方法：提取龟分枝杆菌细菌壁内脂葡聚糖作用于原代培养人角膜上皮细胞(human corneal epithelia，HCE)。实验分为实验组(脂葡聚糖样品处理组)、阳性对照组(LPS处理组)和空白组。脂葡聚糖处理后的0，6，12h各时间点采用RT-PCR法在mRNA水平检测实验组中IL-6和IL-8的表达水平； 脂葡聚糖处理后的0，6，12，24h各时间点采用ELISA法在蛋白水平检测实验组、阳性对照组和空白组中IL-6和IL-8的表达。免疫细胞化学法检测空白组与实验组(时间点24h)HCE细胞内NF-κB的表达与定位。

结果：实验组中，12h内IL-6和IL-8在mRNA水平明显增加，6h时即达到峰值(IL-6是空白组的32.7倍，IL-8是对照组的36.8倍)； 在蛋白质水平，培养上清内IL-6和IL-8含量在24h内呈现时间依赖性上升(6，12，24h各时间点与对照组之间均有统计学差异，P<0.05)，与阳性对照组变化一致。空白组NF-κB定位于细胞浆内，实验组NF-κB被激活，移位至细胞核内。

结论：龟分枝杆菌来源的脂葡聚糖可以促使人角膜上皮细胞过表达IL-6和IL-8，其信号转导通路可能是通过核转录因子NF-κB进行的。

METHODS: Lipoglycans was extracted by the Triton X-114 phase partitioning. Lipoglycans from Che were purified, by successive detergent and phenol extractions. Lipoglycans were separated by gel filtration on a Sephacryl 200 column and Sephacryl 100 column in series, followed by extensive dialisis. Purified Lipoglycans(50μg/mL)were added into culture medium to stimulate primary human corneal epithelial(HCE)cells. Cells and supernatant were collected at 0, 6, 12, 24h after the stimulation. The IL-6 and IL-8 expression at mRNA level was assayed by using real time RT-PCR and the secreted IL-6 and IL-8 in the supernatants was measured by ELISA. Immunochemistry was used to detect the expression and location of NF-κB in HCE cells.

RESULTS: After the treatment of Lipoglycans, the expression of IL-8 and IL-6 at mRNA level obviouly increased within 12h, and reached peak level at 6h(IL-8 was 36.8 times that of the blank control, and IL-6 was 32.7 times). Compared with the blank control group, the expression of IL-8 at protein level in the supernatant increased 2.8 folds at 6h(P>0.05), 13.4 folds at 12h(P<0.05), and 200.7 folds at 24h(P<0.05), and the expression of IL-6 also incresed 3.6 folds at 6h(P<0.05), 6.1 folds at 12h(P<0.05), and 7.0 folds at 24h(P<0.05), which was similar to changes in the positive control group(situmulated by LPS). In the blank control group, NF-κB was localized in the cytoplasm, while in lipoglycans treated group, NF-κB was activated and translocalized to the nucleus in HCE.

CONCLUSION: Lipoglycans from Che can induce HCE cells to produce inflammatory factors(IL-6 and IL-8), and its signal transduction pathway probably is mediated by NF-κB.