【作者】 张文胜；

【导师】 陈槐卿；

【作者基本信息】 四川大学 ， 生物医学工程， 2002， 博士

【摘要】 目的 探讨人脐静脉内皮细胞（Human umbilical vein endothelial cells，HUVECs）在流体切应力作用下IL-8基因的诱导表达以及切应力的作用时间和作用强度对IL-8基因表达影响的变化规律；利用表达谱基因芯片技术研究内皮细胞在低切应力（4.20dyne／cm²，2h）作用下其切应力相关基因的表达情况以及生理浓度(10^-7的17β-雌二醇孵育内皮细胞48h，再经同样条件的切应力处理后对内皮细胞切应力相关基因表达的可能影响。 方法 （1）选择4.20 dyne／cm²的切应力处理内皮细胞，切应力作用时间分别为1h、2h、3h、4h、5h、6h、7h、8h、9h、10h和12h。以未作任何处理的内皮细胞为阴性对照。内皮细胞切应力加载完成后，提取细胞总RNA。应用LightCycler^TM系统的DNA结合染料(SYBR^? GreenI)的定量RT-PCR技术观察切应力作用下内皮细胞IL-8基因表达的变化；（2）分别用2.23、4.20和6.08dyne／cm²的切应力处理内皮细胞，切应力作用时间分别为1h、2h、3h、4h、6h、8h、10h、12h。以未作任何处理的内皮细胞作为对照。内皮细胞切应力加载完成后，提取细胞总RNA。应用同样的定量RT-PCR技术观察切应力作用时间对内皮细胞IL-8基因表达的影响；（3）选择不同强度的切应力（即分别为2.23、4.20、6.08、8.19、9.67、12.15、14.40、16.87、19.29 dyne／cm²）处理内皮细胞。切应力作用时间分别为1h和2h。以未作任何处理的内皮细胞作为对照。内皮细胞切应力加载完成后，提取细胞总RNA。应用同样的定量RT-PCR技术观察切应力作用强度对内皮细胞IL-8基因表达的影响；（4）利用上海联合基因科技集团的包含4096条人体基因的非分类表达谱基因芯片（HGEC-40S）观察内皮细胞在单纯低切应力（4.20dyne／cm²，2h）作用下和生理浓度(10^-7 M)17β-雌二醇孵育48h，再经同样条件的切应力处理后，其切应力相关基因的差异表达情况。 结果 （1）未用切应力处理的内皮细胞没有IL-8基因的表达，基线值为5.261×10⁴拷贝；4.20dyne／cm²的切应力处理内皮细胞后，1h时IL-8mRNA 四川大学博士学位论文—一中文摘要的表达明显增加o．413X10’拷贝)，2 h时ILS InRNA的表达量达到最高值的．“ZX 10‘拷贝)，3 h时 L七 eA的表达量开始下降归．590X旷拷贝)。在 多4－6 h之间 IL－8 InRNA的表达量继续下降，分u为 3．853 X 10’、3．136 X 10’和1．735 X 10’拷贝，与 3 h时的表达量处在同一个数量级；在 7d h之间 h七InRNA的表达量相对稳定，但和4—6 h之间n．8 InRNA的表达量相比下降了一个数量级，分别为9．325X106、4853X106、4＊07X106、4．865X106和 3．812X10‘拷贝。IL七 的表达量具有时相性变化规律；o)未用切应力处理的内皮细胞没有L8基因的表达，基线值分别为5．219X10’、5．308X10’和5．038X 10’拷贝：在切应力分别为 2．23、4．20和 6刀8 dgne／cm’时，内皮细胞均表现出相同的IL－8 mRNA随时间的变化规律；即lh时IL－8 InRNA的表达明显增加（分另为 7．680X 10‘、6．982X 10‘和 5．104X 10‘拷贝），2 h时 n－8 InRNA的表达量至最高值（分别为2．529X10’、1．495X10’和1．468X10’拷贝），3h时几－8dA的表达量开始下降（分别为 6．787X 10‘、1．888X 10‘和 1．639X 10‘拷贝卜在 4术 h之间 IL七 tnRNA的表达量缓慢下降，介于 1．005 X 106《．337 X 10’拷贝之间，与 3 h时的表达量同处一个数量级；在 10－12 h之间，IL．8 InRNA的表达量进一步下降，介于 2．219 XId巧．929 X 10’拷贝之间，和 44 h之间 nEInRNA的表达量相比又下降了一个数量级；③未用切应力处理的内皮细胞没有 IL七基因的表达，基线值分别为 8．864X 10’和 1．Z15X 10‘拷贝；切应力处理内皮细胞后，低切应力c．23 dyne儿m勺时K七 InRNA表达量＊ 时为1．396X 10’拷贝；2 h时为 3．334X 10’拷贝）明显增加，为高切应力（19．29 dyne／cm’）日 IL－8 InRNA表达量（lh时为 2．047X 10’拷贝；2 h时为 6．338X 10’拷贝）白‘约 68或 52倍cn七 InRNA的表达量与内皮细胞所施加的切应力强度呈反变关系：直线回归方程：lh时为 y＝7．57—0．11x，相关系数 r—一0．97；2 h时为y＝7．92—0二 0 x，相关系数 r—一 0．96c式中：y为切应力作用下内皮细胞 hEInRNA的表达量（拷贝数的对数值X X为施加于内皮细胞的切应力强度MyflC儿m勺。不同的切应力作用时间 （ h、2 h）均表现出 IL毛 dA随切q力强度变化的相同规律；p）在低切应力K二0 dyne儿m勺作用内皮细胞2 h后，对基因芯片扫描结果进行分析发现有 108条基因出现差异表达，约占芯片上基因总数的 0刀26％。其中 53条基因表达增加，55条基因表达降低；在 10’M 17D－雌二醇孵育内皮细胞48 h后以及同上条件的切应力作用下，对芯片扫描结果进行分析发现有384条基因出现差异表达，约占芯片上基因总数的0刀94％。其中226条基因表达增加，158条基因表达降低。 结 论 ＊)未用切应力处理的内皮细胞没有IL七基因的表达；流体切应力可以?更多还原

【Abstract】 Objective To investigate the effects of fluid shear stress on IL-8 gene in human umbilical vein endothelial cells (HUVECs) and the roles of time course of low shear stress and intensity of fluid shear stress using LightCycler?system;To investigate the gene expression profiles in HUVECs exposed on low shear stress (4.20 dyne/cm2,2 h) and incubated by 17 P -estradiol (10-7 M) + low shear stress (4.20 dyne/cm2,2 h) using the cDNA microarray approach.Methods Endothelial cells were isolated from human umbilical cord veins by collagenase treatment as described by Jaffe and modified. The cells were grown in Ml99 medium and the cells used were mainly prior to passage 3 or 4. Cell culture was maintained in a humidified 5 %/95 % air incubator at 37 . A flow system was established according to the design previously described. The fluid used to shear HUVECs was the cultured medium. Laminar shear stresses were generated by a peristaltic pump. Cells from static controls or from shear stress experiments were washed twice with ice-cold PBS,and total cellular RNA was isolated by TRIzol reagent according to manufacturer’s manual. RNA quality was insured by gel visualization and spectrophotometric analysis (OD26o/OD28o). Quantitative RT-PCR assay involves LightCycler?technology. Experimental protocol were performed by instruction manual of LightCycler-RNA Amplification Kit SYBR Green I. Human named genes HGEC-40S were used for differential expression screening. The PCR products of 4 096 genes were spotted on a chemical-material-coated-glass plates in array. The DNAs were then fixed on the glass plate by a serial of treatments. Total RNA from normal static cultured HUVECs was labeled by Cy3-dCTP and total RNA of HUVECs from the paired shear stressed experiment was labeled by Cy5-dCTP. The arrays were then hybridized against the cDNA probe mixture and the fluorescent signals were scanned. The expression ratios reported are the average from the two separate experiments. (1) HUVECs exposed to fluid shear stress (4.20 dyne/cm2) for 1 h,2 h,3 h,4 h,5 h,6 h,7 h,8 h,9 h,10 h,or 12 h,respectively. Normal static cultured HUVECs were selected as a control. Quantitative reversal transcription- polymerase chain reaction (qRT-PCR)using LightCycler?system was employed to assay the expression of IL-8 mRNA. (2) HUVECs exposed to low fluid shear stresses (2.23 dyne/cm2,4.20 dyne/cm2,or 6.08 dyne/cm2) for 1 h,2 h,3 h,4 h,6 h,8 h,10 h,or 12 h,respectively. Normal static cultured HUVECs were selected as a control. Quantitative reversal transcription- polymerase chain reaction (qRT-PCR) using LightCycler?system was employed to assay the expression of IL-8 mRNA. (3) HUVECs exposed to different fluid shear stresses (2.23 dyne/cm2,4.20 dyne/cm2,6.08 dyne/cm2,8.19 dyne/cm2,9.67 dyne/cm2,12.15 dyne/cm2,14.40 dyne/cm2,16.87 dyne/cm2,19.29 dyne/cm2,respectively) for 1 h or 2 h. Normal static cultured HUVECs were selected as a control. Quantitative reversal transcription-polymerase chain reaction (qRT-PCR) using LightCycler?system was employed to assay the expression of IL-8 mRNA. (4) HUVECs exposed to low fluid shear stress (4.20 dyne/cm") for 2 h and incubated by 17 P -estradiol (10-7 M) + low shear stress (4.20 dyne/cm2,2 h). Normal static cultured HUVECs were selected as a control. The complementary DNA microarray approach was used to assay the differential gene expression in endothelial cells.Results (1) EL-8 mRNA did not express in HUVECs untreated with fluid shear stress. The baseline value was 5.261 X 104 copies. IL-8 mRNA expression increased (2.413 X 108 copies) when HUVECs exposed to fluid shear stress for 1 h,and it reached the summit (6.642 X 108 copies) when HUVECs exposed to fluid shear stress for 2 h. Then IL-8 mRNA expression gradually decreased (5.590X 107 copies) at 3 h of stimulation by shear stress and remained at a constant level (3.853 X 107-1.735 X107 copies for 4-6 h and 9.325 X 106-3.812X 106 copies for 7-12 h) throughout the time course of the study. The increase of IL-8 mRNA expression by shear stress was time-depen更多还原