摘要
背景与目的:有研究表明不同来源的EB病毒(Epstein-Barrvirus,EBV)潜伏膜蛋白1(latentmembraneprotein1,LMP1)对人高分化鼻咽癌(nasopharyngealcarcinoma,NPC)细胞株CNE1有不同程度的生长促进作用。本研究拟进一步探讨不同来源的LMP1基因转染对转化生长因子β1(transforminggrowthfactorβ1,TGFβ1)抗性的影响及其可能机制。方法:采用脂质体介导法分别将质粒J124(不含LMP1基因的空载体)、J124-B95-8(含B95-8淋巴细胞标准株来源的LMP1基因,简称B95-8-LMP1)和J124-CAO-5(含NPC组织来源的LMP1基因,简称CAO-LMP1)转染CNE1,分别命名三种质粒的转染细胞株为CNE1-V、CNE1-B和CNE1-C;分别用逆转录聚合酶链反应(reversetranscription-polymerasechainreaction,RT-PCR)和蛋白印迹法鉴定LMP1基因和蛋白表达;分别用MTT法和流式细胞术观察TGFβ1处理对细胞生长的抑制效应和细胞周期分布的改变;蛋白印迹法检测转化生长因子β受体Ⅰ(transforminggrowthfactorβreceptorⅠ,TGFβRⅠ)、转化生长因子β受体Ⅱ(TGFβRⅡ)以及cyclinD1蛋白的表达;半定量RT-PCR检测p15mRNA的表达。结果:成功建立稳定表达不同来源LMP1的CNE1细胞株CNE1-B和CNE1-C。5ng/mlTGFβ1对CNE1、CNE1-V、CNE1-B和CNE1-C的生长抑制率分别为31.8%、27.9%、
BACKGROUND &OBJECTIVE: We had proved that different latent memb ra ne protein 1 (LMP1) variants of Epstein-Barr virus (EBV) had different effects upon growth characteristics of a human well-differentiated nasopharyngeal carci noma (NPC) cell strain CNE1. This study was designed to investigate the possible effects of different LMP1 variants on resistance of CNE1 to TGFβ1 and the rela ted mechanism(s) so as to further elucidate the intrinsic mechanisms of their gr owth-promoting effects upon CNE1. METHODS: The plasmids including J124 (served as a control plasmid),124-B95-8 (carried LMP1 gene cloned from B95-8 lymphocy tes,B95-8-LMP1), and J124-CAO-5 (carried LMP1 gene cloned from NPC tissues,C AO-LMP1) were introduced into CNE1 by liposomal transfection. The transfected c ell strains were named CNE1-V,CNE1-B,and CNE1-C,respectively. Gene and protei n expression of LMP1 in CNE1 were identified by reverse transcription-polymeras e chain reaction (RT-PCR) and Western blot analysis,respectively.Then growth in hibition assay using MTT colorimetric method and flow cytometry were conducted t o investigate different effects of the two LMP1 variants on resistance of CNE1 t o TGFβ1. Meanwhile,TGFβRⅠ,TGFβRⅡ, and cyclin D1 were detected by Western bl ot analysis and p15 mRNA was examined by semi-quantitative RT-PCR. RESULTS:Two transfected cell strains (CNE1-B and CNE1-C) stably expressing different LMP1 variants were established successfully. When the treating concentration of TGF β1 was 5 ng/ml, the growth inhibitory rates of CNE1, CNE1-V,CNE1-B,and CNE1- C were 31.8%, 27.9%, 10.94%, and -4.26%, respectively, and the proliferatio n index (PI) were (24.55±2.55)%, (25.43±2.18)%, (46.78±2.56)%, and (54.70 ±3.84)%, respectively. CAO-LMP1 induced complete TGFβ1 resistance in CNE1, w hereas B95-8-LMP1 induced partial resistance. Neither of the two LMP1 variants had effects on TGFβRⅠand TGFβRⅡprotein expression in CEN1,whereas both of t hem induced cyclin D1 expression significantly. CAO-LMP1 induced higher level o f cyclin D1 than B95-8-LMP1 did (P< 0.05). B95-8-LMP1 had no significant eff ect on p15 mRNA expression (P >0.05), whereas CAO-LMP1 down-regulated p15 mR NA level obviously (P< 0.05). CONCLUSION: B95-8-LMP1 could induce partial resi stance of CNE1 to TGFβ1 and the main mechanism was correlated with up-regulati on of cyclin D1 protein, whereas CAO-LMP1 could induce complete resistance to T GFβ1 and the mechanisms were correlated with up-regulation of cyclin D1 protei n as well as down-regulation of p15 mRNA.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2003年第12期1254-1259,共6页
Chinese Journal of Cancer
基金
广东省卫生厅重点科研基金项目(No.卫9803)
关键词
LMP1
基因转染
CNE1细胞
TGFΒ1
抗性
鼻咽肿瘤
Nasopharyngeal neoplasms
Epstein-Barr virus (EBV)
Latent membrane protein 1 (LMP1)
Transforming growth factor β1