不同品系小鼠诱导实验性白色念珠菌性角膜炎的研究

发布时间：2018-05-11 10:53

本文选题：角膜炎 + 不同品系　；参考：《泰山医学院》2012年硕士论文

【摘要】：目的真菌性角膜炎（Fungal Keratitis, FK）是一种致盲率极高的感染性角膜病，其发病机理尚未明确。本研究主要选用两种常见的小鼠品系Balb/c小鼠和C57BL/6小鼠建立实验性真菌性角膜炎模型，通过比较两种小鼠在发病及转归过程中的表现及各相关分子的变化水平，进而研究真菌性角膜炎发病的相关机制。方法选用相同周龄雄性Balb/c小鼠和C57BL/6小鼠，采用角膜基质注射法注射1×10~8CFU/ml白色念珠菌孢子1ul建立小鼠的真菌性角膜炎模型。从感染后第1天起每隔1天裂隙灯下观察、采集图片，并进行临床评分。在感染后的不同时间点收集血浆和受染角膜，组织病理学检查受染角膜组织中炎性细胞的浸润及真菌的生长情况，真菌负荷实验检测受染角膜组织中真菌孢子含量（载菌量），酶联免疫吸附剂测定实验（ELISA）检测血浆和受染角膜匀浆液中IL-17、IFN-γ的含量，实时定量PCR检测受染角膜组织中基质金属蛋白酶及其抑制因子（MMPs，TIMPs）和胶原纤维等分子基因表达水平的变化，免疫荧光技术验证感染后第1天受染角膜内IL-17分子的表达情况。结果两种品系的小鼠在角膜基质注射1×105白色念珠菌孢子后均感染了严重的角膜炎。在感染早期，小鼠角膜水肿、溃疡，后期病情减退。但相比于Balb/c小鼠，C57BL/6小鼠诱导CaK后在相应各时间点的平均疾病分数更高（P＜0.05）；且在感染后期Balb/c小鼠受染角膜逐渐恢复透明性，趋于正常状态；而C57BL/6小鼠直至第30天仍可见较大的角膜斑翳。角膜组织病理学检查结果显示C57BL/6小鼠的白念菌性角膜炎直至感染后的第14天仍有大量的炎性细胞浸润，基质层机化仍很明显，而相应时间点Balb/c小鼠角膜内炎性细胞浸润量已明显减少（P＜0.05），基质层细胞数有所减少，角膜厚度趋于正常。真菌负荷检测结果显示两种品系小鼠受染角膜真菌负荷数随着天数的推移呈现明显的下降趋势，，至感染后第10天，真菌负荷数趋于0。但C57BL/6小鼠在相应各时间点角膜组织菌落计数都较Balb/c小鼠多（P＜0.05）。ELISA结果显示感染早期两种品系小鼠血浆及受染角膜组织中均有较高水平IL-17的表达，中后期IL-17的表达下降，IFN-γ的表达量则明显升高。感染早期，C57BL/6小鼠血浆及受染角膜组织中IL-17的表达量均明显高于Balb/c小鼠（P＜0.05）；而在感染中后期C57小鼠血浆及受染角膜组织中IFN-γ的表达量明显低于Balb/c小鼠（P＜0.05）。免疫荧光技术检测进一步验证了ELISA的检测结果，即在白色念珠菌性角膜炎早期（24h），受染角膜便可检测到大量的IL-17，且C57BL/6小鼠角膜内IL-17的表达明显多于Balb/c小鼠。RT-PCR检测结果发现两种品系小鼠受染角膜组织中大多数基因表达水平存在动态变化，MMP-8，MMP-9，TIMP-1于感染后的第3天迅速升高并达到高峰，这一现象在两种品系小鼠的检测结果中都可以观察到，但在C57BL/6小鼠角膜内这三个因子的表达量明显高于Balb/c小鼠（P＜0.05）；TIMP-2、Col3a1及Col4a1的表达略有增高，其中Balb/c小鼠受染角膜内Col3a1和Col4a1的表达量明显高于C57BL/6小鼠（P＜0.05）；Cak的中期，可见Col3a1和Col4a1的表达量继续升高并达到高峰，且Balb/c小鼠依然明显高于C57BL/6小鼠（P＜0.05），MMP-2、MMP-13和TIMP-2缓慢升高，而MMP-8，MMP-9及TIMP-1的表达量则明显下降（P＜0.05）；后期，除了MMP-2、MMP-13和TIMP-2的表达量升高以外，其他因子的表达水平均较低。结论遗传背景不同的小鼠感染真菌性角膜炎后表现不同，炎性因子IL-17、IFN-γ及损伤修复相关因子MMPs、TIMPs和胶原纤维等在其中发挥重要的作用，关于各种分子在真菌性角膜炎中的确切功能及细胞来源还需进一步的研究。
[Abstract]:Purpose

In this study , two common mouse strains Balb / c mice and C57BL / 6 mice were selected to establish an experimental fungal keratitis model .

method

The expression of IL - 17 and IFN - 纬 in the infected corneal tissue was determined by the method of corneal stroma injection . The levels of IL - 17 and IFN - 纬 were collected from the infected corneal tissue at different time points after infection , and the expression of IL - 17 and IL - 17 in the infected cornea was detected by immunofluorescence technique .

Results

Compared with Balb / c mice , C57BL / 6 mice induced CaK at the corresponding time points higher than those in Balb / c mice ( P < 0.05 ) .
In the later stage of infection , Balb / c mice infected with cornea gradually restored the transparency and tended to be in normal state ;
The results showed that there was a significant decrease of IL - 17 in the cornea of C57BL / 6 mice ( P & lt ; 0.05 ) , and the expression of IL - 17 decreased significantly in both strains of C57BL / 6 mice . The levels of IL - 17 in the plasma and infected corneal tissues of C57BL / 6 mice were significantly higher than those in Balb / c mice ( P < 0.05 ) .
Compared with Balb / c mice , the expression of IL - 17 in cornea of C57BL / 6 mice was significantly higher than that in Balb / c mice ( P < 0.05 ) .
The expressions of TIMP - 2 , Col3a1 and Col4a1 increased slightly , and the expression of Col3a1 and Col4a1 in Balb / c mice was significantly higher than that in C57BL / 6 mice ( P < 0.05 ) .
The expressions of MMP - 2 , MMP - 13 and TIMP - 2 in Balb / c mice were significantly higher than those in C57BL / 6 mice ( P & lt ; 0.05 ) , while MMP - 8 , MMP - 9 and TIMP - 1 decreased significantly ( P & lt ; 0.05 ) .
In the later stage , the expression level of MMP - 2 , MMP - 13 and TIMP - 2 increased , and the expression level of other factors was lower .

Conclusion

The expression of IL - 17 , IFN - 纬 and the related factors of MMP - 3 , TIMPs and collagen fibers play an important role in the pathogenesis of fungal keratitis , and the exact function of various molecules in fungal keratitis and the source of cells need further study .

【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R772.21

【共引文献】