本文選題：細粒棘球蚴 + 體外實驗��； 參考：《石河子大學》2014年碩士論文

【摘要】：目的：目前，囊型包蟲病治療主要以外科手術(shù)為主。但在手術(shù)期間囊腔內(nèi)容物溢出是該病術(shù)后復發(fā)的最主要的原因，向包蟲囊腔內(nèi)灌注局部化療藥物是阻止該病復發(fā)最常用的方法。本研究以體外培養(yǎng)的細粒棘球蚴原頭節(jié)為研究對象，應用小分子干擾RNA(smallinterfer RNA，siRNA)技術(shù)特異性下調(diào)GRP78(glucose regulated protein78)在細粒棘球蚴原頭節(jié)中的表達，探討特異性下調(diào)GRP78對體外細粒棘球蚴原頭節(jié)的作用。 方法：無菌獲取細粒棘球蚴原頭節(jié)，運用Western blot技術(shù)檢測原頭節(jié)中的GRP78蛋白表達。在證實細粒棘球蚴原頭節(jié)中存在GRP78蛋白的基礎上，運用電穿孔技術(shù)將siRNA-GRP78轉(zhuǎn)染至細粒棘球蚴原頭節(jié)內(nèi)，同時設立對照組。用Western blot及RT-PCR技術(shù)檢測原頭節(jié)GRP78mRNA及GRP78蛋白表達水平。用0.1%的伊紅染色溶液按1:1比例加到樣品中，15分鐘后，在光學顯微鏡下根據(jù)顏色的變化觀察siRNA-GRP78轉(zhuǎn)染不同時間原頭節(jié)的活力，活的原頭節(jié)顏色沒有變化，但是死的就會被染成紅色，實驗重復三次，并繪制原頭節(jié)活力曲線。SEM觀察原頭節(jié)表面超微結(jié)構(gòu)；用Caspase-3活性檢測試劑盒檢測原頭節(jié)內(nèi)Caspase-3酶活性；Western blot檢測原頭節(jié)Caspase-12蛋白表達水平。TUNEL檢測siRNA-GRP78轉(zhuǎn)染后對原頭節(jié)凋亡的影響。 結(jié)果：RT-PCR檢測顯示，siRNA-GRP78可降低細粒棘球蚴原頭節(jié)GRP78mRNA表達水平。Western bolt檢測顯示，siRNA-GRP78可降低細粒棘球蚴原頭節(jié)GRP78蛋白表達水平。轉(zhuǎn)染24小時后，正常和對照組原頭節(jié)的形態(tài)和活力幾乎沒有改變，而siRNA-GRP78組原頭節(jié)活力下降約50.19%。隨著siRNA-GRP78轉(zhuǎn)染時間的增加，，siRNA-GRP78對原頭節(jié)的生長抑制作用越明顯。連續(xù)觀察，9d后siRNA-GRP78組原頭節(jié)死亡率達87.65%。掃描電鏡下觀察發(fā)現(xiàn)siRNA-GRP78轉(zhuǎn)染初期，原頭節(jié)體表出現(xiàn)指狀突起、頂端體表出現(xiàn)皺縮，隨著作用時間的增加，漸出現(xiàn)頂突界面缺損，吸盤變形，蟲體表面出現(xiàn)凹陷，指狀突起增多，甚至出現(xiàn)蟲蝕樣損害。Caspase-3活性檢測發(fā)現(xiàn)，較正常對照組，siRNA-GRP78轉(zhuǎn)染后可使原頭節(jié)的caspase-3酶活性增加。TUNEL檢測發(fā)現(xiàn)，siRNA-GRP78轉(zhuǎn)染后可促使原頭節(jié)發(fā)生凋亡。 結(jié)論：1.運用RNAi技術(shù)可以特異性下調(diào)細粒棘球蚴原頭節(jié)GRP78mRNA及蛋白表達。2.抑制細粒棘球蚴原頭節(jié)中GRP78的表達，可抑制細粒棘球蚴原頭節(jié)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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R532.32

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