鸡胰腺干细胞生物学特性及分化机制研究
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摘要
我国家养动物遗传资源丰富，但是对遗传资源的保存亦不容忽视。干细胞作为具有自我更新能力的细胞，其特性恰恰符合畜禽遗传资源保存的要求，故以干细胞形式保存动物遗传资源意义重大。通过开展鸡胰腺干细胞研究以及向β细胞诱导分化机制研究不仅可以以干细胞的形式保存遗传资源，而且可以为β细胞体外分化机制研究提供重要实验数据。本研究成功诱导体外分离培养的鸡胰腺干细胞成为能正常分泌胰岛素的β细胞，然后利用表达谱芯片技术分析了胰腺干细胞与诱导产生的β细胞的表达谱差异，进而为阐明胰腺干细胞向β细胞的分化机制提供依据。得到如下结果：
1、采用酶消化法结合差速贴壁法分离得到的胰腺干细胞扩增培养至P3代，流式细胞仪分选得到Nestin+细胞在体外可连续培养至P23代。所培养细胞多以明显的克隆样生长，细胞核圆形或肾形，胞核较大；冻存前后的细胞活率差异不显著（p>0.05）；细胞表现强的体外增殖能力；细胞免疫荧光、流式细胞术和PCR结果均能检测到胰腺干细胞表面标志物（Pdx-1和Nestin）。结果表明，所采用优化的鸡胰腺干细胞培养、传代、冻存和复苏体系得到了良好的实验结果，所培养的细胞具有胰腺干细胞的特征，符合细胞生物学和胰腺干细胞体外扩增规律，经细胞表面标志物鉴定证明所分离培养的细胞为胰腺干细胞。
2、用β细胞诱导液诱导P3代Nestin+细胞，可见到典型的β细胞囊泡的出现，双硫腙染色呈现阳性；葡萄糖刺激实验证明了诱导得到的细胞具有分泌胰岛素的能力；Fluo-4/AM标记钙离子浓度监测实验也证明诱导得到的细胞在分泌胰岛素的同时伴随了钙离子通道的变化。结果表明，诱导产生的细胞为β细胞具有体外分泌胰岛素的能力。
3、基因芯片分析鸡胰腺干细胞和诱导得到的β细胞的表达谱结果显示，差异表达基因集中分布的20个信号通路中，有10个通路与β细胞发育密切相关；多个转录因子在不同信号通路上存在共同调节；在胰腺干细胞向β细胞分化中研究热点的Wnt、Notch、TGFβ和PDGF四个通路上共得到46个差异基因，24个上调基因，22个下调基因；Real time PCR验证这46个差异基因在诱导过程中的表达，有3个基因（DLC1，FZD3和SFRP2）的表达与Real time PCR结果不一致，吻合度为95.6%。另外还发现了多数差异基因在诱导过程中的表达相对恒定，但JUN、VAV2、MAPK13、BMP6和WNT5A共5个基因在诱导过程中则是从上调转变成下调，而DLL1则是从下调转变为上调。这些基因可能在鸡胰腺干细胞体外诱导分化为胰腺β细胞过程中起到关键性作用或明显受到其他基因的调控。
综上所述，通过本研究的实施，成功体外分离培养了鸡胰腺干细胞并完成了鉴定，利用基因芯片分析了鸡胰腺干细胞和诱导得到的β细胞的表达谱，得到了46个与β细胞分化密切相关的关键基因，并用Real time PCR验证了这些基因在诱导过程中的动态表达，阐释了这些基因在体外诱导过程中的表达变化规律。
In our nation, the genetic resources of domesticated animals are abundant, while theirconservation is far from optimism. Stem cell possesses the renewability and multipotency andmeets the requirements of storing, which make it a seeding cell to preserve the genetic resourcesfrom livestock and poultry with a great significance. In this work, we established a chickenpancreatic stem cell line, then induced them differentiated into beta cells which could produceinsulin, analyzed the difference of expression profile between the natural differentiated beta cellsand induced ones via gene chip technology and explored the differentiation mechanism. Results:
1. The method of enzymic digestion and differential attachment was suitable to derivedpancreatic stem cells in vitro and cell were cultured for3passages, the Nestin+cells sorted byflow cytometry were generated to passage23. The majority of cells presented clone-like growth,the nuclei were round or in kidney-like shape, no significant difference of cell viability before andafter cryopreservation（p>0.05）and the proliferation ability was strong, the pancreatic stem cellspecific surface makers Pdx-1and Nestin were detected by immunofluorescence,flow cytometryand RT-PCR, resulted positively, which suggested the optimized culture system was appropriatefor the cell line, the biological characterization was unique to pancreatic stem cell.
2. The induced beta cells displayed typical vesicles and stain positively by dithizone, andthey were proved the ability of insulin secretion by glucose stimulation experiment, the analysis ofcalcium ion concentration by maker Fluo-4/AM also demonstrated the secretion activities, thesesuggested a success induction.
3. The gene chip showed there are20signal paths concentrating distributed and10areclosely related to the development of beta cells, multiple transcript factors at different paths couldregulated in some sense,46differential genes were discovered among Wnt, Notch, TGFβ andPDGF signal paths including24up-regulated genes and22down-regulated genes, Real time PCRresults showed that only gene DLC1, FZD3and SFRP2was not accordance with gene chipanalysis, the coincide rate was95.6%, besides, JUN, VAV2, MAPK13, BMP6and WNT5A werehighly significant down regulated, gene DLL1highly significant up-regulated. These genes mayplay a key role or significantly regulated by other genes in the differentiation process from PSCsinto pancreatic β cells.
In this study, we obtained a stable chicken pancreatic stem cell line and accomplished thecharacterization, we gained the expression profile of β cells via gene chip and found46relativegenes, the dynamic expression of those genes was accessed by Real time PCR and the expressionpattern was expounded.
1、采用酶消化法结合差速贴壁法分离得到的胰腺干细胞扩增培养至P3代，流式细胞仪分选得到Nestin+细胞在体外可连续培养至P23代。所培养细胞多以明显的克隆样生长，细胞核圆形或肾形，胞核较大；冻存前后的细胞活率差异不显著（p>0.05）；细胞表现强的体外增殖能力；细胞免疫荧光、流式细胞术和PCR结果均能检测到胰腺干细胞表面标志物（Pdx-1和Nestin）。结果表明，所采用优化的鸡胰腺干细胞培养、传代、冻存和复苏体系得到了良好的实验结果，所培养的细胞具有胰腺干细胞的特征，符合细胞生物学和胰腺干细胞体外扩增规律，经细胞表面标志物鉴定证明所分离培养的细胞为胰腺干细胞。
2、用β细胞诱导液诱导P3代Nestin+细胞，可见到典型的β细胞囊泡的出现，双硫腙染色呈现阳性；葡萄糖刺激实验证明了诱导得到的细胞具有分泌胰岛素的能力；Fluo-4/AM标记钙离子浓度监测实验也证明诱导得到的细胞在分泌胰岛素的同时伴随了钙离子通道的变化。结果表明，诱导产生的细胞为β细胞具有体外分泌胰岛素的能力。
3、基因芯片分析鸡胰腺干细胞和诱导得到的β细胞的表达谱结果显示，差异表达基因集中分布的20个信号通路中，有10个通路与β细胞发育密切相关；多个转录因子在不同信号通路上存在共同调节；在胰腺干细胞向β细胞分化中研究热点的Wnt、Notch、TGFβ和PDGF四个通路上共得到46个差异基因，24个上调基因，22个下调基因；Real time PCR验证这46个差异基因在诱导过程中的表达，有3个基因（DLC1，FZD3和SFRP2）的表达与Real time PCR结果不一致，吻合度为95.6%。另外还发现了多数差异基因在诱导过程中的表达相对恒定，但JUN、VAV2、MAPK13、BMP6和WNT5A共5个基因在诱导过程中则是从上调转变成下调，而DLL1则是从下调转变为上调。这些基因可能在鸡胰腺干细胞体外诱导分化为胰腺β细胞过程中起到关键性作用或明显受到其他基因的调控。
综上所述，通过本研究的实施，成功体外分离培养了鸡胰腺干细胞并完成了鉴定，利用基因芯片分析了鸡胰腺干细胞和诱导得到的β细胞的表达谱，得到了46个与β细胞分化密切相关的关键基因，并用Real time PCR验证了这些基因在诱导过程中的动态表达，阐释了这些基因在体外诱导过程中的表达变化规律。
In our nation, the genetic resources of domesticated animals are abundant, while theirconservation is far from optimism. Stem cell possesses the renewability and multipotency andmeets the requirements of storing, which make it a seeding cell to preserve the genetic resourcesfrom livestock and poultry with a great significance. In this work, we established a chickenpancreatic stem cell line, then induced them differentiated into beta cells which could produceinsulin, analyzed the difference of expression profile between the natural differentiated beta cellsand induced ones via gene chip technology and explored the differentiation mechanism. Results:
1. The method of enzymic digestion and differential attachment was suitable to derivedpancreatic stem cells in vitro and cell were cultured for3passages, the Nestin+cells sorted byflow cytometry were generated to passage23. The majority of cells presented clone-like growth,the nuclei were round or in kidney-like shape, no significant difference of cell viability before andafter cryopreservation（p>0.05）and the proliferation ability was strong, the pancreatic stem cellspecific surface makers Pdx-1and Nestin were detected by immunofluorescence,flow cytometryand RT-PCR, resulted positively, which suggested the optimized culture system was appropriatefor the cell line, the biological characterization was unique to pancreatic stem cell.
2. The induced beta cells displayed typical vesicles and stain positively by dithizone, andthey were proved the ability of insulin secretion by glucose stimulation experiment, the analysis ofcalcium ion concentration by maker Fluo-4/AM also demonstrated the secretion activities, thesesuggested a success induction.
3. The gene chip showed there are20signal paths concentrating distributed and10areclosely related to the development of beta cells, multiple transcript factors at different paths couldregulated in some sense,46differential genes were discovered among Wnt, Notch, TGFβ andPDGF signal paths including24up-regulated genes and22down-regulated genes, Real time PCRresults showed that only gene DLC1, FZD3and SFRP2was not accordance with gene chipanalysis, the coincide rate was95.6%, besides, JUN, VAV2, MAPK13, BMP6and WNT5A werehighly significant down regulated, gene DLL1highly significant up-regulated. These genes mayplay a key role or significantly regulated by other genes in the differentiation process from PSCsinto pancreatic β cells.
In this study, we obtained a stable chicken pancreatic stem cell line and accomplished thecharacterization, we gained the expression profile of β cells via gene chip and found46relativegenes, the dynamic expression of those genes was accessed by Real time PCR and the expressionpattern was expounded.
引文
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