Bcl-2核酶诱导SMMC7721细胞凋亡与p16,p21蛋白表达的关系研究 1

来源：医学杂志　2007-01-12 15:33:41　

　　摘要：目的　观察bcl-2核酶对人肝癌细胞株SMMC-7721细胞的作用,并检测p16和p21的表达情况。探讨bcl－2核酶在肝癌治疗中的意义。方法经脂质体介导的方法，将PMTr－neo(正向bcl－2核酶真核表达载体)导入SMMC7721细胞中。细胞克隆转移扩大培养后,采用TUNEL法、免疫组化技术结合图像分析，在检测SMMC7721/PMTr－neo细胞凋亡的同时，检测p16, p21的表达。结果与对照组相比较 ,在 bcl－2核酶诱发SMMC7721细胞发生凋亡的同时,p16和p21基因的表达水平显著增高。结论　bcl-2核酶通过封闭bcl-2的表达可促进SMMC7721细胞凋亡,同时伴发p21和p16表达水平的增高。

　　中图号：R734.7　文献标识码：A　文章编号：1007－8738(2000)03－0219－03

Relationship between apoptosis induced with bcl-2 ribozyme and expression of p16, p21 in SMMC7721 cells

ZHANG Chuan-shan　WANG Wen-liang　　HU Pei-hen　CHAI Yu-bo　ZHAO Yi-ling

　　（Department of Pathology，the Fourth Millitary Medical University, Xi′ an 710032,Shaanxi Province,China）

　　PENG Wei-dan　CHAI Yu-bo

　　（Department of Biochemistry， the Fourth Millitary Medical University, Xi′ an 710032,Shaanxi Province,China）

　　Abstract: Aim To observe the effect of bcl-2 ribozyme on SMMC7721 cells and the expression of p16, p21,so to investigate the role of bcl-2 ribozyme in hepatocarcinoma treatment. Methods PMTr neo (sense bcl-2 ribozyme eucaryotic expression vector) was introduced into SMMC7221 cells through lipofectin mediation. After cloning, TUNEL and IHC in combination with imaging analysis were used to simultaneously determine apoptosis and p16, p21 expressions of the SMMC7721/PMTr neo cells. Results Compared with control, in the course of apoptosis induced by bcl-2 ribozyme p16 and p21 expressions rose significantly in SMMC7721 cells. Conclusion bcl-2 ribozyme can promote SMMC7721 cell apoptosis by blocking bcl-2 expression, which is accompanied by an increased p16 and p21 expressions. This result could be of value for studying hepatocarcinoma genesis and probing therapeutic procedure. neo (sense bcl-2 ribozyme eucaryotic expression vector) was introduced into SMMC7221 cells through lipofectin mediation. After cloning, TUNEL and IHC in combination with imaging analysis were used to simultaneously determine apoptosis and p16, p21 expressions of the SMMC7721/PMTr neo cells. Results Compared with control, in the course of apoptosis induced by bcl-2 ribozyme p16 and p21 expressions rose significantly in SMMC7721 cells. Conclusion bcl-2 ribozyme can promote SMMC7721 cell apoptosis by blocking bcl-2 expression, which is accompanied by an increased p16 and p21 expressions. This result could be of value for studying hepatocarcinoma genesis and probing therapeutic procedure.

　　Keywords: hepatic carcinoma; bcl-2; ribozyme; p16; p21▲

　　抗凋亡蛋白bcl-2在肿瘤发生过程中具有着重要的意义。自从Vaux首次提出bcl-2高表达可诱发肿瘤后，人们深入研究发现，bcl-2可延长细胞的生存期,抑制细胞凋亡〔1－3〕。人们已对bcl-2与肝癌的关系进行了探讨,认为它与肝癌的发生存在密切的关系。我们应用脂质体介导的基因转移方法，将bcl-2核酶导入人肝癌细胞株SMMC7721细胞中,并结合TUNEL原位杂交法和免疫组化等技术，观察了bcl-2核酶对SMMC7721细胞的影响,同时检测了bcl-2,p16,p21的表达情况。目的在于研究bcl-2核酶对肝癌细胞的作用机制,并探讨bcl-2,p16和p21在肝癌发生中的意义,为肝癌的治疗提供理论依据。

　　1　材料和方法

　　1.1材料大肠杆菌JM109由本校生化教研室惠赠；质粒PMTrneo由彭玮丹博士惠赠〔11〕。核酸内切酶购自Promega公司。脂质体转染试剂盒购自Gibico公司；凋亡检测试剂盒购自BoehringerMannheim公司。免疫组化试剂盒EnvisionTM＋System,HRP,mouse(R4001)购自Dako公司。SMMC7721细胞由本室保存培养。

　　1.2方法

　　1.2.1质粒PMTrneo的鉴定及转染细胞以质粒PMTrneo转化大肠杆菌JM109，扩增、提取质粒，经核酸内切酶BamHI和EcoRI双酶切后,电泳鉴定bcl-2核酶片段的大小。把处于对数生长期的SMMC7721细胞分为两组:对照组(SMMC7721细胞)和实验组(转染PMTrneo的7721细胞)。转染方法按脂质体转染试剂盒中的说明书操作步骤进行。培养48h后，传代、加入含G418550mg/L的培养液，筛选抗性细胞。继续培养2wk后，可见细胞克隆形成。

　　1.2.2SMMC7721/PMTrneo细胞的鉴定细胞克隆扩大培养后,制作细胞爬片,加入100μmol/L硫酸锌，诱导bcl-2核酶表达。爬片经丙酮固定后，以免疫组化法检测bcl-2的表达情况。所用抗bcl-2、

　　单抗(mAb)购自中山生物公司。免疫组化的步骤如下：(1)以30mL/LH2O2－甲醇封闭30min；(2)再以30g/LBSA封闭30min；(3)加一抗(抗p16，抗p21和抗bcl-2mAb)4℃过夜；(4)加HRP抗鼠IgGABC复合物(Dako公司产品);(5)显色、苏木精衬染。以上各步骤均用0.01mol/LPBS缓冲液洗涤，光镜下观察结果。

　　1.2.3bcl-2核酶的诱导表达SMMC7721/PMTrneo细胞克隆转移扩大培养后，加入硫酸锌100μmol/L诱导bcl-2核酶表达。24h后换液，继续培养3d后，分别检测细胞凋亡及p16和p21的表达。

　　1.2.4bcl-2核酶的促凋亡作用(1)原位爬片的TUNEL原位杂交检测：检测凋亡方法的步骤参照原位细胞凋亡检测试剂盒说明书进行，简述如下：①细胞爬片用多聚甲醛室温固定30min；②加1mL/LTritonX100和1g/L枸椽酸钠4℃2min；③加TUNEL反应混合液50μL，37℃60min；④加碱性磷酸酶转换液50μL，37℃30min；⑤加50μL底物液，室温10min。以上各操作步骤均用0.01mL/LPBS洗涤，光镜下