前列腺癌靶向FGF8分子探针99mTc-HSQAAVP的制备及生物活性研究

The Preparation and Evaluation of A Molecular Imaging Probe 99mTc-HSQAAVP Targeting FGF8

目的探讨99mTc标记多肽HSQAAVP的最佳条件、研究其生物学活性、体外稳定性及在正常SD大鼠体内的生物学分布。方法应用薄层层析技术(thin layer chromatography,TLC)检测标记产物的放射化学纯度。采用直接标记法对HSQAAVP进行99mTc标记,应用正交叉实验筛选最佳标记条件。用MTT实验法检测99mTc-HSQAAVP对人前列腺癌细胞系PC-3细胞的增殖抑制率,判定99mTc-HSQAAVP的生物学活性。将99mTc-HSQAAVP尾静脉注入正常SD大鼠体内,观察不同脏器内99mTc-HSQAAVP的分布。结果最佳标记条件为室温25℃,在50μL 0.01 mol·L-1、pH7.4的PBS缓冲液中,依次加入HSQAAVP 50μg、氯化亚锡(SnCl2)溶液(1 mg·L-1)10μL、99mTc 74.0Mbq(2mCi),振荡5 min,终止反应。99mTc-HSQAAVP的放射化学纯度为(96.79±1.54)%。其在不同温度的生理盐水中孵育12 h内后放射化学纯度均在95%以上,在人新鲜血清中孵育12 h内的放射化学纯度在85%以上。99mTc-HSQAAVP和HSQAAVP对人前列腺癌PC-3细胞的增殖抑制率差异无统计学意义(P均>0.05)。在正常SD大鼠体内,99mTc-HSQAAVP摄取较高的器官依次是脾脏,肝脏,肺,肾脏,膀胱,血液,股骨。肾脏的每克组织的百分注射剂量率(percentage activity of injection dose per gram of tissue,%/ID/g)随时间延长逐渐增高,其余脏器几乎接近本底值。结论靶向成纤维细胞生长因子8(fibroblast growth factor 8,FGF8)分子探针99mTc-HSQAAVP的标记方法简单易行,标记率高,体外稳定性较好。多肽HSQAAVP经99mTc标记后生物活性未发生改变。

Objective To study the optimal conditions,biological activity,in vitro stability and biological distribution of HSQAAVP labeled with 99mTc in normal SD rats.Methods The radiochemical purity of the labeled products was determined by thin layer chromatography(TLC).The direct labeling method was used to label HSQAAVP with 99mTc,and the positive cross experiment was used to screen the best labeling conditions.The inhibition rate of 99mTc HSQAAVP on the proliferation of PC-3 cell line was detected by MTT assay,and the biological activity of 99mTc HSQAAVP was determined.99mTc HSQAAVP was injected into the tail vein of normal SD rats to observe the distribution of 99mTc HSQAAVP in different organs.Results The best labeling conditions were room temperature 25℃,adding 50μg HSQAAVP,10μL SnCl2 and 99mTc 74.0mbq(2mci)in PBS buffer of 50μL 0.01 mol·L-1 and pH7.4,shaking for 5min to stop the reaction.The radiochemical purity of 99mTc HSQAAVP was(96.79±1.54)%.The radiochemical purity was over 95%after incubation in normal saline of different temperature for 12 hours,and over 85%after incubation in fresh human serum for 12 hours.There was no significant difference between 99mTc HSQAAVP and HSQAAVP in the inhibition rate of PC-3 cell proliferation(P>0.05).In normal SD rats,the organs with high uptake of 99mTc HSQAAVP were spleen,liver,lung,kidney,bladder,blood and femur.The%ID/g of kidney gradually increased with time,and the other organs were almost close to the background value.Conclusion The preparation of molecular probe 99mTc-HSQAAVP is practicable and convenient.The labeled compound can be directly used in experiments without purification.Importantly,99mTc-HSQAAVP had a highly efficiency and the biological activity of the molecular probes remains stability.The molecular probe 99mTc-HSQAAVP metabolized from kidney.In the liver,spleen and lung uptake gradually decrease over time.