胃癌手术前后外周静脉血微转移的研究

以癌胚抗原 (CEA)及角化蛋白 2 0 (CK2 0 )为标志物 ,应用巢式逆转录聚合酶链反应 (Nested,RT-PCR)检测 5 0例胃癌患者术前、后外周静脉血微转移 ,并探讨其与肿瘤生物学因素、手术操作的关系。结果表明 ,胃癌患者术前外周静脉血业已存在微转移 ,可促进微转移的发生 ;检测胃癌术前、后外周静脉血微转移不仅能评估胃癌侵袭转移状态 。

膀胱癌T-24细胞系中细胞角蛋白20的分离纯化及鉴定

目的 分离纯化细胞角蛋白 2 0 (Cytokeratin2 0 ,CK2 0 )并加以鉴定 ,为进一步研制抗CK2 0单克隆抗体奠定基础。方法 培养人膀胱肿瘤T 2 4细胞系并用免疫组化检测CK2 0表达 ,然后大量培养并收集细胞 ,超声波细胞粉碎后 ,先用萃取、高速离心、透析等方式进行蛋白初分离 ,然后利用以DE5 2为介质的阴离子交换层析分离纯化CK2 0蛋白 ,以电泳示踪 ,最后采用SDS PAGE、HLPC和Western blotting加以鉴定。结果 在阴离子交换层析时出现 3组洗脱峰 ,CK2 0主要存在第二组峰中 ,该峰在SDS PAGE和Western blotting上均呈现单一条带 ,HLPC分析显示CK2 0蛋白主峰呈正态分布 ,仅见一个较低峰度的蛋白质杂峰。结论 该纯化方法简便、可行、效果好、获得的蛋白纯度高 ,提取的蛋白可确认为CK2 0并且具有较高的免疫生物活性。

RT-PCR检测膀胱移行细胞癌患者尿CK20 mRNA表达的研究

目的 探讨膀胱移行细胞癌 (TCCB)患者尿细胞角蛋白 2 0 (CK2 0 )mRNA表达及其意义。方法 采用逆转录聚合酶链反应 (RT PCR)检测 6 1例TCCB患者、19例非肿瘤患者及 11例健康志愿者尿脱落细胞CK2 0mRNA。结果 6 1例TCCB患者中 5 2例尿脱落细胞CK2 0阳性表达 ,阳性率为 85 .3% ,对照组 30例中 2例表达 ,阳性率为 6 .7%。各分期尿CK2 0mRNA阳性率T1为 83.9% (2 6 / 31) ,T2 4期 86 .7% (2 6 / 30 ) ,两者差异无统计学意义 ;分级G1为 82 .8% (2 4 / 2 9) ,G2 85 .0 % (17/ 2 0 ) ,G3 91.7% (11/ 12 ) ,三者之间差异无统计学意义。行保留膀胱手术患者 5 1例 ,其中 4 2例术前尿CK2 0mRNA表达阳性 ,术后36例阴性 ,术后转阴率 85 .7%。 5 1例中有 2 9例 (术后尿脱落细胞CK2 0阳性表达 4例 )随访 6个月 ,4例CK2 0阳性表达者有2例 (5 0 % )复发 ,2 5例CK2 0阴性表达有 1例 (4 % )复发 ,两者复发率比较有统计学差异 (P <0 .0 0 5 )。结论 RT PCR检测尿脱落细胞CK2 0mRNA表达是诊断TCCB一种简便方法 ,其敏感性为 85 .3% ,特异性为 93.3% ,有助于膀胱TCCB的早期诊断和预后观察 。

CK20免疫组化检测Ⅱ期直肠癌淋巴结微转移对结局的影响

目的：研究Ⅱ期直肠癌患者术后病理因素和CK20检测淋巴结微转移对患者结局的影响。方法对2006年12月至2009年12月行全直肠系膜切除的70例Ⅱ期直肠癌标本的743枚淋巴结行连续超薄切片结合cytokeratin-20（CK20）免疫组化染色检测，术后平均随访61.11（21~84）个月。根据随访资料分析Ⅱ期直肠癌患者系膜淋巴结微转移的临床意义。结果临床病理因素中，肿瘤的大小、分化程度、肠壁侵润深度、淋巴管浸润与否与CK20的表达呈正相关，其它因素与淋巴结CK20表达无明显相关性。在淋巴结CK20表达阳性、阴性患者中，5年无瘤生存率各为55.0%、75.6%，差异有统计学意义（P=0.010）。临床病理因素与5年无瘤生存率无明显相关性。结论免疫组化检测直肠癌淋巴结CK20表达可对直肠癌的分期、治疗方案的确定、预后的评估更准确合理。

Clinical Significance of Cytokeratin 19 and Cytokeratin 20 in Predicting Recurrence of Bladder Transitional Cell Carcinoma

Objective： To investigate the expressions of cytokeratin 19 （CK19） and cytokeratin 20 （CK20） in bladder transitional cell carcinoma （TCC） and their clinical significance. Methods： The expression of CK19 and CK20 was detected in 54 cases of TCC by immunohistochemical methods and image processing techniques. Results： The expression of CK19 and CK20 was significantly stronger in the recurrent group than in the non-recurrent group （P〈0.01, P〈0.001, respectively）. Conclusion： The expression of CK19 and CK20 was obviously related with biological behaviors of TCC, suggesting that CK19 and CK20 could be used to predict the recurrence of TCC.

细胞角蛋白20单克隆抗体免疫胶体金试纸条的研制

目的研制用于尿液检查的细胞角蛋白20单克隆抗体免疫胶体金试纸条(CK20试条),建立一种无创性筛选诊断和检测膀胱癌复发的新的检测手段。方法将经处理的CK20单抗标记在胶体金颗粒上并活性进行鉴定,合格后吸附在硝酸纤维膜上并固定在长方形硬质塑料胶片一端为测定线;同时将吸附有IgG蛋白胶体金的硝酸纤维膜固定在测定线的一侧为质控线。将制成的CK20试条吸样端浸入尿液测定。结果成功的制备CK20试条,初步临床用于膀胱癌检测,敏感性78%(39/50),特异性为87%。结论采用该方法制备的CK20试条对膀胱癌患者尿液的检测具有较好的灵敏度和特异性,可用于膀胱癌瘤患者筛检诊断和复发的检测。

CEA mRNA、CK20 mRNA在胃肠道肿瘤患者外周血、引流静脉血中的表达及临床意义

目的 :研究胃肠道肿瘤患者外周血、引流静脉血中CEAmRNA、CK2 0mRNA的表达与胃肠道肿瘤的临床病理联系及预后的关系。方法 :以CEAmRNA、CK2 0mRNA为指标 ,用RT PCR方法检测对照组 10例外周血 ,14例胃癌和 11例结直肠癌外周血及腹腔内引流静脉血中的肿瘤细胞。 结果 :对照组 10例无CEAmRNA、CK2 0mRNA表达 ,CEAmRNA、CK2 0mRNA在 2 5例胃肠道肿瘤患者外周血、引流静脉血中的阳性表达率分别为5 / 2 1(2 3.8% )、12 / 2 4 (5 0 % ) ,0 / 2 1(0 % )、0 / 2 4 (0 % )。CEAmRNA阳性表达率高于CK2 0mRNA阳性表达率 (P <0 .0 5 )。CEAmRNA在引流静脉血中的阳性率 (5 0 % )高于外周血中阳性率 (2 3.8% ) ,差异有统计学意义 (P <0 .0 5 )。 3例外周血、引流静脉血CEAmRNA阳性的患者半年内出现复发和转移。 2 5例病例中未发现CEAmRNA阳性表达与年龄、性别、肿瘤的浸润深度、分化、分期及淋巴结转移相关。 结论 :CK2 0mRNA在外周血、引流静脉血中表达非常不稳定 ,以CEAmRNA、CK2 0mRNA为指标监测肿瘤的复发或者作为预后的指标还需要扩大样本进一步的研究。

联合细胞角蛋白20与膀胱肿瘤抗原监测膀胱肿瘤复发的意义

目的:探寻无创、快速、可重复的监测膀胱肿瘤复发的方法。方法:对58例TURBT术后患者,采用免疫组化法检测尿细胞角蛋白20,同时采用单抗免疫分析法测定膀胱肿瘤抗原、膀胱镜检及病理检测有无肿瘤复发。结果:与膀胱镜检及活检结果相比较,尿细胞角蛋白20检测可发现52.4%(11/21)复发病例,尿膀胱肿瘤抗原检测可发现66.7%(14/21)复发病例。2项检测综合分析,检测准确率升至95.2%(20/21)。结论:联合尿细胞角蛋白20与膀胱肿瘤抗原监测膀胱肿瘤复发具有快速、高效、可重复等特点,可以减少膀胱镜检频率。

Expression of cytokeratins in Helicobacter pylori-associated chronic gastritis of adult patients infected with cagA+strains:An immunohistochemical study

AIM： To investigate the expression of different cytokeratins （CKs） in gastric epithelium of adult patients with chronic gastritis infected with Helicobacter pylori （H pylon） cagA ＋ strains. METHODS： The expression of CK 7, 8, 18, 19 and 20 was studied immunohistochemically in antral gastric biopsies of 84 patients. All the CKs were immunostained in cagA＋Hpylori gastritis （57 cases）, non-Hpylori gastritis （17 cases） and normal gastric mucosa （10 cases）. RESULTS： In cagA＋ H pylori gastritis, CK8 was expressed comparably to the normal antral mucosa from surface epithelium to deep glands. Distribution of CK18 and CK 19 was unchanged, i.e. transmucosal, but intensity of the expression was different in foveolar region in comparison to normal gastric mucosa. Cytokeratin 18 immunoreactivity was significantly higher in the foveolar epithelium of H pylori-positive gastritis compared to both Hpylori-negative gastritis and controls. On the contrary, decrease in CK19 immunoreactivity occurred in foveolar epithelium of H pylori-positive gastritis. In both normal and inflamed antral mucosa without Hpyloriinfection, CK20 was expressed stronglyl moderately and homogenously in surface epithelium and upper foveolar region, but in H pylod -induced gastritis significant decrease of expression in foveolar region was noted. Generally, in both normal antral mucosa and H pylori-negative gastritis, expression of CK7 was not observed, while in about half cagA＋ H pylori-infected patients, moderate focal CK7 immunoreactivity of the neck and coiled gland areas was registered, especially in areas with more severe inflammatory infiltrate. CONCLUSION： Alterations in expression of CK 7, 18, 19 and 20 together with normal expression of CK8 occur in antral mucosa of H pylori-associated chronic gastritis in adult patients infected with cagA＋ strains. Alterations in different cytokeratins expression might contribute to weakening of epithelial tight junctions observed in H pylori-infected gastric mucosa.

CK20和Ki-67在膀胱移行细胞癌中的表达及临床意义

目的探讨细胞角质蛋白20(CK20)和细胞核相关抗原(Ki-67)在膀胱移行细胞癌中的表达及临床意义。方法对经临床确诊的膀胱移行细胞癌患者癌组织标本中CK20(66例)和Ki-67(55例)的表达结果进行回顾性分析。结果CK20在膀胱移行细胞癌表达的阳性率为68.2%(45/66)。CK20表达的阳性指数与膀胱移行细胞癌临床分期和细胞分级均呈正相关(P<0.05)。复发膀胱移行细胞癌组CK20阳性表达率92.3%(12/13)高于初发膀胱移行细胞癌组62.3%(33/53)(P<0.05)。Ki-67在膀胱移行细胞癌表达的阳性率为63.6%(35/55)。Ki-67表达的阳性指数与膀胱移行细胞癌细胞分级呈正相关(P<0.05)。复发膀胱移行细胞癌组Ki-67阳性表达率90.9%(10/11)高于初发膀胱移行细胞癌组56.8%(25/44)(P<0.05)。Ki-67和CK20的表达阳性指数呈正相关(r=0.493,P<0.05)。结论研究结果表明CK20和Ki-67的表达与膀胱移行细胞癌的生物学行为相关,因此CK20和Ki-67可作为膀胱移行细胞癌肿瘤病理分级分期以及预后判断的辅助手段。

Multiplex RT-PCR-based detections of CEA, CK20 and EGFR in colorectal cancer patients

AIM: To develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) method detecting cir-culating tumor cells in the peripheral blood of colorectal cancer (CRC) patients. METHODS: Peripheral blood samples were collected from 88 CRC patients and 40 healthy individuals from the blood donors' clinic and subsequently analyzed by multiplex RT-RCR for the expression of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20) and epidermal growth factor receptor (EGFR) mRNA. The analysis involved determining the detection rates of CEA, CK20 and EGFR transcripts vs disease stage and overall survival. Median follow-up period was 19 mo (range 8-28 mo). RESULTS: Rates of CEA, CK20 and EGFR detection in CRC patients were 95.5%, 78.4% and 19.3%, respectively. CEA transcripts were detected in 3 healthy volunteer samples (7.5%), whereas all control samples were tested negative for CK20 and EGFR transcripts. The increasing number of positive detections for CEA, CK20 and EGFR transcripts in each blood sample was positively correlated with Astler-Coller disease stage (P< 0.001) and preoperative serum levels of CEA (P=0.029) in CRC patients. Data analysis using Kaplan-Meier estimator documented signif icant differences in the overall survival of the different CRC patient groups as formed according to the increasing number of positivity for CEA, CK20 and EGFR transcripts. CONCLUSION: These data suggest that multiplex RTPCR assay can provide useful information concerning disease stage and overall survival of CRC patients.

The Implicaiton of the Expression of Three Cytokines in Carcinoma of Bladder

Objective To explore the expression of CK19, CK20 and bFGF in transitional cell carcinoma of the bladder (TCC) and their biological implication.Methods Samples from 54 patients with TCC were examined for expression of CK19, CK20 and bFGF.Results There was a significant difference in CK19 expression between relapse and non-relapse groups ( P < 0.01) ; no significant difference was found among groups with different pathological grades and clinical stages ( P > 0.05). There were significant differences in CK20 expression among groups with different pathological grades ( P < 0.05) and clinical stages ( P < 0.01) and between the relapse and non-relapse groups( P < 0.000 1) . The expression of bFGF was significantly different among groups with different pathological grades and clinical stages and between the relapse and non-relapse groups ( P < 0.05) .Conclusion The expression of CK19, CK20 and bFGF is closely associated with their biological behavior and among them bFGF is primarily secreted by TCC tumor tissues.

Distinction between short-segment Barrett's esophageal and cardiac intestinal metaplasia

AIM： To investigate the roles of mucin histochemistry, cytokeratin 7/20 （CK7/20） immunoreactivity, clinical characteristics and endoscopy to distinguish short- segment Barrett＇s esophageal （SSBE） from cardiac intestinal metaplasia （CIM）. METHODS： High iron diamine/Alcian blue （HID/AB） mucin-histochemical staining and immunohistochemical staining were used to classify intestinal metaplasia （IN） and to determine CK7/20 immunoreactivity pattern in SSBE and CIM, respectively, and these results were compared with endoscopical diagnosis and the positive rate of gastroesophageal reflux disease （GERD） symptoms and H pylori infection. Long-segment Barrett＇ s esophageal and IM of gastric antrum were designed as control. RESULTS： The prevalence of type III IM was significantly higher in SSBE than in CIM （63.33% vs 23.08%, P〈0.005）. The CK7/20 immunoreactivity in SSBE showed mainly Barrett＇s pattern （76.66%）, and the GERD symptoms in most cases which showed Barrett＇ s pattern were positive, whereas H pylori infection was negative. However, the CK7/20 immunoreactivity in CIM was gastric pattern preponderantly （61.54%）, but there were 23.08% cases that showed Barrett＇s pattern. H pylori infection in all cases which showed gastric pattern was significantly higher than those which showed Barrett＇ s pattern （63.83% vs 19.30%, P〈0.005）, whereas the GERD symptoms in gastric pattern were significantly lower than that in Barrett＇s pattern （21.28% vs 85.96%,P〈O.O05）. CONCLUSION： Distinction of SSBE from CIM should not be based on a single method; however, the combination of clinical characteristics, histology, mucin histochemistry, CK7/20 immunoreactivity, and endoscopic biopsy should be applied. Type III IM, presence of GERD symptoms, and Barrett＇s CK7/20 immunoreactivity pattern may support the diagnosis of SSBE, whereas non-type III IM, positive H pylori infection, and gastric CK7/20 immunoreactivity pattern may imply CIM.

A case of mucin producing liver metastases with intrabiliary extension

A 75-year-old man was admitted to our hospital with a diagnosis of liver metastases from colon cancer. He underwent right hemicolectomy for cecal cancer eight years ago, and had a metastatic liver tumor in segment 8 （S8）, which was surgically resected about 4 years after the initial operation. Histopathological examination of the resected specimens from both operations revealed a well-differentiated adenocarcinoma with mucinous carcinoma. Four months after the second operation, computed tomography demonstrated a low-density lesion at the cut surface of the remnant liver. Although it was considered to be a postoperative collection of inflammatory fluid, it formed a cystic configuration and increased in size to approximately 5 cm in diameter. With a tentative diagnosis of a recurrence of metastatic cancer, partial hepatectomy of S8 was performed. Histological examination of the resected specimens also revealed mucinous adenocarci- noma, which had invaded into the biliary ducts, replacing and extending along its epithelium. Immunohistochemi-cally, the tumor cells were positive for cytokeratin （CK） 20, but negative for CK7. Therefore, the tumor was diagnosed as a metastatic adenocarcinoma from colonic cancer. Liver metastases of colorectal adenocarcinoma sometimes invade the Glisson＇s triad and grow along the biliary ducts.

Ck20在大肠癌组织中的表达及其临床意义

目的检测Ck20在大肠癌组织中的表达,探讨其与大肠癌发生、发展的关系。方法采用免疫组织化学法检测在大肠癌组织癌中的表达,研究它的表达与大肠癌恶性程度的关系。结果在大肠癌中,Ck20的阳性表达率以及染色强度均随分化程度的降低而呈现出上升的趋势(P<0.05)。结论在大肠癌组织中Ck20的高表达,对大肠癌的发生发展起一定促进作用。

荧光定量聚合酶链反应检测大肠癌患者外周血癌细胞角蛋白20 mRNA

目的探讨荧光定量聚合酶链反应（FQ—PCR）检测大肠癌患者外周血癌细胞角蛋白20mRNA（CK20mRNA）的表达水平及临床价值。方法采用FQ-PCR法测定136例结直肠癌及其肝脏转移患者外周血癌细胞CK20mRNA的表达水平，系统研究了FQ-PCR法测定CK20mRNA的重复性、定量范围和扩增效率，分析比较其与传统RT—PCR、免疫组化法检测结果的一致性。结果FQ．PCR法测定CK20mRNA的批内CV为3．6％，批间CV为5．3％，定量范围为10^3～10^8 copies／ml，扩增效率为87．4％。与传统RT—PCR和免疫组化法相比较，Kappa值分别为0．87和0．83。结直肠癌患者外周血癌细胞CK20mRNA的表达水平为（3．52×10^4±1．47×10^4）copies／ml，表达阳性率为48．5％；75例对照者均无CK20mRNA表达。Dukes A、B、C、D期大肠癌患者外周血癌细胞CK20mRNA的阳性率分别为9．5％、25．0％、48．8％和87．5％，各组间差异有统计学意义（P〈0．05）。肝脏转移者CK20mRNA阳性率为87．5％，显著高于肝脏无转移者（32．3％，P〈0．05）。35例结直肠癌患者术后1、3、5周内CK20mRNA表达水平呈下降趋势，但术后1、3周与术前比较，差异无统计学意义（P〉0．05），术后5周显著低于术前（P〈0．05）。结论FQ—PCR是较灵敏的快速定量检测CK20mRNA的方法。大肠癌患者外周血癌细胞CK20mRNA的表达与肿瘤的复发和转移有关，CK20mRNA检测将有助于大肠癌血源性播散的监测。

术中美蓝染色及抗细胞角蛋白20检测淋巴结及其微转移在直肠癌患者中的临床应用

目的探讨术中经直肠上动脉注射美蓝行直肠系膜及淋巴结染色检测淋巴结及其微转移方法的临床意义。方法对30例行根治性手术治疗的直肠癌患者,术中经直肠上动脉插管注射美蓝6ml(美蓝染色组),使直肠系膜及各组淋巴结染色以统计淋巴结数目,同时行抗细胞角蛋白20(CK20)免疫组织化学(免疫组化)染色,检测其微转移情况;并与同期32例行直肠癌根治术患者术中常规手检法(常规手检组)检出的淋巴结数目进行对比。结果美蓝染色组肠旁淋巴结、肠系膜淋巴结、肠系膜根部淋巴结检出数目均明显多于常规手检组(P均=0.000)。美蓝染色组淋巴结常规病理检查阴性者,经CK20免疫组化染色,其转移阳性率增加17.7%;14例DukesB期患者(46.7%)经淋巴结CK20免疫组化染色需重新确定为DukesC期,11例患者(36.7%)出现跳跃淋巴结微转移现象。结论术中经直肠上动脉注射美蓝行淋巴结染色能提高淋巴结检出率。对常规病理检查阴性的淋巴结行CK20免疫组化染色检查其微转移灶,能提高淋巴结阳性检出率,使病理分期更准确。

细胞角蛋白20及端粒酶的表达在大肠癌微转移中的意义

目的探讨大肠癌前哨淋巴结(sentinel lymph node,SLN)微转移(micrometastasis,MM)的检测方法及其临床意义。方法前瞻性研究2004年9月至2005年7月在甘肃省人民医院64例行根治性手术的DukesB期大肠癌病人,对其中61例定位成功的122枚SLN应用常规HE染色联合免疫组化SP法检测其前哨淋巴结中细胞角蛋白20(CK20)及端粒酶的表达,随访满3年,记录病人的临床病理参数和生存资料,分析其相关性。结果(1)61例中6例病人9枚SLN经常规HE检测阳性。(2)免疫组化法有27.3%(15/55)病人其SLN存在CK20阳性,21.8%(12/55)的病人SLN存在端粒酶阳性表达。(3)两者联合检测微转移检出率为38.2%(21/55)。(4)DukesB期SLNMM(+)病人癌复发转移率明显高于同期SLNMM(-)组(P<0.05),存活率则降低(P<0.05),与DukesC期对比差异无统计学意义;SLNMM(-)组的复发转移率、存活率与同期DukesC期病人对比,差异有统计学意义(P<0.05)。结论免疫组化法联合检测端粒酶和CK20可提高DukesB期大肠癌SLNMM的检出率,并且对于明确大肠癌病人Dukes分期,指导术后合理的辅助治疗和预后判断有重要的临床意义。

喉癌、喉咽癌患者CK19和CK20的表达及其临床意义

目的探讨喉癌、喉咽癌患者外周血中细胞角蛋白(CK)19mRNA、CK 20mRNA的表达及临床意义。方法采用实时荧光定量RT-PCR检测50例喉癌、喉咽癌患者(A组)和20例正常人群(C组)外周血CK19和CK20mRNA的表达水平。结果 A组外周血中CK19和CK20mRNA表达明显高于C组(0.4626±0.1220vs.0.1712±0.0561和0.4817±0.1401vs.0.2175±0.0704)(P<0.01)。A组晚期患者CK19和CK20mRNA的表达高于早期患者(P<0.05);淋巴结转移患者外周血中CK19和CK20mRNA表达略高于无淋巴结转移患者(P>0.05)。结论检测外周血CK19和CK20mRNA表达有助于喉癌、喉咽癌的诊断。

结直肠癌淋巴结微转移的临床研究

目的探讨结直肠前哨淋巴结(SLN)的定位方法及检测淋巴结微转移的有效方法,并分析其临床意义。方法对60例结直肠癌患者采用亚甲蓝染色法淋巴结示踪,寻找染色的SLN,切除后的SLN行HE染色和细胞角蛋白CK20免疫组化检测;并与前期直接行淋巴结清扫的60例患者对比。结果亚甲蓝组中可识别SLN者54例(90.0%),高于前期直接清扫组的24例(40.0%)(P<0.05);54例中行常规HE染色检出36例阳性,18例阴性。18例SLN阴性者行免疫组化检测,6例(33.3%)检出有微转移灶。结论联合应用亚甲蓝和细胞角蛋白CK20进行结直肠癌SLN定位优于单用其中之一种方法;免疫组化是检测淋巴结微转移的敏感方法。