100023 北京市2345信箱 世界华人消化杂志  2000年9月15日;8(9):983-987
Email: wcjd@public.bta.net.cn 世界华人消化杂志  ISSN 1009-3079  CN  14-1260/R
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⊙研究原著⊙

流式细胞术分析胃癌及癌前病变中细胞凋亡与增殖

郭玉庆1  朱兆华2  黎锦芳2

1深圳市宝安区人民医院消化科  广东省深圳市  518101
2中山医科大学孙逸仙纪念医院消化科  广东省深圳市  510120
郭玉庆，男，1964-11-13生，陕西省西乡县人，汉族. 1987年西安医科大学医疗系本科毕业，学士，1997年中山医科大学硕士，主治医师，主要从事消化系统疾病的临床工作.
项目负责人  郭玉庆，518101，广东省深圳市宝安区人民医院消化科.
1 Department of Gastroenterology, Baoan District Hospital of Shenzhen, Shenzhen 518101, Guangdong Province, China
2 Department of Gastroenterology, Sun Yat-Sen Memorial Hospital, Sun Yat-sen University of Medical Sciences, Guangzhou 510012, Guangdong Province, China
Correspondence to Dr. Yu-Qing Guo, Department of Gastroenterology, Baoan District Hospital of Shenzhen, Shenzhen 518101, Guangdong Province, China
Tel. 0086-755-7788311 Ext.3025
收稿日期  2000-05-18  接收日期  2000-06-02


Flow cytometric analysis of apoptosis and proliferation in gastric cancer and precancerous lesion

Yu-Qing Guo1, Zhao-Hua Zhu2 and Jin-Fang Li2


Abstract

AIM  To investigate the changes and the possible role of the cell apoptosis index (AI), proliferation index (PI), the ratio of AI to PI and the DNA index (DI) in various gastric diseases.

METHODS  The gastric mucosal biopsies taken by endoscopy from 15 cases of  chronic superficial gastritis (CSG), 15 cases of chronic atrofic gastritis (CAG), 16 cases of gastritis epithelial dysplasis (Dys) and the specimens of gastric cancer （GC） tissue from surgically resected stomach in 50 patients with gastric cancer were collected in this study. All specimens were fixed in 10% formalin and embedded in paraffin.The apoptosis cells were labelled by TUNEL technic, the AI was expressed as the percentage of positive TUNEL staining cells. The total cell DNA was labelled by propidium index. The PI was expressed as the percentage of S+G2 M phases cells. The AI and PI ratio of AI to PI and the DI were measured by flow cytometry.

RESULTS  The AI was significantly lower in  GC (6.6%) than that in  CAG (11.2%) and Dys (18.3%,P＜0.05), but not statistically lower than the AI in  CSG (10.2%, P＞0.05). The PI in CSG, CAG, Dys and GC were 14.9%, 20.1%, 24.6% and 31.8% respectively. A statistical difference in PI was found between any two groups  (P＜0.05). The ratio of AI to PI was the lowest in the GC (0.22) compared to CSG (0.65%), CAG (0.57) and Dys (0.72%, P＜0.05). The values of PI was significantly related to the depth of tumor invasion,lymph nodes or distant metastasis, tumor stages as well as the type of DNA ploid (P＜0.05). All of CSG and CAG were diaploid, however, the aneuploid was found in 25% cases of Dys (4/16) and 82% cases of GC (41/50). The gastric cancers with aneuploid had a significantly higher PI (33.6%) and lower ratio  of  AI to PI (0.19%) when compared with that in diaploid tumor (PI=23.4%, AI/PI=0.35), P＜0.05. There was a positive correlation between the AI and PI in both CSG and CAG (r=0.52 and r=0.55, repectively), P＜0.05, but such correlation was not seen in the Dys and GC.

CONCLUSION  The breakdown of the balance between the cell proliferation and apoptosis gradually developed from CSG to GC. The character of cell kinetics in gastric cancer was the superiority of proliferation to apoptosis due to the increase in proliferation and the decrease in apoptosis. The aneuploid DNA was detected earliest in Dys and became preponderant in GC. The gastric cancer  with aneuploid DNA had significantly higher PI and lower ratio of AI to PI. These results suggested that the detection of aneuploid may be useful for a early diagnosis of precarcerous lesion as well as gastric cancer, and for evaluating the malignant degree of tumor.

Subject headings  stomach neoplasms/diagnosis; apoptosis; proliferation; flow cytometry; precancerous conditions;  gastroscopy

Guo YQ, Zhu ZH, Li JF. Flow cytometric analysis of apoptosis and proliferation in gastric cancer and precancerous lesion.
Shijie Huaren Xiaohua Zazhi, 2000;8(9):983-987



摘要

目的  研究慢性浅表性胃炎(CSG)，慢性萎缩性胃炎(CAG)，不典型增生(Dys)和胃癌(GC)病变中，细胞增殖、细胞凋亡和DNA倍体变化的规律及对疾病发生的可能作用.

方法  CSG 15例，CAG 15例，Dys 16例和GC 50例经TUNEL方法标记凋亡细胞和碘化丙啶标记细胞DNA双染色，用流式细胞仪检测细胞凋亡指数（AI）和细胞增殖指数（PI）及DNA指数（DI）.

结果  在CSG，CAG，Dys和GC中，AI分别为10.2%，11.9%，18.3%和6.6%. GC的AI显著低于Dys和CAG(P＜0.05)；PI为14.9%，20.1%，24.6%和31.8%. GC的PI显著地高于其余3组(P＜0.05)，Dys的PI亦显著高于CSG(P＜0.05)；AI/PI比值分别为0.65，0.57，0.72和0.22. GC的AI/PI比值显著地低于其余3组(P＜0.05). PI与胃癌的浸润深度、转移、分期及DNA倍体类型有关. CSG和CAG的DNA均为二倍体，16例Dys中有4例(25%)为异倍体DNA，50例GC有41例(82%)的DNA为异倍体. 异倍体DNA的胃癌，其PI(33.6%)明显增高，而AI/PI(0.19%)却明显低于二倍体胃癌(分别为23.4%和0.35%)，P＜0.05. AI与PI在CSG和CAG中呈明显正相关(r分别为0.52和0.55，P＜0.05). 但在Dys和GC中并无明显的关系(P＞0.05).

结论  胃癌及癌前病变存在着细胞增殖和凋亡平衡的失调，表现为细胞增殖增加和凋亡减少. 细胞增殖的显著优势是胃癌形成的细胞动力学特征. 异倍体DNA最早出现于不典型增生，而在胃癌时最为显著. 异倍体DNA的胃癌有明显高的细胞增殖指数和显著低的凋亡指数与增殖指数比值，提示DNA倍体的检测有助于早期发现癌前病变、早期诊断胃癌和评价肿瘤的恶性程度.

主题词  胃肿瘤/诊断；细胞凋亡；增殖；流式细胞术；癌前状态；胃镜检查

郭玉庆, 朱兆华, 黎锦芳. 流式细胞术分析胃癌及癌前病变中细胞凋亡与增殖. 世界华人消化杂志，2000；8(9)：983-987


0  引言

胃癌(GC)发病原因和机制迄今仍未完全阐明，研究证实GC的发生是一个多步骤、多阶段的过程，部分胃癌的形成过程经历了慢性浅表性胃炎(CSG)、萎缩性胃炎(CAG)、不典型增生(Dys)等几个不同阶段的病变［1］. 长期以来胃癌和其他肿瘤的研究一样多集中于细胞增殖及其影响因素方面，然而很难全面解释肿瘤发生和发展过程中的各种现象. 近年研究发现肿瘤的形成与细胞凋亡也有密切联系［2-21］，细胞增殖和细胞凋亡的相互消长决定了肿瘤的发生、发展及转归. 已有研究表明，胃癌形成过程中也存在着细胞凋亡和细胞增殖的变化［22-30］. 我们应用流式细胞术定量分析了CSG，CAG，Dys和GC病变中的细胞增殖与细胞凋亡，探讨胃粘膜细胞增殖与凋亡在各病变中的变化规律及对疾病发生的可能
作用.

1  材料和方法

1.1  材料  标本均来自中山医科大学孙逸仙纪念医院，CSG，CAG，Dys为内镜下胃粘膜活检，GC为手术切除标本，标本经100mL·L-1福尔马林固定，石蜡包埋，切片经HE染色病理组织学检查确定诊断. 选择病例的一般性资料(表1). Elite型流式细胞仪，美国Coulter公司. TUNEL试剂盒购自德国宝灵曼公司. Dnase Ⅰ(RNase free)购自Promega公司. 碘化丙啶和胃蛋白酶(DNase and RNase free)及RNase(DNase free)购自Sigma公司.

1.2  方法  单细胞悬液的制备［31,32］切取各种组织石蜡包埋标本40μm～50μm厚切片5片，放入试管中，以二甲苯脱蜡. 待蜡脱净后弃二甲苯，依次水化，加5g·L-1胃蛋白酶(pH=1.5)溶液3mL～5mL，37℃水浴30min～60min，每5min摇动一次使其充分消化. 加PBS终止消化，1500r/min离心10min去其上清液，再加PBS 3mL～5mL，用吸管轻轻吹打，300目铜网过滤，离心去上清液. 用PBS洗涤细胞2次，加少量PBS悬浮细胞，并用红血球计数板计数，调整细胞密度107～109·L-1，待染色用.

1.2.1  TUNEL标记及PI一步插入法双重染色［33］ 将细胞悬液移入Eppendoff管(1.5mL)，300g离心10min去上清液. 加1g·L-1柠檬酸钠溶液，置冰浴渗透2min. 加0.01mol·L-1 PBS 250μL×2次，洗去渗透液，300g离心10min去上清液；标记:每管加反应混和液50μL悬浮细胞，置37℃恒温振荡水浴箱避光孵育90min. 阴性对照不加TdT酶，阳性对照用DNase Ⅰ(0.7mg·L-1)处理10min的细胞，余实验步骤均相同. 用PBA 250μL×2次，洗去标记液，300g离心10min去上清液. 每管加0.5mL　PI染液(PI 50mg.L-1)及RNase A　7μL(10g·L-1)，悬浮细胞，置冰浴避光孵育30min，上机前再用300目铜网过滤1次.

1.2.2  流式细胞仪检测  成批进行检测，每例检测10000个细胞，以正常胃粘膜上皮细胞DNA含量作为DNA二倍体(2C)标准.　DNA倍体的判断标准:　DI值在1±0.1范围为二倍体，超过此范围即为异倍体.

细胞增殖指数(PI)

PI=(S+G2M)期细胞数  /(G0/G1+S+G2M)期细胞数    ×100%

细胞凋亡指数的计算(AI)

AI=TUNEL阳性细胞数 /  检测总细胞数  ×100%

   统计学处理  计量资料用均数±标准差表示，均数比较用t检验和方差分析，率的比较用卡方检验，样本量＜40，用Fisher精确计算法；等级相关检验用Spearman法.


2  结果

2.1  PI，AI，AI/PI，以及DNA倍体的类型(表2) GC组的AI明显低于CAG和Dys组(P＜0.05，表2)，而CSG与CAG，Dys及GC组却无显著差异(P＞0.05)，CAG，Dys间也无明显差别(P＞0.05). GC组的PI值均高于CSG，CAG，Dys组(P＜0.05)，且Dys组尚高于CSG，但GC组AI/PI值均显著低于CSG，CAG，Dys任一组(P＜0.05)，而CSG，CAG，Dys三组间无显著差异(P＞0.05). 在CSG和CAG所有DNA均为二倍体，Dys有25%的DNA为异倍体，而胃癌的异倍体DNA占82%，显著地高于CSG，CAG和Dys(P＜0.05).

2.2  胃癌临床病理与AI，PI，AI/PI及DNA倍体的关系  AI，PI，AI/PI与胃癌患者年龄，性别无关(P＞0.05)，也与肿瘤部位无关(P＞0.05). PI在进展期和TNM-Ⅱ,Ⅳ期胃癌中明显升高(P＜0.05，表