Zhang Q L, Lin P X, Chang Y, Webster H D
Laboratory of Experimental Neuropathology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland.
J Neurosci Res. 1994 Apr 1;37(5):612-22. doi: 10.1002/jnr.490370508.
Mouse sciatic nerves were transected and 3 hr to 16 days later proximal segments were removed and homogenized. Supernatants of these segments or of normal sciatic nerves were added to Schwann cells maintained in Dulbecco's modified Eagle's medium (DMEM) + 15% fetal calf serum (FCS). After 6 days, Schwann cells were solubilized and the protein content was measured using a Bio-Rad (Melville, NY) protein assay. Samples containing the same amounts of protein were then applied to microtiter plates and the laminin content was determined by enzyme-linked immunosorbent assay (ELISA). Lysates of cultures treated with 24 hr proximal segment supernatants contained significantly higher levels of laminin than those prepared from other intervals, from distal segments, or from control nerves. Increased surface and cytoplasmic anti-laminin immunoreactivity also was found in Schwann cells treated with 24 hr supernatants. To identify the source(s) of this effect, proximal segments removed 24 hr after transection were bisected; supernatants were prepared from each half and tested. Significant increases in laminin production were produced by supernatants from both halves. When supernatants from proximal and distal halves were compared, the latter produced significantly higher laminin levels. Electron microscopic examination of both halves showed that distal halves contained sprouting neurites and growth cones ensheathed by Schwann cells which had a basal lamina and resembled those seen during development and regeneration. Proximal halves appeared normal. Schwann cell proliferation also was compared in supernatant-treated cultures by using a bromodeoxy-uridine (BrdU) ELISA. The 24 hr and 2 day supernatants increased Schwann cell proliferation significantly; 12 hr, 4 day, and 8 day supernatants produced smaller increases. Our observations suggest that axons undergoing early regenerative changes are one of several possible sources of substance(s) in our proximal segment supernatants which increased Schwann cell proliferation and laminin production.
将小鼠坐骨神经切断，3小时至16天后取出近端节段并匀浆。将这些节段或正常坐骨神经的上清液添加到在杜尔贝科改良伊格尔培养基（DMEM）+ 15%胎牛血清（FCS）中培养的雪旺细胞中。6天后，溶解雪旺细胞，使用伯乐公司（纽约梅尔维尔）的蛋白质测定法测量蛋白质含量。然后将含有等量蛋白质的样品加到微量滴定板上，通过酶联免疫吸附测定法（ELISA）测定层粘连蛋白含量。用近端节段24小时上清液处理的培养物裂解物中，层粘连蛋白水平明显高于从其他时间段、远端节段或对照神经制备的裂解物。在用24小时上清液处理的雪旺细胞中，还发现表面和细胞质抗层粘连蛋白免疫反应性增加。为了确定这种效应的来源，将切断后24小时取出的近端节段一分为二；从每一半制备上清液并进行检测。两半的上清液均使层粘连蛋白产量显著增加。比较近端和远端两半的上清液时，后者产生的层粘连蛋白水平明显更高。对两半进行电子显微镜检查发现，远端半段含有发芽的神经突和被雪旺细胞包裹的生长锥，雪旺细胞具有基膜，类似于发育和再生过程中所见。近端半段看起来正常。还通过使用溴脱氧尿苷（BrdU）ELISA比较了上清液处理的培养物中的雪旺细胞增殖情况。24小时和2天的上清液显著增加了雪旺细胞增殖；12小时、4天和8天的上清液增殖增加幅度较小。我们的观察结果表明，经历早期再生变化的轴突是我们近端节段上清液中几种可能的物质来源之一，这些物质增加了雪旺细胞增殖和层粘连蛋白产生。
