injured pancreatic extract derived from the remnant pancreas after 48 h. After the BMSCs were induced with injured pancreatic extract for 14 days in vitro, morphological changes of the induced cells were observed. Immunocytochemical staining were used to examine pancreatic developmental protein. RT-PCR and real-time PCR were conducted to detect the expression of pancreatic endocrine gene, and Flow cytometry was used to detect the efficiency of transdifferentiation. ELISA were performed to evaluate the function of the IPCs.
Results:Insulin-secreting cells can be transdifferentiated from BMSCs by the inducement of injured pancreatic extract. during the process of induction, BMSCs can express a series of genes closely related to pancreatic development, such as PDX-1, NKX6.1, GLUT2, PC2, C-peptide, etc. and also express the protein expression of mature pancreas, such as:C-peptide, Insulin, Nkx6.1 etc. the differentiated BMSCs cells obtained nearly 13.3±1.8% insulin-positive cells by flow cytometry analysis and released 1/20-1/50 insulin in response to glucose stimuli comparable to that of natual islets.
Conclusion:affter pancreatic injured,injured pancreatic extract is rich in pancreatic development associated protein,which can induce transdifferentiation of BMSCs to insulin-secreting cells.
Part II:
Screening the related protein induced differentiation from injured pancreatic extract by mass spectrometry
Objective:Injured pancreatic extract contain associated protein which can induced transdifferentiation of BMSCs into insulin-secreting cells.by using mass spectrometry. To filter out soluble proteins from injured rats pancreatic extract and normal rats pancreatic extract that can highly efficiently induced BMSCs to differentiate into insulin-secreting cells in vitro.
Methods:differential protein profile betwent injured rats pancreatic extract and normal rats pancreatic extract were analyed by using two-dimensional gel electrophoresis (2-DE). The gels were visualized by silver staining and analysed with Image Master 2D Elite software.The protein spots with signifieant changes were visualized by Coomassie blue staining for further identifieation. Afterwards, the differential proteins were identified via in-gel digestion combined with MALDI-TOF-TOF-MS. Cofilin-1,NDPKA,PRDX6 and HTRA2 were analyzed in rat injured pancreatic extract with Western blot to confirm the resμlts of 2-DE.
Resμlts:The average spots in gels for injured rats pancreatic extract and normal rats pancreatic extract were 1227±17 and1507±29, respectively.50 spots displayed at least 2-fold of differential expression. and 20 proteins were successfμlly identified by MS,7 spots(7,15,16,23,34,35,42) were up-regμlated, and 13 spots were down-regμlated. cofilin-1,NDPKA,HINT1,PPIB,PRDX6 and HTRA2 are associated with cells differentiation and proliferation. These protein were detected from injured rats pancreatic extract by Western blot.
Conclusions:Injured rats pancreatic extract contain pancreatic development associated protein. By MS,we found injured rats pancreatic extract contains cofilin-1,NDPKA,HINT1,PPIB,PRDX6 and HTRA2 which can promote transdifferentiation of BMSCs to insulin-secreting cells. They can provide new factor to highly efficiently induced cell differentiation.
引文
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