PAR-2对食管癌EC109细胞增殖的影响

发布时间：2018-03-25 23:39

本文选题：食管癌　切入点：蛋白酶激活受体2　出处：《河北医科大学》2017年硕士论文

【摘要】：目的:蛋白酶激活受体-2(Proteinase-actived receptors 2,PAR-2)是位于细胞膜表面的G蛋白偶联受体,该受体被激活后可引起一系列关于肿瘤生长、侵袭、转移等方面的生物学行为。食管癌是人类最常见的恶性肿瘤之一,其发生机制目前还不太明确,是当前食管癌研究中重要的一个方面。研究表明,在多种肿瘤组织及细胞中存在PAR-2的高表达,提示PAR-2可能与肿瘤的生物学特性和恶性程度密切相关。关于PAR-2对食管癌EC109细胞增殖的影响及其分子机制,目前尚不清楚。本试验通过体外培养人食管癌细胞株EC109,研究PAR-2对食管鳞状细胞癌EC109细胞增殖的影响,并探讨其调节作用的可能分子机制。方法:细胞培养:用含有10%新生牛血清的1640培养基,置于37℃、5%CO2饱和湿度的孵育箱中培养,待细胞长满培养瓶底部70-80%(约2-3 d)时用胰蛋白酶消化传代,取对数生长期细胞用于实验。细胞转染:显微镜观察细胞生长情况;使用一次性无菌吸管将培养瓶中的废液吸除,使用PBS漂洗2遍;用移液枪加入1 ml Trypsin液,消化3 min(37℃,5%CO2),加入1ml的含血清培养基终止反应,使用一次性无菌吸管吹打培养瓶壁,将培养液装入离心管中,离心;用培养液重悬细胞,细胞计数后选择6×104/ml的密度加入不含抗生素培养基的培养瓶中,将培养瓶转入培养箱中培养,第二天转染;将细胞同上进行消化离心,取1.5ml EP管,标记为1,加入100μl 1640培养基,再加入2μg含PAR-2 shRNA的质粒,静止5 min;另取1.5 ml EP管,标记为2,加入100μl 1640培养基,再加入10μl X-tremeGENE HP DNA Transf,静止5 min;将两管混匀,静止15 min,后再次加入不含抗生素培养基的培养瓶中,培养24-48h后,使用荧光显微镜观察转染效果;使用G418对PAR-2 shRNA转染后的细胞进行筛选,不被转染的细胞不具备G418抗体,会被G418杀死,剩余细胞为PAR-2 shRNA转染成功细胞,用于后续实验。将食管癌细胞按不同处理方法分为空白组、PAR-2激动组(加入SLIGKV)、PAR-2反激动组(加入VKGILS)、PAR-2 shRNA组(shRNA转染)和MAPK抑制组(pd98059)。每组处理前首先使用不含小牛血清的1640培养基饥饿培养24h,在按相应的处理原则加入药品或阻断剂。每组处理结束后,使用rt-pcr检测par-2mrna、erk1mrna和cyclind1mrna的表达情况;使用western-blot检测par-2、erk1、p-erk1、cyclind1和pcna蛋白的表达情况,并且绘制各组细胞的生长曲线。结果:1rt-pcr方法检测基因表达结果显示:(1)与空白组相比较,par-2激动组par-2mrna的表达量为2.651倍(p0.05),par-2shrna组par-2mrna的表达量为0.385倍(p0.05);(2)与空白组相比较,par-2激动组erk1mrna的表达量为2.580倍(p0.05),par-2shrna组erk1mrna的表达量为0.376倍(p0.05),mapk抑制剂组erk1mrna的表达量为0.497倍(p0.05);(3)与空白组相比较,par-2激动组cyclind1mrna的表达量2.012倍(p0.05),par-2shrna组cyclind1mrna的表达量为0.318倍(p0.05),mapk抑制剂组cyclind1mrna的表达量为0.422倍(p0.05)。2western-blot检测蛋白表达结果:(1)par-2激动组par-2蛋白的表达量为空白组的1.863倍(p0.05),par-2shrna组par-2蛋白的表达量为空白组0.373倍(p0.05);(2)par-2激动组p-erk1蛋白的表达量为空白组1.517倍(p0.05),par-2shrna组p-erk1蛋白的表达量为空白组的0.478倍(p0.05),mapk抑制剂组p-erk1蛋白的表达量为空白组的0.410倍(p0.05);(3)par-2激动组cyclind1蛋白的表达量为空白组的1.557倍(p0.05),par-2shrna组cyclind1蛋白的表达量为空白组的0.299倍(p0.05),mapk抑制剂组cyclind1蛋白的表达量为空白组的0.364倍(p0.05);(4)erk1在各组中表达无明显改变(p0.05)。3pcan表达量结果:激动组为空白组的1.738倍(p0.05);转染组为空白组的0.784倍(p0.05);抑制组为空白组的0.683倍(p0.05)。4细胞计数结果显示:与空白对照组相比较,激动组的细胞生长曲线上移,转染组、抑制组下降。结论:1在食管癌细胞ec109中,par-2与erk1及cylind1存在密切联系。2在食管癌细胞EC109中,PAR-2激动后,可以上调其下游通路中ERK1基因及p-ERK1蛋白的表达。3在食管癌细胞EC109中,PAR-2激动后可以上调其下游通路中MAPK ERK1通路,上调CyclinD1 mRNA和CyclinD1蛋白的表达,加快细胞周期,促进食管癌细胞EC109的增殖。4在食管癌细胞EC109中,PAR-2shRNA可以通过调节MAPK ERK1通路,进而调节CyclinD1的表达,进而影响食管癌细胞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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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