Dynamic tracking of stem cells in an acute liver failure model
Dynamic tracking of stem cells in an acute liver failure model
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DOI：
10.3748/wjg.v18.i6.507
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发表时间：
2012-02-14
影响因子：
4.3
通讯作者：
Dannink, Steven W. M. Olde
中科院分区：
文献类型：
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作者：
Ezzat, Tarek;Dhar, Dipok Kumar;Dannink, Steven W. M. Olde
目的：为了研究一种双标记技术，这将使实时监测移植的胚胎干细胞（ESC）的动力学，以及长期tracking.METHODS：C57/BL 6雄性小鼠（n = 40）诱导的肝损伤对乙酰氨基酚（APAP）300 mg/kg腹腔注射。将绿色荧光蛋白（GFP）阳性的C57/BL 6小鼠ESC用近红外荧光亲脂性示踪剂碘化1，1-双十八烷基-3，3，3，3-四甲基吲哚三碳菁（DiR）染色，然后立即移植到脾脏中。细胞治疗组中的每只动物（n = 20）在用APAP治疗后4小时接受5 × 106个ESC。对照组（n = 20）仅接受溶媒。在移植后30 min、3、12、24、48和72 h以及1周和2周后，用IVIS Lumina-2实时监测细胞的分布和动力学。肝组织的免疫组织化学检查用于鉴定GFP和白蛋白的表达。血浆丙氨酸氨基转移酶（ALT）作为肝损伤的指标进行测量。结果：DiR染色的ESCs很容易跟踪与IVIS使用吲哚菁绿色过滤器，由于其高背景通带与最小的背景自发荧光。在移植后30分钟，移植的细胞被限制在脾内，逐渐移动到脾静脉中，并且在3小时时间点在部分肝脏中可检测到。在移植后24小时内，几乎90%的细胞在肝脏中被确认归巢。然而，在第三天，DiR信号开始淡出，并且不同器官的离体IVIS成像允许在信号不能通过体内成像检测到的时间点检测到信号，并且证实最高光子发射在肝脏中（P < 0.0001）。在2周时，DiR信号在体内不再可检测;然而，使用组成型表达的GFP的免疫组织化学分析来提供对细胞分布的了解。在类似肝细胞的组织切片中检测到GFP +ve细胞，并分散在整个肝实质中，存在大量GFP +ve细胞掺入窦状内皮衬里中。在移植的GFP +ve细胞中在72小时检测到非常微弱的白蛋白表达;然而，在2周时，少数GFP阳性的细胞也对白蛋白呈强阳性。与72 h时间点相比，2 wk时两组的血清ALT、白蛋白和胆红素水平均显著改善。在细胞治疗组中，血清ALT显著降低，（P = 0.016）较低，白蛋白（P = 0.009）显著高于对照组，但两组死亡率无差异。结论：双重标记是一种易于使用和廉价的方法，用于纵向监测移植细胞的分布、存活和植入，并且可以用于细胞治疗模型。(C)2012年百世登。All rights reserved.
AIM: To investigate a dual labeling technique, which would enable real-time monitoring of transplanted embryonic stem cell (ESC) kinetics, as well as long-term tracking.METHODS: Liver damage was induced in C57/BL6 male mice (n = 40) by acetaminophen (APAP) 300 mg/kg administered intraperitoneally. Green fluorescence protein (GFP) positive C57/BL6 mouse ESCs were stained with the near-infrared fluorescent lipophilic tracer 1,1-dioctadecy1-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) immediately before transplantation into the spleen. Each of the animals in the cell therapy group (n = 20) received 5 x 10(6) ESCs 4 h following treatment with APAP. The control group (n = 20) received the vehicle only. The distribution and dynamics of the cells were monitored in real-time with the IVIS Lumina-2 at 30 min post transplantation, then at 3, 12, 24, 48 and 72 h, and after one and 2 wk. Immunohistochemical examination of liver tissue was used to identify expression of GFP and albumin. Plasma alanine aminotransferase (ALT) was measured as an indication of liver damage.RESULTS: DiR-stained ESCs were easily tracked with the IVIS using the indocyanine green filter due to its high background passband with minimal background autofluorescence. The transplanted cells were confined inside the spleen at 30 min post-transplantation, gradually moved into the splenic vein, and were detectable in parts of the liver at the 3 h time-point. Within 24 h of transplantation, homing of almost 90% of cells was confirmed in the liver. On day three, however, the DiR signal started to fade out, and ex vivo IVIS imaging of different organs allowed signal detection at time-points when the signal could not be detected by in vivo imaging, and confirmed that the highest photon emission was in the liver (P < 0.0001). At 2 wk, the DiRsignal was no longer detectable in vivo; however, immunohistochemistry analysis of constitutively-expressed GFP was used to provide an insight into the distribution of the cells. GFP +ve cells were detected in tissue sections resembling hepatocytes and were dispersed throughout the hepatic parenchyma, with the presence of a larger number of GFP +ve cells incorporated within the sinusoidal endothelial lining. Very faint albumin expression was detected in the transplanted GFP +ve cells at 72 h; however at 2 wk, few cells that were positive for GFP were also strongly positive for albumin. There was a significant improvement in serum levels of ALT, albumin and bilirubin in both groups at 2 wk when compared with the 72 h time-point. In the cell therapy group, serum ALT was significantly (P = 0.016) lower and albumin (P = 0.009) was significantly higher when compared with the control group at the 2 wk time-point; however there was no difference in mortality between the two groups.CONCLUSION: Dual labeling is an easy to use and cheap method for longitudinal monitoring of distribution, survival and engraftment of transplanted cells, and could be used for cell therapy models. (C) 2012 Baishideng. All rights reserved.