本文選題：食管癌　切入點(diǎn)：蛋白酶激活受體2　出處：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:蛋白酶激活受體-2(Proteinase-actived receptors 2,PAR-2)是位于細(xì)胞膜表面的G蛋白偶聯(lián)受體,該受體被激活后可引起一系列關(guān)于腫瘤生長(zhǎng)、侵襲、轉(zhuǎn)移等方面的生物學(xué)行為。食管癌是人類(lèi)最常見(jiàn)的惡性腫瘤之一,其發(fā)生機(jī)制目前還不太明確,是當(dāng)前食管癌研究中重要的一個(gè)方面。研究表明,在多種腫瘤組織及細(xì)胞中存在PAR-2的高表達(dá),提示PAR-2可能與腫瘤的生物學(xué)特性和惡性程度密切相關(guān)。關(guān)于PAR-2對(duì)食管癌EC109細(xì)胞增殖的影響及其分子機(jī)制,目前尚不清楚。本試驗(yàn)通過(guò)體外培養(yǎng)人食管癌細(xì)胞株EC109,研究PAR-2對(duì)食管鱗狀細(xì)胞癌EC109細(xì)胞增殖的影響,并探討其調(diào)節(jié)作用的可能分子機(jī)制。方法:細(xì)胞培養(yǎng):用含有10%新生牛血清的1640培養(yǎng)基,置于37℃、5%CO2飽和濕度的孵育箱中培養(yǎng),待細(xì)胞長(zhǎng)滿(mǎn)培養(yǎng)瓶底部70-80%(約2-3 d)時(shí)用胰蛋白酶消化傳代,取對(duì)數(shù)生長(zhǎng)期細(xì)胞用于實(shí)驗(yàn)。細(xì)胞轉(zhuǎn)染:顯微鏡觀察細(xì)胞生長(zhǎng)情況;使用一次性無(wú)菌吸管將培養(yǎng)瓶中的廢液吸除,使用PBS漂洗2遍;用移液槍加入1 ml Trypsin液,消化3 min(37℃,5%CO2),加入1ml的含血清培養(yǎng)基終止反應(yīng),使用一次性無(wú)菌吸管吹打培養(yǎng)瓶壁,將培養(yǎng)液裝入離心管中,離心;用培養(yǎng)液重懸細(xì)胞,細(xì)胞計(jì)數(shù)后選擇6×104/ml的密度加入不含抗生素培養(yǎng)基的培養(yǎng)瓶中,將培養(yǎng)瓶轉(zhuǎn)入培養(yǎng)箱中培養(yǎng),第二天轉(zhuǎn)染;將細(xì)胞同上進(jìn)行消化離心,取1.5ml EP管,標(biāo)記為1,加入100μl 1640培養(yǎng)基,再加入2μg含PAR-2 shRNA的質(zhì)粒,靜止5 min;另取1.5 ml EP管,標(biāo)記為2,加入100μl 1640培養(yǎng)基,再加入10μl X-tremeGENE HP DNA Transf,靜止5 min;將兩管混勻,靜止15 min,后再次加入不含抗生素培養(yǎng)基的培養(yǎng)瓶中,培養(yǎng)24-48h后,使用熒光顯微鏡觀察轉(zhuǎn)染效果;使用G418對(duì)PAR-2 shRNA轉(zhuǎn)染后的細(xì)胞進(jìn)行篩選,不被轉(zhuǎn)染的細(xì)胞不具備G418抗體,會(huì)被G418殺死,剩余細(xì)胞為PAR-2 shRNA轉(zhuǎn)染成功細(xì)胞,用于后續(xù)實(shí)驗(yàn)。將食管癌細(xì)胞按不同處理方法分為空白組、PAR-2激動(dòng)組(加入SLIGKV)、PAR-2反激動(dòng)組(加入VKGILS)、PAR-2 shRNA組(shRNA轉(zhuǎn)染)和MAPK抑制組(pd98059)。每組處理前首先使用不含小牛血清的1640培養(yǎng)基饑餓培養(yǎng)24h,在按相應(yīng)的處理原則加入藥品或阻斷劑。每組處理結(jié)束后,使用rt-pcr檢測(cè)par-2mrna、erk1mrna和cyclind1mrna的表達(dá)情況;使用western-blot檢測(cè)par-2、erk1、p-erk1、cyclind1和pcna蛋白的表達(dá)情況,并且繪制各組細(xì)胞的生長(zhǎng)曲線(xiàn)。結(jié)果:1rt-pcr方法檢測(cè)基因表達(dá)結(jié)果顯示:(1)與空白組相比較,par-2激動(dòng)組par-2mrna的表達(dá)量為2.651倍(p0.05),par-2shrna組par-2mrna的表達(dá)量為0.385倍(p0.05);(2)與空白組相比較,par-2激動(dòng)組erk1mrna的表達(dá)量為2.580倍(p0.05),par-2shrna組erk1mrna的表達(dá)量為0.376倍(p0.05),mapk抑制劑組erk1mrna的表達(dá)量為0.497倍(p0.05);(3)與空白組相比較,par-2激動(dòng)組cyclind1mrna的表達(dá)量2.012倍(p0.05),par-2shrna組cyclind1mrna的表達(dá)量為0.318倍(p0.05),mapk抑制劑組cyclind1mrna的表達(dá)量為0.422倍(p0.05)。2western-blot檢測(cè)蛋白表達(dá)結(jié)果:(1)par-2激動(dòng)組par-2蛋白的表達(dá)量為空白組的1.863倍(p0.05),par-2shrna組par-2蛋白的表達(dá)量為空白組0.373倍(p0.05);(2)par-2激動(dòng)組p-erk1蛋白的表達(dá)量為空白組1.517倍(p0.05),par-2shrna組p-erk1蛋白的表達(dá)量為空白組的0.478倍(p0.05),mapk抑制劑組p-erk1蛋白的表達(dá)量為空白組的0.410倍(p0.05);(3)par-2激動(dòng)組cyclind1蛋白的表達(dá)量為空白組的1.557倍(p0.05),par-2shrna組cyclind1蛋白的表達(dá)量為空白組的0.299倍(p0.05),mapk抑制劑組cyclind1蛋白的表達(dá)量為空白組的0.364倍(p0.05);(4)erk1在各組中表達(dá)無(wú)明顯改變(p0.05)。3pcan表達(dá)量結(jié)果:激動(dòng)組為空白組的1.738倍(p0.05);轉(zhuǎn)染組為空白組的0.784倍(p0.05);抑制組為空白組的0.683倍(p0.05)。4細(xì)胞計(jì)數(shù)結(jié)果顯示:與空白對(duì)照組相比較,激動(dòng)組的細(xì)胞生長(zhǎng)曲線(xiàn)上移,轉(zhuǎn)染組、抑制組下降。結(jié)論:1在食管癌細(xì)胞ec109中,par-2與erk1及cylind1存在密切聯(lián)系。2在食管癌細(xì)胞EC109中,PAR-2激動(dòng)后,可以上調(diào)其下游通路中ERK1基因及p-ERK1蛋白的表達(dá)。3在食管癌細(xì)胞EC109中,PAR-2激動(dòng)后可以上調(diào)其下游通路中MAPK ERK1通路,上調(diào)CyclinD1 mRNA和CyclinD1蛋白的表達(dá),加快細(xì)胞周期,促進(jìn)食管癌細(xì)胞EC109的增殖。4在食管癌細(xì)胞EC109中,PAR-2shRNA可以通過(guò)調(diào)節(jié)MAPK ERK1通路,進(jìn)而調(diào)節(jié)CyclinD1的表達(dá),進(jìn)而影響食管癌細(xì)胞EC109的增殖。
[Abstract]:Objective: protease activated receptor -2 (Proteinase-actived receptors 2, PAR-2) is a G protein coupled receptors located on the cell membrane, the receptor activation can cause a series of tumor growth, invasion, metastasis and so on. The biological behavior of esophageal cancer is one of the most common malignant tumor, its pathogenesis is still not too clearly, is one of important current research in esophageal cancer. The study shows that the high expression of PAR-2 in tumor tissues and cells, suggesting that PAR-2 may be associated with tumor biological characteristics and malignancy are closely related. The impact of PAR-2 on the proliferation of esophageal cancer EC109 cells and its molecular mechanism is unclear. This test in vitro culture of human esophageal cancer cell line EC109, the effects of PAR-2 on proliferation of esophageal squamous cell carcinoma cell line EC109, and to explore the possible molecular mechanism of the regulation: Cell culture: the culture medium with 1640 containing 10% newborn calf serum, at 37 C, cultured 5%CO2 saturated humidity incubator. When the cells covered the bottom of the culture flask of 70-80% (about 2-3 d) with trypsin, logarithmic growth phase cells were used for experiments. Cell transfection: microscope cell growth; use of disposable sterile Straw will flask liquid suction, using PBS rinse 2 times; with a pipette with 1 ml Trypsin solution, 3 min digestion (at 37 5%CO2), adding 1ml serum containing medium to stop the reaction, using disposable sterile suction tube and culture bottle wall, liquid culture a centrifuge tube, centrifuge; cell culture liquid suspension, cell count after the selection of 6 * 104/ml density flask with antibiotic free medium, the culture bottle into the culture box, second days will be performed with cell transfection; digested and 1.5ml EP tube, standard Note 1, adding 100 L 1640 medium, adding 2 g PAR-2 containing shRNA plasmid, still 5 min; another 1.5 ml EP tube, marked 2, adding 100 L 1640 medium, adding 10 L X-tremeGENE HP DNA Transf, a 5 min two mixed; well, a 15 min flask join again after antibiotic free medium, cultured 24-48h, the effect of transfection was observed by fluorescence microscope; using G418 on PAR-2 shRNA transfected cells were screened by transfected cells with G418 antibody, G418 will be killed, the remaining cells of PAR-2 shRNA transfected cells that will be used for subsequent experiments. According to the different treatment methods of esophageal carcinoma cells were divided into blank group, PAR-2 group (SLIGKV), excited PAR-2 anti excited group (VKGILS), PAR-2 shRNA group (shRNA transfection) and MAPK inhibition group (PD98059). Each group before the treatment is not the first to use the 1640 medium containing calf serum hungry In the culture of 24h, according to the relevant principle of adding drugs or blocking agents. Each group after the end of treatment, the use of RT-PCR to detect par-2mrna expression of erk1mrna and cyclind1mrna; Western-blot ERK1, detection of PAR-2, p-ERK1, CyclinD1 and PCNA protein expression, and cell growth curve were drawn. Results: 1rt-pcr gene detection method the expression results showed that: (1) compared with the control group, the expression of PAR-2 agonist group par-2mrna was 2.651 times (P0.05), the expression of par-2shrna in group par-2mrna was 0.385 times (P0.05); (2) compared with the control group, the expression of PAR-2 agonist group erk1mrna was 2.580 times (P0.05) expression. The amount of par-2shrna in group erk1mrna was 0.376 times (P0.05), the expression of the MAPK inhibitor erk1mrna was 0.497 times (P0.05); (3) compared with the control group, PAR-2 group, cyclind1mrna excited expression is 2.012 times (P0.05), par-2shrna group cyclind1mrna. As the amount of 0.318 times (P0.05), the expression of the MAPK inhibitor cyclind1mrna was 0.422 times (P0.05) to detect the expression of.2western-blot protein results: (1) the expression of PAR-2 PAR-2 protein excited group was 1.863 times that of the control group (P0.05), the expression of par-2shrna protein in PAR-2 group as blank group 0.373 times (P0.05); (2) the expression of PAR-2 protein in p-ERK1 group was excited as the blank group 1.517 times (P0.05), the expression of par-2shrna protein in p-ERK1 group was 0.478 times that of the control group (P0.05), the expression of MAPK p-ERK1 protein inhibitor group was 0.410 times that of the control group (P0.05); (3) the expression of PAR-2 was CyclinD1 the amount of protein is 1.557 times that of the control group (P0.05), the expression of par-2shrna protein in CyclinD1 group was 0.299 times that of the control group (P0.05), the expression of MAPK cyclinD1 protein inhibitor group was 0.364 times that of the control group (P0.05); (4) the expression of ERK1 in each group had no obvious change in the expression of.3pcan (P0.05) the amount of nodes Results: the excited group was the blank group was 1.738 times (P0.05); 0.784 times the transfection group was the control group (P0.05); the inhibition group was 0.683 times that of the control group (P0.05) the.4 cell count showed that: compared with the control group, excited group cell growth curve shift, transfection group and inhibition group decreased. Conclusion: 1 in esophageal cancer cells Ec109, PAR-2 and ERK1 and cylind1 in close contact with.2 in the presence of esophageal cancer cells EC109, PAR-2 agonist, can up regulate the expression of ERK1 gene in the downstream pathway of p-ERK1 protein and.3 expression in esophageal cancer cell line EC109, PAR-2 can be excited after up regulation of the downstream pathway of MAPK ERK1 pathways, upregulation of the expression of CyclinD1 mRNA and CyclinD1 protein, accelerate cell cycle, promote the proliferation of esophageal cancer cell EC109.4 in esophageal cancer EC109 cells, PAR-2shRNA can regulate MAPK expression and regulation of ERK1 pathway, CyclinD1, and EC109 of esophageal carcinoma cells Proliferation.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 楊麗麗;徐佳瑛;吳偉;祝桂祥;陳啟林;;CyclinD1、p27及PTEN蛋白在口腔鱗狀細(xì)胞癌中的表達(dá)[J];實(shí)用癌癥雜志;2013年05期

2 魯明騫;孔慶志;;絲裂原活化蛋白激酶信號(hào)傳導(dǎo)通路在惡性腫瘤中的研究現(xiàn)狀[J];實(shí)用癌癥雜志;2013年03期

3 陳金梅;鄧全軍;謝立群;趙建業(yè);劉彩虹;鄭艷敏;;蛋白酶激活受體-2基因靶向shRNA表達(dá)質(zhì)粒的構(gòu)建及轉(zhuǎn)染食管癌細(xì)胞致沉默效應(yīng)的研究[J];實(shí)用醫(yī)學(xué)雜志;2013年03期

4 朱寧;原繼榮;王德瑩;;c-fos和c-jun與癌癥關(guān)系的研究進(jìn)展[J];中國(guó)優(yōu)生與遺傳雜志;2012年02期

5 陳建勇;王聰;王娟;曹禮榮;;MAPK信號(hào)通路研究進(jìn)展[J];中國(guó)醫(yī)藥科學(xué);2011年08期

6 趙明哲;劉靖華;李玉花;姜勇;;ERK信號(hào)通路的信號(hào)轉(zhuǎn)導(dǎo)調(diào)控機(jī)制[J];國(guó)際病理科學(xué)與臨床雜志;2009年01期

7 王前;鄧晶;蔣永新;;cyclinD1的研究進(jìn)展[J];現(xiàn)代腫瘤醫(yī)學(xué);2009年02期

8 肖秀英;PCNA在惡性腫瘤中的研究進(jìn)展[J];張家口醫(yī)學(xué)院學(xué)報(bào);2003年06期

，

本文編號(hào)：1665368