国际妇产科学杂志
國際婦產科學雜誌
국제부산과학잡지
JOURNAL OF INTERNATIONAL OBSTETRICS AND GYNECOLOGY
2014年
6期
658-662
,共5页
王宇娟%霍俊玲%李婧%王赞宏
王宇娟%霍俊玲%李婧%王讚宏
왕우연%곽준령%리청%왕찬굉
宫颈肿瘤%人乳头瘤病毒16%内质网%应激%自噬
宮頸腫瘤%人乳頭瘤病毒16%內質網%應激%自噬
궁경종류%인유두류병독16%내질망%응격%자서
Uterine cervical neoplasms%Human papillomavirus 16%Endoplasmic reticulum%Stress%Autophagy
目的：研究宫颈癌C33A细胞中HPV16 E6蛋白过表达对内质网应激－自噬反应的影响。方法：构建HPV16 E6的真核表达载体pcDNA3．1（－）－HPV16 E6，转染C33A细胞[pcDNA3．1（－）－HPV16 E6组]，同时设空载体转染组[pcDNA3．1（－）组]和空白对照组（C33A组），荧光定量聚合酶链反应（PCR）和蛋白质印迹法（Western blot）检测其C33A细胞中HPV16 E6 mRNA及蛋白表达的变化，及其对自噬蛋白Beclin 1、LC3Ⅱ、内质网应激标记蛋白葡萄糖调节蛋白78（GRP78）表达的影响，四甲基偶氮唑盐（MTT）法检测细胞生长的变化；流式细胞仪检测细胞自噬的变化。结果：成功构建了HPV16 E6的真核表达载体pcDNA3．1（－）－HPV16 E6，其可以使C33A细胞中HPV16 E6 mRNA及蛋白表达增加；HPV16 E6在C33A中过表达可促进肿瘤细胞的生长，pcDNA3．1（－）－HPV16 E6转染后C33A的自噬率较pcDNA3．1（－）组和C33A组增高，差异有统计学意义（P＜0．05）；HPV16 E6过表达使Beclin 1、LC3Ⅱ和GRP78蛋白表达增加，阻断内质网应激通路后GRP78表达减少，细胞自噬率降低。结论：内质网应激－自噬反应在HPV16感染导致宫颈癌的过程中具有重要作用，这为宫颈癌的基因治疗提供了新靶点。
目的：研究宮頸癌C33A細胞中HPV16 E6蛋白過錶達對內質網應激－自噬反應的影響。方法：構建HPV16 E6的真覈錶達載體pcDNA3．1（－）－HPV16 E6，轉染C33A細胞[pcDNA3．1（－）－HPV16 E6組]，同時設空載體轉染組[pcDNA3．1（－）組]和空白對照組（C33A組），熒光定量聚閤酶鏈反應（PCR）和蛋白質印跡法（Western blot）檢測其C33A細胞中HPV16 E6 mRNA及蛋白錶達的變化，及其對自噬蛋白Beclin 1、LC3Ⅱ、內質網應激標記蛋白葡萄糖調節蛋白78（GRP78）錶達的影響，四甲基偶氮唑鹽（MTT）法檢測細胞生長的變化；流式細胞儀檢測細胞自噬的變化。結果：成功構建瞭HPV16 E6的真覈錶達載體pcDNA3．1（－）－HPV16 E6，其可以使C33A細胞中HPV16 E6 mRNA及蛋白錶達增加；HPV16 E6在C33A中過錶達可促進腫瘤細胞的生長，pcDNA3．1（－）－HPV16 E6轉染後C33A的自噬率較pcDNA3．1（－）組和C33A組增高，差異有統計學意義（P＜0．05）；HPV16 E6過錶達使Beclin 1、LC3Ⅱ和GRP78蛋白錶達增加，阻斷內質網應激通路後GRP78錶達減少，細胞自噬率降低。結論：內質網應激－自噬反應在HPV16感染導緻宮頸癌的過程中具有重要作用，這為宮頸癌的基因治療提供瞭新靶點。
목적：연구궁경암C33A세포중HPV16 E6단백과표체대내질망응격－자서반응적영향。방법：구건HPV16 E6적진핵표체재체pcDNA3．1（－）－HPV16 E6，전염C33A세포[pcDNA3．1（－）－HPV16 E6조]，동시설공재체전염조[pcDNA3．1（－）조]화공백대조조（C33A조），형광정량취합매련반응（PCR）화단백질인적법（Western blot）검측기C33A세포중HPV16 E6 mRNA급단백표체적변화，급기대자서단백Beclin 1、LC3Ⅱ、내질망응격표기단백포도당조절단백78（GRP78）표체적영향，사갑기우담서염（MTT）법검측세포생장적변화；류식세포의검측세포자서적변화。결과：성공구건료HPV16 E6적진핵표체재체pcDNA3．1（－）－HPV16 E6，기가이사C33A세포중HPV16 E6 mRNA급단백표체증가；HPV16 E6재C33A중과표체가촉진종류세포적생장，pcDNA3．1（－）－HPV16 E6전염후C33A적자서솔교pcDNA3．1（－）조화C33A조증고，차이유통계학의의（P＜0．05）；HPV16 E6과표체사Beclin 1、LC3Ⅱ화GRP78단백표체증가，조단내질망응격통로후GRP78표체감소，세포자서솔강저。결론：내질망응격－자서반응재HPV16감염도치궁경암적과정중구유중요작용，저위궁경암적기인치료제공료신파점。
Objective:To investigate the effects of HPV16 E6 on endoplasmic reticulum (ER) stress-autophagic response in the cervical cancer C33A cells. Methods:The eukaryotic expression vector of HPV16 E6 was constructed and transfected via lipofectamine into C33A cells. Experimental cells was classified into 3 groups: pcDNA3.1 (-)-HPV16 E6 group,pcDNA3.1 (-) group and C33A group. Fluorescent quantitation-ploymerase chain reaction was used for detecting expression of mRNA of HPV16 E6, and Western blot was explored to measure expression of protein of HPV16 E6,Beclin 1,LC3Ⅱ and GRP78 in transfected cells. Flow cytometry(FCM) was employed to observe the effect of transfection on the autophagic of C33A, and proliferation was analyzed by MTT assay. Results:Eukaryotic expression vector pcDNA3.1 (-)-HPV16 E6 was constructed successfully. The eukaryotic expression vector pcDNA3.1 (-)-HPV16 E6 significantly improved the expression of mRNA and protein of HPV16 E6 in C33A cells. The cell proliferations of HeLa cells increased , and FCM investigation showed the autophagic rate of pcDNA3.1 (-)-HPV16 E6 group was higher than that in pcDNA3.1 (-) group and C33A group (P<0.05). The expression of Beclin 1, LC3Ⅱ and GRP78 were significantly improved after transfection with vector pcDNA3.1(+)-HPV16 E6(P<0.05). Furthermore, GRP78 protein level was reduced and the autophagic rate was decreased by treatment with ER stress inhibitor. Conclusions:ER stress-autophagic response plays an important role in the process of HPV16 E6 inducing cervical cancer. So it might be one of the new strategies for gene therapy of cervical carcinoma.
