修回日期: 2002-08-10
接受日期: 2002-08-16
在线出版日期: 2003-07-15
研究肝细胞癌肝动脉化疗栓塞(TACE)对肝肿瘤增生与转移的影响.
1992-07/2000-07不能手术切除的大肝癌经TACE治疗后, 肿瘤缩小, 而后二期手术切除, 本组共施使这类手术72例, 根据性别、年龄、既往史、肿瘤大小共计10项指标, 与同期未经TACE治疗, 直接手术切除的肝癌487例进行配对, 有32对进入本课题研究. 运用免疫组化技术, 对比研究了肝癌细胞中的细胞增生核抗原(PCNA), 和nm23-H1基因的表达.
肝癌经TACE治疗后PCNA的标记指数升高, PCNA标记指数≤25%、26-50%、51-75%、＞75%的对照组分别为75%、14.3%、10.7%、0%, TACE治疗组分别为35.7%、21.4%、28.6%、14.3%, (P<0.01, n = 28). nm23-H1基因表达肝癌细胞与细胞核中均存在, 但治疗组与对照组无显著差异.
肝癌经TACE治疗后残留癌的反应性增生使PCNA标计指数升高, 导致TACE治疗肝癌远期疗效不理想, 所以肝癌TACE后二期手术很有必要.
引文著录: 冯勇, 赵玲, 张爱华, 刘康达, 刘来村, 王彦辉, 尹进强, 杨秉辉. 肝细胞癌肝动脉化疗栓塞后PCNA和nm23-H1/NDPK的研究. 世界华人消化杂志 2003; 11(7): 912-915
Revised: August 10, 2002
Accepted: August 16, 2002
Published online: July 15, 2003
To study the effect of transcatheter arterial Chemoem-bolization (TACE) on the proliferation and metastasis of the hepatocellular carcinoma (HCC).
Seventy-two patients with inoperable huge HCC administered from 1992 to 2000 were treated with TACE initially, and then operated following the shrinkage of the tumor. During same periods of treatment, there were comparable 487 HCC patients who were not treated with TACE, of which 32 pairs of patients who met with the criteria were selected for this study based on the 10 items screening criteria including gender, age, history of the disease, BUS examination. Expression of proliferating of cell nuclear antigen (PCNA) and nm23-H1 were studied by immunohistochemistry.
PCNA label index (PCNA LI) of HCC following TACE was elevated. The distribution of PCNA LI in the range ≤25%, 26-50%, 51-75% and ＞75% is 75%, 14.3%, 10.7% and 0% in control group, and 35.7%, 21.4%, 28.6% and 14.3%, in TACE group (P<0.01, n = 28), respectively. The expression of nm23-H1 gene exists in both cytoplasm and nuclear with no significant difference between treatment and control group.
The proliferative activity of residual tumor of HCC following TACE causes the elevation of PCNA LI implying the poor prognosis of long-term effect of TACE, and therefore, it is necessary to perform two stage-resection after TACE of HCC.
- Citation: Feng Y, Zhao L, Zhang AH, Liu KD, Liu LC, Wang YH, Yin JQ, Yang BH. Expression of PCNA and nm23-H1/NDPK in Hepatocellular Carcinoma following transcatheter arterial chemoembolization therapy. Shijie Huaren Xiaohua Zazhi 2003; 11(7): 912-915
- URL: https://www.wjgnet.com/1009-3079/full/v11/i7/912.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v11.i7.912
肝细胞癌(hepatocellular carcinoma, HCC)是一种严重威胁人类健康的消化系统常见恶性肿瘤[1-10]. 恶性肿瘤的浸润和转移是区别于其他良性肿瘤的一个重要生物学特征, 同时也是一个与预后有直接关系的因素, 是恶性肿瘤患者死亡的重要原因. 了解肿瘤浸润与转移的情况, 是临床医生对预后进行有效估计的关键. 因此, 本实验选择了PCNA和nm23-H1/NDPK这两个与肿瘤浸润和转移有关的蛋白质为研究对象[11-18]. 目的在于了解HCC经TACE治疗后, 对肿瘤生长和转移的影响, 为进一步治疗提供理论指导.
1992-07/2000-07不能手术切除的大肝癌72例, 经肝动脉化疗栓塞(TACE)治疗后肿瘤缩小, 得到了二步手术切除的机会, 与同期未经TACE治疗直接手术切除的487例进行配对. 配对原则: 性别相同; 年龄相差不超过5岁; 有无肝炎史一致, 肝炎病史相差不超过5 a; 饮酒史分为: 无、偶炊、嗜酒三类, 分别对应一致. 持续时间相差不超过5 a; 癌在左右肝叶的位置一致; 术前B超检查肿瘤的最大直径相差不超过0.5 cm; TACE前血清HbsAg与AFP的阳性结果一致; 有无肝硬化一致; 肝癌分化程度一致; 手术切除的方式一致. 共有32对患者符合上述要求. 根据上述配对的病理号, 调出相应的石蜡包埋的肿瘤组织蜡块. 制成4 μm的组织切片, 做为PCNA和nm23/NDPK(H1)免疫组织化学的反应底物. PCNA单抗为鼠抗人单克隆抗体PC-10, Dako公司产品. 兔抗人nm23/NDPK(H1)为Oncogene公司产品, ABC Kit为Vector公司产品.
采用免疫组织化学染色, 细胞核呈棕色颗粒为阳性, 先在低倍镜下观察PCNA染色情况, 寻找染色较密者. 分布均匀区域, 随机取5个高倍镜视野(10×40), 计数500个细胞中PCNA阳性细胞核数, 平均每100个细胞中的阳性细胞核数为PCNA标记指数, 即: PCNA LI = [PCNA(+)细胞核/500个细胞核]×100%. 根据PCNA标记指数的大小, 将阳性患者分为4个等级: PCNA标记指数≤25%为(+), 26-50%为(++), 51-75%为(+++), ＞75%为(++++). nm23/NDPK免疫组织化学 (1)按切片中细胞显色有无及深浅计分(A): 无显色为0分, 浅黄色为1分, 棕黄色为2分, 棕褐色为3分. (2)按切片中显色细胞的比例评分(B): 1/3以下癌细胞显色计1分, 1/3-2/3的癌细胞显色计2分, 2/3以上的癌细胞显色计3分. (3)每例肿瘤的积分 = A×B, 依据积分的高低判断肿瘤的阳性结果: 积分为0阴性(-), 1-4分为弱阳性(+), >4分为强阳性(++). (4)细胞核阳性结果的判断依据下述标准: 连续3个高倍镜(10×40)视野中, 平均有1-5个细胞核显色阳性则为(+); 6-10个为(++); 11-15个为(+++); 超过15个为(+++), 少于一个为(-).
统计学处理 采用符号等级检验(Wilcoxon法).
TACE组32例肝癌患者中有4例由于肿瘤完全坏死, 病理切片中未找到癌细胞, 即将剩余的28例与相应的配对, 对比观察了其PCNA和nm23-H1/NDPK免疫组化检测结果.
治疗组与对照组肝癌PCNA的免疫组化检测结果存在有显著差异(表1, P<0.01), 两组患者肝癌的PCNA标记指数.
| 组别 | -(%) | +(%) | ++(%) | +++(%) | ++++(%) | 合计(%) |
| TACE组 | 10(35.7) | 6(21.4) | 8(28.6) | 4(14.3) | 28(100) | |
| PCNA 对照组 | 21(75) | 4(14.3) | 3(10.7) | 0(0) | 28(100) | |
| 胞质 TACE组 | 8(28.6) | 1 4(50) | 6(21.4) | 28(100) | ||
| nm23-H1/NDPK 对照组 | 8(28.6) | 13(46.4) | 7(25) | 28(100) | ||
| 胞核 TACE组 | 9(32.1) | 11(39.3) | 6(21.4) | 2 (7.2) | 0(0) | 28(100) |
| nm23-H1/NDPK 对照组 | 11(39.3) | 6(21.4) | 7(25) | 3(10.7) | 1(3.6) | 28(100) |
治疗组与对照组肝癌胞质的nm23-H1/NDPK免疫组化阳性率都是71.4%, (表1), 经统计学处理P>0.05, 既肝癌经TACE治疗后细胞中nm23-H1的表达与对照组没有显著差异.
TACE组肝癌细胞中有细胞核nm23-H1/NDPK免疫组化阳性者为67.9%, 对照组为60.7%, (表1), 经统计学处理P>0.05, 两组肝癌nm23-H1/NDPK在细胞核中检测结果没有显著的差别.
细胞增生核抗原(proliferating cell nuclear antigen, PCNA)是聚积在S期在细胞核内复制的细胞周期依赖性蛋白, 是DNA多聚酶δ的辅助蛋白[19]. 他的表达与合成和细胞增生有关, 参与了细胞增生过程中的DNA复制, 在细胞周期性控制中起着一定的作用[20], 可反映细胞增生活性的高低. PCNA在G1期开始增加, S期达到高峰, G2期下降, M期降至最低, 并且与患者预后有关[21-24]. 我们的结果显示, 肝癌经TACE治疗后残留癌的PCNA标记指数增高, 与对照组比较有显著差异, 我们认为可能与TACE治疗后残留癌中新生肿瘤细胞所占比例增大有关. 肝癌患者经TACE治疗后可发生程度不等的肿瘤坏死, 由于肝癌的血供至少80%来源于肝动脉[25], 除了肝动脉外, 还有门静脉, 以及栓塞后新生血管的形成, 侧支循坏的建立. 这主要是由于TACE治疗后使肿瘤缺氧、坏死, 缺氧与坏死组织又可刺激血管内皮生长因子(vascular endothelial growth factor, VEGF)等的分泌, 促进了血管形成[25]. 致使这种坏死不彻底, 在肿瘤边缘可或多或少地有残留癌存在, 他们仍能得到相应的血液供应, 而得以继续生存下去, 这个部位的肿瘤生长最活跃, 使G0期的细胞进入细胞分裂周期中, 处于S期的癌细胞增加, 即相应地PCNA表达量也增加. 所以, 肝癌患者经TACE治疗后肝癌缩小, 症状改善, 疗效是肯定的. 但如本研究所显示, 经TACE后肝癌细胞并不能被完全杀灭, 而残留癌细胞的增生与侵袭能力更强, 致使远期疗效并不理想, 这与其他文献报道是一致的[26-28], 此点应引起临床医师的充分注意, 采取积极的防治措施, 不失时机地进行二步手术切除等.
nm23基因是一种肿瘤抑制基因[29-31], 在肝癌的研究中也是如此[32], 他的表达主要存在于细胞质内, 但在细胞核及细胞膜中也有表达, 即分为胞型、核型与膜型, 核型表达才是评价抑制肿瘤转移的有效指标. 本实验既有胞型, 又有核型, 但没有明显的膜型表达发现, 可能与抗体的来源不同有关. 同时对比观察了TACE治疗组与对照组肿瘤的nm23-H1胞质型与核型的表达情况, 发现这两型均没有差异(P>0.05), 提示TACE治疗对肝癌组织中nm23-H1基因的表达基本上没有影响. 这可能是由于肝癌TACE后到手术切除的时间较长, 原来的nm23-H1产物NDPK已逐渐被代谢, 同时也随着时间的推移nm23-H1基因的表达又恢复了原来的水平, 所以就得到了上述结果.
目前TACE治疗肝癌远期疗效不够理想, 主要原因在于残留癌存在导致的复发与转移. TACE治疗后肝癌的转移主要见于肿瘤坏死不完全的患者, 对于肿瘤完全坏死和并无坏死者则没有影响, 原因是部分坏死的肿瘤中残留癌细胞附着力下降, 易于脱落进入血液循环而发生转移. 另外, 没有经TACE治疗的肝癌患者, 在肿瘤发生自发性不完全性坏死后, 也同样发现发生转移的机会增加. 还应注意, TACE治疗不但促进了VEGF的分泌增加, 使新生血管产生, 侧支循环建立, 同时VEGF还使小血管通透性增加, 再加上肿瘤血管结构不完整, 内皮细胞比较幼稚, 细胞间常有裂隙, 缺乏基底膜等, 使肿瘤细胞易于进入血液循环发生转移[33-35]. 因此, 结合本研究结果可认为, 肝癌经TACE治疗后发生转移的机会增加, 这可能与肿瘤的不完全性坏死有关, 还有PCNA含量的增加, 而与nm23-H1基因的表达可能无关. 因此, 肝癌患者经TACE治疗后适时地进行二步手术切除是非常必要的, 这与国内资料是完全一致的, 不能手术切除的大肝癌经TACE治疗后, 肿瘤缩小再二期切除, 可明显改善患者的远期预后[36,37]. 但是, 对于可切除的肝癌术前进行TACE是不可取的[38], 因为TACE不但延误了手术时机, 增加了手术难度, 还可促进残留癌细胞的转移.
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