基于SHG/TPEF检测小鼠肝脏弹性纤维方法的建立

Establishment of SHG/TPEF method for detection of mouse liver elastic fibers

目的建立二次谐波与双光子激发荧光(SHG/TPEF)显微成像技术检测小鼠肝脏弹性纤维的方法。方法将8周龄雄性Col1α1-GFP转基因小鼠随机分为两组,每组5只,分别腹腔注射CCl4和橄榄油12周建立肝纤维化及对照小鼠模型。小鼠处死后获得肝组织并进行肝脏脱细胞处理,基于SHG/TPEF识别肝细胞外基质骨架结构中的胶原纤维和弹性纤维荧光信号。结果肝组织块脱细胞后骨架结构清晰可见,形态改变较小,弹性纤维伴随胶原纤维沉积,定位于汇管区和纤维间隔中;CCl4腹腔注射12周后,与对照组相比,肝组织胶原纤维面积百分比(12.4%vs.4.2%,P<0.001)和弹性纤维面积百分比(2.8%vs.1.7%,P<0.01)均显著升高。结论 SHG/TPEF可用于脱细胞后肝组织弹性纤维成像,较传统方法具有一定优势。

Objective To establish the approach to detect the localization and distribution of elastic fibers in liver tissues based on SHG/TPEF technology. Methods Eight-week-old male Col1α1-GFP transgenic mice were divided into two groups （n=5 for each group）. CCl 4 and olive oil were intraperitoneally injected, respectively, for 12 weeks to build liver fibrosis mice models and their corresponding controls. After the mice were sacrificed and liver tissues were harvested, decellularization was performed by first removing plasma proteins and liver cells; and then the fluorescence signals of collagenous fibers and elastic fibers in liver extracellular matrix scaffolds were recognized using SHG/TPEF technology. Results After decellularization, the extracellular matrix scaffolds of liver tissues were clearly visible and the morphology only changed a little. Elastic fibers accumulated accompanying with collagenous fibers, both of which were deposited on the portal and fibrous septa areas. In contrast to the control group, 12-week CCl 4 injection intraperitoneally resulted in significant increased percentages of collagenous area （12.4% vs. 4.2%, P 〈0.001） and elastic fiber area （2.8% vs. 1.7%, P 〈0.01）. Conclusion The newly established approach based on SHG/TPEF technology can be well used to detect the liver elastic fibers after decellularization, which was superior to traditional methods.