摘要
背景与目的：HCC形成是多阶段、多步骤、多种基因参与HCC的形成，HCC存在抑癌基因失活。P~(16)抑癌基因编码16KD的蛋白质，抑制cyclinD1与周期素依赖蛋白激酶(cyclin dependent Kinases，CDK4)结合，阻制PRb蛋白磷酸化，从而抑制E2F介异的S期转录活化基因，抑制细胞增殖。P~(16)是细胞增殖的主要调节子之一。不同肿瘤组织P~(16)的失活机制各不相同，HCC的P~(16)基因失活方式主要是P~(16)基因启动子异常甲基化(简称p~(16)甲基化)与纯合性缺失。HCC的P~(16)基因异常率约60％，P~(16)蛋白的缺失与HCC的分化，转移相关。研究表明肿瘤病人外周血浆或血清中含有游离的肿瘤DNA，并能检测到肿瘤组织中的相关异常基因改变，为微创诊断，监测肿瘤复发提供新方法。HCC复发率高是影响HCC预后的主要问题，早期监测HCC复发利于选择治疗方法。本课题检测HCC患者血浆中P~(16)甲基化状况及其作为HCC复发分子标记物的价值。
方法：MSP(methylation-specific-PCR)法是目前检测抑癌基因甲基化改变的最佳方法，我们应用MSP法，原位杂交、免疫组织化学法研究P~(16)基因在35例HCC患者血浆，单个核细胞，肿瘤组织及其癌旁肝炎/肝硬化组织的表达状况。
结果：
1、P~(16)甲基化阳性率HCC癌组织34％明显高于癌旁肝硬化/慢性肝炎组织11％(P＜0.01)，癌组织P~(16)甲基化率在HCC伴有脉管浸润较不伴有脉管浸润者有高的趋势(54％，23％)(P=0.05)。
2、血浆P~(16)甲基化阳性的HCC，其相应的癌组织均呈阳性，血浆P~(16)甲基化阳性检出率为癌组织的50％(6/12)。相反，HCC癌组织P~(16)甲基化阴性其血浆均不能检出P~(16)甲基化。
3、HCC癌组织，血浆P~(16)甲基化阳性在术后一年复发的HCC(64％，36％)均高于术后一年无复发者(14％，5％)(P＜0.05)。
4、P~(16) mRNA原位杂交，P~(16)蛋白免疫组化证实P~(16)甲基化的肿瘤组
织呈现P”帖NA阴性与P”蛋白缺失。
5、外甲基化阳性率在血清AFP呈正常的19例HCC 患者
（AFP＜200fig／l）中为 ZI％（4／19）。
结论：
l、HCC血浆中能检出 P”甲基化异常并与 HCC术后复发相关，可
能是微创监测HCC术后复发的分子标记物。
2、HCC月瘤组织 P”甲基化与 P‘’mRNA转录抑制，P”蛋白表达缺
失密切相关。
3、HCC肿瘤组织P”甲基化可能与HCC浸润转移有关。
4，P‘’甲基化阳性率在血清 AFP呈正常的 HCC患者为 ZI免 检测
血浆 P”甲基化可能对 AFP呈正常的肝癌患者有诊断作用。
Background and aim: Hepatocarcinogenesis is considered to be a long-term process that comprise multiple cumulative genetic alteration, consistent with multistage nature of all late onset cancer. Loss of tumor suppressor genes function has been found in hepatocellular carcinoma (HCC). P16 is reported tumor suppressor gene, which expresses 16 KD protein, the P16 protein inhibits the binding of cyclin Dl to cyclin dependent kinases (CDK4) , preventing the phosphorylation of the retinoblastoma (Rb) protein, the underphosphorylated
Rb inhibits the E2F-mediated transcriptional activation of S phase genes, which is necessary for cell proliferation. P16 is one of the central regulators that controls cell proliferation. Different inactivated mechanism of P16 gene exacts in different tumor. The mechnism of P16 gene is hypermethylation and homozygous deletion in HCC. P16 abnormalities were reported in 60% of HCC. Loss of P16 protein expression is related to the differentiation and metastasis of HCC. Recently showed that free tumor DNA is found in the plasma of cancer patients. And the
presence of related oncogene or tumor suppressor gene mutation that characterized DNA in tumor cells were detected in plasma DNA. Which showed a new minitranmatized methods to diagnose or mornitor tumor, high recurrence rate in HCC mainly affected HCC prognose.monitoring early HCC recurrence helped to choise best procedure to treat HCC.our paper aimed to detect expression of P16 methylation in the plasma in HCC and evaluated P16 methylation in the plasma as molecular maker in HCC.
Methods: methylation-specific-PCR(MSP) is one of best manner to display methylated tumor suppressive gene. Our paper studyed the expression of P16 gene in 35 cases HCC in the plasma,monouncleic cell,tumoral and paratumoral tissue using MSP method ,In situ hybridization and immunohistochemistry manners.
Result:
1. P16 methylation positive rate was detected in tumor tissue 34% and in liver cirrhosis or chronic hepatitis tissue of paratumor 11%(P<0.01). Methylation was detected in HCC with microscopic invasion to hepatic vein or lymph tube (MVT)54% and without MVT 23%(P=0.05).
2. P16 methylation positive in the plasma had the same positive in HCC tumoral tissue, P16 methylation positive rate of 50% HCC tumoral tissues could be detected in the plasmas, on the contrary, P16 methylation negative in HCC tumoral tissues showed the same negative in their plasmas.
3. P16 methylation positive rate of tumoral tissues and plasmas in HCC patients with early recurrence (64%,36%) showed higher than its with later recurrence (14%,5%).
4. The results of P16 mRNA and P16 protein using in situ hybridization and immunohistochemistry methods were closely related to the results of P16 methylation in HCC patients,HCC patients with positive P16 methylation showed negative expression of P16 mRNA and P16 protein.
5. P16 methylation positive rate in plasma was 21% in HCC patients with normal serum AFP (AFP<200ug/l) . Conclusion:
1. P16 methylation could be detected in the plasmas of HCC patients, which related to HCC recurrence after surgical resection and may be useful molecular marker to monitor HCC recurrence.
2. P16 methylation were closely related to P16 mRNA and P16 protein expression in HCC patients.
3. P16 methylation might be related to the invasion and metastasis of HCC.
4. P16 methylation in plasma might help to diagnose HCC patients with normal serum AFP(serum AFP<200ug/l) .
引文
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