探讨热休克蛋白（HSP）70、磷脂酰肌醇3-激酶（PI3K）和蛋白激酶B（Akt）在肝细胞肝癌（肝癌）组织中的表达及意义。

本组回顾性研究标本来源于2005年3月至2008年12月在中国医科大学附属盛京医院接受诊治，经病理学诊断为肝癌的98例患者的手术切除标本。所有患者术前均签署知情同意书，符合医学伦理学规定。男69例，女29例，年龄31~70岁，中位年龄57岁。应用免疫组织化学方法检测HSP70、PI3K、Akt蛋白的特异性表达，用蛋白质印迹法检测肝癌组织中HSP70、PI3K、Akt蛋白的含量。应用实时逆转录-聚合酶链反应（real-time RT-PCR）检测HSP70、PI3K、Akt信使核糖核酸（mRNA）。观察肝癌组织中HSP70、PI3K、Akt蛋白和mRNA表达情况，分析其与患者性别、年龄、病理组织学分化、淋巴结转移和肿瘤、淋巴、转移（TNM）分期等临床病理学参数的关系。采用χ²检验和Fisher确切概率法比较HSP70、PI3K、Akt表达与临床病理学参数关系，采用单因素方差分析和LSD-t检验比较不同组织学分化程度的肝癌组织中HSP70、PI3K、Akt蛋白和mRNA含量差异，采用Pearson法分析HSP70、PI3K、Akt蛋白表达与组织学分化程度之间的关系，采用Kaplan-Meier法和Log-rank检验进行生存分析。

98例患者中，HSP70蛋白阳性表达1级26例（27%），2级41例（42%），3级31例（32%）；PI3K蛋白低表达和高表达分别为40例（41%）、58例（59%），Akt蛋白低表达和高表达分别为42例(43%)、56例（57%）。HSP70、PI3K、Akt蛋白表达与肝癌组织学分化程度有关（χ²=49.99、24.675、32.521，均为P<0.05），而与性别、年龄、淋巴结转移和TNM分期无关（均为P>0.05）。高、中、低分化肝癌组织中HSP70蛋白含量分别为0.5284±0.0056、0.8420±0.0260、0.9810±0.0020；PI3K蛋白含量分别为0.6047±0.0558、0.7027±0.0064、0.9311±0.0019；Akt蛋白含量分别为0.7139±0.0028、0.7756±0.0039、0.9701±0.0010，3组比较差异有统计学意义（F=75.546、76.007、69.693，均为P<0.05），低分化肝癌组织中HSP70、PI3K、Akt蛋白的含量明显高于高分化肝癌组织（t=12.256、11.560、10.532，均为P<0.05）；HSP70、PI3K、Akt蛋白含量与肝癌组织学分化程度有关（r=0.387、0.715、0.713，均为P<0.01）；以高分化肝癌组织中的mRNA相对表达量为1，中、低分化组的肝癌组织中HSP70 mRNA的相对表达量分别为1.674±0.008、2.234±0.006，PI3K分别为2.676±0.006、3.449±0.045，Akt分别为2.754±0.004、3.366±0.006，3组比较差异有统计学意义（F=9.784、16.580、5.933，P<0.05），低分化肝癌组织中HSP70、PI3K、Akt mRNA的相对表达量明显高于高分化的肝癌组织（t=0.126、0.8202、6.497，均为P<0.05）；HSP70、PI3K和Akt mRNA的相对表达量与组织学分化程度有关（r=0.210、0.187、0.397，P=0.001、<0.01、<0.05)。HSP70蛋白的表达与PI3K和Akt蛋白（r=0.715、0.713，均为P=0.001）的表达呈正相关。HSP70蛋白表达为1级、2级和3级患者的术后平均无瘤生存时间分别为31、17和11个月，3组比较差异有统计学意义（均为P=0.001）。PI3K蛋白高表达和低表达患者术后平均无瘤生存时间分别为12、25个月，两组比较差异有统计学意义（P=0.003），Akt蛋白高表达和低表达的患者的术后平均无瘤生存时间分别为11、21个月，两组比较差异统计学意义（P=0.001）。

肝癌组织中存在不同程度的HSP70、PI3K和Akt蛋白和mRNA表达，其表达与组织学分化程度有关；HSP70与PI3K/AKT信号传导通路之间可能具有协同促进肝癌细胞增殖的作用。肝癌组织中HSP70、PI3K和Akt蛋白表达是影响患者无瘤生存期的重要因素，HSP70蛋白表达量越高，患者预后越差。

To investigate the expression and clinical significance of heat shock protein (HSP) 70, phosphatidylinositol 3-kinase (PI3k) and protein kinase(Akt) in hepatocellular carcinoma(HCC).

A total of 98 patients diagnosed hepatocellular carcinoma pathologically were involved in this retrospective study. The patients underwent surgical resection at Shengjing Affiliated Hospital of China Medical University from March 2005 to December 2008. Local ethical committee approval was received and that the informed consent of all participating subjects was obtained. There were 69 males and 29 females with the age range of 31-70 years old and a median age of 57. Immunohistochemistry and Western blot were used to examine the expression and the level of HSP70, PI3K and Akt proteins respectively. Real-time reverse transcription polymerase chain reaction was used to examine the expression of HSP70, PI3K and Akt mRNA. The expression of HSP70, PI3k and Akt protein and mRNA was observed and the relationship between the gender, age, histological differentiation, lymph node metastasis, tumor-node-metastasis (TNM) staging were analyzed and compared by χ² test and Fisher definite probability. The differences of HSP70, PI3K, Akt protein and mRNA content in HCC tissues of diverse differentiation were compared by one-way ANOVA analysis and LSD-t test and the correlation between them were analyzed using Pearson correlation analysis. Kaplan-Meier method and Log-rank test were used for survival analysis.

Twenty-six(27%) cases presented HSP70 positive expression of grade 1, 41(42%) grade 2, and 31(32%) grade 3. The low expression of PI3K and Akt were observed in 40 (41%) and 42(43%) cases, and high expression in 58(59%) and 56 cases (57%) respectively. The expression of HSP70, PI3K, Akt protein were associated with HCC histological differentiation (χ²=49.99, 24.675, 32.521, all in P<0.05) , while were not related with the gender, age, lymph node metastasis, TNM staging (all in P>0.05) . The HSP70 protein levels of well, moderate, poorly differentiated HCC were 0.5284±0.0056, 0.8420±0.0260, 0.9810±0.0020; the PI3K protein content were 0.6047±0.0558, 0.7027±0.0064, 0.9311±0.0019; and the Akt protein levels were 0.7139±0.0028, 0.7756±0.0039, 0.9701±0.0010. There were significant differences between the three groups (F=75.546, 76.007, 69.693; all in P<0.05); The levels of HSP70, PI3K and of Akt protein expression in poorly differentiated HCC tissues were significantly higher than those in well differentiated HCC tissues (t=12.256, 11.560, 10.532; all in P<0.05); The HSP70, PI3K and Akt protein contents were associated with differentiation of HCC(r= 0.387, 0.715, 0.713; P<0.01); When the mRNA relative expression amount of HSP70, PI3K and Akt protein in well differentiated HCC was set as 1, the mRNA expression levels of HSP70 in moderate and poorly differentiated HCC were 1.674±0.008, 2.234±0.006, the PI3K were 2.676±0.006, 3.449±0.045, and the Akt were 2.754±0.004, 3.366±0.006 respectively. There were significant differences among the three groups(F=9.784, 16.580, 5.933; P<0.05). The HSP70, PI3K and of Akt mRNA expression amount in poorly differentiated HCC tissues were significantly higher than that in well differentiated ones (t=0.126, 0.8202, 6.497; P<0.05); The expression of HSP70, PI3K and Akt mRNA were associated with the differentiation grades of HCC (r=0.210, 0.187, 0.397; P=0.001, <0.01, <0.05); Expression of HSP70 was positively associated with the expression of PI3K and Akt protein (r=0.715, 0.713; all in P=0.001); The average tumour-free survival time of patients with HSP70 positive expression at grade 1, 2 and 3 were 31, 17 and 11 months respectively, which showed significant difference among 3 groups(P=0.001); The average tumour-free survival time of patients with high and low PI3K expression were 12 and 25 months, which showed significant difference between 2 groups(P=0.001); The average tumour-free survival time of patients with high and low Akt expression were 11 and 21 months, which also showed significant difference between the 2 groups(P=0.001).

The different expression of HSP70, PI3K, Akt and mRNA in HCC tissues are associated with histological differentiation. HSP70 and PI3K/AKT signaling pathway may have a synergistic role in promoting HCC cell proliferation. The expression of HSP70, PI3K and Akt protein in HCC tissues are important prognostic factors of patients’ tumor-free survival. The higher the HSP70 protein expression is, the worse prognosis the patients have.