【作者】 鲁玉娟；

【导师】 白文元；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 硕士

【摘要】 Barrett食管(Barrett’s esophagus,BE)是指食管鳞柱状上皮结合部移行至胃食管连接部近端且伴有肠化生的疾病。BE是一种癌前病变,与食管腺癌(esophageal adenocarcinoma, EAC)密切相关。在西方国家,由BE转化而来的食管腺癌的发病率明显升高,成为所有恶性肿瘤中增长速度最快的一种。亚洲国家的BE和食管腺癌虽不如西方国家常见,但近年来流行病学资料显示也呈增加趋势,因此我国加快与深入对此领域的研究,并通过干预措施来进行预防的意义重大。胃泌素(Gastrin)是由胃肠道G细胞分泌的一种多肽类激素,具有刺激胃酸分泌,促进胃肠道粘膜生长的作用。胃泌素受体是存在于胃中的胆囊收缩素2受体(CCK2R),属于G蛋白偶联受体超家族,它调节壁细胞的胃酸分泌、肠嗜镉细胞释放组胺以及平滑肌细胞的收缩。胃泌素与其受体结合后,通过激发多条信号传导通路,将有丝分裂信号传递到细胞核,引起细胞增殖。近年来,越来越多的研究表明,胃泌素还参与一些恶性肿瘤的发生和发展。在胰腺和胃中,人们发现CCK2R具有调节生长的作用,而且在许多肿瘤组织中都发现了CCK2R的存在。最近国外研究发现在Barrett化生组织也存在CCK2R,而Barretts食管是食管腺癌的癌前病变。这提示我们:在食管腺癌中可能也有CCK2R的存在,并在食管腺癌的发生和发展中起一定作用。目前对于CCK2R信号传导的机制尚不清楚,从已有的研究结果看,除经典的肌醇磷脂、cAMP信号途径外,还可能有丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)的参与。MAPK是20世纪80年代末期研究生长因子磷酸化产物时发现的一类丝/苏氨酸蛋白激酶家族,MAPK通路是调控炎症、增殖、分化、凋亡等多种细胞生物学效应的关键细胞信号传导通路。其成员ERK1/2即p42/44MAPK,是细胞信号中传递丝裂原信号的关键激酶,活化后可以激活多种核转录因子,影响目的基因的表达,与细胞生长、分化和增殖的调控关系密切。为此,本研究从胃泌素着手,通过对其受体的特异性阻断,研究了胃泌素及其受体在人食管腺癌细胞(SEG-1)增殖过程中的作用,以探讨其作用机制,为今后临床防治工作提供理论依据。目的:探讨胃泌素及其受体在人食管腺癌细胞系SEG-1增殖过程中的作用机制,为今后临床防治工作提供理论依据。方法:1采用体外细胞培养技术,应用MTT法测定不同浓度的胃泌素作用下SEG-1细胞增殖情况;2 RT-PCR方法检测CCK2R mRNA在胃泌素、丙谷胺及胃泌素+丙谷胺三组中的表达水平;3以相同浓度的胃泌素(10-7 mol/L)按时间梯度刺激细胞,Western blot法测定各时间点总p42/44 MAPK、磷酸化p42/44 MAPK蛋白表达情况,以确定p42/44 MAPK最大磷酸化时间点。4 Western blot法测定不同干预组中总p42/44 MAPK、磷酸化p42/44 MAPK蛋白在最大磷酸化时间点表达情况。结果:①胃泌素(G-17)对SEG-1细胞增殖的影响:10-9mol/L~10-5mol/L G17作用于SEG-1细胞24h后,其增殖率分别为108.79%、117.23%、125.38%、138.53%、150.33%,与对照组相比,明显升高,有统计学意义(P<0.05)。②丙谷胺(PGL)对SEG-1细胞增殖的影响:10-8mol/L~10-5mol/L的PGL作用于SEG-1细胞24h后,其生长抑制率分别为14.23%、18.37%、34.24%、55.97%,可显著抑制SEG-1细胞的增殖,有统计学意义(P<0.05)。③胃泌素(G-17)+丙谷胺(PGL)对SEG-1细胞增殖的影响:10-8mol/L~10-5mol/L的PGL作用于含10-7mol/L G-17的SEG-1细胞24h后,其生长抑制率分别为0.57%、3.50%、5.00%、12.22%,可明显抑制G-17对SEG-1细胞的促生长作用。④人食管腺癌细胞系SEG-1CCK2R基因表达:采用RT-PCR检测分析显示,人食管腺癌细胞系SEG-1中有CCK2R基因表达,G-17作用于SEG-1细胞后CCK2R/GAPDH灰度比值为0.78,表达有所下降;PGL作用于细胞后灰度比值为1.59,即出现表达上调;G-17与PGL同时作用于细胞后灰度比值为1.37,仍高于基础表达灰度比值1.03。⑤胃泌素(G-17)、丙谷胺(PGL)作用于SEG-1细胞后p42/44 MAPK磷酸化程度变化:Western blot结果显示,G-17作用于SEG-1细胞后p42/44 MAPK磷酸化程度有所升高;且p42/44 MAPK磷酸化程度随G-17作用时间的变化而变化,0min,5min,10min,20min,40min的p42/p44 MAPK磷酸化的百分比分别为3.92%,19.62%,39.58%,22.41%,14.93%,可见10min的p42/44 MAPK磷酸化水平最高,因此认为该时间点为p42/44 MAPK最大磷酸化时间点。PGL作用于细胞后p42/44 MAPK磷酸化程度有所下降(1.74%);使用PGL+G17共同作用于细胞后,p42/44 MAPK磷酸化程度(21.68%)明显比G-17组(41.32%)为低,仅略高于空白对照组(5.94%),表明G-17使p42/44 MAPK磷酸化作用可被PGL所抑制。结论:1胃泌素能促进人食管腺癌细胞系SEG-1的生长,且呈剂量依赖关系。胃泌素受体拮抗剂丙谷胺能明显抑制胃泌素对SEG-1细胞的促生长作用。2人食管腺癌细胞系SEG-1中有CCK2R基因表达,胃泌素作用于细胞后表达有所下降,丙谷胺作用于细胞后即出现表达上调,胃泌素与丙谷胺同时作用于细胞后其表达仍高于基础表达。3在相同浓度的胃泌素作用下,通过胃泌素受体CCK2R引起p42/44MAPK的磷酸化有时间依赖性。4胃泌素可以诱导人食管腺癌细胞系SEG-1内p42/44MAPK的磷酸化,提示胃泌素对MAPK通路有激活作用。更多还原

【Abstract】 Barrett’s esophagus (BE) is defined as the migration of squamous columnar epithelium of distal esophagus to gastroesophageal junction, with intestinal metaplasia accompanied. BE is an important precancerous change, which is closely correlated with esophageal adenocarcinoma (EAC). Esophageal adenocarcinoma progressed from Barrett’s metaplasia has an obviously increased occurrence rate in Western countries, and it is the malignant tumor with the highest growth rate. Though BE and adenocarcinorma are not so common in Asia, their incidences tend to increase as reported in recent years.Gastrin is a polypeptide hormone secreted from gastrointestinal G-cells, which can stimulate gastric secretion and promote epithelial cell growth. Gastrin receptor is presented as cholecystokinin type-2 (CCK-2) in gaster, a member of G-protein-coupled receptor superfamily. It can regulate gastric acid secretion, release of histamine from enterochromaffin like cell and contraction of contractile fiber cells, so it is named as CCK-2/ gastrin receptor along with CCK2R. After gastrin binds to its receptor, multiple signal transduction pathways would be activated, and mitoschisis signal will be transmitted into cell nucleus to proliferate cells. Recently, more and more evidences indicate that gastrin also participates in the occurrrence and development of some malignant tumors. Previous studies reported the gastrin’s trophic effect on pancreas and gaster, and many tumor tissues expressed cholecystokinin type-2 (CCK-2). Studies abroad also demonstrated the presence of CCK-2 (gastrin) receptor in Barrett’s metaplasia, which is thought to be a precursor of esophageal adenocarcinoma.At present, it is not clear about the mechanism of CCK2R signal transduction. It has been found that, in addition to the classic inositide lipositol and cAMP signal pathways, mitogen-activated protein kinase (MAPK) pathway may also be involved. MAPK is a member of serine streth/threonine protien kinase family, which has been found in the research on growth factor phosphorylation production in the end of 1980s. It is an important intracellular signal transduction pathway for regulating inflammation proliferation, differentiation and apoptosis. Its member ERK1/2 (i.e. p42/44 MAPK) is a key kinase of cell signal to transmit mitogen signal. By activating it, many transcription factors can be stimulated, thus to influence the expression of target gene. It also has a close relationship with cell growth, differentiation and proliferation.Therefore, we set about our research from gastrin. By blocking its specific receptor, the roles of gastrin and its receptor in proliferation of human esophageal adenocarcinoma cells were studied,to explore its mechanism of action, and providding theoretical evidence in clinical prevention and cure.Objective: To explore the role of gastrin and its receptor in the growth of esophageal adenocarcinoma cells, and providding theoretical evidence in clinical prevention and cure.Methods:1 By utilizing in vitro cell culture technique, proliferation of SEG-1 was assayed by MTT.2 Changes of CCK2R mRNA in different interventions were examined by reverse transcription-polymerase chain reaction (RT-PCR).3 Cells were stimulated by gastrin (10-7 mol/L) based on time gradient, expression of total p42/44MAPK and p42/44MAPK phosphorylation were determined by Western blot to identify the time point of maximal phosphorylation of p42/44MAPK.4 Expression of total p42/44MAPK and p42/44MAPK phosphorylation were determined by Western blot.Results:①Effect of gastrin on the proliferation of SEG-1: compared with control group, G-17 at concentration of 10-9 mol/L - 10-5 mol/L promoted the proliferation of SEG-1, and the growth rates were 108.79%, 117.23%, 125.38%, 138.53% and 150.33%, respectively (P<0.05).②Effect of proglumide on proliferation of SEG-1: compared with control group, PGL at concentration of 10-8 mol/L ~ 10-5 mol/L inhibited the proliferation of SEG-1, and the inhibition rates were 14.23%, 18.37%, 34.24% and 55.97%, respectively, (P<0.05).③Effect of gastrin and proglumide on proliferation of SEG-1: compared with control group, PGL at concentration of 10-8 mol/L-10-5 mol/L inhibited the proliferation of SEG-1 incubated in G-17 (10-7 mol/L) for 24h, and the inhibition rates were 0.57%, 3.50%, 5.00% and 12.22%, respectively, (P<0.05).④RT-PCR results: CCK2R mRNA was expressed in normal SEG-1; CCK2R/GAPDH was decreased to 0.78 after G-17 was added, and increased to 1.59 after PGL was added. Under the combined effects of G-17 and PGL, the gray scale ratio was 1.37, still higher than 1.03, the basal expression gray scale ratio.⑤Western blot results: phosphorylation of p42/44 MAPK protein in G-17 groups was increased than that in control group. The phosphorylation level of p42/44 MAPK protein in SEG-1 changed as G-17 action time prolonged. The phosphorylation rates at 0, 5, 10, 20 and 40 min were 3.92%, 19.62%, 39.58%, 22.41% and 14.93%, respectively. Therefore, 10 min is the time point of maximal phosphorylation. Phosphorylation of p42/44 MAPK protein in PGL groups and G-17 + PGL groups were reduced by 1.74% and 21.68% respectivley than that in control group.Conclusions:1 Gastrin could promote the proliferation of SEG-1 in a dose-dependent manner, while proglumide could obviously inhibit the effect.2 CCK2R mRNA expressed in SEG-1, and was downregulated under the effects of G-17, and up-regulated under the effects of PGL.3 The phosphorylation level is in a time-dependent manner. Gastrin-CCK2R-MAPK signal pathway may play an important role in growth of SEG-1.4 Gastrin could induce phosphorylation of p42/44 MAPK in SEG-1, which suggested activation of MAPK pathway.更多还原