【摘要】：目的:探討PTTG1(垂體腫瘤轉(zhuǎn)化基因1)對(duì)人子宮內(nèi)膜癌ISK細(xì)胞增殖、遷移及侵襲能力的影響。方法:1.細(xì)胞培養(yǎng):常規(guī)對(duì)人子宮內(nèi)膜癌ISK細(xì)胞進(jìn)行細(xì)胞復(fù)蘇、細(xì)胞換液、細(xì)胞傳代、細(xì)胞凍存、細(xì)胞計(jì)數(shù)等處理。2.構(gòu)建3條靶向PTTG1基因的shRNA干擾質(zhì)粒(PTTG1-homo-509;PTTG1-homo-922;PTTG1-homo-596)。3.在lipo2000作用下瞬時(shí)轉(zhuǎn)染靶向PTTG1基因的shRNA干擾質(zhì)粒至ISK細(xì)胞中。4.36h后避光條件下觀察瞬時(shí)轉(zhuǎn)染結(jié)果(熒光顯微鏡下進(jìn)行),即觀察有綠色熒光表達(dá)的細(xì)胞所占的比例。5.通過Western blot方法檢驗(yàn)PTTG1蛋白在各組中表達(dá)差異。6.通過MTT實(shí)驗(yàn)檢測降表達(dá)PTTG1后ISK細(xì)胞生長增殖活性的變化。7.通過Transwell小室實(shí)驗(yàn)檢測下調(diào)PTTG1后ISK遷移及侵襲力的變化差異。結(jié)果:1.瞬轉(zhuǎn)ISK細(xì)胞36h后,熒光顯微鏡下觀測到有綠色熒光表達(dá)的細(xì)胞數(shù)量占75%以上,表明瞬轉(zhuǎn)實(shí)驗(yàn)成功。2.Western blot實(shí)驗(yàn)檢測到干擾組-509、干擾組-922、干擾組-596中的PTTG1與內(nèi)參β-actin灰度值比值均明顯小于正常組及陰性對(duì)照組,且差異明顯有統(tǒng)計(jì)學(xué)意義(P0.05)。這就說明3個(gè)干擾組中的PTTG1蛋白表達(dá)均受到了明顯抑制,即構(gòu)建的3條靶向PTTG1基因的shRNA干擾質(zhì)粒均有效。3.在MTT實(shí)驗(yàn)中,干擾組(轉(zhuǎn)染了有效PTTG1 shRNA質(zhì)粒的細(xì)胞)中在各時(shí)間段的OD值均小于正常組及陰性對(duì)照組,說明干擾組中細(xì)胞增長較兩個(gè)對(duì)照組明顯減慢,差異且存在統(tǒng)計(jì)學(xué)意義(P0.05)。4.在Transwell小室實(shí)驗(yàn)中,干擾組(轉(zhuǎn)染了有效PTTG1 shRNA質(zhì)粒的細(xì)胞)中的穿膜細(xì)胞數(shù)較正常及陰性對(duì)照組明顯減少,說明該組細(xì)胞的遷移及侵襲能力較以上兩組細(xì)胞明顯減弱,且差異明顯有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1.PTTG1 sh RNA干擾質(zhì)�？墒谷俗訉m內(nèi)膜癌ISK細(xì)胞中的PTTG1蛋白表達(dá)量明顯降低并可抑制ISK細(xì)胞的增殖、遷移及侵襲力。2.PTTG1在子宮內(nèi)膜癌的發(fā)生發(fā)展過程中起到了重要作用。
[Abstract]:Aim: to investigate the effects of PTTG1 (pituitary tumor transforming gene 1) on the proliferation, migration and invasion of human endometrial carcinoma ISK cells. Methods: 1. Cell culture: human endometrial carcinoma ISK cells were routinely treated with cell resuscitation, cell fluid exchange, cell passage, cell cryopreservation, cell count and so on. 2. Three shRNA interference plasmids (PTTG1-homo-509;PTTG1-homo-922;PTTG1-homo-596) targeting PTTG1 gene were constructed. ShRNA interference plasmids targeting PTTG1 gene were transiently transfected into ISK cells by lipo2000. After 4.36 h, the results of transient transfection (carried out under fluorescence microscope) were observed, that is, the proportion of cells with green fluorescent expression was observed. The expression of PTTG1 protein in each group was detected by Western blot method. 6. 6%. MTT assay was used to detect the growth and proliferation activity of ISK cells after down-expression of PTTG1. 7. 7%. The changes of ISK migration and invasiveness after down-regulation of PTTG1 were detected by Transwell chamber experiment. The result is 1: 1. After transient transfer of ISK cells for 36 h, the number of cells with green fluorescent expression was more than 75% under fluorescence microscope, which indicated that the transient experiment was successful. The ratio of PTTG1 to 尾-actin in interference group-596 was significantly lower than that in normal group and negative control group, and the difference was statistically significant (P0.05). This indicated that the expression of PTTG1 protein was significantly inhibited in the three interference groups, that is, the three shRNA interference plasmids targeting PTTG1 gene were all effective. In the MTT experiment, the OD values in the interference group (the cells transfected with the effective PTTG1 shRNA plasmid) were lower than those in the normal group and the negative control group, indicating that the cell growth in the interference group was significantly slower than that in the two control groups. The difference was statistically significant (P0.05). 4. In the Transwell chamber experiment, the number of perforated cells in the interference group (cells transfected with effective PTTG1 shRNA plasmids) was significantly lower than that in the normal and negative control groups, indicating that the migration and invasion ability of the cells in the interference group was significantly lower than that in the above two groups. The difference was statistically significant (P0.05). Conclusion: 1.PTTG1 sh RNA interference plasmid can significantly decrease the expression of PTTG1 protein in human endometrial carcinoma ISK cells and inhibit the proliferation of ISK cells. Migration and invasiveness. 2.PTTG1 plays an important role in the development of endometrial carcinoma.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.33

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