Development of in vivo biosensors using genetic engineering

1. 使用炎症反应调节元件作为分子传感器，我们建立了一种基于细胞的生物传感器，用于连续、无创地监测体内局部微观炎症。以肾小球肾炎作为局部炎症模型。在KB增强子元件的控制下，用编码分泌碱性磷酸酶（SEAP）的标记基因稳定转染系膜细胞。建立的传感器细胞经肾循环选择性转入大鼠肾小球。急性肾小球肾炎（anti-Thy 1.1肾炎）诱导后，细胞转移肾病大鼠血清SEAP水平短暂升高。血清SEAP动力学与炎症的自然过程密切相关。这些结果表明，无需组织活检等侵入性程序，通过局部创建的基于细胞的生物传感器，可以在体内对微观炎症进行连续、实时监测。2 .更多。内质网（ER）应激的监测我们发现，转染细胞产生的内质网应激反应性碱性磷酸酶（ES-TRAP）的活性在内质网应激下迅速下调，而不依赖于转录调节。这一现象在多种内质网应激诱导剂引发的多种细胞类型中均可观察到，ES-TRAP的下降幅度与内质网应激的程度成正比。在组成性产生ES-TRAP的转基因小鼠中，thapsignargin系统性诱导内质网应激导致血清ES-TRAP活性降低。在这些小鼠中，脂多糖和镉也引起了血清ES-TRAP活性的快速、短暂的降低，这与几个器官内源性内质网应激标志物的上调有关。这些结果首次阐明了内毒素血症和重金属毒性中内质网应激的可能参与，并为ES-TRAP在体内连续、实时监测内质网应激提供了证据。少

1. Monitoring of inflammation Using an inflammation-responsive regulatory element as a molecular sensor, we established a cell-based biosensor for continuous, non-invasive monitoring of local microscopic inflammation in vivo. Glomerulonephritis was used as a model of local inflammation. Mesangial cells were stably transfected with a marker gene encoding secreted alkaline phosphatase (SEAP) under the control of the KB enhancer elements. The established sensor cells were transferred selectively into rat glomeruli via the renal circulation. After induction of acute glomerulonephritis (anti-Thy 1.1 nephritis), the serum level of SEAP was increased transiently in cell-transferred nephritic rats. The kinetics of serum SEAP was closely correlated with the natural course of the inflammation. These results suggested that, without invasive procedures like tissue biopsies, continuous, real-time monitoring of microscopic inflammation is feasible in vivo via locally created, cell-based biosensors.2 … More . Monitoring of endoplasmic reticulum (ER) stress We found that activity of ER stress-responsive alkaline phosphatase (ES-TRAP) produced by transfected cells is rapidly downregulated by ER stress independent of transcriptional regulation. This phenomenon was observed in a wide range of cell types triggered by various ER stress inducers, and the magnitude of the decrease in ES-TRAP was proportional to the extent of ER stress. In transgenic mice constitutively producing ES-TRAP, systemic induction of ER stress by thapsigargin led to reduction in serum ES-TRAP activity. In these mice, administration with lipopolysaccharide and cadmium also caused rapid, transient decrease in serum ES-TRAP activity, and it was correlated with up-regulation of endogenous ER stress markers in several organs. These results elucidated for the first time a possible involvement of ER stress in endotoxemia and toxicity of heavy metal and provided evidence for usefulness of ES-TRAP for continuous, real-time monitoring of ER stress in vivo. Less