CDCA5在裸鼠肝細胞癌中的作用及臨床價值

李德新　李華　李飛　趙曉晨　李茂德　安祥　蘭戴天

摘要：目的 ?探討不同程度CDCA5表達在裸鼠肝細胞癌中的作用及臨床價值。方法 ?取HepG2正常細胞組、CDCA5低表達組、CDCA5高表達組、HepG2肝癌細胞株，將高表達組、低表達組CDCA5高效轉染人肝癌HepG2細胞，并設陰性對照組（NC組）。免疫印跡法檢測轉染24 h后各組CDCA5蛋白的表達水平，CCK-8法檢測轉染24、48、72、96、120 h 各組細胞的增殖能力，Annexin V-FITC/PI雙標記流式細胞術檢測低表達組轉染48 h的凋亡情況;將HepG2正常細胞組，CDCA5低表達組的細胞分別注射于裸鼠成瘤，觀察成瘤時間，第30天處死小鼠，觀察瘤體狀態。免疫組化檢測CDCA5在裸鼠腫瘤中的表達。結果 ?高表達和低表達組的CDCA5蛋白水平均高于NC組（P<0.05）;自轉染48 h起，高表達組和低表達組的相對增殖率均高于NC組（P<0.05）;高表達組和低表達組轉染72 h后的相對增殖率分別為（130.03±0.35）%和（70.10±1.04）%，均高于其他觀察時間點（P<0.05）;低表達組轉染48 h的早期和晚期凋亡率均高于NC組（P<0.05）;裸鼠皮下注射普通HepG2細胞組及CDCA5低表達HepG2細胞，瘤體重量分別為（0.93±0.06）g、（0.69±0.10）g，差異有統計學意義（P<0.05）;CDCA5在低表達組裸鼠腫瘤組織中表達降低。結論 ?CDCA5能促進HepG2癌細胞的增殖，可通過調控CDCA5表達水平，有效抑制肝癌細胞的增殖并促進其凋亡。

關鍵詞：CDCA5;肝癌;裸鼠;預后

中圖分類號：R735.7 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 文獻標識碼：A ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? DOI：10.3969/j.issn.1006-1959.2020.18.015

文章編號：1006-1959（2020）18-0048-03

The Role and Clinical Value of CDCA5 in Nude Mice Hepatocellular Carcinoma

LI De-xin，LI Hua，LI Fei，ZHAO Xiao-chen，LI Mao-de，AN Xiang，LAN Dai-tian

（Sichuan Academy of Medical Sciences/Hepatobiliary Surgery，Sichuan Provincial People's Hospital，

Chengdu 610010，Sichuan，China）

Abstract：Objective ?To explore the role and clinical value of different levels of CDCA5 expression in nude mice hepatocellular carcinoma.Methods ?HepG2 normal cell group， CDCA5 low expression group， CDCA5 high expression group and HepG2 hepatocarcinoma cell lines were selected. The high expression group and low expression group CDCA5 were efficiently transfected into human liver cancer HepG2 cells， and a negative control group （NC group） was established. Western blotting was used to detect the expression level of CDCA5 protein in each group after 24 h of transfection. CCK-8 method was used to detect the proliferation ability of cells in each group after transfection 24， 48， 72， 96， 120 h. Annexin V-FITC/PI double label flow Cytometry was used to detect the apoptosis of the low expression group for 48 h after transfection; the HepG2 normal cell group and the CDCA5 low expression group were injected into nude mice to form tumors， and the tumor formation time was observed. The mice were killed on the 30th day and the tumors were observed body state. Immunohistochemistry was used to detect the expression of CDCA5 in tumors of nude mice.Results ?The CDCA5 protein levels of the high and low expression groups were higher than those of the NC group （P<0.05）; since 48 h after transfection， the relative proliferation rates of the high and low expression groups were higher than those of the NC group （P<0.05）; The relative proliferation rates of high expression group and low expression group 72 h after transfection were （130.03±0.35）% and （70.10±1.04）%， respectively， which were higher than other observation time points （P<0.05）; low expression group was transfected the early and late apoptosis rates at 48 h were higher than those in the NC group （P<0.05）; the nude mice were subcutaneously injected with ordinary HepG2 cells and CDCA5 low-expressing HepG2 cells， and the tumor weights were （0.93±0.06） g and （0.69±0.10）g， the difference was statistically significant （P<0.05）; the expression of CDCA5 in the tumor tissues of nude mice in the low expression group decreased.Conclusion ?CDCA5 can promote the proliferation of HepG2 cancer cells， and could effectively inhibit the proliferation of liver cancer cells and promote their apoptosis by regulating the expression level of CDCA5.

Key words：CDCA5;Liver cancer;Nude mice;Prognosis

肝細胞癌（hepatocellular carcinoma，HCC）是臨床常見的惡性腫瘤，患者就診時往往病情嚴重、預后較差，病死率高，目前主要以手術治療、放療、化療、生物治療等綜合治療為主[1]。研究發現，腫瘤的發生與細胞周期具有重要的相關性，對腫瘤細胞周期異常蛋白的調控成為治療靶點。CDCA5在多種腫瘤組織中表達均增加[2-4]，這提示CDCA5在腫瘤的發生及發展過程中可能起到重要的作用。本研究通過CDCA5細胞實驗、裸鼠成瘤實驗，探討低表達CDCA5在肝癌細胞中的功能，試圖尋找生物治療新的方向提供參考。

1材料與方法

1.1主要試劑與儀器 ?收集HepG2正常細胞組，CDCA5低表達組，CDCA5高表達組，HepG2肝癌細胞株，DMEM 高糖培養基，10%胎牛血清和25%胰蛋白酶，小鼠抗CD-CA5抗體，小鼠抗GAPDH抗體和辣根過氧化物酶標記的山羊抗鼠IgG抗體，電泳……