胃泌素释放肽及其受体在结直肠癌中的表达

目的研究胃泌素释放肽（GRP）及胃泌素释放肽受体（GRPR）在结直肠癌中的表达。方法采用免疫组化方法测定结直肠癌中GRP和GRPR的表达。结果 GRP和GRPR在结直肠癌中阳性表达,阳性表达率分别为65.3%和79.6%,随着病情严重程度增加表达增加。结论 GRP和GRPR的异常表达与结直肠癌有关,可作为结直肠癌治疗的目标。     

胃泌素释放肽及其受体在结直肠癌中的表达

目的探讨结直肠癌中胃泌素释放肽与预后、远处转移的关系,同时分析其在远处转移中的预测价值。方法收集我院62例诊断为结直肠癌患者的临床及病理资料,采用前瞻性研究方法,利用免疫组化技术了解患者中胃泌素释放肽(GRP)、胃泌素释放肽受体(GRPR)蛋白的表达,结合肿瘤体积进行分析,了解二者与结直肠癌患者预后、远处转移的关系。结果 GRP、GRPR蛋白在结直肠癌组织中阳性表达率分别为79.0%和83.2%,二者表达存在相关性。GRP、GRPR蛋白水平与结直肠癌患者的预后及远处转移差异具有一定的相关性。GRP、GRPR能够在一定程度上预测结直肠癌远处转移,敏感度及特异度为69.4%、71.3%及70.1%、65.5%。结论 GRP、GRPR能够较好的预测结直肠癌患者的预后及远处转移,具有较好的敏感性及特异性。     

胃泌素释放肽前体对小细胞肺癌诊治的临床意义

目的探讨血清胃泌素释放肽前体(ProGRP)的生物学特性及其对小细胞肺癌(SCLC)的诊断、病情监测、疗效评估、预后判断的临床意义以及ProGRP与神经元特异性烯醇化酶(NSE)水平之间的相关性,为SCLC的预防和治疗提供科学依据。方法收集肺癌患者1221例,健康对照组500名,采用酶联免疫吸附法检测治疗前后肺癌患者及健康对照者血清ProGRP和NSE水平,同时肺癌患者还进行相关的影像学检查。结果270例治疗前SCLC患者血清ProGRP中位值为544.48pg/ml,敏感性为84.07%,均明显高于非小细胞肺癌(NSCLC)组及健康对照组,差异均有统计学意义(P<0.01)。270例SCLC患者随着临床分期期别的增加,Pro-GRP水平大于界值例数也明显增加,检出敏感性增加(P<0.01)。270例SCLC患者疗效评价获得完全缓解者ProGRP水平明显下降,无变化者及病情进展者ProGRP水平均明显高于界值(P<0.01)。治疗后在24个月之内出现复发、转移患者,血ProGRP水平明显高于治疗后健康生存者,且高于治疗前血ProGRP水平(P<0.01)。ProGRP水平的变化与影像学所示的肿瘤大小具有明显的相关性,ProGRP和NSE水平对SCLC的联合检测呈正相关(r=0.243,P<0.01)。结论血清ProGRP检测可作为SCLC诊断、病情监测、疗效评估、预后判断的敏感和特异性指标,与NSE联合检测可进一步提高敏感性。     

抗胃泌素释放肽单克隆抗体的制备及其在体外对乳腺癌细胞生长的抑制

研制抗胃泌素释放肽(Gastrin-releasing peptide,GRP)单克隆抗体,并且初步评估其在体外对乳腺癌细胞生长的抑制作用。纯化重组的GRP融合蛋白,免疫BALB/c小鼠,取经免疫的小鼠脾细胞与小鼠骨髓细胞SP2/0进行细胞融合,对杂交瘤细胞进行筛选,阳性孔经3次有限稀释法亚克隆,获得稳定分泌GRP抗体的杂交瘤细胞株,进一步从腹水中用硫酸铵沉淀法纯化单抗,并以间接ELISA、Western免疫印迹以及亚类分型等方法进行抗体鉴定,之后在体外用MTT比色法测定该单抗抑制乳腺癌细胞EMT-6生长的作用。结果表明通过筛选获得了一株阳性杂交瘤细胞,命名为W9,这株杂交瘤细胞分泌的单抗具有效价高,特异性好的特点,其亚型为IgG1,体外的细胞实验也表明这种单克隆抗体能够显著地抑制EMT-6细胞的增殖。     

胃泌素释放肽DNA疫苗抑制EMT6乳腺癌生长的实验研究

目的：观察胃泌素释放肽（GRP）DNA疫苗对EMT6小鼠乳腺癌生长的抑制作用。方法：将构建的GRP DNA疫苗pCR3.1-VS-HSP65-TP-GRP6-M2肌肉注射免疫BALB/c雌性小鼠,2周1次,共5次。采用ELISA法对小鼠的血清中抗GRP-IgG类抗体进行检测。最后一次免疫后第2周,接种EMT6小鼠乳腺癌细胞。于肿瘤细胞接种后d14,处死全部动物,称量肿瘤的质量和测量肿瘤的体积。并对瘤组织进行常规HE染色。结果：pCR3.1-VS-HSP65-TP-GRP6-M2免疫BALB/c小鼠,可诱导抗GRP-IgG类抗体的产生。并对随后的EMT6肿瘤细胞攻击有显著的抑制作用（P〈0.01）,抑瘤率为46.53%。病理学检查结果显示,GRP DNA疫苗成功地激发了宿主的抗肿瘤免疫反应;与pCR3.1-VS-HSP65-TP对照组相比,GRP DNA疫苗免疫组小鼠EMT6肿瘤组织浸润性下降。结论：GRP DNA疫苗显著抑制EMT6乳腺癌生长,为此类疫苗应用研究奠定了基础。     

胃泌素释放肽前体融合蛋白的制备及其在肿瘤研究中的应用

目的克隆胃泌素释放肽前体(pro-GRP)基因、构建重组质粒pGEM-T-pro-GRP和表达载体PMS-31b-pro-GRP、在大肠杆菌中热诱导表达并制备融合蛋白。方法从BGC823胃癌细胞中提取总RNA,利用pro-GRP特异引物,扩增人pro-GRP分子的cDNA全长。将pro-GRP基因定向克隆于pGEM-T载体转化JM109,DNA测序鉴定基因序列。将pro-GRP基因定向克隆于原核高效表达载体PMS-31b,转化大肠杆菌POP2136经42℃热诱导表达MS2-pro-GRP融合蛋白。将融合蛋白分离纯化后,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)凝胶鉴定。结果经聚合酶链反应(PCR)扩增成功获得210bp的pro-GRP基因,目的基因序列正确。SDS-PAGE显示热诱导后表达的融合蛋白分子量约为18000。结论成功克隆pro-GRP基因,并表达和纯化了PMS-31b-pro-GRP融合蛋白,为建立pro-GRP检测方法奠定了基础。     

胃泌素释放肽前体在小细胞肺癌诊断及治疗中应用探讨

目的:探究胃泌素释放肽前体在小细胞肺癌诊断及治疗中应用.方法:选取2018年12月～2019年11月某院收治的小细胞肺癌和非小细胞肺癌患者各65例,分为小细胞肺癌患者组和非小细胞肺癌患者组.结果:小细胞肺癌组的ProGRP(胃泌素释放肽前体)、CEA(癌胚抗原)以及NSE(神经元特异度烯醇化酶)等血清指标高于非小细胞肺...     

胃泌素释放肽前体检测对小细胞肺癌诊治的意义

目的探讨血浆胃泌素释放肽前体(ProGRP)对小细胞肺癌(SCLC)的临床诊断价值。方法用化学发光法检测50例健康体检者、49例非小细胞肺癌(NSCLC)及50例初诊SCLC(局限期29例、广泛期21例)患者血浆ProGRP水平,评估血浆ProGRP诊断SCLC的临床价值。结果健康对照组、NSCLC组和SCLC组血浆ProGRP水平分别为34.3、39.1和403.1 ng/L;各组间血浆ProGRP差异均有统计学意义。局限期SCLC(LD-SCLC)组血浆ProGRP高于健康对照组,差别有统计学意义。广泛期SCLC(ED-SCLC)组血浆ProGRP高于LD-SCLC组,差别有统计学意义。以健康组为对照,ROC曲线上取约登指数最大点确定ProGRP临界值为36.9 ng/L,ProGRP的敏感度为88%,特异度为98%;以NSCLC组为对照,ROC曲线上取约登指数最大点确定ProGRP的临界值为62.9 ng/L,ProGRP的敏感度为82%,特异度为96%。结论血浆ProGRP检测可作为SCLC诊断、病情监测的敏感和特异性指标,可广泛应用于临床。     

急性白血病及联合化疗对C肽,可的松和胃泌素的影响

检测急性白血病患者初发时和完全缓解后血清Ｃ肽、可的松和胃泌素含量，显示ＡＬＮＲ和ＡＬＣＲ患者Ｃ肽和可的松含量与正常对照无显著性差异，而ＡＬＣＲ患者胃泌素含量与ＡＬＮＲ和正常对照组有极显著性差异（Ｐ〈０．０１和０．００１），ＡＬＮＲ患者胃泌素含量高于正常对照，但无显著性差异（Ｐ〉０．０５），提示联合化疗可导致胃泌素分泌增加。     

单纯性肥胖儿童胰岛素释放试验中血清瘦素、胰岛素及C肽水平的变化

目的　探讨单纯性肥胖儿童胰岛素释放试验中血清瘦素与胰岛素、C肽、血糖水平的变化及其相关性。方法　随机选取中山市小学单纯性肥胖儿童30名(体块指数≥2 3% )为实验组,30名正常儿童(体块指数18%～2 0 % )为对照组,两组均测定空腹血清瘦素、胰岛素、C肽水平及微机血糖,实验组作胰岛素释放试验,于0、30、6 0、12 0、180min分别测定血清瘦素、胰岛素、C肽水平及微机血糖。结果　实验组空腹血清瘦素、胰岛素、C肽水平均高与对照组(P <0 . 0 5 ) ,微机血糖比较差异无显著意义(P >0 .0 5 ) ;实验组瘦素水平与胰岛素及C肽在0、12 0min及180min时段有相关性,但扣除肥胖因素后无相关性。瘦素的血清水平随着时间的推移有逐渐下降的趋势。结论　单纯性肥胖儿童存在高瘦素、高胰岛素、高C肽血症,进食后胰岛素分泌水平的升高,对瘦素的分泌有抑制作用,且持续时间较长。     

Solid phase synthesis and characterization of two canine gut gastrin-releasing peptides

Two canine gastrin-releasing peptides originally isolated from gut tissue extracts have been synthesized by solid phase methodology and purified by preparative reverse phase high performance liquid chromatography (RP-HPLC). The synthetic gastrin-releasing peptides GRP1-27 and GRP 5-27 were characterized with regard to homogeneity and composition using nine different RP-HPLC systems, mass spectroscopy, amino acid analysis, Edman degradation, methionine oxidation, and peptide mapping with tryptic, Staph. aureus V8 protease and cyanogen bromide cleavage (the latter two systems performed only with GRP 1-27). Although a scarcity of the natural products prevented quantitative biological comparison of the synthetic and natural peptides, they were found to elute identically on RP-HPLC co-chromatography and similar dose dependent biological potencies were observed in canine antral muscle tissue contraction experiments. Indeed, all the peptides containing the bombesin-like car-boxyl terminal decapeptide sequence studied to date have similar biological activities.     

Identification of key amino acids in the gastrin-releasing peptide receptor (GRPR) responsible for high affinity binding of gastrin-releasing peptide (GRP)

The bombesin (Bn) receptor family includes the gastrin-releasing peptide (GRPR) and neuromedin B (NMBR) receptors, Bn receptor subtype 3 (BRS-3) and Bn receptor subtype 4 (BB(4)). They share 50% homology, yet their affinities for gastrin-releasing peptide (GRP) differ. The determinants of GRP high affinity for GRPR and BB(4), and low affinity for BRS-3 are largely unknown. To address this question we made an analysis of structural homologies in Bn receptor members correlated with their affinities for GRP to develop criteria to identify amino acids important for GRP selectivity. Fourteen differences were identified and each was mutated singly in GRPR to that found in hBRS-3. Eleven mutants had a loss of GRP affinity. Furthermore, three of four amino acids in the GRPR selected used a similar approach and previously reported to be important for high affinity Bn binding, were important for GRP affinity. Some GRPR mutants containing combinations of these mutations had greater decreases in GRP affinity than any single mutation. Particularly important for GRP selectivity were K101, Q121, A198, P199, S293, R288, T297 in GRPR. These results were confirmed by making the reverse mutations in BRS-3 to make GRP gain of affinity mutants. Modeling studies demonstrated a number of the important amino acids had side-chains oriented inward and within 6A of the binding pocket. These results demonstrated this approach could identify amino acids needed for GRP affinity and complemented results from chimera/mutagenesis studies by identifying which differences in the extracellular domains of Bn receptors were important for GRP affinity.     

Omeprazole in elderly duodenal ulcer patients: relationship between reduction in gastric acid secretion and fasting plasma gastrin

Summary The effect of omeprazole on acid secretion and gastrin levels has been investigated in 10 elderly duodenal ulcer patients in remission. Doses of 5, 10, 20 and 40 mg omeprazole were given once daily for 7 consecutive days and the basal (BAO) and peak (PAO) acid output and fasting plasma gastrin concentration were measured 24 h after the seventh dose.Omeprazole suppressed PAO significantly and dose-dependently after doses of 10, 20 and 40 mg, the suppression being 42%, 75% and 85%, respectively. No patient showed complete inhibition of PAO and at least 20 mg had to be given to obtain a marked inhibitory effect in all patients. Increasing the dose to 40 mg had only a slight additional effect compared to 20 mg. There was a relationship between degree of acid inhibition and the increase in fasting plasma gastrin. PAO had to be suppressed by more than 80% before a moderate increase in fasting plasma gastrin was observed.The optimal once-daily oral dose of omeprazole for inhibition of acid secretion in elderly patients appears to be 20 mg. Omeprazole 20–40 mg may cause a moderate increase in fasting plasma gastrin.Supported by grants from the Swedish Medical Research Council (project no: 17X-760)     

Function of non-visual arrestins in signaling and endocytosis of the gastrin-releasing peptide receptor (GRP receptor)

Little is known about the role of arrestins in gastrointestinal hormone/neurotransmitter receptor endocytosis. With other G protein-coupled receptors, arrestins induce G protein-uncoupling and receptor endocytosis. In this study, we used arrestin wild-type and dominant-negative mutant constructs to analyze the arrestin dependence of endocytosis and desensitization of the gastrin-releasing peptide receptor (GRP-R). Co-expression of the GRP-R with wild-type arrestin2 and arrestin3 increased not only GRP-R endocytosis but also GRP-R desensitization in arrestin-overexpressing cells. Co-expression of the dominant-negative mutants V53D-arrestin2 or V54D-arrestin3 reduced GRP-R endocytosis. Notably, different trafficking routes for agonist-activated GRP-R-arrestin2 and GRP-R-arrestin3 complexes were found. Arrestin3 internalizes with GRP-R to intracellular vesicles, arrestin2 splits from the GRP-R and localizes to the cell membrane. Also, the recycling pathway of the GRP-R was different if co-expressed with arrestin2 or arrestin3. Using different GRP-R mutants, the C-terminus and the 2nd intracellular loop of the GRP-R were found to be important for the GRP-R-arrestin interaction and for the difference in GRP receptor trafficking with the two arrestin subtypes. Our results show that both non-visual arrestins play an important role in GRP-R internalization and desensitization.     

Stimulation of proliferation and migration of a colorectal cancer cell line by amidated and glycine-extended gastrin-releasing peptide via the same receptor

Although amidated forms of gastrin-releasing peptide (GRP) have been identified as autocrine growth factors in small cell lung cancer, their role in the development and progression of colorectal carcinoma is less clear. In addition, the biological activity of non-amidated gastrin-releasing peptide has not been investigated in colorectal carcinoma cells. We therefore investigated the effect of bombesin (a homologue of gastrin-releasing peptide) on proliferation, migration and inositol phosphate production in the human colorectal carcinoma cell line DLD-1, and determined the ability of gastrin-releasing peptide receptor antagonists to inhibit these effects. We also compared the biological activities of amidated and non-amidated GRP in the same assays. Treatment with either bombesin, or amidated or non-amidated GRP resulted in significant increase in proliferation, and in migration in a wound-healing assay. Both the mitogenic and migratory effects of amidated and non-amidated forms were inhibited by the GRP receptor antagonist [d-Phe⁶, Leu-NHet¹³, des-Met¹⁴]-bombesin(6-13). The presence of GRP receptor mRNA and GRP binding sites in three colorectal carcinoma cell lines was demonstrated by RT-PCR and by binding of radiolabelled bombesin, respectively. Transfection of DLD-1 cells with a dominant negative phosphatidylinositol 3-kinase did not affect bombesin-stimulated cell proliferation, but inhibited bombesin-stimulated cell migration. Bombesin and GRPgly activated phospholipase C, mitogen-activated protein kinase and focal adhesion kinase. We conclude that both amidated and non-amidated forms of gastrin-releasing peptide accelerate proliferation and migration of DLD-1 human colorectal carcinoma cells via the gastrin-releasing peptide receptor, but that phosphatidylinositol 3-kinase is only involved in the cell migration signalling pathway. Our results suggest a potential role for gastrin-releasing peptide receptor antagonists in the management of colorectal carcinoma.     

肝硬化患者血浆降钙素基因相关肽和内皮素-1含量分析

目的　观察肝硬化患者血浆降钙素基因相关肽 (CGRP)和内皮素 (ET 1)含量变化。方法　选择肝硬化患者 6 0例 (其中腹水患者 4 3例 )、正常对照 30例 ,用放射免疫法检测其血浆CGRP和ET 1含量。结果　肝硬化组血浆CGRP和ET 1明显高于对照组 (P <0 0 1) ;肝硬化腹水患者高于无腹水患者 (P <0 0 1) ;肝功能Child Pugh分级中C级高于B级、B级高于A级。结论　肝硬化患者血浆CGRP和ET 1水平增高 ,CGRP及ET 1在肝功能损伤及肝硬化高动力循环状态的发生中具有重要作用     

Effect of repeated doses of netazepide,a gastrin receptor antagonist,omeprazole and placebo on 24 h gastric acidity and gastrin in healthy subjects

Aim

To administer repeated oral doses of netazepide to healthy subjects for the first time, to assess safety, tolerability, pharmacokinetics and effect on 24 h gastric pH and plasma gastrin.

Method

We did two randomized, double-blind, parallel group studies. The first compared netazepide 25 and 100 mg 12 hourly, omeprazole 20 mg once daily and placebo for 7 days. On day 7 only, we measured pH and assayed plasma gastrin. The second study compared netazepide 5, 10 and 25 mg and placebo once daily for 14 days. We measured pH on days 1, 7 and 14 and assayed plasma gastrin on days 1 and 14. We compared treatments by time gastric pH ≥ 4 during 0–4, 4–9, 9–13 and 13–24 h after the morning dose, and by plasma gastrin. P < 0.05 was significant.

Results

Netazepide was well tolerated. On day 7 of the first study, netazepide increased pH significantly only during 9–13 h after the 100 mg dose, whereas omeprazole raised pH significantly during all periods. Both netazepide and omeprazole increased plasma gastrin significantly. Netazepide had linear pharmacokinetics. In the second study, netazepide caused dose-dependent, sustained increases in pH on day 1, but as in the first study, netazepide had little effect on pH on days 7 and 14. Again, netazepide increased plasma gastrin significantly.

Conclusion

Although repeated doses of netazepide led to tolerance to its effect on pH, the accompanying increase in plasma gastrin is consistent with continued inhibition of acid secretion, via gastrin receptor antagonism and gene up-regulation.     

Visualisation of the gastrin receptor within rat mucosa using a biotinylated gastrin antagonist

We have previously demonstrated that the peptide Boc-l -Trp-l -Leu-β-Ala is a potent and specific antagonist of pentagastrin-stimulated acid secretion in both the rat and the dog. Using conventional solution phase methodology, the analogue biotinyl-l -Trp-l -Leu-β-Ala was prepared in reasonable yield and purity and applied to cryostat sections of rat intestinal and other tissues. The sections were exposed to 5–10 μg of peptide and the bound analogue was visualised using streptavidin-fluorescein. The binding of the analogue was demonstrated in sections from fundus, duodenum, ileum, colon, and lung. However, the analogue failed to bind to tissue from the pancreas, heart, kidney, or liver. The binding of the probe was greatly reduced or completely inhibited by preincubation with Boc-l -Trp-l -Leu-β-Ala, pentagastrin, or gastrin 1–17. The distribution of the cells recognised by the probe was consistent with the distribution of histamine-containing enterochromaffin-like cells. The results of this study may have some bearing on current theories of the mechanism of gastrin-stimulated acid release.