抑制GRP78表达对细粒棘球蚴原头节生长的影响

发布时间：2018-05-13 10:11

本文选题：细粒棘球蚴 + 体外实验　；参考：《石河子大学》2014年硕士论文

【摘要】：目的：目前，囊型包虫病治疗主要以外科手术为主。但在手术期间囊腔内容物溢出是该病术后复发的最主要的原因，向包虫囊腔内灌注局部化疗药物是阻止该病复发最常用的方法。本研究以体外培养的细粒棘球蚴原头节为研究对象，应用小分子干扰RNA(smallinterfer RNA，siRNA)技术特异性下调GRP78(glucose regulated protein78)在细粒棘球蚴原头节中的表达，探讨特异性下调GRP78对体外细粒棘球蚴原头节的作用。方法：无菌获取细粒棘球蚴原头节，运用Western blot技术检测原头节中的GRP78蛋白表达。在证实细粒棘球蚴原头节中存在GRP78蛋白的基础上，运用电穿孔技术将siRNA-GRP78转染至细粒棘球蚴原头节内，同时设立对照组。用Western blot及RT-PCR技术检测原头节GRP78mRNA及GRP78蛋白表达水平。用0.1%的伊红染色溶液按1:1比例加到样品中，15分钟后，在光学显微镜下根据颜色的变化观察siRNA-GRP78转染不同时间原头节的活力，活的原头节颜色没有变化，但是死的就会被染成红色，实验重复三次，并绘制原头节活力曲线。SEM观察原头节表面超微结构；用Caspase-3活性检测试剂盒检测原头节内Caspase-3酶活性；Western blot检测原头节Caspase-12蛋白表达水平。TUNEL检测siRNA-GRP78转染后对原头节凋亡的影响。结果：RT-PCR检测显示，siRNA-GRP78可降低细粒棘球蚴原头节GRP78mRNA表达水平。Western bolt检测显示，siRNA-GRP78可降低细粒棘球蚴原头节GRP78蛋白表达水平。转染24小时后，正常和对照组原头节的形态和活力几乎没有改变，而siRNA-GRP78组原头节活力下降约50.19%。随着siRNA-GRP78转染时间的增加，，siRNA-GRP78对原头节的生长抑制作用越明显。连续观察，9d后siRNA-GRP78组原头节死亡率达87.65%。扫描电镜下观察发现siRNA-GRP78转染初期，原头节体表出现指状突起、顶端体表出现皱缩，随着作用时间的增加，渐出现顶突界面缺损，吸盘变形，虫体表面出现凹陷，指状突起增多，甚至出现虫蚀样损害。Caspase-3活性检测发现，较正常对照组，siRNA-GRP78转染后可使原头节的caspase-3酶活性增加。TUNEL检测发现，siRNA-GRP78转染后可促使原头节发生凋亡。结论：1.运用RNAi技术可以特异性下调细粒棘球蚴原头节GRP78mRNA及蛋白表达。2.抑制细粒棘球蚴原头节中GRP78的表达，可抑制细粒棘球蚴原头节的活力，激活Caspase-3及Caspase-12酶的活性。
[Abstract]:Objective: at present, the main treatment of cystic hydatid disease is surgery. However, during the operation, the most important reason for the recurrence of the disease was the overflow of the contents of the cyst. The most common way to prevent the recurrence of the disease was to infuse local chemotherapeutic drugs into the cyst cavity of the hydatid worm. In this study, the expression of GRP78(glucose regulated protein 78 was down-regulated by small molecular interference (RNA(smallinterfer) siRNAs in the procephalic ganglia of echinococcus granulosus in vitro. To investigate the effect of down-regulation of GRP78 on the procephalic ganglion of Echinococcus granulosus in vitro. Methods: Western blot technique was used to detect the expression of GRP78 protein in Echinococcus granulosus. On the basis of confirming the existence of GRP78 protein in the proto ganglia of Echinococcus granulosus, siRNA-GRP78 was transfected into the original ganglia of Echinococcus granulosus by electroporation, and the control group was set up at the same time. Western blot and RT-PCR techniques were used to detect the expression of GRP78mRNA and GRP78. After adding 0.1% eosin dye solution to the sample at 1:1 for 15 minutes, we observed the activity of siRNA-GRP78 transfection at different time according to the change of color under the optical microscope. The color of the living original head did not change, but the dead one was dyed red. The experiment was repeated three times and the surface ultrastructure of the original cephalic ganglia was observed by SEM. Caspase-3 activity assay kit was used to detect the activity of Caspase-3 in the original cephalic ganglion. The expression level of Caspase-12 protein was detected by Western blot. Tunel was used to detect the effect of siRNA-GRP78 transfection on the apoptosis of the proto-cephalic ganglion. Results the expression level of GRP78mRNA in the protopectoma of Echinococcus granulosus was decreased by RT-PCR. Western bolt analysis showed that GRP78 could decrease the expression of GRP78 protein in Echinococcus granulosus. After 24 hours of transfection, the morphology and activity of the normal and control groups were almost unchanged, while the activity of the former cephalic ganglia in siRNA-GRP78 group was decreased by 50.19%. With the increase of siRNA-GRP78 transfection time, the inhibitory effect of siRNA-GRP78 on the growth of protocephalus was more obvious. After 9 days of continuous observation, the mortality of the original head ganglion in siRNA-GRP78 group was 87.65. Scanning electron microscope (SEM) showed that in the early stage of siRNA-GRP78 transfection, the surface of the original head ganglion appeared digital protuberance, the top of the body surface shrank, with the increase of the time, the apical process interface defect appeared gradually, the sucker was deformed, the surface of the worm appeared depression, and the finger protuberance increased. It was found that the activity of caspase-3 increased after transfection of siRNA-GRP78. Tunel assay showed that siRNA-GRP78 could induce apoptosis of the original head ganglion after transfection. Conclusion 1. The expression of GRP78mRNA and protein in echinococcus granulosus can be specifically down-regulated by RNAi. Inhibition of GRP78 expression and activation of Caspase-3 and Caspase-12 enzyme activity in procephalus of echinococcus granulosus were observed.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R532.32

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