【作者】 王奭骥；

【导师】 柳忠辉；

【作者基本信息】 吉林大学 ， 免疫学， 2007， 博士

【摘要】 卵泡抑素(follistatin,FS)是一种单链糖蛋白,具有广泛的组织分布及多种生物学活性。同时,FS作为激活素(Activin,ACT)结合蛋白,参与多种生理、病理功能的调节,如在胚胎发育、卵巢颗粒细胞分化、成骨细胞功能的调节、红细胞生成、维持神经细胞存活、肝纤维化、脑损伤、动脉粥样硬化、生殖及肿瘤形成中均具有重要的作用。粘蛋白-1(mucins-1,MUC1)是广泛分布于机体多种组织、器官粘膜表面的高分子量糖蛋白,具有多种功能,如其粘附和抗粘附作用,免疫活化和免疫抑制作用等,且与肿瘤抗原递呈、肿瘤的侵袭和转移、肿瘤的预后等密切相关。本研究采用荧光定量RT-PCR检测临床中常见的一些疾病,如肿瘤(肺癌、卵巢癌、肝癌、胃癌、乳腺癌、肾癌及良性肿瘤)、非肿瘤(糖尿病、高血压、急性心梗、肺炎、肺结核)及妊娠妇女和流产患者外周血循环FS、MUC1 mRNA表达水平,探讨其临床意义;并通过免疫组织化学染色对FS、MUC1成熟蛋白在组织中的表达进行研究。实验结果显示,良性疾病组糖尿病患者外周血循环FS mRNA表达水平明显升高,与健康对照组比较具有显著性差异,p<0.01。肿瘤患者组卵巢癌及肺癌患者外周血循环FS mRNA表达水平升高明显,与健康对照组比较具有统计学差异,p<0.01。健康成年人外周血循环FS mRNA表达水平在老年组轻度升高,但与青壮年组比较无统计学意义,p>0.05。正常早期与晚期妊娠女性外周血循环FS mRNA表达水平亦无明显变化。先兆流产患者外周血循环FS mRNA表达明显增加,与正常早期妊娠组比较具有显著性差异,p<0.05。ELISA检测结果显示健康成年人外周血FS蛋白水平在老年组明显升高,统计学比较差异显著,p<0.05;正常晚期妊娠女性外周血FS蛋白水平明显高于早期妊娠女性组,p<0.01。上述结果提示成年人外周血循环FS mRNA表达水平受年龄及妊娠状态影响较小,在肿瘤诊断上可能较外周血FS蛋白检测更有意义。检测不同疾病患者外周血循环MUC1 mRNA结果显示,良性疾病组心肌梗塞患者外周血循环MUC1 mRNA表达水平明显升高,与健康对照组比较,具有统计学差异,p<0.05。肿瘤患者中,卵巢癌、乳腺癌和肺癌患者外周血循环MUC1 mRNA水平明显高于对照组,统计学比较差异显著,分别为p<0.01,p<0.05,p<0.01;而肝癌、胃癌、肾癌及良性肿瘤患者外周血循环MUC1 mRNA水平无明显变化。健康老年组与青壮年组外周血循环MUC1 mRNA表达水平比较无统计学差异,p>0.05。正常晚期妊娠与早期妊娠女性外周血循环MUC1 mRNA表达水平比较明显升高,p<0.01。先兆流产患者外周血循环MUC1 mRNA表达水平也明显升高,与正常早期妊娠组比较具有统计学差异,p<0.01。为了进一步研究肿瘤患者及妊娠女性外周血循环FS、MUC1 mRNA的来源,采用免疫组织化学染色观察部分肿瘤及胎盘绒毛组织FS、MUC1成熟蛋白表达情况。抗FS及MUC1抗体组化染色结果显示,肺癌、乳腺癌及卵巢癌组织均呈阳性染色;胎盘绒毛组织FS、MUC1成熟蛋白也有明显表达。上述结果提示肿瘤患者血循环FS及MUC1 mRNA表达增加可能直接来源于肿瘤细胞;妊娠期MUC1 mRNA升高可能来自胎盘组织。综合上述资料,FS作为激活素结合蛋白可能参与糖尿病的形成过程,循环FS、MUC1 mRNA的检测有助于临床肿瘤诊断,胎盘组织FS及MUC1的表达可能与生理及病理妊娠有关。更多还原

【Abstract】 FS (Follistatin, FS) is a single polypeptide chain glycoprotein isolated from ovarian follicular fluid which directly inhibits the secretion of FSH from anterior pituitary cells. FS has a widely histological distribution especially has a high expression in ovary, pituitary, kidney and embyro which indicates that FS has a different function on different kind of cells. As an activin (ACT) binding protein, FS participates in various kinds of physiological and pathological functional regulation by blocking the biological activities of ACT. The studies on gene–knocked out mice suggest that FS is involved in the process from embryogenesis to maturity. FS and ACT co-exist and express in one organ as a balance system to maintain its normal function. The high expression or low expression of either of FS and ACT can lead to functional unbalance of the organ. Current studies suggest that FS plays an important role in diseases including hepatic fibrosis, pulmonary fibrosis, renal injury, skin injury, brain injury, atherosclerosis and so on. Mucins-1 (MUC1) is a kind of macromolecule glucoprotein which widely distribute on the surface of mucous membrane in several kinds of tissues and organs. 13 different kinds of MUC have been found (MUC1-MUC13). MUC1 mucoprotein normally expresses in epithelial cell proximal end and glandular cavosurface which is polarly distributes in various kinds of tissues, organs. Abnormity expression of MUC1 is involved in many kind of cancer so that MUC1 are regarded as a cancer marker. In recent years, numerous vaccines based on MUC1 immunogen and immune targeting vector based on MUV1 antigen has been used to cancer biotherapy research and many have been used in clinical experiment phase.1. ObjectiveTo investigate the application value of FS, MUC1 mRNA examination in clinical disease diagnosis2. Methods(1) Flurescent quantity RT-PCR was used to examine the peripheral blood circulating FS and MUC1 mRNA of several diseases, such as cancers (lung cancer, ovarian carcinoma, liver cancer, breast cancer, renal carcinoma and benign tumor), non-tumors (diabetes mellitus, hypertension, acute myocardial infarction, pneumonia, pulmonary tuberculosis) and normal pregnant women and threatened abortion patients(2) Immunohistochemistry stain was used to examine the FS and MUC1 protein expression on ovarian carcinoma, cancer of breast, lung cancer and placental villus tissue.(3) Two site ELISA was used to examine the expression of mature protein expressions of FS and MUC1 in healthy adults and pregnant women peripheral blood.3. Results(1) The results showed that the level of circulating FS mRNA in sera of the diabetic among the benign disease groups had been detected obviously increased. There’s a statistical significance (p<0.01) compared with that of the healthy control group, while other benign disease groups possessed no statistical difference with the healthy adults group (p>0.05). The peripheral blood circulating FS mRNA level in the ovarian carcinoma and lung cancer patients in the cancer group had been detected obviously increased and there’s a statistical significance compared with that of healthy control group (P<0.01). The peripheral blood circulating FS mRNA level in healthy old-aged group increased slightly, whereas there is no statistical difference compared with the healthy youth group (p>0.05). The peripheral blood circulating FS mRNA level in early and late pregnant group women had no obvious difference (p>0.05). The peripheral blood circulating FS mRNA level in threatened abortion patients increased evidently. There is a statistical significance (p<0.05) compared with normal early pregnant group. The peripheral blood circulating FS mRNA level examined by ELISA in late pregnant women are obviously higher than that in early pregnant group (P<0.01). The above data revealed that the peripheral blood circulating FS mRNA level was not closely related with age and pregnancy, but played much more significant role in cancer diagnosis than the examination of FS protein in peripheral blood. (2) Investigation of the peripheral blood circulating MUC1 mRNA in various diseases patients demonstrated that the MUC1 mRNA level of myocardial infarct patients in benign group increased obviously and there was a statistical significance (p<0.05) compared with the control group whereas there was no significant difference among the diabetics, hypertension, pneumonia and pulmonary tuberculosis groups. The peripheral blood circulating MUC1 mRNA level in ovarian carcinoma, lung cancer and breast cancer were obviously higher than that in control group and there were statistical significances (p<0.01, p<0.01 and p<0.05 respectively) whereas the peripheral blood circulating MUC1 mRNA level in liver cancer, stomach carcinoma, renal carcinoma and benign cancer patients had no significance changes. There was no statistic difference (p>0.05) between the peripheral blood circulating MUC1 mRNA level in healthy gerontism group and healthy adult group. Compared with early pregnant women, the peripheral blood circulating MUC1 mRNA level in late pregnant group increased obviously (p<0.01).4. Conclusion(1) The peripheral blood circulating FS mRNA level in lung cancer and ovarian cancer patients are highly expression. There is a significant difference compared with normal control group which suggests that the examination of the peripheral blood circulating FS mRNA may act as an assistant diagnosis for clinical cancer. The peripheral blood circulating FS mRNA level in adult is not closely related with age and pregnancy so that FS mRNA expression level plays a much more significant role in cancer diagnosis than the examination of peripheral blood circulating FS protein. But whether the abnormal high expression is the result of cancer genesis or cancer formation requires further investigation. The peripheral blood circulating FS mRNA expression level of threatened abortion patients increased significantly compared with normal early pregnant group which suggests that the expression of peripheral blood circulating FS mRNA may be associate with pathological pregnancy. What’s more, the expression of peripheral blood circulating FS mRNA also might play certain part in diabetic diagnosis and assessment of therapy effect, but the relationship with the formation of diabetic still need further study.(2) The peripheral blood circulating MUC1 mRNA levels in ovarian carcinoma, breast cancer and lung cancer patients are obviously increased highly and there is a statistical significance which reveals that the examination of peripheral blood circulating MUC1 mRNA may give an assistant diagnosis for clinical cancer. Compared with early pregnant women, the peripheral blood circulating MUC1 mRNA levels in late pregnant group increased obviously and peripheral blood circulating MUC1 mRNA level in threatened abortion patients also increased evidently which indicates that MUC1 might have some relationship with physiological and pathological pregnancy. The research approves that peripheral blood circulating MUC1 mRNA level in myocardial infarct patients increased obviously. However, the mechanism is still unclear. Whether MUC1 could act as a kind of available marker in acute myocardial serum still or not need further investigation. (3) The experiment results show that peripheral blood circulating MUC1 mRNA level increased obviously in cancer, physiological and pathological pregnancy and myocardial infarct patients which indicates that as an assistant index for cancer diagnosis, the examination of peripheral blood circulating FS mRNA may has much more speciality than that of MUC1.更多还原