Shetty A K, Rapoza D, Madison R D, Turner D A
Duke University Medical Center, Durham VAMC, NC 27710, USA.
Neuroscience. 1995 Nov;69(2):407-16. doi: 10.1016/0306-4522(95)00279-r.
Selective removal of grafted tissue is critical to assess the functional role of that tissue in the host, yet is technically difficult for well-dispersed neural grafts. We labeled fetal hippocampal cells with both a nuclear marker (5'-bromodeoxyuridine) and a cytoplasmic marker (latex microspheres) before grafting into normal adult hippocampus. A nontoxic chromophore, chlorin e6, was conjugated on to the surface of latex microspheres of the treatment group. Chlorin e6 produces cytotoxic singlet oxygen only during photoactivation. Grafted animals received transcranial exposure to various intensities of near-infrared laser light. Following laser exposure, grafts were observed in all groups except in transplants prelabeled with chlorin e6 latex microspheres. An optimal laser exposure of 50-100 J/cm2 (4-8 min) was found to selectively remove only the chlorin e6-containing grafted cells. With increasing doses of laser illumination, non-specific lesions of the host tissue surrounding the graft were induced. Quantitative graft analysis, in the absence of laser exposure, indicated that the survival of grafted cells was similar between control transplants labeled with latex microspheres alone and grafts labeled with latex microspheres plus chlorin e6. This is further evidence that chlorin e6 by itself was not toxic without laser exposure. The results clearly demonstrate that singlet oxygen-induced cell photolysis can result in selective, non-invasive removal of dispersed grafted cells located in adult hippocampus. This technique may facilitate defining specific mechanisms of action of grafted cells which mediate functional recovery in different host conditions.
选择性去除移植组织对于评估该组织在宿主中的功能作用至关重要，但对于分散良好的神经移植来说，在技术上具有挑战性。我们在将胎儿海马细胞移植到正常成年海马之前，用核标记物（5'-溴脱氧尿苷）和细胞质标记物（乳胶微球）对其进行标记。将一种无毒发色团二氢卟吩e6偶联到治疗组乳胶微球的表面。二氢卟吩e6仅在光激活过程中产生细胞毒性单线态氧。移植后的动物接受不同强度近红外激光的经颅照射。激光照射后，除了预先用二氢卟吩e6乳胶微球标记的移植组外，在所有组中均观察到移植组织。发现50 - 100 J/cm2（4 - 8分钟）的最佳激光照射能选择性地仅去除含二氢卟吩e6的移植细胞。随着激光照射剂量的增加，移植周围宿主组织会出现非特异性损伤。在没有激光照射的情况下进行的定量移植分析表明，仅用乳胶微球标记的对照移植组和用乳胶微球加二氢卟吩e6标记的移植组中，移植细胞的存活率相似。这进一步证明在没有激光照射的情况下，二氢卟吩e6本身无毒。结果清楚地表明，单线态氧诱导的细胞光解可导致对位于成年海马中的分散移植细胞进行选择性、非侵入性去除。该技术可能有助于确定移植细胞在不同宿主条件下介导功能恢复的具体作用机制。
