Gene therapy for diabetes using liganded-thyroid hormone receptor

糖尿病再生医学的一个目标是将成熟的胰腺外分泌细胞转化为胰岛素产生细胞。我们最近报道了结合型甲状腺激素受体α(TrRα)在出生后发育过程中对β细胞团的扩张起关键作用。Adtrα是一种在巨细胞病毒启动子控制下表达人Trα1的重组腺病毒载体。为了分析重组α基因转移是否诱导胰腺外分泌细胞重编程为胰岛素分泌细胞，将重组α注入免疫缺陷小鼠胰腺内。用AdtRα感染具有外分泌和神经内分泌特性的大鼠胰腺AR42J细胞。用定量RT-PCR、免疫印迹或免疫细胞化学方法分析参与胰腺内分泌细胞分化的转录因子的表达。为了探讨α诱导的胰腺外分泌细胞重编程是直接作用还是间接作用，用AdtRα感染的AR42J细胞同时转染NGN3或MafA的小干扰RNA，观察到Adtrα感染的小鼠有少量表达脂肪酶的胰岛素分泌细胞群。与未处理的AR42J细胞相比，感染AdtRα并经激活素A预处理的AR42J细胞经T3处理后，Ngn3和MafAmRNA和蛋白的表达水平均增加。在激活素A处理的AR42J细胞中，过表达TRα和T3处理也诱导了胰岛素的表达。小干扰RNA抑制NGN3或MafA的表达可显著抑制Adtrα诱导的AR42J细胞向胰岛素分泌细胞的重编程。结论：配体结合α和激活素A可通过诱导NGN3和MafA将胰腺外分泌细胞重编程为胰岛素产生细胞。我们的发现也支持这一假设，即连接的α在出生后发育过程中对β细胞的再生起着关键作用。

One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that liganded thyroid hormone receptorα(TRα) plays a critical role in expansion of theβ-cell mass during postnatal development.AdTRαis a recombinant adenoviral vector that expresses human TRα1 under the control of the cytomegalovirus promoter. To analyze whether TRαgene transfer induces reprogramming of pancreatic exocrine cells to insulin-producing cells, AdTRαwere injected into the pancreas of immunodeficient mice. Rat pancreatic AR42J cells that possess exocrine and neuroendocrine properties were infected with AdTRα. The expression of transcription factors that are involved in the differentiation of pancreatic endocrine cells was then analyzed by quantitative RT-PCR, western blot or immunocytochemistry. To explore whether liganded-TRα-induced reprogramming of pancreatic exocrine cells is direct or indirect effect, AdTRα-infected AR42J cells were concomitantly transfected with siRNA of Ngn3 or MafA.Small scattered clusters of insulin-producing cells, which also expressed lipase, were observed in AdTRα-infected mice. T3-treatment of AR42J cells that were infected with AdTRαand pretreated with activin A increased the mRNA and protein expression levels of Ngn3 and MafA, compared to no T3-treatment. Overexpression of TRαtogether with T3-treatment also induced insulin expression in activin A-treated AR42J cells. The siRNA-induced inhibition of expression of Ngn3 or MafA significantly inhibited AdTRα-induced reprogramming of AR42J cells into insulin-producing cells.Conclusions : These results suggested that combination of liganded-TRαand activin A leads to reprogramming pancreatic exocrine cells to insulin-producing cells via induction of Ngn3 and MafA. Our findings also support the hypothesis that liganded-TRαplays a critical role inβ-cell regeneration during postnatal development.