探讨脂筏在Toll样受体(TLR)4促进宫颈癌低氧诱导因子(HIF)-1α高活性中的作用及机制。

采用体外细胞实验，对按照计算机随机法分为4组宫颈癌Siha细胞，分别采用4种方法进行处理：对于甲基-β-环糊精(MβCD)＋脂多糖组进行MβCD处理及脂多糖刺激，siTLR4＋脂多糖组进行siTLR4转染及脂多糖刺激，空白对照组不进行上述处理及刺激，脂多糖对照组仅进行脂多糖刺激。对4组宫颈癌Siha细胞，进行以下项目检测和观察：①采用MTT比色法测定宫颈癌Siha细胞悬液光密度(OD)值，并绘制Siha细胞生长抑制曲线，通过软琼脂集落培养实验，观察各组Siha细胞集落形成情况。②采用免疫细胞化学染色法和Western印迹法检测宫颈癌Siha细胞内HIF-1α蛋白阳性表达及含量变化。③光泽精化学发光法检测还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶活性，DCFH-DA探针检测宫颈癌Siha细胞内活性氧含量。④采用免疫荧光染色法检测脂筏与HIF-1α蛋白的共定位关系。统计学比较上述各项检测，于4组间或处理后不同时间点(0、12、24、36、48 h)的差异。

① MTT比色法和软琼脂集落培养实验结果显示，脂多糖对照组宫颈癌Siha细胞活性和集落数，均较空白对照组高或多，而MβCD＋脂多糖组及siTLR4＋脂多糖组，则较空白对照组或脂多糖对照组低或少，并且差异均有统计学意义(P<0.01)。②免疫细胞化学染色和Western印迹法结果显示，脂多糖对照组宫颈癌Siha细胞HIF-1α蛋白相对表达量，较空白对照组多，而MβCD＋脂多糖组及siTLR4＋脂多糖组，则较空白对照组或脂多糖对照组少，并且差异均有统计学意义(P<0.05)。③ MβCD＋脂多糖组及siTLR4＋脂多糖组Siha细胞经过相应处理后12、24、36、48 h的NADPH氧化酶活性和活性氧含量，均分别较空白对照组或脂多糖对照组相应时间点低；脂多糖对照组Siha细胞经过相应处理后24、36、48 h的NADPH氧化酶活性和活性氧含量，均分别较空白对照组相应时间点高，并且上述差异均有统计学意义(P<0.05)。④免疫荧光染色结果显示，脂筏定位于细胞表面，呈绿色荧光，HIF-1α蛋白主要定位于细胞质内，呈红色荧光，并且脂筏增多的细胞，HIF-1α蛋白表达亦增强。

TLR4信号可能通过激活脂筏，刺激NADPH氧化酶的氧化还原信号，产生活性氧来维持宫颈癌细胞HIF-1α高活性。

To investigate the role and mechanism of lipid rafts in Toll like receptor(TLR) 4 in promoting the high activity of hypoxia inducible factor(HIF)-1α in cervical cancer.

In vitro cell experiments have been conducted. Cervical cancer Siha cells were randomly divided into 4 groups according to the computer randomized method, and then treated with the following methods, methyl beta cyclodextrin(MβCD)+ lipopolysaccharide group was treated with MβCD and lipopolysaccharide stimulation, siTLR4+ lipopolysaccharide group was treated with siTLR4 transfection and lipopolysaccharide stimulation.The blank control group was not treated and stimulated, and lipopolysaccharide control group only treated with lipopolysaccharide stimulation.Detected and observed the following items in 4 groups: ①MTT colorimetric method was used to measure the optical density(OD) value of cervical cancer Siha cells suspension and drawn the Siha cells growth inhibition curves. Colony formation was observed in soft agar colony culture. ②The intracellular positive expression and content variations of HIF-1α protein of cervical cancer Siha cells were detected by immunocytochemical staining and Western blotting. ③Lucigenin nicotinamide adenine dinucleotide phosphate(NADPH) oxidase activity in cervical cancer Siha cells was detected with chemiluminescence and intracellular reactive oxygen species with DCFH-DA probe. ④The colocalization of lipid rafts and HIF-1α protein was detected by immunofluorescence staining.The differences of these items above mentioned among groups and at different time points(0, 12, 24, 36, 48 h) after threated and stimulation were compared by statistical method.

① The MTT assay and soft agar experiments showed that, the activity of cervical cancer Siha cells and colony numbers of lipopolysaccharide control group were higher or more than those of blank control group, while those of MβCD + lipopolysaccharide group and siTLR4 + lipopolysaccharide group were lower or less than those of lipopolysaccharide control group or blank control group, and all the differences were statistically significant (P<0.01). ② The immunocytochemical staining and Western blotting showed that, the relative expression of HIF-1α protein of cervical cancer Siha cells in lipopolysaccharide control group was more than that of blank control group, while that of MβCD + lipopolysaccharide group and siTLR4 + lipopolysaccharide group were less than that of blank control group or lipopolysaccharide control group, and all the differences were statistically significant (P<0.05). ③ NADPH oxidase activity and reactive oxygen content in cervical cancer Siha cells in MβCD+ lipopolysaccharide group and siTLR4 + lipopolysaccharide group after treated at 12, 24, 36, 48 h were lower than those of blank control group or lipopolysaccharide control group at the same time point. The activity of NADPH oxidase and reactive oxygen content in cervical cancer Siha cells in lipopolysaccharide control group after treated at 24, 36, 48 h were higher than those of blank control group at the same time point, and the differences were statistically significant(P<0.05). ④ The immunofluorescence staining showed that, lipid rafts number located on the cell surface with green fluorescence, and HIF-1α protein was mainly located in the cytoplasm with red fluorescence. The cells with the increased lipid rafts number were also showed the high expression level of HIF-1α protein.

TLR4 signal may activate the lipid raft and stimulate the redox signal of NADPH oxidase, and then produce reactive oxygen to maintain the high activity of HIF-1α in cervical cancer cells.