CAJ | 학술논문

目的采用基因工程技术成功构建出肿瘤抑素相关肽T42肽,然后用基因工程重组菌pgex-4t-t使其高效表达,并提取纯化T42肽,观察其抗肝癌细胞活性.方法用人工合成T42基因,将其链接到pgex-4t-1表达载体中,构建表达载体pgex-4t-t42,将该质粒转化到大肠杆菌BL21(DE3)菌株中培养,采用几丁质柱亲和层析纯化T42肽.在体外对肝癌细胞进行噻唑蓝(MTT)实验,实验分4组:T42肽组、磷酸盐缓冲液(PBS)阴性对照组、氟尿嘧啶阳性对照组和空白对照组.T42肽组与氟尿嘧啶组分别加入不同浓度的肿瘤抑素T42肽和氟尿嘧啶,浓度梯度为10、20、30、40、50 mmol/L,PBS组加入等体积的PBS,比较不同浓度T42肽作用下细胞存活率的变化,观察T42肽对人肝癌HepG2细胞、LO2细胞活性的影响.结果成功提取纯度在90％以上的T42肽,其相对分子质量与理论值基本一致.MTT实验结果显示:PBS对照组细胞的存活率为(96.86±1.12)％,T42肽组及氟尿嘧啶组的细胞存活率明显高于与PBS组,差异均有统计学意义(P＜0.01),而T42肽组与氟尿嘧啶组间的差异无统计学意义(P＞0.05).随着T42肽药物浓度的升高HepG2细胞的存活率逐渐下降.吖啶橙/溴化乙锭(AO/EB)荧光染色结果显示,浓度为30 mmol/L的T42肽作用48 h后,HepG2细胞出现核皱缩、胞膜受损等细胞凋亡的特征性变化.结论 T42肽可明显抑制人肝癌细胞HepG2的生长增殖,并促进其凋亡,显示出较强的抗肿瘤细胞活性.
목적 채용기인공정기술성공구건출종류억소상관태T42태,연후용기인공정중조균pgex-4t-t사기고효표체,병제취순화T42태,관찰기항간암세포활성.방법 용인공합성T42기인,장기련접도pgex-4t-1표체재체중,구건표체재체pgex-4t-t42,장해질립전화도대장간균BL21(DE3)균주중배양,채용궤정질주친화층석순화T42태.재체외대간암세포진행새서람(MTT)실험,실험분4조:T42태조、린산염완충액(PBS)음성대조조、불뇨밀정양성대조조화공백대조조.T42태조여불뇨밀정조분별가입불동농도적종류억소T42태화불뇨밀정,농도제도위10、20、30、40、50 mmol/L,PBS조가입등체적적PBS,비교불동농도T42태작용하세포존활솔적변화,관찰T42태대인간암HepG2세포、LO2세포활성적영향.결과 성공제취순도재90％이상적T42태,기상대분자질량여이론치기본일치.MTT실험결과현시:PBS대조조세포적존활솔위(96.86±1.12)％,T42태조급불뇨밀정조적세포존활솔명현고우여PBS조,차이균유통계학의의(P＜0.01),이T42태조여불뇨밀정조간적차이무통계학의의(P＞0.05).수착T42태약물농도적승고HepG2세포적존활솔축점하강.아정등/추화을정(AO/EB)형광염색결과현시,농도위30 mmol/L적T42태작용48 h후,HepG2세포출현핵추축、포막수손등세포조망적특정성변화.결론 T42태가명현억제인간암세포HepG2적생장증식,병촉진기조망,현시출교강적항종류세포활성.
Objective To construct anti-tumor peptide of tumor chalone T42 peptide,and make it high expression from gene engineering bacteria pgex-4t-t,so as to extract and purify its anti-hepatoma cell activity for a preliminary study.Methods A synthetically designed gene T42 was inserted into pgex-4t-1 expression vector to construct pgex-4t-t42 expression vectors.The pgex-4t-t42 in E.coli was cultivated and fusion protein was produced.T42 peptide was extracted by the method of affinity chromatograph.The methyl thiazol tetrazolium (MTY) assay was used to the effect of peptide T42 on human hepatoma HepG2 cells,LO2 cells activity in vitro.Following group were set up:T42 peptide group,phosphate buffer (PBS) negative control group,fluorouracil (Fu) positive control group and blank control group.T42 peptide group and Fu positive control group were given different concentrations of T42 peptide and Fu respectively:10,20,30,40,and 50 mmol/L.PBS group was given an equal volume of PBS.The changes of cell viability were compared.Results T42 peptide (more than 90％ purity) was constructed successfully,which was consistent with the theoretical values in relative molecular mass.The result of MTT showed that the cell survival rate was (96.86 ± 1.12)％ in PBS control group.The cell survival rate in T42 group and Fu group was significantly lower than that in PBS control group (P ＜ 0.05),but these was no significant difference between T42 group and Fu group.The survival rate of HepG2 cells was decreased with the increase of T42 peptide concentrations.The result of acridine orange/ethidium bromide (AO/EB) indicated that the HepG2 cells displayed typical characteristics of apoptosis after treated with T42 peptide for 48 h.Conclusion T42 peptide showing strong anti-tumor activity,can evidently inhibit the proliferation of HepG2 cells,and induce apoptosis of HepG2 cells.