铼-188标记分子探针的实验研究
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摘要
第一部分~(188)Re标记胰岛素样生长因子-1类似物(IGF-1A)的实验研究一、~(188)Re-IGF-1A的制备
目的研究标记率高且稳定的~(188)Re标记胰岛素样生长因子-1类似物(insulin-like growth factor 1 analogue, IGF-1A)的方法学。方法1.采用直接标记法标记经修饰的IGF-1A,改变标记条件:Tween80的用量从2～10μL、SnCl_2.2H_2O浓度变化从0.75～25mg/mL、IGF-1A的用量从20～100μg、淋洗液的体积从10～500μL;分别在不同时间点测定标记率。2.标记物中加入生理盐水或人血清后不同时间点测定其标记率。结果1.最佳~(188)Re标记方法为:将100μLSnCl_2.2H_2O(10mg/mL)加入50μL IGF-1A(2mg/mL);300μL Na_3PO_.4和10μL0.1%Tween80混匀后加入50μL ~(188)ReO_4~-新鲜洗脱液;在IGF-1A体系中加入洗脱液体系,混匀,室温下反应30min;加入500μL NaH_2PO_4将pH值调节到7.0左右,标记完成。最高标记率为(94.07±0.32)%,胶体含量为(5.50±1.50)%。2.室温下放置6h标记率为(85.50±1.21)%,加入人血清放置24h后标记率为(76.57±9.96)%。结论用直接标记法进行~(188)Re标记IGF-1A,标记率高且稳定性良好。
二、~(188)Re-IGF-1A与胰腺癌细胞结合及细胞杀伤效应实验
目的研究~(188)Re-IGF-1A与胰腺癌细胞的特异性结合及其对胰腺癌细胞的杀伤效果。方法1.测定~(188)Re-IGF-1A与胰腺癌Patu8988细胞的结合率。2.用MTT实验检测给药后细胞增殖情况,将1×10~4/孔细胞接种于96孔板上,细胞分为对照组、IGF-1A组(1、5、10、20μg)、~(188)ReO_4~-组(0.37、1.85、3.70、7.40MBq)和~(188)Re-IGF-1A组(0.37、0.74、1.85MBq),另设空白调零组。3.~(188)ReO_4~-组和~(188)Re-IGF-1A组接种后,分别在给药后1～7d,每天采用MTT比色法测定其OD值,IGF-1A组分别在给药后1～6d,每天采用MTT比色法测定其OD值。根据各组OD值,计算生存率和抑制率。4.将细胞接种于培养瓶中,分~(188)ReO_4~-组和~(188)Re-IGF-1A组(1.85、3.70、7.40MBq),在给药后3d用流式细胞仪检测细胞凋亡率。结果1.~(188)Re-IGF-1A与5×10~6个/50μL胰腺癌细胞的最高总结合率为(24.13±2.03)%,特异性结合为12.68%。2.加入不同剂量~(188)Re-IGF-1A后,在1～3d内,随时间增加而抑制率增高,第3d时0.37、0.74、1.85MBq组抑制率分别为(64.48±4.18)%、(66.89±1.39)%和(89.71±1.27)%;第6天时,1.85MBq组抑制率达到(93.20±1.93)%。3.加入与~(188)Re-IGF-1A同量的IGF-1A或相同放射性活度的~(188)ReO_4~-后,~(188)Re-IGF-1A对细胞的抑制率在各时间点均高于单独用~(188)ReO_4~-,两者差异有统计学意义(P<0.05);与IGF-1A组比较,第1d差异无统计学意义,在第2～6d,~(188)Re-IGF-1A对细胞的抑制率在各时间点均高于单独用IGF-1A,两者差异有统计学意义。4.给药1.85、3.70、7.40MBq3d后,~(188)ReO_4~-组和~(188)Re-IGF-1A组细胞漂浮率分别为(16.58±3.57)%、(24.58±6.50)%、(34.12±7.39)%和(16.56±0.95)%、(33.39±5.93)%、(43.76±1.38)%,漂浮细胞的凋亡率分别为(9.27±1.80)%、(16.00±1.15)%、(15.47±0.65)%和(12.70±2.27)%、(17.80±1.51)%、(23.23±1.22)%。结论~(188)Re-IGF-1A能与胰腺癌Patu8988细胞特异性结合,并对胰腺癌细胞生长有明显的抑制作用,可诱导细胞凋亡。
三、~(188)Re-IGF-1A在荷瘤裸鼠体内分布、显像和吸收剂量估算
目的研究~(188)Re-IGF-1A在荷人胰腺癌裸鼠体内的分布、显像和剂量估算。方法1.建立荷人胰腺癌Patu8988裸鼠模型。2.将66只荷人胰腺癌裸鼠随机分2组,~(188)Re-IGF-1A组36只,~(188)ReO_4~-组30只。采取瘤内注射给药方式,注射~(188)Re-IGF-1A或~(188)ReO_4~- 0.03mL,同时取0.05mL~(188)ReO_4~-洗脱液做标准源。在瘤内注射后即刻行SPECT平面显像,通过裸鼠全身与标准源的ROI计数比值,换算每个荷瘤裸鼠总注射剂量。3.~(188)Re-IGF-1A组于注射后15min、1、4h、1、3、5d显像并处死裸鼠。~(188)ReO_4~-组于注射后15min、1、2、4、24h显像并处死裸鼠。解剖后取所需组织称量并测量其60s的放射性计数,计算各组织的每克组织百分注射剂量率(%ID/g),并计算肿瘤与血、心、脑、甲状腺、肝、脾、肺、肾、胃、肠、胰、骨、肌肉的比值(T/NT)。4.根据各组织不同时间点的平均%ID/g,通过放射性药物内照射剂量计算软件计算出吸收剂量(mGy/MBq)。结果1.瘤内注射~(188)Re-IGF-1A后肿瘤、肾脏、肝脏、脾脏内放射性较高,注射后15min肿瘤摄取为(39.30±17.98)%,肾脏摄取为(19.77±10.75)%,肝脏摄取为(2.03±2.28)%,脾脏摄取为(1.04±0.92)%。瘤内注射后1、4h、1、3、5d肿瘤内摄取分别为(35.03±23.37)%、(41.22±23.88)%、(10.59±9.39)%、(5.32±1.53)%、(5.30±2.28)%;肿瘤部位放射性积聚量4h内差异无统计学意义(P>0.05),且随时间延长,肿瘤与其它脏器的T/NT呈上升趋势,肿瘤与肾脏的T/NT在5d时达到5.64±7.50。肿瘤/肌肉在5d时达到2610.65±1534.02。2.瘤内注射~(188)ReO_4~-后,在体内开始主要分布于甲状腺、胃、肿瘤、血液。胃内放射性高峰出现在1h,为(23.14±12.08)%,甲状腺放射性高峰出现在2h,为(100.27±19.67)%,随时间延长,肿瘤部位计数迅速下降。肿瘤部位在24h摄取为(0.09±0.03)%,与肾脏差异无统计学意义(P=0.27)。3.在瘤内注射后各时间点,~(188)Re-IGF-1A组肿瘤及肾脏内摄取比~(188)ReO_4~-组高,两者有统计学差异(P<0.01)。两组肿瘤内摄取比值在24h达到117.67倍。4.瘤内注射~(188)Re-IGF-1A后,SPECT平面显像见瘤内浓聚,5d时仅见肿瘤部位显影。5.肿瘤内吸收剂量为5165.8mGy/MBq。结论瘤内注射~(188)Re-IGF-1A后,在肿瘤部位积聚较高,可望作为胰腺癌经动脉灌注治疗的药物,并能在体外观察其分布。
第二部分~(188)Re标记反基因肽核酸(AGPNA)的实验研究
一、k-ras-AGPNA抑制k-ras基因表达实验
目的探索自主设计的与胰腺癌k-ras点突变基因特异性结合的反基因肽核酸(antigene peptide nucleic acid, AGPNA)的生物活性。方法1.设计并合成与胰腺癌k-ras点突变基因特异性结合的k-ras-AGPNA。2.应用RT-PCR检测转染k-ras-AGPNA前后胰腺癌Patu8988细胞k-ras癌基因的mRNA表达水平,分对照组、k-ras-AGPNA组、脂质体转染k-ras-反基因寡核苷酸(antigene oligonucleotides, AGON)组、脂质体转染k-ras-反义寡核苷酸(antisense oligonucleotides, ASON)组。3.应用流式细胞仪测定转染k-ras-AGPNA前后胰腺癌Patu8988细胞的k-ras蛋白表达水平。结果1.转染1nmol/mL k-ras-AGPNA组和k-ras-AGON组的细胞的k-ras突变基因mRNA的灰度比分别为(1.00±0.39)和(1.22±0.31),比对照组(1.86±0.07)低,差异有显著统计学意义(P<0.01)。k-ras-AGPNA、k-ras-AGON和k-ras-ASON组内不同剂量之间差异无统计学意义(P>0.05)。2.转染1nmol/mL k-ras-AGPNA、k-ras-AGON和k-ras-ASON组的细胞其k-ras蛋白表达分别为(15.05±5.07)%、(10.20±2.63)%、(8.80±4.31)%,比对照组(24.38±5.40)%低,差异有显著统计学意义(P<0.01)。k-ras-AGPNA、k-ras-AGON和k-ras-ASON组内不同剂量之间差异无统计学意义(P>0.05)。结论自主设计并合成的k-ras-AGPNA能抑制k-ras基因在mRNA和蛋白水平的表达。
二、~(188)Re-k-ras-AGPNA的制备及其胰腺癌细胞内化实验研究
目的制备~(188)Re-k-ras-AGPNA并研究其与胰腺癌Patu8988细胞结合的特性。方法1.用~(188)Re直接标记经修饰的k-ras-AGPNA。2.预实验中其他标记条件不变,将SnCl2.2H2O浓度改为20mg/mL,k-ras-AGPNA的用量改为20μL(40μg)后分别进行实验。3.在标记后不同时间点15、30min、1、2、4、6h测定标记率。4.测定标记物在人血清和生理盐水中5min～24h不同时间点的标记率。5.胰腺癌Patu8988细胞摄取~(188)Re-k-ras-AGPNA的内化实验。结果1.~(188)Re-k-ras-AGPNA的标记率最高为(89.61±0.91)%,放射性胶体含量为(9.40±0.55)%。2.~(188)Re-k-ras-AGPNA放置血清中24h后标记率为(89.14±0.63)%,放置生理盐水中1h后标记率为(79.12±5.26)%。3.~(188)Re-k-ras-AGPNA转染胰腺癌Patu8988细胞,最高细胞结合率为(38.16±2.17)%,最高核内化率为(22.41±0.86)%。结论~(188)Re直接标记k-ras-AGPNA方法简便、标记率较高,能与胰腺癌细胞结合并转染入细胞核。
三、~(188)Re-k-ras-AGPNA诱导细胞凋亡和荷瘤裸鼠体内分布实验
目的研究~(188)Re-k-ras-AGPNA的诱导凋亡效应和在荷人胰腺癌裸鼠体内的分布、显像和剂量估算。方法1.胰腺癌Patu8988细胞给予~(188)Re-k-ras-AGPNA和~(188)ReO_4~- 3～5d后,用流式细胞仪检测细胞凋亡率。2.将28只荷人胰腺癌裸鼠分7组,每组4只。采取瘤内注射给药方式,注射~(188)Re-k-ras-AGPNA 0.03mL。3.注射后15min、1、4h、1、3、5、7d显像并处死裸鼠,解剖后取所需组织称量并测量其60s的放射性计数,计算各组织的%ID/g和肿瘤与其他组织的比值(T/NT),并根据各组织不同时间点的平均%ID/g计算出吸收剂量(mGy/MBq)。结果1.给药3、4、5d后,~(188)ReO_4~-组和~(188)Re-k-ras-AGPNA组细胞漂浮率分别为(5.68±0.82)%、(8.14±0.12)%、(11.87±0.17)%和(5.99±3.59)%、(25.66±8.51)%、(29.59±4.92)%,漂浮细胞的凋亡率分别为(3.88±2.10)%、(8.75±3.11)%、(16.87±5.85)%和(5.28±1.12)%、(26.30±7.45)%、(27.90±10.38)%。2.瘤内注射~(188)Re-k-ras-AGPNA后肿瘤、肾脏、肺、甲状腺、肝脏内放射性较高,注射后15min肿瘤摄取为(53.23±16.64)%,肾脏摄取为(12.60±1.53)%,肺内摄取为(2.32±1.35)%,甲状腺摄取为(1.95±1.22)%,肝脏摄取为(1.78±0.63)%。瘤内注射后1、4h、1、3、5、7d肿瘤内摄取分别为(37.47±21.31)%、(34.84±6.46)%、(35.96±7.80)%、(17.43±5.46)%、(11.09±3.52)%、(15.46±4.93)%;肿瘤部位放射性积聚量24h内差异无统计学意义(P>0.05),且随时间延长,肿瘤与肾脏的T/NT呈上升趋势,在7d时达到11.08±9.22。肿瘤/肌肉在3d时达到870.86±1079.02。3.瘤内注射~(188)Re-k-ras-AGPNA后,SPECT平面显像见瘤内浓聚,7d时仅见肿瘤部位显影。4.肿瘤内吸收剂量为15569mGy/MBq。结论瘤内注射~(188)Re-k-ras-AGPNA后,肿瘤部位摄取高、清除缓慢,肿瘤吸收剂量高,可望作为胰腺癌治疗的药物。
第三部分~(188)Re-锡硫胶体介入治疗荷VX2肝癌兔的体内生物分布研究
目的研究~(188)Re-锡硫胶体(tin sulfur colloid,TSC)的制备及其经肝动脉灌注给药后在兔VX2肝肿瘤模型体内的生物学分布特征,探讨其作为放射性介入治疗剂的可行性。方法1.改变标记条件制备~(188)Re-TSC和~(188)Re-聚合白蛋白(Macroaggregated albumin, MAA),检测标记物在人血清中的稳定性,测定~(188)Re-TSC的颗粒大小。2.建立32只VX2肝肿瘤模型,均经CT或DSA证实,1例行病理学检查。3.31只荷瘤兔分~(188)Re-TSC(n=12)、~(188)Re-MAA(n=15)和~(188)ReO4-(n=4)3组,将放射性药物经肝动脉注射入肿瘤内,分别在注射后15min、1h和24h分别进行SPECT平面显像,画感兴趣区进行肿瘤/肝脏计数的半定量分析。4.在注射后1h和24h处死模型兔后取感兴趣脏器进行称量、计数,计算各脏器的每克组织百分注射剂量。5.根据灌注~(188)Re-TSC后肿瘤内%ID/g进行吸收剂量估算。结果1.~(188)Re-TSC和~(188)Re-MAA标记率最高分别为(99.94±0.04)%和(99.95±0.03)%,在人血清中放置72h后分别为(95.77±0.54)%和(92.13±1.21)%。~(188)Re-TSC的颗粒大小分布为10.83±6.60μm (D10)、44.91±14.46μm (D50)和235.29±126.61μm (D90)。2.在平面显像中,62.96%(17/27)模型兔的肿瘤显像清晰,肿瘤/肝脏感兴趣区放射性计数的比值为2.15±0.80。3.~(188)Re-TSC组中肿瘤摄取在1h和24h分别为(24.32±11.93)%和(21.88±18.29)%,肿瘤与肝脏的放射性比值在1h和24h分别为70.89±19.58和17.42±13.96。4.~(188)Re-MAA组中肿瘤摄取在1h和24h分别为(38.78±30.23)%和(15.98±26.64)%,肿瘤与肝脏的比值在1h和24h分别为39.71±25.06和8.13±4.61。5.灌注~(188)Re-TSC后肿瘤内24h的放射吸收剂量为0.24 Gy/MBq。结论~(188)Re-TSC可望成为有临床应用前景的放射介入治疗药物。
PartⅠradiosynthesis and biological experiment of ~(188)Re-IGF-1A
Background The insulin-like growth factor-1 receptor (IGF-1R) is a cellular receptor found to be overexpressed in many tumor cell lines from different anatomical sites. It can be used as molecular targets by which radiolabeled insulin-like growth factor 1 analogue (IGF-1A) can localize cancers for peptide receptor radiation therapy.
1. Investigations of labeling insulin-like growth factor 1 analogue with ~(188)Re
Objective To establish a useful method for labeling IGF-1A with ~(188)Re. Methods Direct labeling method was adopted to label IGF-1A. Several labeling conditions were tested. The volume of Tween80 was changed from 2 to 10μL; the concentration of SnCl_2.2H_2O was changed from 0.75 to 25mg/mL; the amount of IGF-1A was changed from 20 to 100μg and the volume of ~(188)Re perrhenate was changed from 10 to 500μL. The labeling efficiency was analyzed from 15min to 8h after labeling. The in vitro stabilities of ~(188)Re-IGF-1A was analyzed in human serum or sodium chloride medium, and the labeling efficiency was determined from 2 to 24h after adding the medium. Results The optimum labeling condition was 100μL stannous chloride (10mg/mL) dissolved in sodium gluconate, 50μL IGF-1A (2mg/mL), 300μL Na3PO4, 10μL 0.1% Tween80, 50μL ~(188)Re perrhenate added and the incubation time was 30 min at room temperature, then added 500μL NaH_2PO_4 to adjust the pH about 7.0. The labeling efficiency of ~(188)Re-IGF-1A could reach (94.07±0.32)% and the amount of radiocolloid was (5.50±1.50)%. It was (85.50±1.21)% after incubation for 6h at room temperature, and was (76.57±9.96)% after incubation for 24h with human serum. Conclusion This method of labeling IGF-1A with ~(188)Re using SnCl_2.2H_2O is stable and high labeling efficiency can be obtained.
2. Experimental study of ~(188)Re-IGF-1A on cell combination and proliferation inhibition effect to pancreatic carcinoma cell
Objective To investigate cell combination and proliferation inhibition effect of ~(188)Re-IGF-1A to Patu8988 human pancreatic carcinoma cell. Methods (1)The combinative efficiency of ~(188)Re-IGF-1A with Patu8988 cell was determined. (2)Patu8988 cell were seeded onto 96-well plate and divided into blank control group, IGF-1A group (1, 5, 10, 20μg), ~(188)ReO4- group (0.37, 1.85, 3.70, 7.40MBq) and ~(188)Re-IGF-1A group (0.37, 0.74, 1.85MBq). (3)The proliferation inhibition effect of ~(188)Re-IGF-1A group and ~(188)ReO4- group on cell growth was detected every day by MTT test from 1d to 7d after administration. The proliferation inhibition effect of IGF-1A group was detected every day from 1d to 6d, and inhibition rates were calculated. (4) At 3d after treatment with ~(188)ReO4- and ~(188)Re-IGF-1A (1.85, 3.70, 7.40 MBq), the cell’s apoptosis was detected by flow cytometry. Results (1)The total combinative efficiency of ~(188)Re-IGF-1A with Patu8988 cell was (24.13±2.03)%, and the special combinative efficiency was 12.68%. (2) Inhibition rates of Patu8988 cell with ~(188)Re-IGF-1A group (0.37, 0.74, 1.85MBq) were (64.48±4.18)%,(66.89±1.39)% and (89.71±1.27)% after 3d, and the inhibition rates was higher when the radioactive dose higher. After 6d, inhibition rate of Patu8988 cell with ~(188)Re-IGF-1A group (1.85MBq) was (93.20±1.93)%. (3) Inhibition rates of Patu8988 cell with ~(188)Re-IGF-1A group were always higher than ~(188)ReO4- group and IGF-1A group. The difference was significant (P < 0.05). (4) At 3d after treated with ~(188)ReO4- and ~(188)Re-IGF-1A, float cell ratio were (16.58±3.57)%, (24.58±6.50)%, (34.12±7.39)% and (16.56±0.95)%, (33.39±5.93)%, (43.76±1.38)%, respectively. Float cell’s apoptosis ratio of Patu8988 cell with ~(188)ReO4- and ~(188)Re-IGF-1A group were (9.27±1.80)%, (16.00±1.15)%, (15.47±0.65)% and (12.70±2.27)%, (17.80±1.51)%, (23.23±1.22)%, respectively. Conclusion Proliferation of human pancreatic carcinoma cell Patu8988 can be inhibited and apoptosis can be promoted with ~(188)Re-IGF-1A.
3. Biodistribution characteristics and dosimetry measurement of ~(188)Re-IGF-1A in nude mice bearing the Patu8988 human pancreatic cancer xenografts
Objective To investigate the biodistribution characteristics and measure dosimetry of ~(188)Re-IGF-1A in nude mice bearing human pancreatic cancer xenografts. Methods (1)66 nude mice model bearing human pancreatic carcinoma cell Patu8988 xenografts were established and devided into the ~(188)Re-IGF-1A group (n=36) and the ~(188)ReO4- control group (n=30). (2)In the ~(188)Re-IGF-1A group, at 15min, 1, 4, 24, 72, and 120h after intratumor injection with ~(188)Re-IGF-1A, the mice were killed and the organs of interest were excised, weighted and counted on a gamma counter. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/g) and the ratios of tumor to normal tissue (T/NT) were calculated. (3) In the ~(188)ReO_4~- control group, organs of interest were excised and counted. %ID/g and T/NT were calculated at 15min, 1, 2, 4 and 24h after intratumor injection. (4) Scintigraphy imaging for each nude mouse was performed at 15min after intratumor injection with radiopharmaceuticals,in which serial planar scintigraphy imaging were performed for 6 nude mice at 15min, 1, 4, 24, 72, and 120h after intratumor injection with ~(188)Re-IGF-1A. (5) Radiation dosimetry in organs of interest were estimated on the basis of the mean %ID/g. Results (1) ~(188)Re-IGF-1A was major distributed in tumors and the largest uptake of tumors was (41.22±23.88)% at 4h after injection. The uptake of tumor were (10.59±9.39)% and (5.30±2.28)% at 24h and 120h after injection, respectively. (2)~(188)ReO_4~- was major distributed in thyroid glands, stomachs, tumors and blood in nude mice after injection at first. Then uptake of tumor decreased rapidly, but slowly in stomachs and thyroid glands. The uptake of tumor were (0.09±0.03)% at 24h after injection. (3) The tumor in nude mice could be seen clearly in scintigraphy images until 120h after intratumoral injection with ~(188)Re-IGF-1A. Conclusion ~(188)Re-IGF-1A was major distributed in tumors after intratumor injection in nude mice bearing human pancreatic cancer xenografts, and we propose that ~(188)Re-IGF-1A may be a potential tumor transarterial radionuclide therapeutic agent used in clinic.
PartⅡradiosynthesis and biological experiment of ~(188)Re-k-ras-AGPNA
Background We hypothesized that antigene peptide nucleic acid (AGPNA) hybridization probes targeted k-ras 12 point mutation oncogene, with an IGF-1A peptide loop on the C-terminus and ~(188)Re chelator peptide on the N-terminus, could hybridize with k-ras 12 point mutation oncogene and decrease the multiplication of pancreatic cancer cell Patu8988.
1. Experiment of k-ras-AGPNA on depress the k-ras gene expression in human pancreatic cancer cell Patu8988
Objective To investigate the effect of AGPNA on depress the k-ras gene expression and the k-ras protein expression of human pancreatic cancer cell Patu8988. Methods (1)AGPNA hybridizing k-ras 12 point mutation DNA was designed and synthesized. (2)Before and after transfected AGPNA, the expression level of k-ras mRNA in human pancreatic cancer cell Patu8988 were detected by reverse transcription- polymerase chain reaction (RT-PCR). (3)The expression ratio of k-ras protein in cell were detected by flow cytometry. Results (1)After transfected 1nmol/mL AGPNA and antigene oligonucleotides (AGON), the k-ras mRNA gray scale ratio were (1.00±0.39) and (1.22±0.31). Its was lower than (1.86±0.07) of the control group, and the difference was significant (P<0.01). Among the difference dose of AGPNA, AGON, and antisense oligonucleotides (ASON) group, the difference of the expression level of k-ras mRNA was not significant (P>0.05). (2)After transfected 1nmol/mL AGPNA, AGON, and ASON, the expression ratios of k-ras protein were (15.05±5.07)%, (10.20±2.63)%, and (8.80±4.31)%. Its was lower than (24.38±5.40)% of the control group, and the difference was significant (P<0.01). Compared with the difference dose of AGPNA, AGON, and ASON group, the difference of the expression ratios of k-ras protein was no significant (P>0.05). Conclusion AGPNA can depress the k-ras gene expression on the mRNA level and depress the k-ras protein expression of human pancreatic cancer cell Patu8988.
2. Radiosynthesis of ~(188)Re labeled k-ras-AGPNA for binding with pancreatic carcinoma cell
Objective To establish a method for labeling k-ras-AGPNA with ~(188)Re, and investigate its binding with pancreatic cancer cell Patu8988. Methods The directly labeling method was adopted, and several labeling conditions were tested, such as the concentration of SnCl2.2H2O and the amount of k-ras-AGPNA. The labeling efficiency was determined from 15min to 6h after labeling. The in vitro stability of ~(188)Re-k-ras-AGPNA was analyzed by using human serum or sodium chloride as medium, and the labeling efficiency was determined from 2 to 24h after labeling. The total binding efficiency of ~(188)Re-k-ras-AGPNA with pancreatic cancer cell Patu8988 and the bound of ~(188)Re-k-ras- AGPNA on the cellular nucleus were determined at various time points. Results The optimum labeling conditions were 100μL stannous chloride (20mg/mL) dissolved in sodium gluconate, and 20μL k-ras-AGPNA (2mg/mL), 100μL ~(188)Re perrhenate, the pH about 7.0, and incubated 30 min at room temperature. The labeling efficiency of ~(188)Re-k-ras-AGPNA could reach (89.61±0.91)% and the amount of radiocolloid was (9.40±0.55)%. The labeling efficiency was (89.14±0.63)% after incubated for 24h in human serum. The highest total binding efficiency of ~(188)Re-k-ras-AGPNA with Patu8988 cell was (38.16±2.17)%, and the highest binding efficiency on the nucleus was increased to (22.41±0.86)% at the 24th hour. Tumor in nude mice could be seen clearly in scintigraphy images until 7d after injection. Conclusion This method of labeling k-ras-AGPNA with ~(188)Re is stable and high labeling efficiecy can be obtained. ~(188)Re-k-ras-AGPNA can bind with the pancreatic cancer cell Patu8988 and penetrated into the Patu8988 cellular nucleus.
3. Inducing apoptosis effect on Patu-8988 cell and biodistribution characteristics of the ~(188)Re-k-ras-AGPNA in nude mice bearing xenografts
Objective To investigate the inducing apoptosis effect on Patu8988 cell and biodistribution characteristics, determine the dosimetry in nude mice bearing xenografts of ~(188)Re-k-ras-AGPNA. Methods (1) At 3d to 5d after treatment with Patu8988 cell in culture flask with 925KBq ~(188)ReO_4~- or ~(188)Re-k-ras-AGPNA, the cell’s apoptosis was detected everyday by flow cytometry. (2) 28 nude mice model bearing human pancreatic carcinoma cell Patu8988 xenografts were established. At 15min, 1, 4, 24, 72, 120 and 148h after intratumor injection with ~(188)Re-k-ras-AGPNA, the mice were killed and organs of interest were excised, weighted and counted on a gamma counter. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/g) and the ratios of tumor to normal tissue (T/NT) were calculated. (3) Scintigraphy imaging was performed for each nude mice at 15min after intratumor injection with radiopharmaceuticals, in which serial planar scintigraphy imaging were performed for 6 nude mice at 15min, 1, 4, 24, 72, 120 and 148h after intratumor injection with ~(188)Re-k-ras-AGPNA. (4) According the mean %ID/g of organs in different time point, the absorbed dose of organs were calculated. Results (1)At 3d to 5d after treated with ~(188)ReO_4~- and ~(188)Re-k-ras-AGPNA, float cell ratio were (5.68±0.82)%, (8.14±0.12)%, (11.87±0.17)% and (5.99±3.59)%, (25.66±8.51)%, (29.59±4.92)%, respectively. Float cell’s apoptosis ratios of ~(188)ReO_4~- and ~(188)Re-k-ras- AGPNA group were (3.88±2.10)%, (8.75±3.11)%, (16.87±5.85)%, and (5.28±1.12)%, (26.30±7.45)%, (27.90±10.38)%, respectively. (2) ~(188)Re-k-ras-AGPNA was major distributed in tumors and the largest uptake of tumors was (53.23±16.64)% at 15min after injection. The uptake of tumor were (35.96±7.80)% and (15.46±4.93)% at 24h and 148h after injection, respectively. (3) Tumor in nude mice could be seen clearly in scintigraphy images until 148h after intratumoral injection with ~(188)Re-k-ras-AGPNA. (4) The absorbed dose of tumor was 15569mGy/MBq. Conclusion ~(188)Re-k-ras-AGPNA was major distributed in tumors after intratumor injection in nude mice bearing human pancreatic cancer xenografts, and we propose that ~(188)Re-k-ras-AGPNA may be a potential tumor transarterial radionuclide therapeutic agent used in clinic.
PartⅢThe biodistribution characteristics of ~(188)Re-tin sulfur colloid after transhepatic arterial embolization in rabbits bearing VX2 liver tumor
Objective To investigate the method for labeling tin sulfur colliod (TSC) with rhenium-188 and its biodistribution after transhepatic arterial embolization in rabbits bearing VX2 liver tumor. Methods The direct method was adopted to label TSC or macroaggregated albumin(MAA) with ~(188)Re-perrhenate, and labeling efficiency was determined at the different time point. The in vitro stabilities of ~(188)Re-TSC and ~(188)Re-MAA were analyzed by in the medium of sodium chloride or human serum (v:v=1:10), and the labeling efficiency was determined by Xinhua 1# paper chromatography (PC) analysis from 30min to 72h after added to medium. Particle size of ~(188)Re-TSC was measured (n=32) by laser scatterance/diffraction method used a Mastersizer 2000. 31 rabbits bearing VX2 liver tumor were performed transcatherter hepatic arterial injection of ~(188)Re-TSC (n=12), ~(188)Re-MAA (n=15), and Na~(188)ReO_4~- solution (n=4), respectively. Planar scans with IRIX3 SPECT (PHILIPS, Holander) were obtained at 1 and 24h after administration. The biodistribution characteristics of ~(188)Re-TSC, ~(188)Re-MAA, and ~(188)Re-perrhenate in rabbits at 1 and 24h after administration were evaluated. Organs were excised, washed with saline, weighed, and counted on a gamma counter. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/g). Tumor retention was calculated and compared between ~(188)Re-TSC and ~(188)Re-MAA. Results The labeling efficiencies of ~(188)Re-TSC and ~(188)Re-MAA were reached (99.94±0.04)% and (99.95±0.03)%, and radiopharmaceuticals were stable for 72h in human serum. The particle size of ~(188)Re-TSC was 10.83±6.60μm (D10), 44.91±14.46μm (D50), and 235.29±126.61μm (D90), respectively. In 17 of the 27 rabbits, TAE was performed successfully. The radioactive ratio of tumor/liver in ROI of SPECT images was 2.15±0.80 and the tumor can be seen clearly in planar imaging. Tumor uptake of ~(188)Re-TSC at 1h and 24h were (24.32±11.93)% and (21.88±18.29)%, and the radioactive ratio of tumor/liver were 70.89±19.58 and 17.42±13.96, respectively. Tumor uptake of ~(188)Re-MAA at 1h and 24h were (38.78±30.23)% and (15.98±26.64)%, and the radioactive ratio of tumor/liver were 39.71±25.06 and 8.13±4.61, respectively. The retention ratio of 24h to 1h of ~(188)Re-TSC in tumor was 89.95%, and it was 41.22% of ~(188)Re-MAA. Conclusion ~(188)Re-TSC may be a potential radiopharmaceutical for the therapy of tumors.
目的研究标记率高且稳定的~(188)Re标记胰岛素样生长因子-1类似物(insulin-like growth factor 1 analogue, IGF-1A)的方法学。方法1.采用直接标记法标记经修饰的IGF-1A,改变标记条件:Tween80的用量从2～10μL、SnCl_2.2H_2O浓度变化从0.75～25mg/mL、IGF-1A的用量从20～100μg、淋洗液的体积从10～500μL;分别在不同时间点测定标记率。2.标记物中加入生理盐水或人血清后不同时间点测定其标记率。结果1.最佳~(188)Re标记方法为:将100μLSnCl_2.2H_2O(10mg/mL)加入50μL IGF-1A(2mg/mL);300μL Na_3PO_.4和10μL0.1%Tween80混匀后加入50μL ~(188)ReO_4~-新鲜洗脱液;在IGF-1A体系中加入洗脱液体系,混匀,室温下反应30min;加入500μL NaH_2PO_4将pH值调节到7.0左右,标记完成。最高标记率为(94.07±0.32)%,胶体含量为(5.50±1.50)%。2.室温下放置6h标记率为(85.50±1.21)%,加入人血清放置24h后标记率为(76.57±9.96)%。结论用直接标记法进行~(188)Re标记IGF-1A,标记率高且稳定性良好。
二、~(188)Re-IGF-1A与胰腺癌细胞结合及细胞杀伤效应实验
目的研究~(188)Re-IGF-1A与胰腺癌细胞的特异性结合及其对胰腺癌细胞的杀伤效果。方法1.测定~(188)Re-IGF-1A与胰腺癌Patu8988细胞的结合率。2.用MTT实验检测给药后细胞增殖情况,将1×10~4/孔细胞接种于96孔板上,细胞分为对照组、IGF-1A组(1、5、10、20μg)、~(188)ReO_4~-组(0.37、1.85、3.70、7.40MBq)和~(188)Re-IGF-1A组(0.37、0.74、1.85MBq),另设空白调零组。3.~(188)ReO_4~-组和~(188)Re-IGF-1A组接种后,分别在给药后1～7d,每天采用MTT比色法测定其OD值,IGF-1A组分别在给药后1～6d,每天采用MTT比色法测定其OD值。根据各组OD值,计算生存率和抑制率。4.将细胞接种于培养瓶中,分~(188)ReO_4~-组和~(188)Re-IGF-1A组(1.85、3.70、7.40MBq),在给药后3d用流式细胞仪检测细胞凋亡率。结果1.~(188)Re-IGF-1A与5×10~6个/50μL胰腺癌细胞的最高总结合率为(24.13±2.03)%,特异性结合为12.68%。2.加入不同剂量~(188)Re-IGF-1A后,在1～3d内,随时间增加而抑制率增高,第3d时0.37、0.74、1.85MBq组抑制率分别为(64.48±4.18)%、(66.89±1.39)%和(89.71±1.27)%;第6天时,1.85MBq组抑制率达到(93.20±1.93)%。3.加入与~(188)Re-IGF-1A同量的IGF-1A或相同放射性活度的~(188)ReO_4~-后,~(188)Re-IGF-1A对细胞的抑制率在各时间点均高于单独用~(188)ReO_4~-,两者差异有统计学意义(P<0.05);与IGF-1A组比较,第1d差异无统计学意义,在第2～6d,~(188)Re-IGF-1A对细胞的抑制率在各时间点均高于单独用IGF-1A,两者差异有统计学意义。4.给药1.85、3.70、7.40MBq3d后,~(188)ReO_4~-组和~(188)Re-IGF-1A组细胞漂浮率分别为(16.58±3.57)%、(24.58±6.50)%、(34.12±7.39)%和(16.56±0.95)%、(33.39±5.93)%、(43.76±1.38)%,漂浮细胞的凋亡率分别为(9.27±1.80)%、(16.00±1.15)%、(15.47±0.65)%和(12.70±2.27)%、(17.80±1.51)%、(23.23±1.22)%。结论~(188)Re-IGF-1A能与胰腺癌Patu8988细胞特异性结合,并对胰腺癌细胞生长有明显的抑制作用,可诱导细胞凋亡。
三、~(188)Re-IGF-1A在荷瘤裸鼠体内分布、显像和吸收剂量估算
目的研究~(188)Re-IGF-1A在荷人胰腺癌裸鼠体内的分布、显像和剂量估算。方法1.建立荷人胰腺癌Patu8988裸鼠模型。2.将66只荷人胰腺癌裸鼠随机分2组,~(188)Re-IGF-1A组36只,~(188)ReO_4~-组30只。采取瘤内注射给药方式,注射~(188)Re-IGF-1A或~(188)ReO_4~- 0.03mL,同时取0.05mL~(188)ReO_4~-洗脱液做标准源。在瘤内注射后即刻行SPECT平面显像,通过裸鼠全身与标准源的ROI计数比值,换算每个荷瘤裸鼠总注射剂量。3.~(188)Re-IGF-1A组于注射后15min、1、4h、1、3、5d显像并处死裸鼠。~(188)ReO_4~-组于注射后15min、1、2、4、24h显像并处死裸鼠。解剖后取所需组织称量并测量其60s的放射性计数,计算各组织的每克组织百分注射剂量率(%ID/g),并计算肿瘤与血、心、脑、甲状腺、肝、脾、肺、肾、胃、肠、胰、骨、肌肉的比值(T/NT)。4.根据各组织不同时间点的平均%ID/g,通过放射性药物内照射剂量计算软件计算出吸收剂量(mGy/MBq)。结果1.瘤内注射~(188)Re-IGF-1A后肿瘤、肾脏、肝脏、脾脏内放射性较高,注射后15min肿瘤摄取为(39.30±17.98)%,肾脏摄取为(19.77±10.75)%,肝脏摄取为(2.03±2.28)%,脾脏摄取为(1.04±0.92)%。瘤内注射后1、4h、1、3、5d肿瘤内摄取分别为(35.03±23.37)%、(41.22±23.88)%、(10.59±9.39)%、(5.32±1.53)%、(5.30±2.28)%;肿瘤部位放射性积聚量4h内差异无统计学意义(P>0.05),且随时间延长,肿瘤与其它脏器的T/NT呈上升趋势,肿瘤与肾脏的T/NT在5d时达到5.64±7.50。肿瘤/肌肉在5d时达到2610.65±1534.02。2.瘤内注射~(188)ReO_4~-后,在体内开始主要分布于甲状腺、胃、肿瘤、血液。胃内放射性高峰出现在1h,为(23.14±12.08)%,甲状腺放射性高峰出现在2h,为(100.27±19.67)%,随时间延长,肿瘤部位计数迅速下降。肿瘤部位在24h摄取为(0.09±0.03)%,与肾脏差异无统计学意义(P=0.27)。3.在瘤内注射后各时间点,~(188)Re-IGF-1A组肿瘤及肾脏内摄取比~(188)ReO_4~-组高,两者有统计学差异(P<0.01)。两组肿瘤内摄取比值在24h达到117.67倍。4.瘤内注射~(188)Re-IGF-1A后,SPECT平面显像见瘤内浓聚,5d时仅见肿瘤部位显影。5.肿瘤内吸收剂量为5165.8mGy/MBq。结论瘤内注射~(188)Re-IGF-1A后,在肿瘤部位积聚较高,可望作为胰腺癌经动脉灌注治疗的药物,并能在体外观察其分布。
第二部分~(188)Re标记反基因肽核酸(AGPNA)的实验研究
一、k-ras-AGPNA抑制k-ras基因表达实验
目的探索自主设计的与胰腺癌k-ras点突变基因特异性结合的反基因肽核酸(antigene peptide nucleic acid, AGPNA)的生物活性。方法1.设计并合成与胰腺癌k-ras点突变基因特异性结合的k-ras-AGPNA。2.应用RT-PCR检测转染k-ras-AGPNA前后胰腺癌Patu8988细胞k-ras癌基因的mRNA表达水平,分对照组、k-ras-AGPNA组、脂质体转染k-ras-反基因寡核苷酸(antigene oligonucleotides, AGON)组、脂质体转染k-ras-反义寡核苷酸(antisense oligonucleotides, ASON)组。3.应用流式细胞仪测定转染k-ras-AGPNA前后胰腺癌Patu8988细胞的k-ras蛋白表达水平。结果1.转染1nmol/mL k-ras-AGPNA组和k-ras-AGON组的细胞的k-ras突变基因mRNA的灰度比分别为(1.00±0.39)和(1.22±0.31),比对照组(1.86±0.07)低,差异有显著统计学意义(P<0.01)。k-ras-AGPNA、k-ras-AGON和k-ras-ASON组内不同剂量之间差异无统计学意义(P>0.05)。2.转染1nmol/mL k-ras-AGPNA、k-ras-AGON和k-ras-ASON组的细胞其k-ras蛋白表达分别为(15.05±5.07)%、(10.20±2.63)%、(8.80±4.31)%,比对照组(24.38±5.40)%低,差异有显著统计学意义(P<0.01)。k-ras-AGPNA、k-ras-AGON和k-ras-ASON组内不同剂量之间差异无统计学意义(P>0.05)。结论自主设计并合成的k-ras-AGPNA能抑制k-ras基因在mRNA和蛋白水平的表达。
二、~(188)Re-k-ras-AGPNA的制备及其胰腺癌细胞内化实验研究
目的制备~(188)Re-k-ras-AGPNA并研究其与胰腺癌Patu8988细胞结合的特性。方法1.用~(188)Re直接标记经修饰的k-ras-AGPNA。2.预实验中其他标记条件不变,将SnCl2.2H2O浓度改为20mg/mL,k-ras-AGPNA的用量改为20μL(40μg)后分别进行实验。3.在标记后不同时间点15、30min、1、2、4、6h测定标记率。4.测定标记物在人血清和生理盐水中5min～24h不同时间点的标记率。5.胰腺癌Patu8988细胞摄取~(188)Re-k-ras-AGPNA的内化实验。结果1.~(188)Re-k-ras-AGPNA的标记率最高为(89.61±0.91)%,放射性胶体含量为(9.40±0.55)%。2.~(188)Re-k-ras-AGPNA放置血清中24h后标记率为(89.14±0.63)%,放置生理盐水中1h后标记率为(79.12±5.26)%。3.~(188)Re-k-ras-AGPNA转染胰腺癌Patu8988细胞,最高细胞结合率为(38.16±2.17)%,最高核内化率为(22.41±0.86)%。结论~(188)Re直接标记k-ras-AGPNA方法简便、标记率较高,能与胰腺癌细胞结合并转染入细胞核。
三、~(188)Re-k-ras-AGPNA诱导细胞凋亡和荷瘤裸鼠体内分布实验
目的研究~(188)Re-k-ras-AGPNA的诱导凋亡效应和在荷人胰腺癌裸鼠体内的分布、显像和剂量估算。方法1.胰腺癌Patu8988细胞给予~(188)Re-k-ras-AGPNA和~(188)ReO_4~- 3～5d后,用流式细胞仪检测细胞凋亡率。2.将28只荷人胰腺癌裸鼠分7组,每组4只。采取瘤内注射给药方式,注射~(188)Re-k-ras-AGPNA 0.03mL。3.注射后15min、1、4h、1、3、5、7d显像并处死裸鼠,解剖后取所需组织称量并测量其60s的放射性计数,计算各组织的%ID/g和肿瘤与其他组织的比值(T/NT),并根据各组织不同时间点的平均%ID/g计算出吸收剂量(mGy/MBq)。结果1.给药3、4、5d后,~(188)ReO_4~-组和~(188)Re-k-ras-AGPNA组细胞漂浮率分别为(5.68±0.82)%、(8.14±0.12)%、(11.87±0.17)%和(5.99±3.59)%、(25.66±8.51)%、(29.59±4.92)%,漂浮细胞的凋亡率分别为(3.88±2.10)%、(8.75±3.11)%、(16.87±5.85)%和(5.28±1.12)%、(26.30±7.45)%、(27.90±10.38)%。2.瘤内注射~(188)Re-k-ras-AGPNA后肿瘤、肾脏、肺、甲状腺、肝脏内放射性较高,注射后15min肿瘤摄取为(53.23±16.64)%,肾脏摄取为(12.60±1.53)%,肺内摄取为(2.32±1.35)%,甲状腺摄取为(1.95±1.22)%,肝脏摄取为(1.78±0.63)%。瘤内注射后1、4h、1、3、5、7d肿瘤内摄取分别为(37.47±21.31)%、(34.84±6.46)%、(35.96±7.80)%、(17.43±5.46)%、(11.09±3.52)%、(15.46±4.93)%;肿瘤部位放射性积聚量24h内差异无统计学意义(P>0.05),且随时间延长,肿瘤与肾脏的T/NT呈上升趋势,在7d时达到11.08±9.22。肿瘤/肌肉在3d时达到870.86±1079.02。3.瘤内注射~(188)Re-k-ras-AGPNA后,SPECT平面显像见瘤内浓聚,7d时仅见肿瘤部位显影。4.肿瘤内吸收剂量为15569mGy/MBq。结论瘤内注射~(188)Re-k-ras-AGPNA后,肿瘤部位摄取高、清除缓慢,肿瘤吸收剂量高,可望作为胰腺癌治疗的药物。
第三部分~(188)Re-锡硫胶体介入治疗荷VX2肝癌兔的体内生物分布研究
目的研究~(188)Re-锡硫胶体(tin sulfur colloid,TSC)的制备及其经肝动脉灌注给药后在兔VX2肝肿瘤模型体内的生物学分布特征,探讨其作为放射性介入治疗剂的可行性。方法1.改变标记条件制备~(188)Re-TSC和~(188)Re-聚合白蛋白(Macroaggregated albumin, MAA),检测标记物在人血清中的稳定性,测定~(188)Re-TSC的颗粒大小。2.建立32只VX2肝肿瘤模型,均经CT或DSA证实,1例行病理学检查。3.31只荷瘤兔分~(188)Re-TSC(n=12)、~(188)Re-MAA(n=15)和~(188)ReO4-(n=4)3组,将放射性药物经肝动脉注射入肿瘤内,分别在注射后15min、1h和24h分别进行SPECT平面显像,画感兴趣区进行肿瘤/肝脏计数的半定量分析。4.在注射后1h和24h处死模型兔后取感兴趣脏器进行称量、计数,计算各脏器的每克组织百分注射剂量。5.根据灌注~(188)Re-TSC后肿瘤内%ID/g进行吸收剂量估算。结果1.~(188)Re-TSC和~(188)Re-MAA标记率最高分别为(99.94±0.04)%和(99.95±0.03)%,在人血清中放置72h后分别为(95.77±0.54)%和(92.13±1.21)%。~(188)Re-TSC的颗粒大小分布为10.83±6.60μm (D10)、44.91±14.46μm (D50)和235.29±126.61μm (D90)。2.在平面显像中,62.96%(17/27)模型兔的肿瘤显像清晰,肿瘤/肝脏感兴趣区放射性计数的比值为2.15±0.80。3.~(188)Re-TSC组中肿瘤摄取在1h和24h分别为(24.32±11.93)%和(21.88±18.29)%,肿瘤与肝脏的放射性比值在1h和24h分别为70.89±19.58和17.42±13.96。4.~(188)Re-MAA组中肿瘤摄取在1h和24h分别为(38.78±30.23)%和(15.98±26.64)%,肿瘤与肝脏的比值在1h和24h分别为39.71±25.06和8.13±4.61。5.灌注~(188)Re-TSC后肿瘤内24h的放射吸收剂量为0.24 Gy/MBq。结论~(188)Re-TSC可望成为有临床应用前景的放射介入治疗药物。
PartⅠradiosynthesis and biological experiment of ~(188)Re-IGF-1A
Background The insulin-like growth factor-1 receptor (IGF-1R) is a cellular receptor found to be overexpressed in many tumor cell lines from different anatomical sites. It can be used as molecular targets by which radiolabeled insulin-like growth factor 1 analogue (IGF-1A) can localize cancers for peptide receptor radiation therapy.
1. Investigations of labeling insulin-like growth factor 1 analogue with ~(188)Re
Objective To establish a useful method for labeling IGF-1A with ~(188)Re. Methods Direct labeling method was adopted to label IGF-1A. Several labeling conditions were tested. The volume of Tween80 was changed from 2 to 10μL; the concentration of SnCl_2.2H_2O was changed from 0.75 to 25mg/mL; the amount of IGF-1A was changed from 20 to 100μg and the volume of ~(188)Re perrhenate was changed from 10 to 500μL. The labeling efficiency was analyzed from 15min to 8h after labeling. The in vitro stabilities of ~(188)Re-IGF-1A was analyzed in human serum or sodium chloride medium, and the labeling efficiency was determined from 2 to 24h after adding the medium. Results The optimum labeling condition was 100μL stannous chloride (10mg/mL) dissolved in sodium gluconate, 50μL IGF-1A (2mg/mL), 300μL Na3PO4, 10μL 0.1% Tween80, 50μL ~(188)Re perrhenate added and the incubation time was 30 min at room temperature, then added 500μL NaH_2PO_4 to adjust the pH about 7.0. The labeling efficiency of ~(188)Re-IGF-1A could reach (94.07±0.32)% and the amount of radiocolloid was (5.50±1.50)%. It was (85.50±1.21)% after incubation for 6h at room temperature, and was (76.57±9.96)% after incubation for 24h with human serum. Conclusion This method of labeling IGF-1A with ~(188)Re using SnCl_2.2H_2O is stable and high labeling efficiency can be obtained.
2. Experimental study of ~(188)Re-IGF-1A on cell combination and proliferation inhibition effect to pancreatic carcinoma cell
Objective To investigate cell combination and proliferation inhibition effect of ~(188)Re-IGF-1A to Patu8988 human pancreatic carcinoma cell. Methods (1)The combinative efficiency of ~(188)Re-IGF-1A with Patu8988 cell was determined. (2)Patu8988 cell were seeded onto 96-well plate and divided into blank control group, IGF-1A group (1, 5, 10, 20μg), ~(188)ReO4- group (0.37, 1.85, 3.70, 7.40MBq) and ~(188)Re-IGF-1A group (0.37, 0.74, 1.85MBq). (3)The proliferation inhibition effect of ~(188)Re-IGF-1A group and ~(188)ReO4- group on cell growth was detected every day by MTT test from 1d to 7d after administration. The proliferation inhibition effect of IGF-1A group was detected every day from 1d to 6d, and inhibition rates were calculated. (4) At 3d after treatment with ~(188)ReO4- and ~(188)Re-IGF-1A (1.85, 3.70, 7.40 MBq), the cell’s apoptosis was detected by flow cytometry. Results (1)The total combinative efficiency of ~(188)Re-IGF-1A with Patu8988 cell was (24.13±2.03)%, and the special combinative efficiency was 12.68%. (2) Inhibition rates of Patu8988 cell with ~(188)Re-IGF-1A group (0.37, 0.74, 1.85MBq) were (64.48±4.18)%,(66.89±1.39)% and (89.71±1.27)% after 3d, and the inhibition rates was higher when the radioactive dose higher. After 6d, inhibition rate of Patu8988 cell with ~(188)Re-IGF-1A group (1.85MBq) was (93.20±1.93)%. (3) Inhibition rates of Patu8988 cell with ~(188)Re-IGF-1A group were always higher than ~(188)ReO4- group and IGF-1A group. The difference was significant (P < 0.05). (4) At 3d after treated with ~(188)ReO4- and ~(188)Re-IGF-1A, float cell ratio were (16.58±3.57)%, (24.58±6.50)%, (34.12±7.39)% and (16.56±0.95)%, (33.39±5.93)%, (43.76±1.38)%, respectively. Float cell’s apoptosis ratio of Patu8988 cell with ~(188)ReO4- and ~(188)Re-IGF-1A group were (9.27±1.80)%, (16.00±1.15)%, (15.47±0.65)% and (12.70±2.27)%, (17.80±1.51)%, (23.23±1.22)%, respectively. Conclusion Proliferation of human pancreatic carcinoma cell Patu8988 can be inhibited and apoptosis can be promoted with ~(188)Re-IGF-1A.
3. Biodistribution characteristics and dosimetry measurement of ~(188)Re-IGF-1A in nude mice bearing the Patu8988 human pancreatic cancer xenografts
Objective To investigate the biodistribution characteristics and measure dosimetry of ~(188)Re-IGF-1A in nude mice bearing human pancreatic cancer xenografts. Methods (1)66 nude mice model bearing human pancreatic carcinoma cell Patu8988 xenografts were established and devided into the ~(188)Re-IGF-1A group (n=36) and the ~(188)ReO4- control group (n=30). (2)In the ~(188)Re-IGF-1A group, at 15min, 1, 4, 24, 72, and 120h after intratumor injection with ~(188)Re-IGF-1A, the mice were killed and the organs of interest were excised, weighted and counted on a gamma counter. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/g) and the ratios of tumor to normal tissue (T/NT) were calculated. (3) In the ~(188)ReO_4~- control group, organs of interest were excised and counted. %ID/g and T/NT were calculated at 15min, 1, 2, 4 and 24h after intratumor injection. (4) Scintigraphy imaging for each nude mouse was performed at 15min after intratumor injection with radiopharmaceuticals,in which serial planar scintigraphy imaging were performed for 6 nude mice at 15min, 1, 4, 24, 72, and 120h after intratumor injection with ~(188)Re-IGF-1A. (5) Radiation dosimetry in organs of interest were estimated on the basis of the mean %ID/g. Results (1) ~(188)Re-IGF-1A was major distributed in tumors and the largest uptake of tumors was (41.22±23.88)% at 4h after injection. The uptake of tumor were (10.59±9.39)% and (5.30±2.28)% at 24h and 120h after injection, respectively. (2)~(188)ReO_4~- was major distributed in thyroid glands, stomachs, tumors and blood in nude mice after injection at first. Then uptake of tumor decreased rapidly, but slowly in stomachs and thyroid glands. The uptake of tumor were (0.09±0.03)% at 24h after injection. (3) The tumor in nude mice could be seen clearly in scintigraphy images until 120h after intratumoral injection with ~(188)Re-IGF-1A. Conclusion ~(188)Re-IGF-1A was major distributed in tumors after intratumor injection in nude mice bearing human pancreatic cancer xenografts, and we propose that ~(188)Re-IGF-1A may be a potential tumor transarterial radionuclide therapeutic agent used in clinic.
PartⅡradiosynthesis and biological experiment of ~(188)Re-k-ras-AGPNA
Background We hypothesized that antigene peptide nucleic acid (AGPNA) hybridization probes targeted k-ras 12 point mutation oncogene, with an IGF-1A peptide loop on the C-terminus and ~(188)Re chelator peptide on the N-terminus, could hybridize with k-ras 12 point mutation oncogene and decrease the multiplication of pancreatic cancer cell Patu8988.
1. Experiment of k-ras-AGPNA on depress the k-ras gene expression in human pancreatic cancer cell Patu8988
Objective To investigate the effect of AGPNA on depress the k-ras gene expression and the k-ras protein expression of human pancreatic cancer cell Patu8988. Methods (1)AGPNA hybridizing k-ras 12 point mutation DNA was designed and synthesized. (2)Before and after transfected AGPNA, the expression level of k-ras mRNA in human pancreatic cancer cell Patu8988 were detected by reverse transcription- polymerase chain reaction (RT-PCR). (3)The expression ratio of k-ras protein in cell were detected by flow cytometry. Results (1)After transfected 1nmol/mL AGPNA and antigene oligonucleotides (AGON), the k-ras mRNA gray scale ratio were (1.00±0.39) and (1.22±0.31). Its was lower than (1.86±0.07) of the control group, and the difference was significant (P<0.01). Among the difference dose of AGPNA, AGON, and antisense oligonucleotides (ASON) group, the difference of the expression level of k-ras mRNA was not significant (P>0.05). (2)After transfected 1nmol/mL AGPNA, AGON, and ASON, the expression ratios of k-ras protein were (15.05±5.07)%, (10.20±2.63)%, and (8.80±4.31)%. Its was lower than (24.38±5.40)% of the control group, and the difference was significant (P<0.01). Compared with the difference dose of AGPNA, AGON, and ASON group, the difference of the expression ratios of k-ras protein was no significant (P>0.05). Conclusion AGPNA can depress the k-ras gene expression on the mRNA level and depress the k-ras protein expression of human pancreatic cancer cell Patu8988.
2. Radiosynthesis of ~(188)Re labeled k-ras-AGPNA for binding with pancreatic carcinoma cell
Objective To establish a method for labeling k-ras-AGPNA with ~(188)Re, and investigate its binding with pancreatic cancer cell Patu8988. Methods The directly labeling method was adopted, and several labeling conditions were tested, such as the concentration of SnCl2.2H2O and the amount of k-ras-AGPNA. The labeling efficiency was determined from 15min to 6h after labeling. The in vitro stability of ~(188)Re-k-ras-AGPNA was analyzed by using human serum or sodium chloride as medium, and the labeling efficiency was determined from 2 to 24h after labeling. The total binding efficiency of ~(188)Re-k-ras-AGPNA with pancreatic cancer cell Patu8988 and the bound of ~(188)Re-k-ras- AGPNA on the cellular nucleus were determined at various time points. Results The optimum labeling conditions were 100μL stannous chloride (20mg/mL) dissolved in sodium gluconate, and 20μL k-ras-AGPNA (2mg/mL), 100μL ~(188)Re perrhenate, the pH about 7.0, and incubated 30 min at room temperature. The labeling efficiency of ~(188)Re-k-ras-AGPNA could reach (89.61±0.91)% and the amount of radiocolloid was (9.40±0.55)%. The labeling efficiency was (89.14±0.63)% after incubated for 24h in human serum. The highest total binding efficiency of ~(188)Re-k-ras-AGPNA with Patu8988 cell was (38.16±2.17)%, and the highest binding efficiency on the nucleus was increased to (22.41±0.86)% at the 24th hour. Tumor in nude mice could be seen clearly in scintigraphy images until 7d after injection. Conclusion This method of labeling k-ras-AGPNA with ~(188)Re is stable and high labeling efficiecy can be obtained. ~(188)Re-k-ras-AGPNA can bind with the pancreatic cancer cell Patu8988 and penetrated into the Patu8988 cellular nucleus.
3. Inducing apoptosis effect on Patu-8988 cell and biodistribution characteristics of the ~(188)Re-k-ras-AGPNA in nude mice bearing xenografts
Objective To investigate the inducing apoptosis effect on Patu8988 cell and biodistribution characteristics, determine the dosimetry in nude mice bearing xenografts of ~(188)Re-k-ras-AGPNA. Methods (1) At 3d to 5d after treatment with Patu8988 cell in culture flask with 925KBq ~(188)ReO_4~- or ~(188)Re-k-ras-AGPNA, the cell’s apoptosis was detected everyday by flow cytometry. (2) 28 nude mice model bearing human pancreatic carcinoma cell Patu8988 xenografts were established. At 15min, 1, 4, 24, 72, 120 and 148h after intratumor injection with ~(188)Re-k-ras-AGPNA, the mice were killed and organs of interest were excised, weighted and counted on a gamma counter. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/g) and the ratios of tumor to normal tissue (T/NT) were calculated. (3) Scintigraphy imaging was performed for each nude mice at 15min after intratumor injection with radiopharmaceuticals, in which serial planar scintigraphy imaging were performed for 6 nude mice at 15min, 1, 4, 24, 72, 120 and 148h after intratumor injection with ~(188)Re-k-ras-AGPNA. (4) According the mean %ID/g of organs in different time point, the absorbed dose of organs were calculated. Results (1)At 3d to 5d after treated with ~(188)ReO_4~- and ~(188)Re-k-ras-AGPNA, float cell ratio were (5.68±0.82)%, (8.14±0.12)%, (11.87±0.17)% and (5.99±3.59)%, (25.66±8.51)%, (29.59±4.92)%, respectively. Float cell’s apoptosis ratios of ~(188)ReO_4~- and ~(188)Re-k-ras- AGPNA group were (3.88±2.10)%, (8.75±3.11)%, (16.87±5.85)%, and (5.28±1.12)%, (26.30±7.45)%, (27.90±10.38)%, respectively. (2) ~(188)Re-k-ras-AGPNA was major distributed in tumors and the largest uptake of tumors was (53.23±16.64)% at 15min after injection. The uptake of tumor were (35.96±7.80)% and (15.46±4.93)% at 24h and 148h after injection, respectively. (3) Tumor in nude mice could be seen clearly in scintigraphy images until 148h after intratumoral injection with ~(188)Re-k-ras-AGPNA. (4) The absorbed dose of tumor was 15569mGy/MBq. Conclusion ~(188)Re-k-ras-AGPNA was major distributed in tumors after intratumor injection in nude mice bearing human pancreatic cancer xenografts, and we propose that ~(188)Re-k-ras-AGPNA may be a potential tumor transarterial radionuclide therapeutic agent used in clinic.
PartⅢThe biodistribution characteristics of ~(188)Re-tin sulfur colloid after transhepatic arterial embolization in rabbits bearing VX2 liver tumor
Objective To investigate the method for labeling tin sulfur colliod (TSC) with rhenium-188 and its biodistribution after transhepatic arterial embolization in rabbits bearing VX2 liver tumor. Methods The direct method was adopted to label TSC or macroaggregated albumin(MAA) with ~(188)Re-perrhenate, and labeling efficiency was determined at the different time point. The in vitro stabilities of ~(188)Re-TSC and ~(188)Re-MAA were analyzed by in the medium of sodium chloride or human serum (v:v=1:10), and the labeling efficiency was determined by Xinhua 1# paper chromatography (PC) analysis from 30min to 72h after added to medium. Particle size of ~(188)Re-TSC was measured (n=32) by laser scatterance/diffraction method used a Mastersizer 2000. 31 rabbits bearing VX2 liver tumor were performed transcatherter hepatic arterial injection of ~(188)Re-TSC (n=12), ~(188)Re-MAA (n=15), and Na~(188)ReO_4~- solution (n=4), respectively. Planar scans with IRIX3 SPECT (PHILIPS, Holander) were obtained at 1 and 24h after administration. The biodistribution characteristics of ~(188)Re-TSC, ~(188)Re-MAA, and ~(188)Re-perrhenate in rabbits at 1 and 24h after administration were evaluated. Organs were excised, washed with saline, weighed, and counted on a gamma counter. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/g). Tumor retention was calculated and compared between ~(188)Re-TSC and ~(188)Re-MAA. Results The labeling efficiencies of ~(188)Re-TSC and ~(188)Re-MAA were reached (99.94±0.04)% and (99.95±0.03)%, and radiopharmaceuticals were stable for 72h in human serum. The particle size of ~(188)Re-TSC was 10.83±6.60μm (D10), 44.91±14.46μm (D50), and 235.29±126.61μm (D90), respectively. In 17 of the 27 rabbits, TAE was performed successfully. The radioactive ratio of tumor/liver in ROI of SPECT images was 2.15±0.80 and the tumor can be seen clearly in planar imaging. Tumor uptake of ~(188)Re-TSC at 1h and 24h were (24.32±11.93)% and (21.88±18.29)%, and the radioactive ratio of tumor/liver were 70.89±19.58 and 17.42±13.96, respectively. Tumor uptake of ~(188)Re-MAA at 1h and 24h were (38.78±30.23)% and (15.98±26.64)%, and the radioactive ratio of tumor/liver were 39.71±25.06 and 8.13±4.61, respectively. The retention ratio of 24h to 1h of ~(188)Re-TSC in tumor was 89.95%, and it was 41.22% of ~(188)Re-MAA. Conclusion ~(188)Re-TSC may be a potential radiopharmaceutical for the therapy of tumors.
引文
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1.张学宏,高玉堂.胰腺癌的流行病学.胰腺病学, 2005, 5(3): 180-183.
2. Yang GY, Wagner TD, Fuss M, et al. Multimodality approaches for pancreatic cancer. CA Cancer J Clin 2005, 352-367.
3. Lowenfels AB, Maisonneuve P. Epidemiology and prevention of pancreatic cancer. Jpn J Clin Oncol, 2004, 34(5): 238-244.
4. Howard A Reber. Pancreatic cancer-pathogenesis, diagnosis, and treatment.世界图书出版西安公司,西安,1999.
5.詹文华.胰腺癌基因诊断及研究的最近进展.国外医学.外科学分册, 1995, 1: 4-7.
6.杨尹默,黄筵庭,刘强. ras基因与胰腺肿瘤.中华医学杂志, 1994, 74(6): 386-388.
7.黄筵庭,杨尹默,刘强.胰腺癌组织中K-ras基因点突变的研究.中华医学杂志, 1994, 74(2): 77-79.
8.于观贞.胰腺癌的分子变化基础及分子诊断.国外医学.生理病理科学与临床分册, 2003, 23(1): 64-66.
9.Guerrero S, Casanova I, Farre L, et al. K-ras coden 12 mutation induces higher level of resistance to apoptosis and predisposition to anchorage-independent growth than coden
13 mutation or proto-oncogene overexpression. Cancer Research, 2000, 60(Dec): 6750-6756.
10.Song MM, Nio Y, Dong M, et al. Ki-ras point mutations and p53 expression in human pancreatic cancer: a comparative study among Chinese, Japanese, and Western patients. Cancer Epidemiol Biomarkers Prev, 2000, 9: 279-284.
11.苏震东. K-ras基因突变在胰腺癌中的作用研究进展.国外医学消化系疾病分册, 2005, 25(1): 48-51.
12.雷小勇,张洹.从反义技术到反基因技术:基因治疗的新突破.医学与哲学, 2001, 22(9): 10-12.
13.盛世乐,黄钢.肿瘤反基因放射治疗的研究进展.中华核医学杂志, 2004, 24(6): 377-378.
14.Ratilainen T, Lincoin P, Norden B. A simple model for gene targeting. Biophysical Journal, 2001, 81: 2876-2885.
15.龚镭,诸玲,胥明.胰腺癌基因治疗的现状与前景.胰腺病学, 2005, 5(1): 56-57.
16.陈熹,王永向,纪宗正,等. K-ras基因点突变的反义寡脱氧核苷酸对人胰腺癌靶基因表达的抑制作用.西安交通大学学报(医学版), 2003, 24(2): 147-149.
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