CAJ | 학술논문

目的以精氨酸-甘氨酸-天冬氨酸(RGD)多肽重复序列作为截短组织因子(tTF)的载体,表达(RGD)_3-tTF融合蛋白,研究(RGD)_3-tTF融合蛋白对大肠癌的治疗作用.方法用3个串联的RGD多肽作为tTF的载体,构建(RGD)_3-tTF融合基因,在大肠杆菌BL21(DE_3)上表达并用镍柱纯化(RGD)_3-tTF融合蛋白.在体外通过凝血实验检测(RGD)_3-tTF融合蛋白的凝血活性.建立大肠癌小鼠动物模型,异硫氰酸罗丹明B(RBITC)标记(RGD)_3-tTF、tTF融合蛋白,运用活体成像技术和共聚焦显微镜观察分析融合蛋白在大肠癌小鼠模型体内的定位.观察检测(RGD)_3-tTF融合蛋白抑制肿瘤生长的能力.结果在Ca~(2+)存在时,随着(RGD)_3-tTF融合蛋白浓度的增加,凝血时间相应缩短,浓度为6μmoL/L时凝血时间是(8.6±0.2)min,而浓度为0μmol/L时凝血时间＞30 min(P＜0.05).(RGD)_3-tTF融合蛋白的凝血活性与tTF相仿(F=0.09,P＞0.05).活体成像技术和共聚焦显微镜观察结果显示,注射标记了RBITC荧光的(RGD)_3-tTF融合蛋白富集在小鼠的肿瘤血管腔.抑瘤实验中,(RGD)_3-tTF融合蛋白能诱导肿瘤血管形成血栓,给药后第1、3、5天,(RGD)_3-tTF组的肿瘤体积分别为(120.8±4.8)mm~3、(93.8±3.4)mm~3、(132.2±7.7)mm~3,均显著小于tTF组[(181.4±13.8)mm~3、(333.0±32.0)mm~3、(514.0±11.5)mm~3]和磷酸缓冲液(PBS)组[(182.6±11.5)mm~3、(332.8±21.0)mm~3、(524.2±16.7)mm~3](P＜0.05),而tTF组和PBS组之间的肿瘤体积差异无统计学意义(P＞0.05).结论成功制备的(RGD)_3-tTF融合蛋白保留了组织因子的凝血活性并靶向定位在肿瘤血管,能诱导肿瘤血管形成血栓从而抑制肿瘤生长,有望成为治疗大肠癌的一种新方法.
목적 이정안산-감안산-천동안산(RGD)다태중복서렬작위절단조직인자(tTF)적재체,표체(RGD)_3-tTF융합단백,연구(RGD)_3-tTF융합단백대대장암적치료작용.방법 용3개천련적RGD다태작위tTF적재체,구건(RGD)_3-tTF융합기인,재대장간균BL21(DE_3)상표체병용얼주순화(RGD)_3-tTF융합단백.재체외통과응혈실험검측(RGD)_3-tTF융합단백적응혈활성.건립대장암소서동물모형,이류청산라단명B(RBITC)표기(RGD)_3-tTF、tTF융합단백,운용활체성상기술화공취초현미경관찰분석융합단백재대장암소서모형체내적정위.관찰검측(RGD)_3-tTF융합단백억제종류생장적능력.결과 재Ca~(2+)존재시,수착(RGD)_3-tTF융합단백농도적증가,응혈시간상응축단,농도위6μmoL/L시응혈시간시(8.6±0.2)min,이농도위0μmol/L시응혈시간＞30 min(P＜0.05).(RGD)_3-tTF융합단백적응혈활성여tTF상방(F=0.09,P＞0.05).활체성상기술화공취초현미경관찰결과현시,주사표기료RBITC형광적(RGD)_3-tTF융합단백부집재소서적종류혈관강.억류실험중,(RGD)_3-tTF융합단백능유도종류혈관형성혈전,급약후제1、3、5천,(RGD)_3-tTF조적종류체적분별위(120.8±4.8)mm~3、(93.8±3.4)mm~3、(132.2±7.7)mm~3,균현저소우tTF조[(181.4±13.8)mm~3、(333.0±32.0)mm~3、(514.0±11.5)mm~3]화린산완충액(PBS)조[(182.6±11.5)mm~3、(332.8±21.0)mm~3、(524.2±16.7)mm~3](P＜0.05),이tTF조화PBS조지간적종류체적차이무통계학의의(P＞0.05).결론 성공제비적(RGD)_3-tTF융합단백보류료조직인자적응혈활성병파향정위재종류혈관,능유도종류혈관형성혈전종이억제종류생장,유망성위치료대장암적일충신방법.
Objective To explore the therapy effects of(arginine-glycine-aspartic,RGD)_3truncated tissue factor(tTF)fusion protein on colorectal carcinoma in mice.Methods The(RGD)_3-tTF fusion gene,constructed with tTF and three series-wound peptides RGD,was expressed in Escherichia coli BL21(DE_3).The fusion protein was purified through Nickel affinity chromatography column.The coagulation activity of the(RGD)_3-tTF fusion protein was detected by clotting assay in vitro.Mice colorectal cancer cells line CT26 were inoculated subcutaneously into mice to establish colorectal cancer model Four mice were randomly divided into two groups to be injected with the(RGD)_3-tTF or tTF fusion protein labeled with rhodamine B isothiocyanate(RBITC)at a single dose of 50 μg respectively.The location of the(RGD)_3-tTF fusion protein in the colorectal carcinoma bearing mice tissue was analyzed by using in vivo optical imaging one hour after the injection and confocal microscopy twenty-four hours after the injection.Fifteen mice bearing colorectal carcinoma were randomly divided into three groups for injection with the(RGD)_3-tTF,tTF fusion protein or phosphate buffered saline(PBS)at a single dose of 50μg respectively.The tumor size was measured daily to calculate the tumor volume.Five days after the injection,the mice were killed to harvest tumor tissues,hearts,livers,spleens,lung,kidneys and brains to observe valid thrombogenesis and tumor necrosis.Results With the concentration of the(RGD)_3-tTF fusion protein increased,the clotting time was shorten correspondingly under the conditions of Ca~(2+),and the clotting time was(8.6±0.2)min when the concentration was 6 μmol/L,and it was＞30 min in the group of 0 μmol/L(P＜0.05).The coagulation activity of(RGD)_3-tTF and tTF fusion protein was alike(F=0.09,P＞0.05).The in vivo optical imaging and confocal microscopy analyses showed that RBITC fluorescence labeling(RGD)_3-tTF fusion protein was assembled in the tumor vasculature.On the first,third,fifth day after injection,the tumor volume of(RGD)_3-tTF fusion protein group was(120.8±4.8)mm3,(93.8±3.4)mm~3,(132.2±7.7)nun~3 respectively,which was significantly smaller than that of the tTF group[(181.4±13.8)mm~3,(333.0±32.0)mm3,(514.0±11.5)mm~3]and PBS group[(182.6±11.5)mm~3,(332.8±21.0)mm~3,(524.2±16.7)mm3](both P＜0.05).However,there was no significant difference in the tumor volume between the latter two groups(P＞0.05).Conclusion The(RGD)_3-tTF fusion protein is capable of targeting to tumor vasculature and inducing thrombogenesis for suppressing the tumor growth in the colorectal carcinoma mice model,and it's expected to be a new therapy for colorectal cancer.