摘要：目的为了解肠癌患者外周血(PB)中粘附分子(CD44)水平的临床意义。方法用流式细胞术检测了105例肠癌患者术前PB中CD44水平和免疫细胞亚群，进行临床病理分析与100例正常人PB中CD44％作统计学处理比较。结果正常人PB中CD44％为46．14±13.41，CD44％与年龄、性别比较无统计学意义．直肠癌患者PB中CD44％(54．17±16．62)与正常人比较差异有非常显著性(P＜0．01)．外生型癌患者PB中CD44％(55．10±18．52)与正常人比较差异有非常显著性(P＜0．01)．肿瘤＞4cm～8cm(53.48±16．97)和＞9cm(63．67+12．99)患者PB中CD44％与正常人比较差异有非常显著性(P＜0．01)．中分化腺癌患者PB中CD44％(51．83±18．15)与正常人比较差异有显著性(P＜0．05)，低分化腺癌患者PB中CD44％(58．31±14．66)与正常人比较差异有非常显著性(P＜0．01)．有淋巴结转移者PB中CD44％(53±19．51)与正常人比较差异有显著性(P＜0．05)．DukesA+B期患者PB中CD44％(51．09±15．83)与正常人比较差异有显著性(P＜0．05)，D期患者PB中CD44％(63．93~13．9)与正常人比较差异有非常显著性(P＜0．01)．女性肠癌患者PB中m4％(55．07±17．17)与正常女性比较差异有非常显著性(P＜0．01)．＜59岁肠癌患者PB中CD44％(54．76±16．39)与正常人比较差异有非常显著性(P＜0．01)，结论肠癌患者PB中CD44％升高与肿瘤发生部位、肿瘤大体类型、肿瘤大小、肿瘤分化程度、淋巴结转移、临床分期、性别、年龄均有相关性．用流式细胞术检测肠癌患者术前PB中CD44水平，可为临床提供判断患者预后的参考依据。

Relationship between CD44 in the peripheral blood of patients with colorectal cancer and clinico-pathological features

Shen Hua Xu ,Jian Guo feng(Zhejiang Cancer Research linstituteDepartment of Surgery of Zhejiang Cancer hospital,310022 Hangzhou,Zhjiang Province,China)

de Chuan Li(Department of Surgery of Zhejiang Cancer Hospital,310022 Hangzhou,Zhjiang province,China)

han Zhou Mou(Zhejiang Cancer Research linstituteDepartment of Surgery of Zhejiang Cancer hospital,310022 Hangzhou,Zhjiang Province,China)

rong Can Lou(Department of Surgery of Zhejiang Cancer Hospital,310022 Hangzhou,Zhjiang province,China)

Abstract：AIM to understand the clinical significance of call adheeive molecules (CD44) in the peripheral blood (PB) of patients with colorectal cancer.METHODS The level of cD44 in PB of 105 patients with colorectal cancer was examined by flow cytometry and the results were clinicopatbelogically and statistically analyzed.RESULTS the mean of the CD44% in PB of the normal individuals was 46.14±13.41 and there were no statistical differences for their age and sex. The significant difference (P＜0.01) was present between the patients with the rectal cancer cD44% (54.17±16.62) and normal individuals. There was significant difference(P＜0.01) between the patients with expophytic type colorental cancer CD44%(55.10±18.52) and normal individuals,between the patients with ＞ 4 cm-8cm(53.48±16.97)and ＞gcm (63.67±12.99) mass CD44% and normal individuals(P＜ 0.01 ), between the patients with maderatdy differentiated adenocarcinoma cD44% ( 51.83±18.15) and normal individuals (P＜0.05), between the patients with lowly differentiated edenocarcinorna CD44% (58.31±14.66) and normal individuals (P＜0.01),between the patients with the metastatic lymph node cD44% (53±19.51 ) and normal individuals (P＜0.05),between the patients with dukes A+ B stage CD44% (51.09±15.83)and normal individuals (P＜0.05), between the patients with Dukes D stage CD44% (63.93±13.92) and normal individuals(P＜0.01), between the female patients CD44% (55.07±17.17)and normal femaleindividuals (P＜0.01) and between the patients with age ＜59 CD44%(54.76±16.39) and normal individuals (p＜0.01).CONCLUSION The increased level of CD44 in PB of patients with colorectal cancer may be closely linked with site of tumor growth, type of tumor mass, size of tumor mass, degree of tumor differentiation, metastatic lymph node, clinical stage, sex, age of petiants. examining level of CD44% in the periphery blood of patients with colorectal cancer by flow cytometry can suggest prognosis of patients.

Keywords：intestinal neoplasms;immunity, cellular; flow cytometry; pathology;adenocarcinoma; rectal neoplasms;lymphatic metastasis; research

0引言粘附分子(CD44)与恶性肿瘤的转移有关[1-3]，但大多数报道都是检测恶性肿瘤细胞CD44，的表达，少见用流式细胞仪检测肠癌患者外周血(PB)中CD44水平的报道．为了解肠癌患者PB中CD44水平的临床意义，作者检测105例肠癌患者术前PB中CD44水平，并进行临床病理分析及与100例正常人进行比较。1材料和方法1．1材料1997-07-15／1998-05-07浙江省肿瘤医院外科住院的肠癌患者，术前检测了外周血中CD44水平的患者共105例，均经复查病理诊断和病历资料．男62例，女43例，年龄24岁～78岁，平均年龄52．83岁±1．15岁．对照组取本院正常健康职工100例，男2l例，女79例，年龄19岁～66岁，千均38．66岁±2．35岁．试剂：本研究使用的抗体均为荧光异硫氰酸荧光素(FITC)或藻红蛋白(PE)标记的鼠抗人单克隆抗体．CD44-FITC，CD56-PE(NK细胞)，CD19-FITC(美国B-D公司产品)，CD3-FITC，CD44-FITC，CD8-PE(北京医科大学免疫室产品)。1．1方法取正常人或患者外周血2mL加入到含有抗凝剂(25μ／Ml肝素)的试管中．取20uL抗体加入到塑料小管中，每例做5管，A管CDl9-FITC，B管CD4-FITC/CD8-PE，C管CD3-FITC／CD56-PE，D管CD44-FITC，E管阴性对照，在每管中加100μL新鲜图1正常人PB中CD44表达的直方图(a)阴性对照(b)。一个50岁男性健康人，CD44为47%。图2(a)CD44表达直方图(b)阴性对照，一个男性直肠溃疡型低分化腺癌有22个淋巴结转移患者，CD44为79%。的抗凝全血，充分混匀后在室温暗处静置15min～30min，每管加2mL溶血素(0．159mol／LNH4C1，0．01mol／LKHOC3，0，lmmoL／L．Na2-EDTA)，充分混匀后，在室温于暗处静置(10～12)min，离心(300×g)5min，倾去上清，加2mLPBS(含1g／L叠氮钠)震荡，混匀，离心(200×g)5min，倾去上清，每管加0．5mL10mL／L的副甲醛固定液，当天上机检测分析。流式细胞仪(FACSCalibur美国B-D公司)采集窗内2000个以上细胞，用CellGuest软件分析阳性标记细胞百分率．CD44分析用直方图(图1，2)，免疫细胞亚群分析用点阵图。统计学处理采用t检验。2结果2．1正常人PB中CD44范围正常人PB中CD44平均46．14±13．41，根据总体均数99％可信限为±2．58s公式，所得值范围为42．7％～49．6％．2．2肿瘤发生部位105例肠癌患者均有发生部位记载．肠癌患者PB中CD44％与肿瘤发生部位的关系(表1)。表l肠癌患者PU中CD44％与肿瘤发生部位的关系2．3肿瘤大体类型91例肠癌患者有肿瘤大体类型记录，肠癌患者PB中CD44％与肿瘤大体类型的关系(表2)。表2肠癌患者PB中CD44％与肿瘤大体类型的关系Ap＜0.01t=3．16vs正常人组.注：外生型中包括窿起型．2．4肿瘤大小99例肠癌患者有肿瘤大小记载．肠癌患者PB中CD44％与肿瘤大小的关系(表3)。表3肠癌患者PB中CD44％与肿瘤大小的关系Ap＜0．01，t=3．03，t=3．89vs正常人组。2．5肿瘤分化程度105例肠癌患者均有病理报告，肠癌患者PB中CD44％与肿瘤分化程度的关系(表4)。表4肠癌患者PB中CD44％与肿瘤分化程度的关系Ap＜0．01，t=2．84，Bp＜0．05t=2．42vs正常人组。注：高分化包括癌变和粘液腺癌；低分化包括印戒细胞癌；胨型类癌和神经内分泌癌各1例未统计。2．6淋巴结转移情况95例有手术标本肠癌患者PB中CD44％与淋巴结转移的关系(表5)。表5肠癌患者PB中CD44％与淋巴结转移的关系Ap＜0.05t=2．4vs正常人组。2．7肠癌患者PB中CD44％与临床分期的关系表6。表6肠癌患者PB中CD44％与临床分期的关系Ap＜0．01t=4．55，bp＜0．05t=2．04vs正常人组。2．8肠癌患者PB中CD44％与性别关系表7。表7肠癌患者PB中CD44％与性别的关系Ap＜0．01t=4．82vs正常人组。2．9肠癌患者PB中CD44％与年龄关系表8．表8肠癌患者PB中CD44％与年龄的关系Ap＜0．01t=3.81vs正常人组。2．10肠癌患者和正常人的免疫细胞亚群比较表9。表9肠癌患者和正常人的免疫细胞亚群比较(±s)Ap＜0．0lt=4．46，t=3．55vs正常人组；bp＜0．05t=2．15vs正常人组．3讨论M80000～90000的CD44是分布极为广泛的细胞表面跨膜糖蛋白．CD44基因位于人11号染色体短臂上．CD44、包含10个组成型外显子，其转录片段存在于所有CD44产物中，主要在体内造血细胞表达，也可称为CD44H，CD44v包含10个变异性拼接外显子，主要在上皮源性细胞表达，也称为CD44E·CD4蛋白属细胞表面的粘附分子，主要参与细胞-细胞、细胞-基质之间的特异性粘连过程，CD44基因的异常表达在肿瘤早期诊断、转移潜能判断、治疗和估计预后方面有很大潜在价值[1]，Matsumuraetal[2]用PCR技术检测转移性和无转移结肠癌及正常结肠黏膜中CD44v基因表达，发现转移性结肠癌细胞CD44v表达明显增强，与其他样本比较有显著差异，Wielengaetal[3]认为CD44基因异<