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甲状腺髓样癌细胞(medullary thyroid carcinoma cell,MTC)来源于不表达钠碘共同转运体的甲状腺滤泡旁细胞(又称C细胞),不能直接摄取131I并对其产生相应的生物学效应[1]。有报道指出MTC在病程中易向肺、骨等远处器官转移,因此手术也难以将MTC肿瘤组织彻底切除[2]。聚酰胺-胺 (polyamidoamine,PAMAM)是纳米分子树枝状聚合物中应用最广泛的一种,它具有稳定、无免疫原性、五代以下使用剂量无毒性的优点,其因分散性低、通透性高、分子表面具有大量官能团而易于修饰的特点,已应用于包括药物、DNA、siRNA的递送以及MRI探针的研究等领域。同时PAMAM容易有效地通过细胞小窝蛋白内吞而被摄入,且随着浓度的升高,PAMAM还可进入细胞器和细胞核[3-4]。MTC表面能表达丝氨酸-精氨酸-谷氨酸-丝氨酸-脯氨酸-组氨酸-脯氨酸(Ser-Arg-Glu-Ser-Pro-His-Pro,SRESPHP)(简称SR)特异性受体,且有学者发现多肽SR作为靶向肽连接在腺病毒上可以特定靶向和杀伤MTC细胞[5]。因此,我们对第五代聚酰胺-胺(PAMAM(G5.0))表面进行修饰使其便于连接靶向肽SR,并用131I标记形成靶向和诊治MTC的新型探针,现报道如下。
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131I-PAMAM(G5.0)-SR的标记率为73.21%,131I-PAMAM(G5.0)的标记率为77.48%,纯化后的产物经TLC法检测放化纯度,结果显示标记物集中在原点(图 1中A、B),而游离的Na131I随溶剂迁移到层析纸前沿(图 1中C),TLC法检测131I-PAMAM(G5.0)-SR和131I-PAMAM(G5.0)的放化纯度分别是94.4%和93.3%。
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探针131I-PAMAM(G5.0)-SR和131I-PAMAM (G5.0)在PBS中放置24 h后的放化纯度均在85%以上(图 2)。
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131I-PAMAM(G5.0)-SR的脂水分配系数P为0.085,logP为-1.07;131I-PAMAM(G5.0)的脂水分配系数P为0.046,logP为-1.34,说明131I标记物表现出良好的水溶性。
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在不同质量浓度的实验前体PAMAM(G5.0)-SR和PAMAM(G5.0)作用下,细胞存活率均大于95%,说明上述两种实验前体对细胞的毒性较小(图 3)。
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通过GraphPad Prism 5.01软件计算得出,加入不同放射性活度的131I-PAMAM(G5.0)-SR和131I-PAMAM(G5.0)作用细胞24 h后,当131I-PAMAM- (G5.0)-SR 的放射性活度为513.6 kBq/mL时,可导致细胞生长抑制率达50%;当131I-PAMAM(G5.0)的放射性活度为596.8 kBq/mL时,可导致细胞生长抑制率达50%(图 4)。
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随着时间的推移,PAMAM(G5.0)-SR阻断组的摄取率逐渐降低,2 h时的摄取率达最低,为5.83%;而131I-PAMAM(G5.0)-SR组及PAMAM (G5.0)阻断组的摄取率则随时间的推移保持在7%以上(图 5)。结果显示,PAMAM(G5.0)-SR阻断组的131I-PAMAM(G5.0)-SR细胞摄取率与131I-PAMAM(G5.0)-SR组相比明显降低,差异均有统计学意义(t=7.315、22.590和22.570,均P<0.01),而PAMAM(G5.0)阻断组的131I-PAMAM(G5.0)-SR细胞摄取率与131I-PAMAM (G5.0)-SR组相比无明显降低(t=0.937、4.304和1.115,P分别为>0.05、<0.05和>0.05)。
图 5 PAMAM(G5.0)-SR 和 PAMAM(G5.0)对细胞摄取 131I-PAMAM(G5.0)-SR 的阻断作用 图中, PAMAM(G5.0): 第五代聚酰胺-胺; SR: 丝氨酸-精氨酸-谷氨酸-丝氨酸-脯氨酸-组氨酸-脯氨酸(SRESPHP)的简称; 与 131I-PAMAM(G5.0)-SR 组比较, 差异有统计学意义, *: t=4.304, P<0.05; **: t= 7.315、 22.590 和 22.570, 均 P<0.01。
Figure 5. Effects of blocking the uptake of 131I-PAMAM(G5.0)-SR by PAMAM(G5.0)-SR or PAMAM(G5.0)on cells
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6~48 h时TT细胞对131I-PAMAM(G5.0)的摄取高于131I-PAMAM(G5.0)-SR,说明131I-PAMAM(G5.0)-SR对TT细胞的影响较大,易导致细胞死亡,但随着时间的推移,TT细胞对131I标记物的摄取均降低,48 h时,TT细胞对131I标记物的摄取均出现上升的现象(表 1)。
组别 细胞摄取率(%) 6h 12h 24h 48h 72h 131I-PAMAM (G5.0)-SR组
131I-PAMAM (G5.0)
Na131I组0.52±0.0002
0.85±0.0001
0.12±0.00010.43±0.0000
0.76±0.0000
0.13±0.00030.40±0.0000
0.53±0.0000
0.17±0.00000.61±0.0002
0.84±0.0000
0.16±0.00000.42±0.0001
0.26±0.0003
0.15±0.0000注: 表中, PAMAM(G5.0): 第五代聚酰胺-胺; SR: 丝氨酸-精氨酸-谷氨酸-丝氨酸-脯氨酸-组氨酸-脯氨酸(SRESPHP)的简称。 表 1 甲状腺髓样癌 TT 细胞对半数致死剂量131I 标记物的摄取(x±s)
Table 1. Uptake of the median lethal dose of 131I tracers in medullary thyroid carcinoma TT cells(x±s)
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在半数致死剂量下,随着时间的延长,Na131I组的TT细胞存活率差异不大,但131I-PAMAM (G5.0)-SR组及131I-PAMAM (G5.0)组的TT细胞存活率随时间的延长而逐渐下降,说明只加入Na131I对TT细胞生长影响不大,但131I-PAMAM(G5.0)-SR及131I-PAMAM(G5.0)对TT细胞的生长存在不同程度的抑制作用(表 2)。
组别 细胞存活率(%) 6h 12h 24h 48h 72h 131I-PAMAM (G5.0)-SR组 72.62 ±0.003 59.71 ±0.018 57.89±0.032 58.40 ±0.009 59.21 ±0.054 131I-PAMAM (G5.0) 组 71.38 ±0.009 58.27 ±0.012 59.90 ±0.043 58.90 ±0.013 60.53 ±0.033 Na131I组 93.10 ±0.009 84.17 ±0.006 86.22 ±0.007 83.46 ±0.061 82.02 ±0.025 注:表中,PAMAM (G5.0):第五代聚酰胺-胺;SR:丝氨酸-精氨酸-谷氨酸-丝氨酸-脯氨酸-组氮酸-脯氨酸(SRESPHP)的简称。 表 2 半数致死剂量的探针处理甲状腺髓样癌 TT 细胞不同时间对细胞生存的影响(x±s)半数致死剂量的探针处理甲状腺髓样癌 TT 细胞不同时间对细胞生存的影响(x±s)
Table 2. Effect on half lethal dose of probes on medullary thyroid carcinoma cell survival at different times after treatment(x±s)
靶向肽结合131I-PAMAM(G5.0) 抑制甲状腺髓样癌细胞增殖的研究
Effects of targeted peptide-conjugated 131I-PAMAM (G5.0) on the inhibition of medullary thyroid carcinoma cells proliferation
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摘要:
目的研究131I标记靶向肽丝氨酸-精氨酸-谷氨酸-丝氨酸-脯氨酸-组氮酸-脯氨酸(SRESPHP)(简称SR)修饰的第五代聚酰胺-胺(PAMAM(G5.0))的体外性质及其作为甲状腺髓样癌细胞靶向探针的可行性。 方法用氯胺T法进行PAMAM(G5.0)-SR和PAMAM(G5.0)的131I标记,通过薄层层析法分别测定所制备的两种探针的标记率及稳定性,并考察131I标记物的脂水分配系数;通过阻断摄取实验分别考察两种探针的靶向性;计算两种探针对细胞的半数致死剂量并分析其对细胞生长的影响。采用GraphPad Prism 5.01分析软件对符合正态分布及方差齐性的数据进行样本t检验。 结果 131I-PAMAM(G5.0)-SR和131I-PAMAM(G5.0)的标记率均大于70%,纯化后的放化纯度均大于90%。两种探针在体外PBS体系中的稳定性好,且均显示出良好的水溶性。细胞阻断实验结果显示,加入PAMAM(G5.0)-SR阻断的131I-PAMAM(G5.0)-SR细胞摄取率明显降低,差异均有统计学意义(t=7.315、22.590和22.570,均P < 0.01),提示131I-PAMAM(G5.0)-SR对细胞具有较好的靶向性。131I-PAMAM(G5.0)-SR的细胞半数致死剂量为513.6 kBq/mL。细胞摄取实验结果显示,随着时间的延迟,细胞对半数致死剂量下的131I-PAMAM(G5.0)-SR的摄取逐渐降低,但在48 h细胞摄取出现上升的现象,随后细胞摄取再次下降。 结论 131I-PAMAM(G5.0)-SR具有良好的生物学性质,可靶向甲状腺髓样癌细胞并抑制细胞增殖。 -
关键词:
- 碘放射性同位素 /
- 分子探针 /
- 甲状腺髓样癌 /
- 聚酰胺-胺 /
- 丝氨酸-精氨酸-谷氨酸-丝氨酸-脯氨酸-组氨酸-脯氨酸
Abstract:Objective To incorporate 131I i nto the fifth generation polyamidoamine(PAMAM (G5.0)) with the targeting peptide Ser-Arg-Glu-Ser-Pro-His-Pro(SRESPHP)(SR for short) and observe the in vitro properties for the targeting probe of medullary carcinoma cells (MTCs). Methods PAMAM (G5.0)-SR and PAMAM(G5.0) were radiolabeled with 131I by chloramine T. Labeling yield and stability were determined by thin layer chromatography. Lipid -water partition coefficients were also evaluated. The targeting of the two types of 131I-radiotracers(131I-PAMAM(G5.0)-SR and 131I-PAMAM(G5.0)) was determined in a blocking uptake study where TT tumor cells were used. The median lethal dose of the two probes was then calculated. GraphPad Prism 5.01 analysis software was used to conduct a t-test for the data that fit the normal distribution and homogeneity of variance. Results The labeling yields of the two types of 131I radiotracers all exceeded 70%, and the radiochemical purity levels were higher than 90% after purification. The stability of the two probes in the PBS system was satisfactory, and both probes showed excellent water solubility. The results of the blocking uptake study on the TT cells showed that the cell uptake rate decreased significantly (t=7.315, 22.590, 22.570, all P < 0.01) after the PAMAM (G5.0)-SR blocked the 131I-PAMAM (G5.0)-SR. This result indicated that 131I-PAMAM (G5.0)-SR achieved excellent targeting and that its median lethal dose was only 513.6 kBq/mL. The cell uptake results showed that the cell uptake rate of 131I-PAMAM (G5.0)-SR with a median lethal dose gradually decreased with time. However, cell uptake rate increased for 48 h before it decreased again. Conclusion 131I-PAMAM (G5.0)-SR can target medullary thyroid carcinoma cells and thus inhibit cell proliferation. -
图 5 PAMAM(G5.0)-SR 和 PAMAM(G5.0)对细胞摄取 131I-PAMAM(G5.0)-SR 的阻断作用 图中, PAMAM(G5.0): 第五代聚酰胺-胺; SR: 丝氨酸-精氨酸-谷氨酸-丝氨酸-脯氨酸-组氨酸-脯氨酸(SRESPHP)的简称; 与 131I-PAMAM(G5.0)-SR 组比较, 差异有统计学意义, *: t=4.304, P<0.05; **: t= 7.315、 22.590 和 22.570, 均 P<0.01。
Figure 5. Effects of blocking the uptake of 131I-PAMAM(G5.0)-SR by PAMAM(G5.0)-SR or PAMAM(G5.0)on cells
表 1 甲状腺髓样癌 TT 细胞对半数致死剂量131I 标记物的摄取(x±s)
Table 1. Uptake of the median lethal dose of 131I tracers in medullary thyroid carcinoma TT cells(x±s)
组别 细胞摄取率(%) 6h 12h 24h 48h 72h 131I-PAMAM (G5.0)-SR组
131I-PAMAM (G5.0)
Na131I组0.52±0.0002
0.85±0.0001
0.12±0.00010.43±0.0000
0.76±0.0000
0.13±0.00030.40±0.0000
0.53±0.0000
0.17±0.00000.61±0.0002
0.84±0.0000
0.16±0.00000.42±0.0001
0.26±0.0003
0.15±0.0000注: 表中, PAMAM(G5.0): 第五代聚酰胺-胺; SR: 丝氨酸-精氨酸-谷氨酸-丝氨酸-脯氨酸-组氨酸-脯氨酸(SRESPHP)的简称。 表 2 半数致死剂量的探针处理甲状腺髓样癌 TT 细胞不同时间对细胞生存的影响(x±s)半数致死剂量的探针处理甲状腺髓样癌 TT 细胞不同时间对细胞生存的影响(x±s)
Table 2. Effect on half lethal dose of probes on medullary thyroid carcinoma cell survival at different times after treatment(x±s)
组别 细胞存活率(%) 6h 12h 24h 48h 72h 131I-PAMAM (G5.0)-SR组 72.62 ±0.003 59.71 ±0.018 57.89±0.032 58.40 ±0.009 59.21 ±0.054 131I-PAMAM (G5.0) 组 71.38 ±0.009 58.27 ±0.012 59.90 ±0.043 58.90 ±0.013 60.53 ±0.033 Na131I组 93.10 ±0.009 84.17 ±0.006 86.22 ±0.007 83.46 ±0.061 82.02 ±0.025 注:表中,PAMAM (G5.0):第五代聚酰胺-胺;SR:丝氨酸-精氨酸-谷氨酸-丝氨酸-脯氨酸-组氮酸-脯氨酸(SRESPHP)的简称。 -
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