131I-K237对人前列腺癌LNCaP细胞的凋亡诱导作用

Apoptosis-inducing effect of 131I-K237 on human prostate cancer LNCaP cells

目的鉴定131I-K237多肽（H-组氨酸-苏氨酸-蛋氨酸-酪氨酸-酪氨酸-组氨酸-组氨酸-酪氨酸-谷氨酰胺-组氨酸-组氨酸．亮氨酸-OH）在体外与人前列腺癌LNCaP细胞的亲和性和对其的凋亡诱导作用。方法应用Iodogen法对K237多肽进行131I标记，用TLC测定标记率及经SephadexG25层析柱分离纯化后的131I-K237放化纯。于96孔板接种LNCaP细胞，分4组，每组设3个复孔：实验组加入15kBq 131I-K237；阴性对照组加入Na131I，设5、10、15kBq3个亚组；竞争组加入K237（质量浓度分别为1、2、4、8、16μg/μl）后加入15kBq 131I-K237；空白对照组加入PBS。48h后测定放射性计数并计算各组细胞结合率。在24孔板接种LNCaP细胞，分3组：131I-K237干预组分别加入5、10、15kBq的131I-K237，K237干预组分别加入100μl质量浓度为1、2、4μg/μl的K237，空白对照组加100IxlPBS；温育48h后，分别应用光学显微镜观察细胞形态变化，荧光显微镜鉴别凋亡细胞，再行核酸凝胶电泳，并以流式细胞术（FCM）检测LNCaP细胞的凋亡率。采用单因素方差分析和最小显著差异t检验比较数据差异。结果131I-K237标记率达（73．7±3．2）％，分离纯化后放化纯为（96．7±0．6）％。实验组、阴性对照3个亚组和空白对照组的LNCaP细胞结合率分别为（95．8±1．5）％、（8．2±0．4）％、（8．3±0．6）％、（8．6±0．5）％和0。竞争组实验结果显示，131I-K237与LNCaP细胞的结合率随未标记多肽K237量的增加而显著下降（t=4．71，P〈0．01）。LNCaP细胞经131I-K237干预后，光学显微镜和荧光显微镜下观察到凋亡细胞，DNA电泳可见明显的梯形条带。5、10、15kBq 131I-K237组LNCaP细胞凋亡率分别为（34．1±2．9）％、（37．3±3．4）％、（41．7±3．6）％；质量浓度为1、2、4μg/μl的未标记K237组凋亡率分别为（10．8±1．0）％、（12．5±2．1）％、（13．1±2．4）％；空白对照组中自然凋亡率为（2．9±0.3）％，差异有统计学意义（F=76．31，P〈0．05）；131I-K237组各亚组间差异有统计学意义（t=3．09、3．27和4．52，均P〈0．05）。结论 131I-K237能与人前列腺癌LNCaP细胞特异性结合，对LNCaP细胞具有显著的凋亡诱导作用。

Objective To assess the in vitro affinity and apoptosis-inducing effect of 131I-K237 pep- tide （H-His-Thr-Met-Tyr-Tyr-His-His-Tyr-Gln-His-His-Leu-OH） to LNCaP prostate cancer cell line. Methods The K237 peptide was radiolabeled with 131I by the Iodogen method. The radiolabeling efficiency and radiochemical purity after purification were then characterized by TLC in vitro. LNCaP cells were inoculated in 96-well cell plate and divided into following groups （3 duplicate wells for each group） ： 15 kBq 1311-K237 was added in the experimental group, different doses of Na131I （5, 10, 15 kBq） were added in 3 negative control groups, 15 kBq 131I-K237 with different doses of unlabeled K237 （ 1, 2, 4, 8, 16 μg/μl） were added in 3 blocking groups, and PBS was added in blank control group. The cellular binding ratios were cal- culated after 48 h. LNCaP cells were inoculated in 24-well cell plate and divided into 3 groups ： 131I-K237 group ,which including 3 different dose subgroups （5, 10, 15 kBq） ; unlabeled K237 group, which inclu- ding 3 different dose subgroups （ 1, 2, 4 μg/μl） ; blank control group with 100 txl PBS. All the cells were cultured for 48 h, then optical microscopy （OM） and fluorescence microscopy （FM） were used to observe the cell morphology; DNA gel electrophoresis was conducted and flow cytometry （FCM） was used to esti- mate the apoptotic rate of LNCaP cells. One-way analysis of variance and the least significant difference （LSD）-t test were used to analyze the data. Results The labeling efficiency of 131I-K237 was （73.7±3.2） % and the radiochemical purity was （96.7±0.6） % after purification. The binding ratio of experimental group was （95.8± 1.5）%, whereas the ratio of negative groups with 5, 10, 15 kBq Na131I and PBS group was （8.2±0.4） %, （8.3±0.6） %, （8.6±0.5） % and 0, respectively. The binding ratio of 131I-K237 and LNCaP significantly declined with the increased dose of unlabeled K237 （ t = 4.71, P〈0.01 ）. The apoptosis of LNCaP cells cultured with 131 I-K237 was observed. Typical DNA ladder was found by DNA gel electrophoresis. The apoptotic rates of 5, 10, 15 kBqTM I-K237 groups were （34.1±2.9） %, （37.3±3.4） % and （41.7±3.6） %, respectively; whereas those of unlabeled K237 groups and blank control group were （10.8± 1.0）%, （12.5± 2.1 ） %, （ 13.1± 2.4） % and （2.9±0.3） %, respectively. There were significant differences of apoptotic rate among groups （F= 76.31 ,P〈0.05）. The difference among 5, 10, 15 kBq 131 I-K237 groups was statistically significant （t = 3.09, 3.27, 4.52, all P〈0.05）. Conclusion 131I-K237 can bind to LNCaP cells with highly affinity and has significant apoptosis-inducing efficacy on the prostate cancer cell line.