MR靶向对比剂前体——VEGF165-适配体与VEGF165体外结合实验研究

目的应用酶联免疫吸附实验(ELISA)检测MR靶向对比剂前体——血管内皮生长因子165-适配体(VEGF165-aptamer)与VEGF165的体外结合能力,以了解其对VEGF的靶向性。方法实验组采用亲和素96孔酶标板,包被生物素化VEGF165-aptamer,设置递减的浓度梯度(共9组)和空白对照组,采用组间方差分析。结果实验组包被生物素化VEGF165-aptamer浓度在50～0.78pmol/μL时,实验组与空白组及实验各相邻组间差异有统计学意义(P<0.05);实验组包被浓度为0.39、0.195pmol/μL时,两组与空白组间及两组间差异均无统计学意义(P>0.05)。结论采用ELISA检测技术验证了VEGF165-aptamer与VEGF165间的体外结合能力,其有效最低小包被浓度为0.78pmol/μL,VEGF165-aptamer对VEGF165有良好的靶向性。     

MR对比剂荧光VEGF165-USPIO合成及其活性的体外实验

目的：探讨荧光 VEGF165-USPIO（血管内皮生长因子165-超顺磁性氧化铁）作为 MR 对比剂的可行性,检测该对比剂体外对 VEGF165抗原的靶向性。方法：通过氨基-羧基偶联方式将 VEGF165单克隆抗体和 USPIO 连接,然后将 Cy5．5标记到单克隆抗体表面,构建 Cy5．5-anti-VEGF165-USPIO 靶向对比剂。以离心和磁分离器的洗涤手段进行改良 ELISA 实验检测其体外活性,实验组加入0．25ml 的荧光-anti-VEGF165-USPIO,对照组加入相同量的荧光 USPIO,每组各设5个离心管,两样本为计量资料,采用独立 t 检验比较不同组间吸光值差异有无显著性。结果：通过分光光度计验证对比剂连接成功,VEGF165单克隆抗体偶联率达到了71．7％,CY5．5的偶联率为83．3％。ELISA 实验表明实验组和对照组各自吸光值差异有显著性意义（P ＜0．05）,实验组 OD（吸光值）明显高于对照组。结论：荧光 anti-VEGF165-US-PIO 体外有结合 VEGF165的活性,为进一步的 MR 肿瘤靶向对比剂研究奠定了实验基础。     

顺磁性对比剂Gd-DTPA-DG 的制备及其在肺癌裸鼠模型活体MR实验研究

目的 合成一种顺磁性脱氧葡萄糖类MR对比剂二乙三胺五乙酸-脱氧葡萄糖钆盐(Gd-DTPA-DG)并探讨其在荷瘤裸鼠体内肿瘤信号改变的规律.方法 首先合成DTPA-DG ,再与Gd2O3螯合,制得Gd-DTPA-DG.采用荷瘤裸鼠体内实验模型,将裸鼠随机分成实验组与对照组(n=10),前者尾静脉注射Gd-DTPA-DG,后者尾静脉注射含相同Gd3+浓度(0.1 mmol/kg)的Gd-DTPA, 测量SE T1WI平扫及引入对比剂30 s、2 min、5 min、 10 min、20 min、30 min、45 min、1 h、2 h后瘤体信号强度,并计算对比度噪声比(CNR).将右侧前肢肌肉组织信号变化作为参照物,并进行统计分析.结果 Gd-DTPA-DG比Gd-DTPA在肿瘤组织内表现为更强且更持久的强化.注射对比剂30 s后,实验组裸鼠瘤体信号增加与扫描前差异没有统计意义(P=0.171),在30 min左右差异性最大(P<0.001).对照组5 min时前后差异最大(P<0.001),但2 h时即与注射前没有显著差异性(P=0.057).结论 Gd-DTPA-DG 可在肺癌模型活体内起到肿瘤强化作用,是一种新型顺磁性Gd(III)类糖代谢MR对比剂.     

对比剂铁羧葡胺在正常兔MR脑灌注的初步实验研究

目的 探讨铁羧葡胺(Resovist)应用于脑MR灌注加权成像(PWI)的可行性、给药方法以及最佳剂量.方法 健康新西兰大白兔30只,随机数字法平均分为A、B、C、D、E组.其中A、B、C、D 4组分别给予4、8、16、32 μmol Fe/kg;E组设为对照,给予0.2 mmol/kg的钆喷替酸葡甲胺(Gd-DTPA).所有动物均行MR PWI,获得相应信号强度一时间曲线图,并分别计算脑灰质、白质的最大信号下降百分比(SRRmax)、局部脑血容量(rCBV)和灰质与白质rCBV之比(QRCBV)、SRRmax之比(QRR max).所得数据,根据资料性质,行配对t检验和单因素方差分析.结果 Resovist能快速团注,4组均获得满意的信号强度一时间曲线图;4种剂量的Resovist对实验兔脑灰质和白质均有良好的分辨率.A、B、C、D、E 5组脑灰质和白质的rCBV分别为(50.48±3.84)、(25.57±2.10),(94.69±2.60)、(45.33±3.14),(141.13±6.26)、(67.67±4.65),(243.75±5.90)、(162.06±5.14),(84.60±3.60)、(41.36±2.18)ml/100 g;灰质和白质的SRRmax分别为:(13.70±1.50)%、(7.38±0.41)%,(31.01±4.06)%、(16.49±2.35)%,(43.81±3.42)%、(21.64±4.14)%,(64.49±5.35)%、(43.61±5.78)%,(27.78±2.98)%、(14.42±2.25)%;各组脑灰质与白质检测数据比较差异均有统计学意义(P值均<0.01).A、B、C、D、E 5组QrCBV分别为1.98±0.07、2.09±0.11、2.09±0.07、1.50±0.01、2.05±0.03;QSRRmax分别为:1.85±0.11、1.88±0.06、2.06±0.25、1.49±0.09、1.94±0.12;5组间差异均有统计学意义(QrCBV的F值为85.076,QSRR max的F值为13.915,P均<0.01).A、B、C 3组QrCBV值和QSRR max值差异无统计学意义(P>0.05);而D组QrCBV值和QSRR max值显著低于A组(P<0.01).结论 Resovist应用于MR脑灌注是可行的,适宜剂量4～16 μmol Fe/kg.     

MRI监测兔VX2瘤脑脊液源性转移与VEGF表达的相关性研究

目的:分析兔VX2瘤脑脊液源性转移动物模喇中血管内皮生长因子(VEGF)的表达及其变化规律;MRI动态监测动物模型的形态学变化,分析其参数与肿瘤VEGF表达水平的相关性.方法:30只新两兰大白兔分两组.每次MRI检查前.分别抽取实验兔的脑脊液和血液样品做VEGF的酶联免疫吸附法(ELISA)测定.MRI扫描后处死所扫描实验兔,获取病理标本,行HE染色及VEGF免疫组化染色.结果:经t检验,参数SLE及MER在VEGF表达分组中有显著统计学差异.实验组免疫组化在肿瘤细胞的细胞质和细胞膜以及部分细胞核中均可见VEGF阳性颗粒,对照组呈阴性.DCE-MRI参数与VEGF表达经Spearman's等级相关法分析,SLE与肿瘤组织免疫组化VEGF表达呈正相关(P<0.01),而MER与肿瘤组织免疫组化VEGF表达旱负相关(P<0.05).结论:在兔VX2瘤脑脊液源性转移中,VEGF呈高表达,动态增强磁共振成像可有效地临测肿瘤的生物学行为,为评价脑脊液源性转移是否采用抗血管治疗及判定治疗效果提供可靠的影像学检测手段.     

新型MR对比剂长循环超顺磁氧化铁脂质体纳米微粒的体内外实验研究

目的 评价自制新型MR对比剂:长循环超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)脂质体纳米微粒的药代动力学.方法 以加入SPIO为对照组,加入长循环SPIO脂质体纳米微粒为实验组.(1)巨噬细胞的体外吞噬实验:将巨噬细胞株RAW 264.7经复苏、培养,细胞达到80%～90%融合后,以每孔2.5×105个细胞接种于培养板,对照组和实验组细胞与不同浓度药物孵化后,分别测定细胞中Fe浓度和Fe染色情况,并用析因设计的方差分析对结果进行统计学处理.(2)小鼠体内药物分布实验:C57BL/6J雄性小鼠6只,每组2只,设为空白对照组、对照组、实验组,经尾静脉分别注射生理盐水、SPIO以及长循环SPIO脂质体后牺牲动物,经病理检查观察2组药物在体内的分布情况.(3)荷肺癌裸鼠MR成像实验:建立20只BALB/c裸鼠移植型肺癌模型,分为对照组和实验组,每组10只,分别经尾静脉注入SPIO和长循环SPIO脂质体纳米微粒后行MR扫描,测量增强前后2组实验动物各时间点肝脏和肿瘤ROI的信号强度,绘制信噪比(SNR)时间动态变化曲线,采用协方差分析比较2组动物肝脏及肿瘤增强12 h 后SNR差异有无统计学意义.(4)细胞毒性实验(噻唑蓝,MTT法):检查2种药物对人肝细胞株HL-7702细胞的毒性作用,用析因设计的方差分析对结果进行统计学处理.结果 (1)巨噬细胞的体外实验:析因分析结果表明,对照组细胞内Fe含量明显高于实验组,差异有统计学意义(F=296 839.9,P=0.000).普鲁士蓝染色显示对照组巨噬细胞染色较深,实验组巨噬细胞染色较浅.(2)小鼠体内药物分布实验:对照组肝、脾、肺、肾组织内蓝染颗粒多于实验组.(3)荷肺癌裸鼠MR成像实验:平扫对照组肝脏SNR为31.47±0.56,实验组为30.89±1.41;增强扫描12 h后对照组为17.00±0.96,实验组为22.29±0.73.平扫对照组肿瘤SNR为58.41±0.61,实验组为58.44±1.08;增强扫描12 h后对照组为58.50±0.63,实验组为52.47±1.18.经协方差分析表明增强12 h后对照组肝脏SNR显著低于实验组(F=167.022,P=0.000);而实验组肿瘤 SNR显著低于对照组(F=266.106,P=0.000).(4)细胞毒性实验:随着培养液中Fe浓度的增加,HL-7702细胞生存率均有下降趋势,但析因分析结果表明,实验组药物的细胞毒性较对照组低(F=2256.204,P=0.000).结论 长循环SPIO脂质体纳米微粒在体内外均有较好的抗巨噬系统吞噬功能,在体内实现了长循环,对非网状内皮系统的肿瘤也具有良好的T2负性被动靶向强化效果;同时还降低了SPIO对细胞的毒性作用.

Abstract：

Objective To evaluate pharmacodynamics of prepared long-circulating superparamagnetic iron oxide (SPIO) liposomes. Methods Control and experimental groups were established after adding SPIO or long-circulating SPIO liposomes as agents. (1)Macrophages experiment in vitro: the RAW 264.7 macrophage cell strains were recovered, cultured and seeded in the culture plate at a density of 2.5×105 cells/well until they reached 80%-90% confluence. The intracellular Fe uptake of control and experimental group cells were quantified by Ferrozine assay after incubation with different concentrations of drugs. Factorial design analysis of variance was used as statistics method. Prussian blue staining method was used to detect staining of experimental cells.(2)Drug biodistribution in mice: C57BL/6J(n=6) were classified into blank control group (n=2), control group(n=2) and experimental group(n=2).Saline, SPIO and long circulating SPIO were injected via the tail vein in the blank control group, control group and experimental group respectively. Then distribution of drugs in the body was observed by pathological examination.(3) MR imaging of tumor-bearing nude mice: 20 BALB/c nude mice bearing lung cancer models were established and classified into control group and experimental group. After administration of drugs, all animals underwent MR scanning. Signal intensities of livers and tumors were measured, SNR-time dynamic curves were drew. Covariance analysis was used to compare post-enhanced SNR at the 12th hour. (4)Cytotoxicity studies (MTT): cytotoxicity of both drugs on human liver cell line HL-7702 was studied, and statistically analyzed using factorial design analysis of variance. Results (1) Macrophages experiment in vitro: The nanoparticle uptake by macrophage cells evaluated by ferrozine assay showed the uptake of blank SPIO was higher than long-circulating SPIO liposomes. Compared with the blank control group, there was strong blue staining in the macrophages with Prussian staining in the control group and little blue staining in the experimental group. (2) Drug biodistribution in mice: for blue stained cells composed of iron particles, the amounts in the liver, spleen, lung, kidney of the control group were more than those in the experimental group. (3) MR imaging of tumor-bearing nude mice: the non-enhanced SNR of livers and tumors in the control group and experimental group were 31.47 ± 0.56, 30.89 ± 1.41, 58.41 ± 0.61, 58.44 ± 1.08, respectively. After injecting of contrast agents, SNR of livers and tumors in the control group and experimental group were 17.00 ± 0.96, 22.29 ± 0.73, 58.50 ± 0.63, 52.47 ± 1.18, respectively. The covariance analysis showed that SNR of the livers in the control group after 12 hours was significantly lower than the experimental group (F=167.022, P=0.000); while the SNR of the tumors in the experimental group was significantly lower than the control group (F=266.106, P=0.000).(4) Cytotoxicity of nanoparticles by MTT method: the viability of HL-7702 cells tend to decrease with the increase of Fe concentration. Cytotoxicity in the long-circulating SPIO liposomes was lower than the SPIO(F=2256.204,P=0.000). Conclusions Long-circulating SPIO liposomes we prepared reveal suitable sizes, even distribution, and good anti-macrophage ability in vitro and in vivo. They have long circulation characteristic and T2 negative enhancement effect in the transplanted lung cancers, while they still maintain low cytotoxicity.     

钆-乙二胺二乙酸-肼基烟酰胺-多肽螯合物MR分子探针的构建及在体肿瘤显像实验研究

目的 构建一种含有精氨酸、甘氨酸、天冬氨酸(RGD)基序多肽的钆(Gd)类小分子MR分子探针,以特异性显示高表达整合素avβ3受体的肿瘤.方法 肼基烟酰胺(HYNIC)-RGD在乙二胺二乙酸(EDDA)存在的情况下,用三氯化钆(GdCl3)进行标记,用固相萃取(SPE)方法进行分离,得到分子探针Gd-EDDA-HYNIC-RGD.对分子探针进行紫外和MRI信号检测,测量弛豫率.进行3-(4,5-二甲基噻唑)-2,5-二苯基四氮唑溴盐(MTT)细胞毒性实验,将分子探针稀释成0.40、0.20、0.10、0.05 μmol/ml 4个浓度梯度,每个浓度20μl加入到含有人肝癌细胞的培养板内,测量吸光度值,并与未加分子探针的对照组吸光度值进行独立样本t检验.用逆转录聚合酶链反应(RT-PCR)检测BEL-7402人肝癌细胞系和肿瘤组织avβ3受体表达,用免疫荧光观察受体-配体结合情况.建立裸鼠肿瘤模型,将荷瘤裸鼠分为3组,分别由尾静脉注射0.2 ml分子探针、钆喷替酸葡甲胺(Gd-DTPA)和生理盐水,平扫加注药后即刻、30、60和90 min以及24 h分别进行图像采集,对0和90 min信号变化数据进行配对资料t检验.结果 Gd-EDDA-HYNIC-RGD分子探针被成功分离,MR T1 WI信号较强;分子探针弛豫率为3.31 mmoL/s;分子探针浓度在0.1 μmoL/ml以下比较安全;BEL-7402人肝癌细胞系和肿瘤组织表达avβ3受体;受体-配体在体外可以特异性结合;体内实验表明,肿瘤部位平扫和增强90 rain后的平均信号强度分别为2247.6±39.0和2820.9±35.2,增强25%,差异有统计学意义(t=-38.031,P＜0.05);肌肉部位则为1824.2±32.8和1845.8±27.2,信号变化无统计学意义(t=-1.424,P＞0.05);肿瘤信号与肌肉信号两者差值(423.4±56.7和975.1±32.1)有统计学意义(t=-24.650,P＜0.05).肿瘤在注射分子探针、Gd-DTPA和生理盐水后具有不同的增强特点:分子探针组在0～90 min内逐步增强,在90 min信号最强;Gd-DTPA在注药后30 min信号最强,其后迅速减低;生理盐水组基本无增强;组织学检查显示,肿瘤为典型的恶性形态,血管丰富,可见大量坏死灶.结论 Gd-EDDA-HYNIC-RGD有望成为一种新的MR分子探针,显示高表达avβ3受体肿瘤,为临床提供一种早期诊断和鉴别诊断的可能手段.     

减少脊椎术后影像的金属伪影:IDEAL对比剂增强的T1和T2加权MR成像的作用——模体和临床研究

目的前瞻性比较使用迭代分解水和脂肪的回声不对称与最小二乘法估计技术(IDEAL)的T2加权和对比剂增强T1WMRI对于减小模体和病人脊柱手术植入金属后的金属伪影的能力。材料与方法本研究获得机构审查委员会的批准和病人的知情同意。将6枚钛合金椎弓根     

磁共振钆靶向对比剂的研究现状及展望

特异性MR靶向对比剂通过与体内特定的靶点结合显示活体分子靶点状态,为临床提供分子水平的信息。选择针对特定靶点的特异性运载体和良好的MR显像剂,是构建良好分子探针的一个关键因素。钆剂作为临床常用的MR对比剂,在构建分子探针上具有诸多优势,目前在MR分子成像领域进行了大量实验研究。就目前研究常用的分子靶点和分子探针,对MR钆靶向对比剂的研究现状及展望进行综述。     

Minimizing contrast agent dose during intraarterial gadolinium-enhanced MR angiography: in vitro assessment.

PURPOSE: To minimize contrast agent dosage for intra-arterial (IA) contrast-enhanced magnetic resonance angiography (CE-MRA) by examining the effects of encoding order (elliptical vs. sequential) and injection duration (100% to 30% of the acquisition time). MATERIALS AND METHODS: Catheter-based IA gadolinium (Gd) injections were performed in an arterial flow phantom. Blood flow rates, injection rates, and injection durations were systematically varied. Signal-to-noise (SNR) measurements were obtained in the aorta, renal artery, and common iliac artery. RESULTS: No significant SNR losses were observed for any of the vessels with 75% injection duration, or for the aorta and iliac artery with 50% injection duration. Excellent images of all vessels were obtained at 50% injection duration. There was no significant SNR difference between encoding schemes. CONCLUSION: Contrast agent dosage can be substantially reduced without loss of SNR by limiting injection to part of the imaging acquisition time.     

Gadolinium-DTPA-dextran: a macromolecular MR blood pool contrast agent

RATIONALE AND OBJECTIVES: Magnetic resonance (MR) imaging blood pool agents offer numerous advantages for vascular and tumor imaging. The purpose of this study was to test gadolinium-diethylenetriaminepentaacetate-dextran ([Gd]DTPA-dextran) as a new water soluble macromolecular blood pool agent for MR imaging. MATERIALS AND METHODS: [Gd]DTPA-dextran (187 gadolinium atoms per dextran, molecular weight 165 kD, diameter 17.6 nm) was synthesized. Fifteen anesthetized New Zealand White rabbits with thigh VX2 tumors were scanned in a knee coil at 1.5T. Coronal 3D MR angiographic sequences were obtained before and at several time points up to 72 hours after the intravenous bolus injection of [Gd]DTPA-dextran providing gadolinium at either 0.05 (n = 4) or 0.1 mmol/kg (n = 8) or [Gd]DTPA-bismethylamide (BMA) providing gadolinium at 0.1 mmol/kg (n = 3). Time enhancement curves for aorta, cava, and tumor rim were compared by univariate General Linear Model. RESULTS: Contrast enhancement of cava and aorta relative to a water phantom were significantly greater at all time points after either dose of [Gd]DTPA-dextran than after [Gd]DTPA-BMA (P < 0.01). Tumor rim enhancement was less intense for either dose of [Gd]DTPA-dextran at peak than for [Gd]DTPA-BMA (P < 0.05). Tumor rim enhancement with both doses of [Gd]DTPA-dextran became equivalent to that of [Gd]DTPA-BMA at one hour and was greater at 24 hours (P < 0.05). CONCLUSION: [Gd]DTPA-dextran is a new macromolecular MR contrast agent that can be synthesized to carry a high density of gadolinium atoms without intra-molecular cross-linking. It provides significantly greater vascular residence time than a conventional gadolinium chelate and shows promise for MR blood pool imaging.     

Gadolinium-DTPA as a contrast agent in MR imaging--theoretical projections and practical observations

The theoretical basis for the use of paramagnetic agents to enhance proton relaxation is described. Factors of importance in the design of contrast agents are considered. Measurements of changes in T1 and T2 in vitro due to Gd3+-diethylenetriamine pentaacetic acid (DTPA) are used to predict changes in the intensity of transverse magnetisation seen with different sequences in magnetic resonance imaging. The use of Gd3+-DTPA in clinical cases is illustrated.     

Axonal transport and MR imaging: Prospects for contrast agent development

Axonal transport plays a critical role in the physiology and pathology of neurons, yet there have been virtually no clinical tools for its evaluation in human subjects. A wide variety of molecules that can act as axonal transport facilitators have been discovered and, in many cases, used to deliver labels detectable with histologic methods. Recently a number of investigators have reported preliminary success in developing intraneural contrast agents based on various versions of dextran-coated magnetite that may render magnetic resonance imaging capable of depicting axonal transport. It is not yet clear whether any clinically useful agents will eventually be developed, but there has been considerable progress in identifying design factors for such a pharmaceutical agent.