构建可模拟亚致死热应激状态的体外细胞及活体小鼠原位肝癌不完全热消融模型。

将悬浮的单层H22细胞分为4组（37℃、42℃、47℃、50℃）分别置于6孔培养板中培养，热处理前8 h更换新鲜完全培养基，随后恒温水浴15 min进行热处理以构建亚致死热应激体外模型并置于37℃培养箱中继续培养。48 h后进行Western Blot实验检测EMT表型，并通过Cell Counting Kit-8（CCK-8）试剂盒检测细胞活力。沿小鼠腹中线开腹后暴露肝脏并于肝包膜下注射H22细胞（5×10⁶个），待瘤体长径约等于8 mm后将小鼠随机分为完全消融组（complete ablation group，cMWA）、不完全消融组（incomplete ablation group，iMWA）及假手术组，并在开腹直视下沿瘤体长轴行微波消融以建立原位肝癌不完全热消融模型。消融后第14天复查MRI并采集肝脏标本进行组织病理学检查。

接种H22细胞约14天后小鼠肿瘤达到预定肿瘤长径范围，肿瘤平均长径为（9.32±0.83）mm。消融术后14天，不完全消融组肿瘤平均长径（12.93±1.51）mm，且较消融前明显增大。免疫荧光染色结果显示iMWA组残瘤内见大量中性粒细胞浸润。Western blot结果显示亚致死热应激状态下肝癌细胞EMT表型的恶性转变，其中波形蛋白（Vimentin）、α-平滑肌肌动蛋白（α-SMA）表达上调，E钙黏附蛋白（E-cadherin）表达下调。

亚致死热应激状态的体外细胞及活体小鼠原位肝癌不完全热消融模型成功构建，该状态下残瘤内大量中性粒细胞浸润，同时肝癌细胞可能转变为更具侵袭性的间充质表型并导致肿瘤进展。

To construct hepatocellular carcinoma (HCC) cells lines that can mimic sub-lethal heat shock (HS) in vitro and orthotopic HCC mouse model that received incomplete thermal ablation.

The suspension H22 cells were grown in 6-well plates, and the medium was replaced with fresh complete medium 8 hours before heat treatment. Then, the cells were divided into 4 groups for constant temperature water bath at 37°C, 42°C, 47°C, 50°C for 15 min, respectively. After the heat treatment was completed, the cells were maintained in a 37℃ incubator until detection. The phenotype of epithelial-mesenchymal transition (EMT) was detected by Western blot 48 hours later, and the cell viability was detected by Cell Counting Kit-8 (CCK-8) kit. The liver of mice was exposed by laparotomy along the abdomen midline and H22 cells (5×10⁶) were injected through the hepatic capsule to conduct orthotopic HCC model. When the longest diameter of the tumor reached approximately 8 mm, the mice were randomly divided into complete ablation group (cMWA), incomplete ablation group (iMWA) and Control group treated with sham operation. Microwave ablation along the long axis of tumor was performed to establish the incomplete thermal ablation model of orthotopic HCC. MRI was performed for tumor measurement and liver specimens were collected for histopathological assessment at day 14 after microwave ablation.

14 daysafter inoculation of H22 cells, the tumors reached average tumor diameter of 9.32 ± 0.83 mm which was predetermined to be suitable for microwave ablation. 14 days after microwave ablation, the average tumor diameter reached 12.93 ± 1.51mm in iMWA group, which were significantly bigger than preoperative period. In addition, immunofluorescence staining showed a large number of infiltrated neutrophils in the residual tumor of iMWA group. Western blot was used to observe the change in EMT-phenotype, results showed that the expressions of Vimentin and α-SMA were up-regulated and the expressions of E-cadherin were down-regulated.

HCC cells lines that can mimic Sub-lethal heat shock (HS) in vitro and orthotopic HCC mouse model that received incomplete thermal ablation were successfully constructed. There was substantial staining for neutrophils infiltration after incomplete ablation. Incomplete ablation may allow HCC cells to become more prone to migrate and to become more invasive.