大麻制剂HU210对实验性急性胰腺炎的干预作用及其与Toll样受体4信号通路的可能关系

目的:研究大麻类制剂HU210对雨蛙肽(caerulein,CAE)诱导的野生型(wild-type,WT)和Toll样受体4基因敲除(tlr4~(-/-))小鼠急性胰腺炎(acute pancreatitis,AP)的干预作用,并探讨其作用机制。方法:将成年C57BL/10J小鼠及相同背景的tlr4~(-/-)小鼠各随机分成3组:AP组、AP+HU210组及正常对照组。小鼠腹腔注射CAE(50μg·kg~(-1)·h~(-1))6次及脂多糖(10 mg/kg)1次复制AP模型;AP+HU210组在造模前及造模后各腹腔注射1次HU210(50μg/kg);对照组注射生理盐水替代CAE和脂多糖。造模处理后3 h,取血处死小鼠,取胰腺、肺组织及肠道Peyer’s结。结果:与对照组相比,无论在WT或tlr4~(-/-)小鼠,AP造模后胰腺病理评分,血浆淀粉酶活性,血浆IL-6、TNF-α及MCP-1水平,以及肺MPO活性均明显升高(P0.05),P38蛋白表达明显上调(P0.05)。同时,AP造模后,与WT小鼠相比,tlr4~(-/-)小鼠血浆IL-6、TNF-α及MCP-1水平,以及胰腺P38及p-P38蛋白表达均明显降低(P0.05),Peyer’s结CD3~+、CD4~+T淋巴细胞百分比及CD4~+/CD8~+比值明显降低(P0.05)。HU210干预使2种小鼠AP模型的胰腺病理学评分和肺MPO活性的升高明显得到改善(P0.05);在WT小鼠,而非tlr4~(-/-)小鼠,AP引起的血浆淀粉酶活性和胰腺P38及p-P38蛋白表达的变化在HU210干预后明显逆转(P0.05)。结论:TLR4主要参与AP全身炎症反应,其机制可能依赖TLR4-P38 MAPK信号通路;大麻制剂HU210对AP的干预主要通过抑制炎症细胞的浸润发挥组织保护作用,与TLR4信号通路的关系似乎不明显。     

川芎嗪对急性胰腺炎大鼠细胞凋亡的影响

目的:研究川芎嗪(TMP)对急性胰腺炎(AP)血栓形成、组织病理变化、氧自由基和细胞凋亡的影响机制。方法:采用十二指肠胆胰管逆行加压注射5%牛磺胆酸钠的方法制备大鼠AP模型,动态观察TMP治疗前后大鼠血栓烷A2/前列环素代谢产物血栓烷B2/6-酮-前列腺素F1α(TXB2/6-Keto-PGF1α)比值(T/P)、血浆超氧化物岐化酶(SOD)、丙二醛(MDA)、淀粉酶(AMY)等各项指标;通过观察胰腺组织的病理形态进行病理评分;采用TUNEL法评价胰腺细胞凋亡指数(AI)。结果:经TMP治疗后,大鼠T/P值明显降低,血清SOD水平升高,MDA水平降低;胰腺组织病理评分为4.85±0.98(6h),AI为9.88±0.98(6h),与AP组比较差异有显著性意义(P<0.05)。结论:TMP对AP的治疗作用与其纠正血栓烷A2/前列环素I2失衡,改善AP大鼠微循环,减少自由基造成的损伤,诱导细胞凋亡,减少胰腺细胞坏死有关。     

清胰汤Ⅱ号冲剂对急性胰腺炎小鼠的保护作用及机制

目的:探讨清胰汤Ⅱ号冲剂(QYT)对急性胰腺炎(acute pancreatitis,AP)小鼠的保护作用及机制。方法:成年C57BL/6小鼠24只,雌雄各半,随机均分为3组。AP组和AP+QYT组首先经腹腔注射雨蛙肽(50μg/kg)及脂多糖(10 mg/kg)复制重症AP模型,AP组小鼠给予饮用水灌胃,AP+QYT组给予QYT灌胃;正常对照组小鼠给予等量生理盐水注射及饮用水灌胃。于末次注药后3 h麻醉处死动物。检测和分析胰腺组织的病理改变、肠道细菌总数和分类、Peyer’s结T淋巴细胞亚群的变化、血浆淀粉酶、白细胞介素6(IL-6)、单核细胞趋化蛋白1(MCP-1)水平以及胰腺和肺脏组织髓过氧化物酶(MPO)活性等。结果:与对照组相比,AP小鼠的胰腺组织病理学评分、肠道细菌数量、血浆淀粉酶活性、IL-6及MCP-1水平、胰和肺组织的MPO活性都有明显升高(P0.05);QYT可在一定程度上逆转AP时相关指标的变化(P0.05)。小肠Peyer’s结数量在各组无明显差异,但AP组的CD3+T淋巴细胞百分比较对照组明显降低(P0.05),而且,AP组和AP+QYT组,尤其是后者CD4+T淋巴细胞百分比和CD4+/CD8+比值较对照组明显升高(P0.05)。结论:清胰汤II号冲剂对雨蛙肽和脂多糖诱导的小鼠急性胰腺炎具有明显的拮抗作用,其机制可能与其抑制炎症反应、促进肠内细菌的清除和调节肠道T淋巴细胞功能有关。     

用抗α_2-M单克隆抗体ELISA技术测定尿液中α_2-M(摘要)

有关生理与病理情况下尿液中的α_2-M含量,至今未见正式报告。我们应用本科自制鼠抗人α_2-M单克隆抗体,建立ELISA技术,对尿液中α_2-M含量进行测定,发现此法敏感性高,可直接从尿液中测出低迷26ng/ml的α_2-M,批内误差(cv)为11％,现将结果报告如下: 料材和方法 1.鼠抗人α_2-M单克隆抗体、羊抗人α_2-M多克隆抗体、辣根过氧化物酶标记兔抗羊IgG均由本科自制。     

硝普钠对急性胰腺炎大鼠治疗作用初探

目的与方法:为探索硝普钠对急性胰腺炎(AP)的治疗作用,采用胰管内注射牛磺胆酸钠溶液的方法建立大鼠AP模型,并用外源性一氧化氮(NO)供体硝普钠(SNP)实验性治疗,动态观察AP模型复制后(8、16、24、48h)血浆NO、超氧化物歧化酶(SOD)和淀粉酶含量的变化及胰腺组织学改变。结果:AP组血浆NO、SOD明显下降(P＜0.05),并持续24h以上,血浆淀粉酶含量明显升高(P＜0.05),而治疗组血浆NO、SOD明显高于非治疗组(P＜0.05),淀粉酶含量明显下降(P＜0.05)。治疗组胰腺组织病理损害明显轻于模型组。结论:大鼠实验性急性胰腺炎时,血浆NO含量、SOD活性明显下降,早期腹腔注射硝普钠能显著升高血浆NO、SOD含量,降低血浆淀粉酶含量,减轻胰腺病损。本实验为临床急性胰腺炎的早期治疗提供一新途径。     

分泌抗α_2-巨球蛋白单克隆抗体的杂交瘤细胞系建立

α_2-巨球蛋白(α_2-Macroglobulin,α_2-M)是人血清中α球蛋白的一种重要成分。含量约占血清总蛋白量的3％,分子量82万左右,α_2-M有抑制多种蛋白分解酶和活化生长激素与胰岛素的作用。参予免疫调控,具有抗放射性损伤的作用。最近我们用细胞融合技术建立了分泌抗α_2-M单克隆抗体的杂交瘤细胞系,本文就这一细胞系的建立情况及初步鉴定结果报告如下:     

血浆ET与ANF在急性胰腺炎早期发病中的作用及意义

目的探讨内皮素(ET)与心钠素(ANF)在急性胰腺炎(AP)早期发病中的作用及临床意义.方法采用放射免疫分析测定急性发病期(24～48)h轻症急性胰腺炎(MAP)35例,重症急性胰腺炎(SAP)17例和正常对照组(NC)30例的血浆内皮素与心钠素的含量.Imrie评分＞3分,APACHE Ⅱ评分≥8分者为SAP.结果SAP患者Imrie评分和APACHE Ⅱ评分明显高于MAP患者(P＜0.05,P＜0.01).SAP患者血浆ET水平(59.20±10.69)pg/ml明显增高,MAP患者血浆ET水平(34.20±9.44)pg/ml明显降低,两组之间比较以及与NC组比较具有非常显著差异(P＜0.001).SAP和MAP患者血浆ANF分别为(155.76±34.24)pg/ml,(181.59±58.15)pg/ml,与NC组(165.51±57.16)pg/ml比较无明显变化(P＞0.05).结论ET可能参与了SAP早期胰腺微循环障碍,对胰腺组织具有内源性损伤作用.提示早期给予拮抗ET或ET-A受体的药物,可能有利于减轻胰腺组织损害,改善胰腺微循环,防止胰腺出血坏死性改变.ANF可能对AP早期的胰腺血循环无明显影响.     

急性胰腺炎大鼠腹水白细胞cPKC-α水平的变化及汉防己甲素等的影响

目的:观察急性胰腺炎(AP)大鼠腹水白细胞传统型蛋白激酶C-α(cPKC-α)的水平变化及汉防己甲素(Tet)等药物的影响,并对其机制进行探讨。方法:取56只SD大鼠随机分为4组:假手术组(SO+NS组),AP对照组(AP+NS组),AP+阿司匹林(ASP)组,AP+Tet组,采用牛磺脱氧胆酸钠复制AP模型,模型制成后10min,AP+Tet组腹腔注入Tet(80mg/kg·BW);AP+ASP组经食管灌入ASP混悬液(125mg/kg·BW)。各组动物在相应处理后1h、5h,提取腹水白细胞,制备白细胞裂解液,用免疫印迹(Westernblot)和化学发光法检测其cPKC-α水平;同时分别取动物血浆测定淀粉酶(AMY)活性;取5h大鼠胰腺组织作病理检查。结果:ASP和Tet组胰腺病理损伤轻于AP+NS组,血浆AMY活性低于AP+NS组,腹水白细胞cPKC-α水平高于AP+NS组(P＜0.05或P＜0.01)。结论:ASP、Tet可增加AP大鼠腹水白细胞cPKC-α的水平;ASP、Tet可明显减轻AP时胰腺组织及功能损伤。     

急性胰腺炎胰酶胞内激活机制中Cpn60的可能作用

目的:进一步探讨急性胰腺炎(AP)时胰酶胞内激活的机制及蛋白伴侣因子(Cpn60)在其中的可能作用。方法:用去氧胆酸钠逆行注入大鼠胰胆管复制AP模型,5h、10h分批采血、取胰腺,测血清及胰腺组织中淀粉酶活性及炎症介质TNF-α的含量;取5h胰腺作光镜、电镜切片,检测胰腺组织和腺泡细胞各细胞器形态学的变化;用蛋白金标免疫细胞化学技术检测各细胞器中Cpn60及胰酶含量的变化。结果:血清淀粉酶活性及胰腺组织匀浆TNF-α含量在5h明显高于正常对照组,持续达10h;AP5h的光镜病理检查见胰腺水肿、出血、坏死;电镜检查见腺泡内溶酶体增多,形态各异,散在于各处,甚至位于高尔基体中;内质网、高尔基体、酶原颗粒等细胞器中Cpn60明显减少,然而胰脂肪酶含量升高,糜蛋白酶原保持在高浓度水平。结论:AP时腺泡细胞内溶酶体增多,分布异常,尤其胰脂肪酶以及蛋白酶含量增多,Cpn60含量减少,提示蛋白伴侣相对不足,这些异常变化可能引起胰酶在腺泡内的聚积和激活,从而参与AP的发生和发展。     

新型大麻制剂O-1602和CBD减轻小鼠 实验性急性胰腺炎的机制研究

目的: 观察2种新型大麻类制剂O-1602和大麻二酚(CBD)对雨蛙肽(CAE)诱导的小鼠急性胰腺炎(AP)的抗炎作用,并通过其对热休克蛋白60(HSP60)表达的影响,探讨其可能的机制。方法: 以CAE腹腔注射(50 μg·kg^-1·h^-1,共6次)复制小鼠AP模型,对照组用生理盐水(NS)替代CAE;给AP小鼠腹腔注射O-1602或CBD作为治疗组。组织学检查评估不同处理组胰腺组织形态学改变;测定血浆淀粉酶及脂肪酶活性(生化法)、血浆细胞因子如肿瘤坏死因子α(TNF-α)及白细胞介素6(IL-6)的水平(ELISA法),以及肺脏中髓过氧化物酶(MPO)活性的变化(生化法);同时,用 real-time PCR和Western blotting测定胰腺组织热休克蛋白(HSP60) mRNA和蛋白的表达特点。结果: AP组小鼠胰腺组织出现明显损伤及炎症表现;此类表现在AP+O-1602及AP+CBD治疗组得到明显改善。AP组血浆淀粉酶及脂肪酶活性、TNF-α及IL-6水平、肺脏MPO水平较NS组均有显著升高(P<0.05);而在AP+O-1602及AP+CBD组,这些指标均较AP组明显降低(P<0.05)。同时,胰腺组织HSP60 mRNA和蛋白的表达量在AP组均降低(P<0.05), O-1602或CBD处理可一定程度提高HSP60的表达(P<0.05)。结论: CAE可成功诱导小鼠AP的发生,并伴有一定程度的全身炎症反应,O-1602和CBD可改善胰腺局部损伤程度及全身炎症程度,其机制可能与大麻类物质对炎症介质、细胞因子的抑制以及提高细胞保护因子HSP60的表达有关。     

Variability of the arterial system of the human pancreas

The basic feature related to survival and the ultimate functioning of a transplanted segment or whole pancreas is the preservation and continuation of an adequate blood flow. The current study examined the anatomical variability of the arterial vascularization in 126 pancreases following their removal form cadavers by the technique routinely used for transplantation. Arteriography was conducted initially by the injection of either 75% uropolin or barium sulfate in 70 specimens following removal of the specimen. All 126 specimens were injected with latex, and corrosion casts were prepared through maceration with either sodium hydroxide or hydrochloric acid. The dorsal pancreatic artery showed the greatest variability with respect to the mode of origin and division into branches. Also 42.1% of the cases were found to have poor anastomoses between the body and tail segments. These observations suggest that it is necessary to carefully assess the origins of the vascular supply of the donor organ 1) as resection at the celiac rather than the splenic artery can increase preservation of the dorsal pancreatic artery; 2) whole organ transplantation may be indicated to ensure adequate vascularization; and 3) the presence of a separate arterial supply to the caudal segment in 42.1 % of the cases allows the consideration of its use as an independent transplant from a live donor. © 1993 Wiley-Liss, Inc.     

Transthyretin, identified by proteomics, is overabundant in pancreatic juice from pancreatic carcinoma and originates from pancreatic islets

Analyses of pancreatic juice by proteomics have identified many proteins that are overabundant in pancreatic cancer (PC) juice. The mechanism by which secretion of these proteins occur remains unclear. Pancreatic juice was collected from patients with three pancreatic diseases: PC, chronic pancreatitis (CP), and simple choledocholithiasis (CDS), and analyzed by 2-DE, MALDI-TOF/MS, and Western blot. Five PC cell lines, 30 PC tissues and their corresponding adjacent pancreatic tissues were used to validate the expression of genes which code for overabundant proteins in PC juice. The mRNA and protein levels were measured by RT-PCR and immunohistochemistry, respectively. Using proteomics, it was demonstrated that the protein transthyretin (TTR) was upregulated more than 2-fold in PC juice compared with CP and CDS, while apolipoprotein A-I, lithostathine, and regenerating islet-derived 1 beta precursor were downregulated more than 2-fold. Western blots confirmed that TTR was overabundant in the PC juice. However, TTR mRNA was not detected in any of the five PC cell lines, and was only detected in islet cells. By microscopy, it was shown that islet architecture was almost completely destroyed, and the islet's maximum diameter appeared larger in PC tissues than in normal. Some overabundant proteins in PC juice, such as TTR expressed only in islets, leak into the pancreatic ductal system due to hyperplasia and architectural damage in PC tissues. The destruction of organ and tissue architecture by tumor growth may result in novel tumor markers even if the markers are not secreted directly by tumor cells.     

Trend Toward Individualization of the Endocrine and Exocrine Portions of the Giant Anteater Pancreas (Myrmecophaga Tridactyla,Xenarthra)

Considering the physiological importance of the pancreas as an endocrine and exocrine organ, this study described the characteristics of the gross and microscopic morphology of this organ using 16 Myrmecophaga tridactyla individuals. The pancreas was located in the left antimere of the body, was pale in colour and exhibited an elongated shape with a central body and lobulated surface. It was positioned in the abdomen, following the curvatura ventriculi major of the stomach, and was attached to the initial portion of the duodenum. The corpus pancreatis was elongated and showed a caudal curvature of 45°. The pancreas exhibited a facies dorsalis (related to the spleen and stomach) and a facies ventralis (related to the renal capsule and intestine). Macroscopically, a craniodorsal, medial, and caudoventral regions were identified, in addition to the left lobe. Structurally, the organ exhibited two distinct parts: the first had exocrine characteristics, consisting of acini and ducts; the second, which was the endocrine portion, consisted of the pancreatic islets, which were located in the medial, caudoventral and left lobe regions. Ultrastructural analysis identified secretory vesicles containing zymogen granules, mitochondria, Golgi apparatus and rough endoplasmic reticulum in pancreatic centroacinar cells. Morphological data on the anatomy of members of the Xenarthra have revealed important peculiarities of several organs and systems, adding great biological value to the representatives of this group. In addition, these studies significantly contribute not only to knowledge of the biology, taxonomy and, consequently, preservation of these animals but also to the discovery of new experimental models. Anat Rec, 300:1104–1113, 2017. © 2016 Wiley Periodicals, Inc.     

Pathology and genetics of pancreatic neoplasms with acinar differentiation

Pancreatic neoplasms with acinar differentiation, including acinar cell carcinoma, pancreatoblastoma, and carcinomas with mixed differentiation, are distinctive pancreatic neoplasms with a poor prognosis. These neoplasms are clinically, pathologically, and genetically unique when compared to other more common pancreatic neoplasms. Most occur in adults, although pancreatoblastomas usually affect children under 10 years old. All of these neoplasms exhibit characteristic histologic features including a solid or acinar growth pattern, dense neoplastic cellularity, uniform nuclei with prominent nucleoli, and granular eosinophilic cytoplasm. Exocrine enzymes are detectable by immunohistochemistry and, for carcinomas with mixed differentiation, neuroendocrine or ductal lineage markers are also expressed. The genetic alterations of this family of neoplasms largely differ from conventional ductal adenocarcinomas, with only rare mutations in TP53, KRAS, and p16, but no single gene or neoplastic pathway is consistently altered in acinar neoplasms. Instead, there is striking genomic instability, and a subset of cases has mutations in the APC/β-catenin pathway, mutations in SMAD4, RAF gene family fusions, or microsatellite instability. Therapeutically targetable mutations are often present. This review summarizes the clinical and pathologic features of acinar neoplasms and reviews the current molecular data on these uncommon tumors.     

胰腺导管腺癌中胰腺星形细胞的研究进展

胰腺星形细胞(pancreatic stellate cells,PSC)是胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDAC)肿瘤微环境中最重要的成分,在PDAC发生发展中具有非常关键的作用.目前大量研究关注于PSC与胰腺癌细胞(pancreatic cancer cells,PCC)之间的相互作用及PSC在PDAC微环境中发挥的作用.PSC在许多情况下发生活化,如乙醇、氧化应激和高血糖等.PDAC早期即可出现PSC的活化,PCC可以诱导刺激PSC发生活化,活化的PSC可以产生大量胶原纤维,形成适宜PCC生长的间质微环境,促进PCC的增殖,减少化疗药物对肿瘤细胞的杀伤作用.另外,PSC还可以与间质中各种细胞成分如内皮细胞和各种免疫细胞相互作用,在血管生成、免疫逃逸和神经侵犯等方面协助肿瘤进展.因此,阐明PSC与肿瘤细胞以及其他间质成分之间复杂的相互作用至关重要.     

EGFR expression in pancreatic intraepithelial neoplasia and ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignant tumor with poor prognosis. Epidermal growth factor receptor (EGFR) is an important cell adhesion and signaling pathway mediator. The aim of this study was to evaluate the expression of EGFR in both pancreatic intraepithelial neoplasia (PanIN) and PDA and their relationship to clinicopathologic characteristics. Formalin-fixed, paraffin-embedded tissues including 81 cases with pancreatic ductal adenocarcinoma, 27 with normal pancreas, 16 with PanIN-1A, 18 with PanIN-1B, 11 with PanIN-2, and 24 with PanIN-3 were used for construction of tissue microarrays. Imunohistochemistry for EGFR was performed. Normal pancreatic ducts, PanIN-1A, and PanIN-1B did not show EGFR overexpression. EGFR overexpression was observed in 18.2% (2/9) of PanIN-2, 41.7% (10/14) of PanIN-3, and 64.2% (52/81) of PDA, respectively. Significantly higher EGFR overexpression was observed in PDAs than in PanIN lesions (P<0.05). No statistically significant correlation was observed between EGFR overexpression and patient age, sex, tumor location, size, histological grade, vascular invasion, lymph node metastasis and stage at presentation, respectively. In conclusion, EGFR expression increased from PanIN to PDA. EGFR may be involved in early stage in development of PDA.     

Epigenetics and Epigenetic Alterations in Pancreatic Cancer

Pancreatic cancer remains a major therapeutic challenge. In 2008, there will be approximately 37,680 new cases and 34,290 deaths attributable to pancreatic cancer in the United States (U.S.), making it the fourth leading cause of cancer-related death. Recent comprehensive pancreatic cancer genome project found that pancreatic adenocarcinomas harbored 63 intragenic mutations or amplifications/homozygous deletions and these alterations clustered in 12 signaling pathways. In addition to widespread genetic alterations, it is now apparent that epigenetic mechanisms are also central to the evolution and progression of human cancers. Since epigenetic silencing processes are mitotically heritable, they can drive neoplastic progression and undergo the same selective pressure as genetic alterations. This review will describe recent developments in cancer epigenetics and their importance in our understanding of pancreatic adenocarcinomas.     

Concentrations of trace elements and KRAS mutations in pancreatic ductal adenocarcinoma

Trace elements are a possible risk factor for pancreatic ductal adenocarcinoma (PDAC). However, their role in the occurrence and persistence of KRAS mutations remains unstudied. There appear to be no studies analyzing biomarkers of trace elements and KRAS mutations in any human cancer. We aimed to determine whether patients with KRAS mutated and nonmutated tumors exhibit differences in concentrations of trace elements. Incident cases of PDAC were prospectively identified in five hospitals in Spain. KRAS mutational status was determined through polymerase chain reaction from tumor tissue. Concentrations of 12 trace elements were determined in toenail samples by inductively coupled plasma mass spectrometry. Concentrations of trace elements were compared in 78 PDAC cases and 416 hospital-based controls (case–control analyses), and between 17 KRAS wild-type tumors and 61 KRAS mutated tumors (case–case analyses). Higher levels of iron, arsenic, and vanadium were associated with a statistically nonsignificant increased risk of a KRAS wild-type PDAC (OR for higher tertile of arsenic = 3.37, 95% CI 0.98–11.57). Lower levels of nickel and manganese were associated with a statistically significant higher risk of a KRAS mutated PDAC (OR for manganese = 0.34, 95% CI 0.14–0.80). Higher levels of selenium appeared protective for both mutated and KRAS wild-type PDAC. Higher levels of cadmium and lead were clear risk factors for both KRAS mutated and wild-type cases. This is the first study analyzing biomarkers of trace elements and KRAS mutations in any human cancer. Concentrations of trace elements differed markedly between PDAC cases with and without mutations in codon 12 of the KRAS oncogene, thus suggesting a role for trace elements in pancreatic and perhaps other cancers with such mutations. Environ. Mol. Mutagen., 60:693–703, 2019. © 2019 Wiley Periodicals, Inc.     

Analysis of K-ras oncogene mutation directly applied to atypical cell clusters on cytologic smear slides of bile and pancreatic juice

To develop an objective reference for the cytological evaluation of atypical cells in bile and pancreatic juice, we analyzed K-ras oncogene mutation In atypical cell clusters, which were collected directly from cytological smear slides; 50 samples (cell clusters) from 31 smear slides of 21 patients with carcinomas of the pancreatic head region, and nine samples from eight cases of benign disease. These cell clusters (5–1000 cells/cluster) were selectively suspended In buffer containing proteinase K, and subjected to DNA extraction. K-ras codon 12 mutation was determined by polymerase chain reaction amplification, followed by digestion with BstNI. The K-ras gene was amplified in 20 of 21 cases with carcinoma (34/50 samples), and In seven of eight cases with non-neoplastic disease (8/9 samples). Among the cases of which primary tumors showed K-ras mutation, amplification was successful in 10 of 11 cases; mutation was demonstrated in three of seven cases with cytologically atypical cells (4/11 samples), and in three of three cases with cytologically malignant cells (5/7 samples). No mutation was Identified in the 10 cases of carcinoma without K-ras mutation (0/15 samples), or in eight cases of non-neoplastlc disease (0/8 samples). Cytological details could be comparatively evaluated between atypical cell clusters with or without mutation on the same smear slides in two cases. This type of direct analysis of atypical cell clusters may be useful in the self-assessment of cytological diagnosis of bile and pancreatic juice.