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载STEAP-1抗体前列腺癌靶向超声微泡的制备及体外寻靶实验研究
作者:刘莹  江峰  袁韵 
单位:皖南医学院弋矶山医院 超声医学科, 安徽 芜湖 241001
关键词:前列腺跨膜上皮抗原-1 靶向超声微泡 前列腺癌 生物素-亲和素 
分类号:R737.25
出版年·卷·期(页码):2018·37·第三期(445-451)
摘要:

目的:采用生物素-亲和素法制备载前列腺跨膜上皮抗原-1(STEAP-1)抗体的靶向超声微泡,检测其结合率、稳定性及体外寻靶能力。方法:(1)体外培养人前列腺癌细胞LNCaP和PC3;(2)采用生物素-亲和素法将STEAP-1抗体与普通声诺维微泡结合,显微镜下观察靶向微泡荧光情况,通过流式细胞术检测其结合率与稳定性;(3)检测所制备的靶向微泡体外结合能力。结果:(1)荧光显微镜下两种前列腺癌细胞表面均发出绿色荧光,即呈阳性表达;蛋白免疫印迹实验结果显示,LNCaP细胞中STEAP-1呈相对高水平表达。(2)载STEAP-1抗体靶向微泡涂片后荧光显微镜下观察,微泡表面呈现环状绿色荧光,普通对照组镜下视野丢失;流式细胞术检测显示,所制备的靶向微泡大小均一,结合率明显高于对照组(P<0.05),振荡后结合率略小于振荡前,但差异无统计学意义(P>0.05)。(3)靶向微泡与前列腺癌细胞孵育反应后出现微泡向细胞区域聚集的现象;靶向微泡于细胞周边呈花环状黏附,LNCaP细胞的黏附率高于PC3细胞(P<0.05);两种细胞的前、后黏附率比较差异均无统计学意义(P>0.05)。结论:(1) STEAP-1蛋白在人前列腺癌细胞LNCaP和PC3中均呈阳性表达,且前列腺癌分化早期代表细胞株LNCaP细胞呈相对高水平表达;(2)载STEAP-1抗体的靶向微泡可经生物素-亲和素法制备,STEAP-1抗体与普通微泡的结合率高且稳定性好;(3)载STEAP-1抗体靶向微泡体外寻靶可与人前列腺癌细胞牢固结合,且LNCaP细胞较PC3细胞结合率高。

Objective: To prepare the six-transmembrane epithelial antigen of the prostate(STEAP-1) targeted novel ultrasound contrast agent via biotin-streptavidin binding and to evaluate the binding rate, stability and searching target function in vitro.Methods: Human prostate cancer cells LNCaP and PC3 were cultured in vitro. Human STEAP-1 antibody was conjugated to the Sono Vue microbubbles using avidin-biotin complex technology and then physiochemical properties of the targeted microbubbles were observed using optical and fluorescence microscope. The binding rate and stability of microbubbles were examined using flow cytometry. In vitro binding capacity of targeted microbubbles was detected.Results: (1)Under fluorescence microscope the membranes of prostate cancer cells, LNCaP and PC3 exhibited green fluorescence. The western blotting showed a higher expression of STEAP-1 in LNCaP than that in PC3. (2)Under fluorescence microscope the surface of STEAP-1 targeted microbubbles seemed like green circles, while no green fluorescence was observed on the surface of the control microbubbles. The flow cytometry showed a higher binding rate of prepared targeted microbubbles with the same size than the control one(P<0.05). The STEAP-1-carrying rate had no obvious change before and after shaking in vitro, the difference being not statistically significant(P>0.05). (3)The targeted microbubbles showed a concentration in the cell regions after the reaction with the prostate cancer cells. A large amount of the targeted microbubbles adhered to the surface of LNCaP and PC3, which seemed like garlands. No adhesion of control microbubble was observed around the cells. The LNCaP had a higher adherence rate in comparison with PC3(P<0.05). And their rates had no significant change(P>0.05).Conclusions: (1) The expression of STEAP-1 is both positive in LNCaP and PC3, and LNCaP, the representative cell line of early prostate cancer, shows a higher expression. (2) The STEAP-1 targeted novel microbubbles can be prepared via avidin-biotin complex technology with a high binding rate and a good stability. (3) In vitro the STEAP-1 targeted novel microbubble is combined with the prostate cancer cells firmly, and the LNCaP also has a higher binding rate compared with PC3.

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