TSHR蛋白在宫颈癌的表达及其与HPV-16感染的关系

目的研究促甲状腺激素受体（TSHR）在子宫颈癌组织的表达及其与乳头瘤病毒（HPV－16）的关系。方法应用免疫组织化学链霉菌抗生物素过氧化物酶（SP）法检测79例子宫颈癌和30例子宫颈炎组织HPV－16与TSHR蛋白表达。79例癌症患者中病理分级〈Ⅱ级33例,≥Ⅱ级46例;病理分期〈Ⅱ期56例,≥Ⅱ期23例;无淋巴结转移66例,有淋巴结转移13例;肿瘤大小〈3cm44例,肿瘤大小≥3cm35例。结果HPV－16在子宫颈癌表达率55．70％明显高于宫颈炎5％（P〈0．05）,TSHR在子宫颈癌表达率68．35％明显高于宫颈炎26．67％（P〈0．05）。HPV－16表达与肿瘤的大小、肿瘤分级、分期、淋巴结转移不相关。TSHR表达与肿瘤的大小呈正相关,P〈0．05,与肿瘤分级、分期及淋巴结转移不相关。HPV－16与TSHR在宫颈癌表达呈正相关。结论HPV感染对宫颈癌病变起到强烈的预警作用。TSHR不仅在甲状腺滤泡上皮细胞表达,在子宫颈癌细胞也表达,TSHR过表达能促进宫颈细胞的异常增殖,其异常功能可能是恶性肿瘤特定的临床表型。HPV与TSHR在子宫颈癌变过程中起协同作用。     

人乳头瘤病毒16型 E7蛋白在子宫颈癌细胞内的定位

应用重组质粒在大肠杆菌中表达人乳头瘤病毒(HPV)16 型 E7 基因.以所产生的 E7 融合蛋白为抗原免疫家兔,制得抗 E7 蛋白抗血清.在子宫颈癌组织切片中用此抗血清作免疫组化染色(胶体金标记染色法).在光学显微镜下可观察到癌细胞中存在 E7 抗原黑色颗粒,位于细胞核内.主要附着于核膜,可证明 E7 基因在 HPV16 感染的子宫颈癌细胞中有强烈表达;提示 E7 基因可能即为 HPV16的癌基因.     

Ezrin蛋白在眼睑基底细胞癌组织中的表达及其意义

目的探讨Ezrin蛋白在眼睑基底细胞癌组织中的表达。方法收集武汉市中心医院和武汉大学人民医院病理科2002—2009年手术切除及活检的眼睑基底细胞癌（basalcellcarcinoma,BCC）标本共20例,另取癌周围组织5例作对照。采用免疫组织化学方法观察各组细胞内Ezrin蛋白表达。利用图像分析系统测定Ezrin蛋白在以上各组中表达的平均光密度和平均阳性面积率。结果眼睑基底细胞癌组织中Ezrin蛋白呈高表达;癌旁组织中Ezrin蛋白呈低表达。图像分析结果显示：眼睑基底细胞癌组织与癌旁组织之间Ezrin蛋白的平均光密度及阳性面积率的差异有显著性意义（P〈0．05）。结论Ezrin蛋白在眼睑基底细胞癌的发生、发展过程中起了重要作用。     

Gli1蛋白和PDGFRα在大肠癌组织中的表达及临床意义

Gli1蛋白和血小板衍生生长因子受体α(PDGFRα)的表达在大肠癌的发生发展过程中的作用还不清楚.通过免疫组化法检测60例大肠癌及30例正常组织中Gli1和PDGFRα的表达,并与临床病理因素进行相关性分析,研究两者在大肠癌中的作用.研究发现,Gli1和PDGFRα蛋白在大肠癌组织中的阳性率分别为73.7%和78.3%,明显高于正常组织的表达(P<0.05).Gli1和PDGFRα蛋白表达与患者年龄、肿瘤大小、组织学类型等均无关(P>0.05),但是Gli1蛋白和PDGFRα表达与淋巴结转移状况和Duke分期相关(P<0.05).Spear-man相关分析显示,Gli1与PDGFRα蛋白表达正相关(r=0.298,P<0.05).结果表明Gli1和PDGFRα在大肠癌发生、发展中呈协同和相互调节作用,Gli1和PDGFRα的表达参与大肠癌的侵袭和转移过程.联合检测可作为判断大肠癌预后,筛选高危转移患者的有效指标,同时也可用于指导大肠癌的靶向药物治疗.     

子宫内膜癌组织中survivin蛋白和VEGF表达的意义及相关性分析

目的：探讨子宫内膜癌组织中survivin（存活素）蛋白和VEGF（血管内皮生长因子）表达及其意义。方法：运用免疫组织化学S-P法,检测20例正常子宫内膜组织、20例不典型增生子宫内膜和63例子宫内膜癌组织中survivin蛋白和VEGF的表达,并结合临床病理特点进行分析。结果：survivin蛋白、VEGF的阳性表达率在正常子宫内膜组织、不典型增生子宫内膜、子宫内膜癌中逐渐升高,三者之间有显著性差异（x2=-24．97,P〈0．01;x2=18．65,P〈0．01）。在子宫内膜癌中,survivin蛋白的表达与组织学分级和手术-病理分期密切相关（X2=9．20,P〈0．05;X2=20．60,P〈0．01）;VEGF的表达与组织学分级无关（X2=4．93,P〉0．05）,而与手术-病理分期分期密切相关（x2=-38．10,P〈0．01）。survivin表达与VEGF表达呈显著正相关（r=0．257;p〈0．05）。结论：survivin、VEGF分别通过抑制细胞凋亡、诱导新生血管的形成而影响子宫内膜癌的发生和发展,其在子宫内膜癌的发生中可能存在协同作用。     

乳腺癌p53蛋白过度表达和雌激素受体免疫组化检测及意义

应用免疫组化方法,对３２例乳腺癌组织中肿瘤抑制基因ｐ５３蛋白的表达和雌激素受体的状态分别进行了检测。结果３２例中ｐ５３蛋白阳性１８例,阳性率５６．３％,１８例伴有同侧腋下淋巴结转移者阳性１３例（７２．２％）３而１４例无转移者阳性５例（３５．７％）二组阳性率差异显著（ＰＭＯ．０５）。３２例中雌激素受体阳性１７例,１８例伴转移者阳性８例（４４．４％）;１４例无转移者阳性９例（６４．３％）。结果表明,ｐ５３蛋白的异常表达与乳腺癌的浸润转移有关,结合雌激素受体的检查,可作为临床预后判断一个有价值的标志。     

诱导受体3（DcR3）在胆管癌中的表达及意义

目的:观察胆管癌组织及血清中诱导受体3(decoy receptor 3,DcR3)蛋白的表达及其临床价值。方法:采用免疫组化S-P法检测45例胆管癌、15例癌旁胆管正常组织中DcR3蛋白的表达,ELISA法检测31例胆管癌及18例胆道良性疾病患者和28例正常人外周血清中DcR3的水平。结果:45例胆管癌组织中DcR3阳性表达29例,阳性率为64.4%,胆管正常组织中无阳性表达。DcR3的表达与肿瘤临床分期、肿瘤浸润和转移有关(P<0.05)。胆管癌患者及胆管良性疾病患者血清DcR3水平分别为152.2535.94 pg/ml,98.35 14.27 pg/ml,均高于正常人。胆管癌患者与胆道良性疾病患者血清DcR3水平相比差异有显著性(P<0.01)。结论:DcR3在胆管癌组织中表达增高。DcR3的表达与胆管癌的发生、发展以及转移有关,可成为治疗胆管癌的一个新靶点。血清DcR3的检测对胆管癌的诊断有一定的临床价值。     

诱导受体3（DcR3）在胆管癌中的表达及意义

目的：观察胆管癌组织及血清中诱导受体3（decoy receptor 3,DcR3）蛋白的表达及其临床价值。方法：采用免疫组化S-P法检测45例胆管癌、15例癌旁胆管正常组织中DcR3蛋白的表达,ELISA法检测31例胆管癌及18例胆道良性疾病患者和28例正常人外周血清中DcR3的水平。结果：45例胆管癌组织中DcR3阳性表达29例,阳性率为64.4%,胆管正常组织中无阳性表达。DcR3的表达与肿瘤临床分期、肿瘤浸润和转移有关（P〈0.05）。胆管癌患者及胆管良性疾病患者血清DcR3水平分别为152.2535.94 pg/ml,98.35 14.27 pg/ml,均高于正常人。胆管癌患者与胆道良性疾病患者血清DcR3水平相比差异有显著性（P〈0.01）。结论：DcR3在胆管癌组织中表达增高。DcR3的表达与胆管癌的发生、发展以及转移有关,可成为治疗胆管癌的一个新靶点。血清DcR3的检测对胆管癌的诊断有一定的临床价值。     

胆管癌组织中层粘连蛋白及67KDa层粘连蛋白受体的表达及其临床意义

目的 探讨层粘连蛋白及67KDa层粘连蛋白受体的表达与胆管癌临床病理学行为的关系。方法 应用S-P免疫组化法对52例人胆管癌组织中层粘连蛋白及67KDa层粘连蛋白受体的表达水平进行研究。结果 层粘连蛋白及67KDa层粘连蛋白受体的高表达与胆管癌淋巴结转移呈明显相关,层粘连蛋白及67KDa层粘连蛋白受体阳性肿瘤淋巴结转移率（83％,55％）明显高于层粘连蛋白及67KDa层粘连蛋白受体阴性肿瘤（15％,20％,P〈0．05）,且层粘连蛋白及67KDa层粘连蛋白受体的表达与胆管癌组织学类型、分化程度亦存在明显相关（P〈0．05）。结论 层粘连蛋白及67KDa层粘连蛋白受体的表达在胆管癌淋巴结转移中起协同作用。     

Fas受体与FasL在胃癌细胞株的表达及意义

观察Fas受体与FasL在胃癌细胞株的表达,以及Fas和FasL的功能提供形态学依据。采用敏感的免疫组织化学SABC法,结果提示所有胃癌细胞均呈Fas受体免疫反应阳性,阳性物质定位于胸质,胞核为阴性反应;同样所有胃癌细胞亦呈FasL免疫反应性,阳性信号位于胞质,胞核未见阳性信号。这提示Fas可能参与了胃癌细胞生长的调节,胃癌细胞的凋亡在自我调控机制。     

甲状腺癌患者血清促甲状腺激素和甲状腺激素表达水平及临床意义

目的:探讨甲状腺癌患者血清促甲状腺激素和甲状腺激素表达水平及临床意义。方法:应用电化学发光方法检测甲状腺癌组、甲状腺良性病变组和正常对照组血清促甲状腺激素(TSH)和甲状腺激素(TT3、FT3、TT4、FT4)水平。结果:①血清TSH在三组中比较有统计学意义(P〈0.001),甲状腺癌组血清TSH水平(3.56±0.93ulU/ml)明显高于甲状腺良性病变组(2.82±0.70ulU/ml)和正常对照组(2.04±0.56ulU/ml);TSH与肿瘤病理分期和肿瘤大小呈正相关(P<0.05)。②血清FT3、FT4水平在三组中有统计学意义(均P〈0.001),甲状腺癌组FT3、FT4水平处于较低水平,二者均明显低于甲状腺良性病变组和正常对照组(P<0.001);FT3与肿瘤病理分期和淋巴结转移呈负相关(P<0.05)。③TT3和TT4水平在三组之间比较均无统计学意义(P>0.05)。结论:高水平TSH可增加甲癌复发的危险性。低甲状腺激素水平在甲状腺癌形成中可能起到一定的作用,因此可以将其作为预测甲癌复发的重要指标之一。     

Extended hormone binding site of the human thyroid stimulating hormone receptor: distinctive acidic residues in the hinge region are involved in bovine thyroid stimulating hormone binding and receptor activation

The human thyroid stimulating hormone receptor (hTSHR) belongs to the glycoprotein hormone receptors that bind the hormones at their large extracellular domain. The extracellular hinge region of the TSHR connects the N-terminal leucine-rich repeat domain with the membrane-spanning serpentine domain. From previous studies we reasoned that apart from hormone binding at the leucine-rich repeat domain, additional multiple hormone contacts might exist at the hinge region of the TSHR by complementary charge-charge recognition. Here we investigated highly conserved charged residues in the hinge region of the TSHR by site-directed mutagenesis to identify amino acids interacting with bovine TSH (bTSH). Indeed, the residues Glu-297, Glu-303, and Asp-382 in the TSHR hinge region are essential for bTSH binding and partially for signal transduction. Side chain substitutions showed that the negative charge of Glu-297 and Asp-382 is necessary for recognition of bTSH by the hTSHR. Multiple combinations of alanine mutants of the identified positions revealed an increased negative effect on hormone binding. An assembled model suggests that the deciphered acidic residues form negatively charged patches at the hinge region resulting in an extended binding mode for bTSH on the hTSHR. Our data indicate that certain positively charged residues of bTSH might be involved in interaction with the identified negatively charged amino acids of the hTSHR hinge region. We demonstrate that the hinge region represents an extracellular intermediate connector for both hormone binding and signal transduction of the hTSHR.     

Development of serum-free bioreactor production of recombinant human thyroid stimulating hormone receptor

For the detection of autoantibodies to thyroid stimulating hormone receptors (TSH-R) in Graves' disease based on a novel coated tube assay system, human TSH-R is needed in large amounts. Whereas expression of TSH-R in bacteria, yeast, or insect cells results in nonfunctional, denaturated receptor, mammalian cells such as COS, CHO, and HeLa are able to express functional TSH-R, but only in very low amounts. Furthermore, for all of these cultivations expensive standard media containing 10% fetal calf serum are needed to obtain functional receptor. Here we report on the development of a serum-free production-scale process based on a stable transformed and highly productive human leukemia cell line K562 (1). Starting with K562-TSH-R cells growing in medium containing 10% fetal calf serum the cell line was adapted to serum-free medium. The adaptation medium was optimized in regards to amino acid and protein concentrations, since the use of unadjusted medium caused cell death after 2 days. The adapted cells were stable and could be cultivated without antibiotics for more than 50 cell doublings without losing their productivity. The obtained receptor showed improved TSH binding. The process development was based on cultivations in a 2-L bench-scale bioreactor. Cultivations in batch mode and chemostat mode and perfusion cultivation with the usage of an internal microfiltration device and a spin-filter device were compared. After process optimization a continuous process using spin-filter was set up and run in a 20 L-pilot-scale bioreactor. The presented results were the prerequisite for the production of the novel assay for the diagnosis of autoantibodies to TSH-R in Graves' disease.     

Assessment of the biological stability of human thyroid stimulating hormone prepared by immunoaffinity chromatography

This paper reports a comprehensive study of the biological stability of an immunoaffinity purified preparation of human thyroid stimulating hormone (TSH) and provides a reference against which future natural or synthetic preparations may be compared. The stability of the hormone preparation was investigated using the accelerated degradation method. The bioassay of the TSH was carried out in mice using a modified McKenzie method. Analysis of the results showed that the preparation was as stable as other TSH preparations purified by conventional methods.     

Modulation of nuclear statin expression in rat thyroid follicle cell following administration of thyroid stimulating hormone.

This study was designed to examine the state of proliferation in the rat thyrocyte following the administration of thyroid stimulating hormone (TSH). An immunohistochemical technique involving the use of a monoclonal antibody to statin, a nonproliferation-specific nuclear antigen, was developed to measure the subpopulation of cells that have ceased to divide. Following the random assignment of young male Sprague-Dawley rats into various groups, the rats in the control group received a single intraperitoneal (i-p) injection of normal saline, whereas the experimental groups received single i-p injections of TSH at doses of 0.25, 0.50, and 1.0 IU, respectively. All rats were subsequently sacrificed in groups of three at 1, 2, 4, and 24 hours. The statin antibody label was readily identified within the follicle cell nucleus. Results revealed a statistically significant transient decrease in the mean percent statin-positive nuclei in the TSH-treated groups. The time- and dose-dependent effect of TSH was maximal at 2 hours and no longer discernible at 24 hours. A second experiment involving the chronic administration of TSH (i-p 0.25 IU twice daily) resulted in a cumulative response with a statistically significant progressive decrease in the mean percent of statin-positive nuclei at 5 and 10 days, returning to near normal values 5 days following the cessation of treatment. Determination of the nuclear optical density of the statin reaction product by image analysis techniques revealed that a single injection of TSH resulted in a rapid disappearance of the statin nuclear protein. This result suggests that the disappearance of statin in the nucleus appears to reflect the event of cells leaving the nondividing quiescent state to resume the cell cycle traverse following the administration of TSH. The disappearance of statin appears as an early nuclear event that parallels the earliest known cytoplasmic pinocytotic response to TSH in the rat thyroid follicle cell.