【作者】 初照成；

【导师】 于成国；

【作者基本信息】 中国医科大学 ， 临床检验诊断学， 2002， 硕士

【摘要】 前言 人内因子（intrinsic factor IF）是由胃粘膜的壁细胞分泌的一种糖蛋白。其有两个活性部位，在摄入维生素B₁₂吸收的过程中，IF通过其中一个活性部位与摄入的维生素B₁₂结合形成复合物，从而保护维生素B₁₂不被小肠内水解酶破坏。然后再通过另一个活性部位与远端回肠上皮细胞膜上的受体结合，从而使维生素B₁₂被吸收。众所周知A型萎缩性胃炎患者因胃IF缺乏可引起恶性贫血，但有关血清IF水平与胃粘膜功能状态的研究报道不多。为了探讨血清IF水平与胃粘膜功能状态的关系，本研究分离纯化了人胃IF，并制作了放免试剂盒，最后采用放射免疫分析方法测定正常人及胃病患者血清中IF的含量。 材料与方法 1 实验材料 正常人胃组织来自中国医科大学法医学院，雄性家兔（大耳白）：两只，体重2kg，来自中国医科大学实验动物中心，血清标本来自青海省人民医院2000年10月至2001年8月住院患者，其中胃癌患者49例（术后2例），年龄32—86岁；胃溃疡患者22例，年龄22—67岁；萎缩性胃炎患者25例，年龄20—65岁，均经内镜或病理检查证实。所有患者于清晨空腹安静状态下采血，分离血清，于-20℃冻存。正常血清标本来自中国医科大学附属一院献血员，共40例，年龄20—49岁。 主要试药：DEAE-Sephadex A50、Sephadex G-75、CNBr Activa-t。d Seph删sc 4B（Ph。cia公司产品），Ac…咖de和伍s砒ylam－ide石DSu 公司产品X低分子量标准蛋白质(上海丽珠生物有限公司产品X弗氏完全佐剂(本院免疫教研室提供入 Na”‘Iu京原子能研究所X其它常规试剂为化学纯。 2实验方法 半冰冻条件下刮取胃粘膜，用滤纸吸干，称重，剪碎，于 0．01pH7．0磷酸缓冲液中制匀浆，经过两次离心，制成粗提液。 取粗提液30ml加人用0．01M pH7．0磷酸缓冲液预平衡的DEAE－SePhadex A50层析柱（3cm x 60cm），连续盐梯度洗脱，280urn波长下测各管吸光度，绘制吸光度曲线，将各个蛋白峰分别收集起来，经缩水透析后，于－20℃保存。 取上述第4峰透析液IInl加人已用0．01M pH7．0磷酸缓冲液预平衡的 SePhadex GJS层析柱（Icm x 60cm），用上述缓冲液洗脱，280run波长下测各管吸光度，绘制吸光度曲线，收集蛋白峰，经缩水过夜后，于－20℃保存。 再取凝胶过滤层析第互峰浓缩液0．6rnl于偶联Vi风。的滇化氰活化的 Sepharose 4B亲和层析柱（0．6cm x 20cm）上，4℃条件下过夜，然后以高浓度盐酸肌梯度作为最后洗脱步骤洗脱，280urn波长下测各管吸光度，绘制吸光度曲线，收集蛋白峰。经缩水透析后，于一20℃保存。 各蛋白峰蛋白质浓度测定采用酚试剂法。其分子量估计采用不连续SDS聚丙烯酚氨凝胶电泳法。 用部分纯化的IF兔疫家兔制备抗血清，以双向兔疫扩散法勿．定抗体效价。纯化的 IF采用 lodosen法，用放射性‘’l标记。 最后采用放免方法测定各组血清IF水平，其数据值范围均以X。Sk表示，统计学差异性检验采用t检验。 ·2· 结 果 胃粘膜匀浆经离子交换层析共分离出5个蛋白峰，其第4峰经凝胶过滤层析的第二峰再经亲和层析共分离2个蛋白峰，第二峰经皿S－PAGE显示为一条带，分子量约为66 00。免疫家兔所得血清IF的抗体效价为二：32。 各组血清IF放免测定结果：对照组为1．0584。0．4636pg／ml；胃癌组为 0．6784。0．2508 pgilnl；萎缩性胃炎组为 0．7040 L 0．2220 pg／tnl；胃溃疡组为 0．9279。0．4898 pgllnl。胃癌组和萎缩性胃炎组患者血清IF水平明显低于对照组h＜0．ofh胃溃疡组与对照组比较血清 IF水平无统计学差异h＞0．05人胃癌组与萎缩性胃炎组比较血清 IF水平无明显差异帅＞0．05人 胃溃疡组与胃癌组和萎缩性胃炎组比较差异不明显N．01＜p＜0．05人 讨 论 IF是摄人维生素B；。吸收所必需的辅因子，在体内其合成与分泌受多种物质影响。由于IF的含量较低，分离比较困难使其在临床方面的应用受到限制。为了探讨血清IF与胃粘膜功能状态的关系，特别是在胃癌时的变化，本研究采用离子交换层析、凝胶过滤层析和亲和层析相结合的方法，成功分离了人的胃IF，经不连续SDS聚丙烯酚凝胶电泳显示为一条带，分子量大约为66 000。对胃病患者血清IF水平测定结果表明胃癌组患者和萎缩性胃炎组患者血清IF水平明显低于对照组，这可能是由于此两种疾病时，患者胃粘膜损坏导致壁细胞大量减少，从而使IF的合成与分泌减少引起的。众所周知，萎缩性胃炎是胃癌的癌前病变，二者血清IF水平无统计学差异，因此该结果表明较低的血清IF水平很 ·3·可能是胃癌的高危指标，对于血清IF水平较低的体检者应该进一步做胃镜检查。在胃溃疡患者，血清IF水平与对照组比较无统计学差异，可能是由于胃溃疡时同时伴有广泛的胃粘膜炎性增生使壁细胞分泌IF的量增加，所以，尽管壁细胞数更多还原

【Abstract】 Intrinsic factor (IF) is a transport glycoprotein secreted by the parietal cells of the gastric fundic mucosa, which is necessary cofactor for the absorption of orally ingested vitaminB12 ( VitB12). IF has two binding sites, one each for Vit B12 and for the ileal receptor recognizing the IF-VitB12 complex that could not be hydrolyzed by hydrolase in the intestinal tract. It is known that IF deficiency induces malab-sorption of VitB12 which leads to subsequent megaloblastic changes as pernicious anemia in A type atrophic gastritis. Reports that reflected the correlation between the level of serum IF and gastric mucosal functional status is few. To study the relation, we separated and purified human IF, and maked the radio-immunity kits of IF. Eventually, we determined the content of serum IF by the method of radioimmunoas-say.Materials and MethodsTissue samples. Human normal gastric mucosa were gained fromCollege of Forensic Medicine of China Medical University. Two New Zealand male white rabbits with 2 kilogram. Samples of patients serum were gained from The People Hospital of Qinghai province from October, 2000 to August, 2001. These patients included 49 cases of gastric cancer ( age from 32 - 86 years) , 22 cases of stomach ulcer ( age from 22 - 67 years) , 25 case of atrophic gastritis ( age from 20 - 65 years). All of patients were diagnosised by endoscopy and/or histo-logic examination. Fasting sera from 96 patients obtained in the early morning were stored at -20℃. 40 cases normal serum samples were gained from healthy volunteers to donate blood.Chemicals. DEAE-Sephadex A50, Sephadex G-75 and CNBr activated Sephrose 4B were purchased from Pharmacia Corp. Acrylam-ide, Bis-acrylamide and SDS were purchased from Sigma Corp. 131I was a product of Institute of Atomic Energy in Beijing. Protein marker was purchased from Huamei Corp. All other chemicals were of reagent grade.Preparation of gastric mucosal extract. The mucosa that was thawed was separated by sharp dissection, drained on filter paper, weighed,, minced and homogenized in cold 0. 01M phosphate buffer, pH7.0(2ml/g tissue) , after two centrifuge, the supernatant was the extract.A column of 3cm x60cm was packed with diethylaminethvl (DE-AE)-Sephadex A50 and equilibrated with the above buffer. A-30 ml portion of mucosal extract was applied to the top of the column. The column was eluated with a linear NaCl gradient The protein peaks were pooled, concentrated, dialyzed and stored at - 20℃, respectively.Gel filtraton chromatography of partially purified IF on Sephadex G-75. 1-ml portion of fourth peak of above chromatography was appliedto the column. The column was eluted with 0. 01M phosphate buffer, pH7. 0. Protein peaks were pooled, concentrated and stored at -20℃.Affinity chromatography of partially purified IF on VitB12-Sepha-rose 4B. VitB12 were covalently coupled to CNBr-activated Sepharose 4B resulting in 1.17mg of B12 coupled per ml of Sepharose. Partially purified IF was passed over the column. A linear gradient of guanidine HC1 (0 to 4.0M) was used to separate IF.Protein determination. Protein was determined by the method of Lowry et al. with bovin serum albumin as a standard.The identification of Protein peaks and the evaluation of molecular weight of them were performed on Sodium Dodecyl Sulfate- Polyac-rylamide Gel Electrophoresis (SDS-PAGE)Preparation of the antiserum to IF and determination of valence. Rabbits were immunized by injecting the partially purified IF together with an equal volume of Freund s complete adjuvant into the back intramuscularly and subcutaneously at 2 week intervals. Valence of antibodies was determined by a double immunodiffusion method.The purified IF was labeled with I in accordance with the Iodo-gen method. A SephadexG-25 column was used to remove free 131I from 131I-IF.Serum IF level of groups was determined by the method of Radio-immunoassay.Statistical analysis The significance of differences in mean values was determined by Student’s t test for unpaired更多还原