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首页> 外文期刊>World Journal of Gastroenterology >Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method
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Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method

机译：使用pax4-核转染法增强从胚胎干细胞中产生胰岛素的细胞分化

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摘要

AIM: To enhance the differentiation of insulin producing cell (IPC) ability from embryonic stem (ES) cells in vitro. METHODS: Four-day embryoid body (EB)-formatted ES cells were dissociated as single cells for the followed plasmid DNA delivery. The use of Nucleofector™ electroporator (Amaxa biosystems, Germany) in combination with medium-contained G418 provided a high efficiency of gene delivery for advanced selection. Neucleofected cells were plated on the top of fibronectin-coated Petri dishes. Addition of Ly294002 and raised the glucose in medium at 24 h before examination. The differentiation status of these cells was monitored by semi-quantitative PCR (SQ-PCR) detection of the expression of relative genes, such as oct-4, sox-17, foxa2, mixl1, pdx-1, insulin 1, glucagons and somatostatin. The percentage of IPC population on d 18 of the experiment was investigated by immunohistochemistry (IHC), and the content/secretion of insulin was estimated by ELISA assay. The mice with severe combined immunodeficiency disease (SCID) pretreated with streptozotocin (STZ) were used to eliminate plasma glucose restoration after pax4~+ ES implantation. RESULTS: A high efficiency of gene delivery was demonstrated when neucleofection was used in the present study; approximately 70% cells showed DsRed expression 2 d after neucleofection. By selection of medium-contained G418, the percentage of DsRed expressing cells kept high till the end of study. The pancreatic differentiation seemed to be accelerated by pax4 nucleofection. When compared to the group of cells with mock control, foxa2, mixl1, pdx1, higher insulin and somatostatin levels were detected by SQ-PCR 4 d after nucleofection in the group of pax4 expressing plasmid delivery. Approximately 55% of neucleofected cells showed insulin expression 18 d after neucleofection, and only 18% of cells showed insulin expression in mock control. The disturbance was shown by nucleofected pax4 RNAi vector; only 8% of cells expressed insulin 18 d after nucleofection. A higher IPC population was also detected in the insulin content by ELISA assay, and the glucose dependency was demonstrated in insulin secretion level. In the animal model, improvement of average plasma glucose concentration was observed in the group of pax-4 expressed ES of SCID mice pretreated with STZ, but no significant difference was observed in the group of STZ-pretreated SCID mice who were transplanted ES with mock plasmid. CONCLUSION: Enhancement of IPC differentiation from EB-dissociated ES cells can be revealed by simply using pax4 expressing plasmid delivery. Not only more IPCs but also pancreatic differentiation-related genes can be detected by SQ-PCR. Expression of relative genes, such as foxa 2, mixl 1, pdx-1, insulin 1 and somatostatin after nucleofection, suggests that pax4 accelerates the whole differentiation progress. The higher insulin production with glucose dependent modulation suggests that pax4 expression can drive more mature IPCs. Although further determination of the entire mechanism is required, the potential of pax-4-nucleofected cells in medical treatment is promising.
机译：目的：增强体外从胚胎干（ES）细胞分化胰岛素产生细胞（IPC）的能力。方法：将四天胚状体（EB）格式的ES细胞解离为单个细胞，用于随后的质粒DNA递送。 Nucleofector™电穿孔仪（德国Amaxa生物系统公司）与中等含量的G418的结合使用可为高效基因选择提供高效的基因选择。将经神经检测的细胞铺板在纤连蛋白包被的培养皿的顶部。在检查前24小时，添加Ly294002并使培养基中的葡萄糖升高。通过半定量PCR（SQ-PCR）检测相关基因（例如oct-4，sox-17，foxa2，mixl1，pdx-1，胰岛素1，胰高血糖素和生长抑素）的表达来监测这些细胞的分化状态。在实验的第18天，通过免疫组织化学（IHC）研究IPC群体的百分比，并通过ELISA分析评估胰岛素的含量/分泌。接受链脲佐菌素（STZ）预处理的患有严重合并免疫缺陷病（SCID）的小鼠用于消除pax4〜+ ES植入后的血浆葡萄糖恢复。结果：在本研究中使用核转染证明了基因传递的高效率；核转染后2 d约有70％的细胞显示DsRed表达。通过选择中等含量的G418，DsRed表达细胞的百分比一直保持高水平，直到研究结束。 pax4核转染似乎促进了胰腺分化。当与具有模拟对照的细胞组比较时，在表达质粒的pax4组中，经核转染后4 d，通过SQ-PCR检测到了foxa2，mixl1，pdx1，较高的胰岛素和生长抑素水平。在模拟转染后18 d，大约55％的经神经检测的细胞显示胰岛素表达，而在模拟对照中只有18％的细胞显示胰岛素表达。核转染的pax4 RNAi载体显示了这种干扰。核转染后18 d只有8％的细胞表达胰岛素。通过ELISA分析还检测到较高的IPC人群的胰岛素含量，并且在胰岛素分泌水平上证明了葡萄糖依赖性。在动物模型中，在用STZ预处理的SCID小鼠的pax-4表达的ES组中观察到平均血浆葡萄糖浓度的改善，但是在用ST预处理的ESZ预处理的SCID小鼠组中未观察到显着差异。质粒。结论：通过简单地使用表达pax4的质粒传递可以揭示从EB分离的ES细胞中IPC分化的增强。通过SQ-PCR不仅可以检测到更多的IPC，而且可以检测到与胰腺分化相关的基因。核转染后相关基因的表达，例如foxa 2，mixl 1，pdx-1，胰岛素1和生长抑素，表明pax4加速了整个分化过程。具有葡萄糖依赖性调节作用的较高胰岛素产生表明pax4表达可以驱动更成熟的IPC。尽管需要进一步确定整个机制，但pax-4-核转染细胞在医学治疗中的潜力是有希望的。

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