摘要
目的:构建反义RNA干扰组织因子(TF)在猪胰岛细胞的表达,观察抑制TF表达的胰岛细胞对移植后IBMIR发生程度的影响。
方法:采用反义技术,设计5条反义RNA转染猪胰岛细胞,运用半定量逆转录-聚合酶链反应(RT-PCR)及实时定量RT-PCR方法筛选对TF沉默效果最佳反义RNA;应用IBMIR体外模型,对沉默TF后的猪胰岛细胞在IBMIR体外模型中各项指标进行检测,从而了解IBMIR发生程度。
结果:经PCR检测在5条反义RNA中抽取有沉默效果的oligoTF-1006、oligoTF-1009、oligoTF-114;该三种反义RNA转染胰岛细胞后,通过实时定量RT-PCR方法检测发现转染胰岛细胞后4天对TFmRNA的沉默效果达到最佳,分别为71.95%、59.63%、19.05%;根据PCR结果采用转染后4天的胰岛细胞加入体外IBMIR模型中,形成血凝块的质量较轻,血液各项指标显示这三组IBMIR的发生程度较空白转染对照组减轻。
结论:TF作为启动凝血途径的重要因素,与胰岛细胞移植后IBMIR的发生存在一定关系,用反义RNA转染胰岛细胞抑制其表达,可以减低胰岛细胞移植后IBMIR的发生程度,有望在将来应用在临床上,减低移植物的丢失,提高移植后胰岛细胞存活率。
Objective: Antisense RNA was constructed to inhibit expression of TF in porcine islet cell,then The Effect of inhibition of Expressing TF in islet cell was observed for IBMIR after transplantation.
Methods:5 antisense RNA were designed to transfect porcine islet cell with antisense technology,then the semiquantitative reverse transcription-PCR and realtime-quantitative reverse transcription-PCR were applied to select the best Antisense RNA for TF silencing; the indexes were detected in vitro model of IBMIR in the porcine islet cell after TF silencing, to known the level of development of IBMIR.
Result:The oligoTF-1006、oligoTF-1009、oligoTF-114 which had effect of silence were selected by PCR detection;the realtime- quantitative PCR showed that the results of TF-mRNA seliencing after 4 days by transfection of these three Antisense RNA in islet cells,which are 71.95%、59.63%、19.05% respectively; the weight of blood clot was lighter when the islet cells which were transfected 4days ago had been added in IBMIR models in vitro according to PCR,and the blood indexes showed that the development of IBMIR in these tthree groups were lighter than ones in blank transfection group.
Conclusion: There is a relation between TF which is the important factor to start blood cloting path and IBMIR after replantaion of islet cells,and antisense RNA can inhibit the espression of TF and reduce the development of IBMIR after replantaion of islet cells.This method can be used to lower the lost of grafts and increase the survival rate of islet cells clinically in the future.
引文
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