HK6和B7-H4在卵巢癌诊断价值的探讨

目的:分别检测卵巢正常和卵巢良性病变和卵巢癌组织及血清中HK6、B7-H4表达水平,探讨HK6和B7-H4联合诊断卵巢癌的临床意义。方法:免疫组织化学SP法和酶联免疫吸附实验检测卵巢组织及血清中HK6和B7-H4表达水平,并结合临床病理资料分析28例卵巢癌中HK6和B7-H4在组织表达与血清浓度之间相关性。结果:HK6在卵巢癌组织中呈阳性表达,但B7-H4在黏液性癌中呈阴性表达。HK6和B7-H4在卵巢正常患者和卵巢良性病变患者血清中低表达,在卵巢癌患者血清中相对较高表达。卵巢癌患者血清中HK6和B7-H4两种蛋白的表达有明显的正相关关系(P<0.01)。结论:HK6和B7-H4在卵巢癌组织及血清中均较高表达,两者联合可指导卵巢癌诊断。

hK6单克隆抗体制备及在胃和乳房肿瘤组织中的初步应用

目的:原核表达人组织激肽释放酶6(human kallikrein 6,hK6),采用杂交瘤技术制备其单克隆抗体,建立免疫组织化学方法,检测hK6在胃癌、乳腺癌组织中的表达情况。方法:通过原核表达hK6融合蛋白免疫BALB/c小鼠,采用杂交瘤技术制备特异性单克隆抗体,并用间接ELISA、SDS-PAGE及Western-blot鉴定抗体,protein A agarose亲和层析柱纯化抗体,并以此抗体建立免疫组织化学方法检测人胃癌、乳腺癌组织中hK6表达情况。结果:成功筛选出4株高分泌型特异性杂交瘤细胞株,其制备抗体可与hK6融合蛋白发生特异性结合;免疫组织学显示,hK6主要分布在胃癌细胞胞浆中,成阳性表达,而在癌旁胃黏膜中成阴性表达;在乳腺癌组织中定位于胞浆,成弱阳性表达,癌旁组织成阴性表达。结论:成功制备了高纯度、特异性的抗hK6单克隆抗体;该抗体为深入研究hK6功能和建立高特异性、高敏感性检测组织和体液中hK6的方法奠定基础。

The relationship between the expression of hK6 in ovarian neoplasm and the clinicopathologic variables and prognosis

Objective: The aim of this study was to approach the relationship between the expression of human kallikrein 6 (hK6) in ovarian neoplasm and the clinicopathologic variables and prognosis for finding a new tumor marker for ovarian cancer. Methods: Through immunohistochemistry to examine the expression of hK6 in 19 cases with benign, 11 cases with borderline and 45 cases with malignant ovarian neoplasms and statistically analyzed whether the expression of hK6 correlated with the clinicopathologic variables and prognosis in patients with ovarian cancer. Results: The positive rate of hK6 in ovarian cancer tissues (60.0%) was significantly higher than that in the benign (15.8%) and borderline (27.3%) ovarian neoplasm tissues (P < 0.01). The expression of hK6 in low-grade ovarian cancer tissues was higher than that in high-grade (68.4% vs 14.3%; P < 0.05); hK6 in late–stage (stage III) was more frequently expressed than that in early-stage (stage I or II) (76.7% vs 26.7%; P < 0.01); and it significantly higher in ovarian carcinomas with lymph node metastases than those without lymph node metastases (77.8% vs 33.3%; P < 0.01 ); moreover, the expression of hK6 in the cancer tissues that the patients died or their pathogenetic condition recurred or their tumor metastasized within 3 years after surgery was higher (75.0%) than that in the cancer tissues that the pathogenetic condition of the patients was stable (42.9%; P < 0.05). Conclusion: The expression of hK6 in ovarian cancer tissues was higher than that in the benign and borderline ovarian neoplasm tissues, and high hK6 expression correlated with late–stage, low-grade, node metastasis and poor prognosis of patients. hK6 could potentially be a novel tumor marker for ovarian cancer that can predict the prognosis of the patients.

联合检测肿瘤标志物CA125、OPN、hK6和hK10对卵巢癌早期诊断的意义

目的:探讨血清癌抗原125(CA125)、骨桥蛋白(OPN)、人激肽释放酶6(h K6)和人激肽释放酶10(h K10)联合检测对卵巢上皮癌早期诊断的临床价值。方法:选取卵巢上皮癌患者44例、卵巢上皮类良性肿瘤患者50例及健康妇女50例的血清样品,分别用酶联免疫吸附法及微粒子化学发光法测定OPN、CA125、h K6和h K10含量。结果:卵巢上皮癌组血清OPN、CA125、h K6和h K10水平明显高于卵巢上皮类良性肿瘤组及对照组,组间比较,差异具有统计学意义(P<0.05);Ⅲ期和Ⅳ期卵巢上皮癌患者OPN、CA125、h K6和h K10水平明显高于I期和Ⅱ期患者,组间比较,差异具有统计学意义(P<0.05)。OPN、CA125、HK6和h K10检测的敏感度分别为59.1%、65.9%、56.8%、61.3%;联合检测的敏感度为95.45%。结论:血清OPN、CA125、h K6和h K10联合检测可提高卵巢上皮癌早期诊断的敏感性。

人组织激肽释放酶6的原核表达载体的构建及表达

目的:构建人组织激肽释放酶6原核表达系统。方法:采用RT-PCR技术从人乳腺癌组织中扩增出KLK6基因片段,克隆至原核表达载体pET28b中,经酶切和序列测定后,转化至E.coli BL21,IPTG进行诱导表达,表达产物经SDS-PAGE检测,Ni-NTA亲和层析柱纯化,其纯化产物进行SDS-PAGE和Western-blot检测。结果:pET28b-KLK6原核表达载体构建成功,经IPTG诱导高效表达hK6蛋白,纯化后获得大量高纯度蛋白。结论:成功构建了KLK6的原核表达系统并在E.coli BL21中高效表达,获得高纯度的hK6融合蛋白。

人组织型激肽释放酶6在卵巢上皮性肿瘤中的表达及其与临床病理特征和预后的关系

目的探讨人组织型激肽释放酶6（hK6）在卵巢肿瘤组织中的表达与临床病理特征和预后的关系。方法通过免疫组化SP法检测hK6在19例良性卵巢上皮性肿瘤、11例交界性卵巢上皮肿瘤和45例卵巢癌中的表达，分析其表达与卵巢癌患者年龄、原发肿瘤大小、肿瘤组织学类型、分化程度、临床分期和淋巴结转移等临床病理特征和患者预后的关系，并探讨其临床意义。结果hK6在良性、交界性和恶性卵巢肿瘤组织中的阳性表达率分别为15．8％、27．3％和60．0％，卵巢癌组织中的hK6阳性表达率明显高于良性和交界性肿瘤（P〈0．01）。hK6的阳性表达与患者年龄、原发肿瘤大小以及肿瘤组织学类型无关，而与肿瘤组织分化程度、临床分期及淋巴结转移密切相关。hK6在中、低分化卵巢癌组织中的表达率（68．4％）明显高于高分化者（14．3％，P〈0．05），在Ⅲ期卵巢癌组织中的阳性表达率（76．7％）显著高于Ⅰ-Ⅱ期（26．7％，P〈0．01），在有淋巴结转移者的卵巢癌组织中的阳性表达率（77．8％）明显高于无淋巴结转移者（33．3％，P〈0．01），在术后3年内复发、转移、死亡患者的卵巢癌组织中的阳性表达率（75．0％）高于术后3年内病情稳定者（42．9％，P〈0．05）。结论hK6在卵巢癌组织中的表达明显高于良性和交界性卵巢肿瘤组织，且在临床晚期、组织分化差、有淋巴结转移、预后差的卵巢癌中高表达，提示hK6可能是一种新的反映卵巢癌恶性程度和预后的肿瘤标志物。