中国医学创新
中國醫學創新
중국의학창신
MEDICAL INNOVATION OF CHINA
2014年
9期
18-20
,共3页
Beclin1%真核表达载体%细胞%增殖
Beclin1%真覈錶達載體%細胞%增殖
Beclin1%진핵표체재체%세포%증식
Beclin1%Eukaryotic expression vector%Cells%Multiplication
目的：构建自噬基因Beclin1的真核表达载体pcDNA3.1-Beclin1，检测人子宫内膜癌HEC细胞转染pcDNA3.1-Beclin1后，外源性基因Beclin1在蛋白水平的表达，并观察其对HEC细胞增殖的影响。方法：通过RT-PCR和T/A克隆等方法，构建自噬基因Beclin1的真核表达载体pcDNA3.1-Beclin1，并通过脂质体介导的方法将外源性Beclin1基因导入人类子宫内膜癌细胞株HEC中，通过实时PCR检测转染后Beclin1在HEC中表达的情况，并设立实验组（转染pcDNA3.1-Beclin1真核载体）、空质粒对照组（转染pcDNA3.1空载体）、空白脂质体对照组，用Western blot法检测子宫内膜癌HEC细胞中Beclin1的表达情况，MTT法检测Beclin1对HEC细胞体外生长的影响等，研究Beclin1基因与子宫内膜癌的关系。结果：经限制性内切酶鉴定及测序，证明Beclin1基因真核质粒的序列完全正确，将其转染HEC细胞，转染后的HEC细胞在体外能继续增殖，但增殖能力明显下降。结论：Beclin1基因pcDNA3.1-Beclin1真核表达载体构建成功，并能在HEC细胞中表达，转染HEC细胞体外增殖能力明显降低，Beclin1基因用于子宫内膜癌基因治疗具有重要靶向价值。
目的：構建自噬基因Beclin1的真覈錶達載體pcDNA3.1-Beclin1，檢測人子宮內膜癌HEC細胞轉染pcDNA3.1-Beclin1後，外源性基因Beclin1在蛋白水平的錶達，併觀察其對HEC細胞增殖的影響。方法：通過RT-PCR和T/A剋隆等方法，構建自噬基因Beclin1的真覈錶達載體pcDNA3.1-Beclin1，併通過脂質體介導的方法將外源性Beclin1基因導入人類子宮內膜癌細胞株HEC中，通過實時PCR檢測轉染後Beclin1在HEC中錶達的情況，併設立實驗組（轉染pcDNA3.1-Beclin1真覈載體）、空質粒對照組（轉染pcDNA3.1空載體）、空白脂質體對照組，用Western blot法檢測子宮內膜癌HEC細胞中Beclin1的錶達情況，MTT法檢測Beclin1對HEC細胞體外生長的影響等，研究Beclin1基因與子宮內膜癌的關繫。結果：經限製性內切酶鑒定及測序，證明Beclin1基因真覈質粒的序列完全正確，將其轉染HEC細胞，轉染後的HEC細胞在體外能繼續增殖，但增殖能力明顯下降。結論：Beclin1基因pcDNA3.1-Beclin1真覈錶達載體構建成功，併能在HEC細胞中錶達，轉染HEC細胞體外增殖能力明顯降低，Beclin1基因用于子宮內膜癌基因治療具有重要靶嚮價值。
목적：구건자서기인Beclin1적진핵표체재체pcDNA3.1-Beclin1，검측인자궁내막암HEC세포전염pcDNA3.1-Beclin1후，외원성기인Beclin1재단백수평적표체，병관찰기대HEC세포증식적영향。방법：통과RT-PCR화T/A극륭등방법，구건자서기인Beclin1적진핵표체재체pcDNA3.1-Beclin1，병통과지질체개도적방법장외원성Beclin1기인도입인류자궁내막암세포주HEC중，통과실시PCR검측전염후Beclin1재HEC중표체적정황，병설립실험조（전염pcDNA3.1-Beclin1진핵재체）、공질립대조조（전염pcDNA3.1공재체）、공백지질체대조조，용Western blot법검측자궁내막암HEC세포중Beclin1적표체정황，MTT법검측Beclin1대HEC세포체외생장적영향등，연구Beclin1기인여자궁내막암적관계。결과：경한제성내절매감정급측서，증명Beclin1기인진핵질립적서렬완전정학，장기전염HEC세포，전염후적HEC세포재체외능계속증식，단증식능력명현하강。결론：Beclin1기인pcDNA3.1-Beclin1진핵표체재체구건성공，병능재HEC세포중표체，전염HEC세포체외증식능력명현강저，Beclin1기인용우자궁내막암기인치료구유중요파향개치。
Objective:To build the eukaryotic expression vector pcDNA3.1-Beclin1 of the autophagy gene Beclind1 and to examin the pcDNA3.1-Beclin1 transfected by the human endometrial carcinoma HEC cells,the expression of exogenous gene Beclin1 in protein,together with its effect to the HEC cell multiplication will be examined.Method:By way of RT-PCR&T/A cloning,built the eukaryotic expression vector pcDNA3.1-Beclin1,transferred the exogenous genes Beclin1 into the human endometrial carcinoma cell line HEC based on the lipidosome mediation,examined the expression of the transfected Beclin1 in HEC based on RT-PCR,set up the experimental group(transfected eukaryotic vector pcDNA3.1-Beclin1),empty plasmid control group(transfected empty blank load group pcDNA3.1)and lipidosome control group, Western Blot and MTT were employed to research the interrelationship between Beclin1 and endometrial carcinoma,which Western Blot was employed to examine the expression of Beclin1 in endometrial carcinoma HEC cell and MTT was employed to examine the effect of Beclin1 to HEC cell external growth.Result:Being identified and sequenced by restriction enzyme, DNA sequence in the eukaryotic plasmid of Beclin1 was proved to be exactly correct.The transfected HEC cells could continue to multiply in vitro,but the multiplication capabilities remarkably declined.Conclusion:Beclin1 gene is successfully built in eukaryotic expression vector pcDNA3.1-Beclin1 and can be expressed in HEC cells.The multiplication capabilities of transfected HEC cells remarkably decline in vitro.It is quite significant to apply Beclin1 genes into the endometrial carcinoma gene treatment.
