Tagouri Y M, Sanders P W, Picken M M, Siegal G P, Kerby J D, Herrera G A
Vaughan Health Care, Selma, Alabama, USA.
Lab Invest. 1996 Jan;74(1):290-302.
AA-amyloid has been produced experimentally in animal models, allowing the study of mechanisms involved in AA-amyloidogenesis, but those involved in renal AL-amyloidogenesis have not been adequately investigated due, in part, to lack of appropriate in vitro models. Rat and human mesangial cells were grown on a human extracellular matrix (Amgel) derived from normal tissues and on coverslips in the presence of 10 microliters of amyloid enhancing factor (AEF) per milliliter of media and 10 micrograms/ml monoclonal lambda light chains (LCs) obtained from two patients with AL-amyloidosis. Two additional lambda LCs derived from the urine of patients with myeloma and tubulointerstitial renal disease were used as controls. To verify amyloid deposition, light and electron microscopic examination, as well as Congo red and thioflavin T staining, were performed on samples incubated under different experimental conditions. Intracellular and extracellular amyloid was identified in samples incubated for 24 hours with human mesangial cells (for 48 hours with rat mesangial cells), amyloidogenic monoclonal LCs, and AEF. The amount of amyloid detected, which increased with longer incubation times, was found to be most abundant at 14 days. Amyloid was not present in cultures of mesangial cells incubated with amyloidogenic LCs alone or in the absence of mesangial cells. Likewise, incubation of mesangial cells with amyloidogenic LC or AEF separately or amyloidogenic LC in the presence of AEF but without mesangial cells did not result in amyloid formation. Amyloid was not seen when LCs obtained from the urine of patients with tubulointerstitial renal disease were incubated with AEF and mesangial cells. AL-amyloid production requires all three components--mesangial cells, amyloidogenic LCs, and AEF. In addition, amyloid was detected intracellular in mesangial cells, supporting the hypothesis that the production of AL-amyloid in the kidney requires intracellular processing by these cells. This system provides a unique experimental model to study renal AL-amyloidogenesis and a platform to explore mesangial cell-matrix interactions.
AA淀粉样蛋白已在动物模型中通过实验产生，这使得对AA淀粉样蛋白生成所涉及机制的研究成为可能，但肾AL淀粉样蛋白生成所涉及的机制尚未得到充分研究，部分原因是缺乏合适的体外模型。大鼠和人系膜细胞在源自正常组织的人细胞外基质（Amgel）上以及盖玻片上生长，培养基中每毫升含有10微升淀粉样蛋白增强因子（AEF）以及从两名AL淀粉样变性患者获得的10微克/毫升单克隆λ轻链（LCs）。另外两种源自骨髓瘤和肾小管间质性肾病患者尿液的λ轻链用作对照。为了验证淀粉样蛋白沉积，对在不同实验条件下孵育的样本进行了光镜和电镜检查以及刚果红和硫黄素T染色。在与人系膜细胞孵育24小时（与大鼠系膜细胞孵育48小时）、淀粉样蛋白生成性单克隆LCs和AEF的样本中鉴定出细胞内和细胞外淀粉样蛋白。检测到的淀粉样蛋白量随孵育时间延长而增加，在14天时最为丰富。单独用淀粉样蛋白生成性LCs孵育的系膜细胞培养物或在没有系膜细胞的情况下均未出现淀粉样蛋白。同样，分别用淀粉样蛋白生成性LC或AEF孵育系膜细胞，或在有AEF但没有系膜细胞的情况下用淀粉样蛋白生成性LC孵育，均未导致淀粉样蛋白形成。当将源自肾小管间质性肾病患者尿液的LCs与AEF和系膜细胞一起孵育时，未见到淀粉样蛋白。AL淀粉样蛋白的产生需要所有三个成分——系膜细胞、淀粉样蛋白生成性LCs和AEF。此外，在系膜细胞内检测到淀粉样蛋白，支持了肾中AL淀粉样蛋白的产生需要这些细胞进行细胞内加工的假说。该系统为研究肾AL淀粉样蛋白生成提供了独特的实验模型，也为探索系膜细胞与基质的相互作用提供了一个平台。
