基于Endoglin靶的胶质瘤血管生成的MRI显像研究
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摘要
目的:(1)合成靶向Endoglin的空间稳定型脂质体MRI对比剂并评价其基本特性;(2)探讨靶向Endoglin的空间稳定型脂质体MRI对比剂评价胶质瘤血管生成的价值,从而为客观准确评价肿瘤血管生成建立活体的MRI方法。
材料与方法:(1)以叔丁氧碳基保护的双氨基聚乙二醇(Boc-NH-PEG-NHS)、氢化蛋黄磷酯酰乙醇胺(HEPE)为原材料,首先合成Boc-NH-PEG-HEPE,然后去除叔丁氧碳基(Boc基团),合成NH-PEG-HEPE。通过NH-PEG-HEPE和SPDP发生反应,得PDP-PEG-HEPE,用于桥接抗体和脂质体。(2)配制8个不同浓度的磁显葡胺溶液,用日立F-3000型荧光分光光度仪测定其荧光,建立标准曲线。并对其稳定性、回收率和精密度进行考察。(3)先制备含有PDP-PEG-HEPE的空间稳定脂质体(PDP-SLs),再通过二硫苏糖醇(DTT)将其还原为表面带有巯基的脂质体(HS-SLs),最后与SMPB衍生化后的IgG或抗Endoglin抗体相连接即可得空间稳定免疫脂质体(IgG-SLs或Endoglin-SLs),并用激光散射粒度分析仪行粒径分析。用钼蓝法测定磷脂浓度;用~(125)I标记法检测空间稳定免疫脂质体中抗体的连接效率、脂质体表面抗体密度;用酶联免疫吸附实验(ELISA)检测空间稳定免疫脂质体中抗体的免疫活性。(4)采用薄膜法和冻融法制备基于Endoglin靶的钆空间稳定型免疫脂质体对比剂(Endoglin-Gd-SLs),并用同样方法制备IgG-Gd-SLs、Gd-SLs。采用透射电镜观察脂质体的形态。并对其粒径大小及粒径分布进行分析。利用荧光法测定脂质体中Gd的包封率,并计算其载药量。分析不同HEPC/Chol/mPEG-DSPE/PDP-PEG-HEPE比例、Gd-DTPA浓度、pH值、脂质体的粒径等因素对Gd-DTPA包封率和载药量的影响。并对其粒径稳定性、药物泄漏率进行考察。(5)40只皮下接种F98胶质瘤的Fischer344大鼠随机分为四组,分别静脉注射本研究合成的Endoglin-Gd-SLs、IgG-Gd-SLs、Gd-SLs及Gd-DTPA。于注射前、注射后即刻、5min、30min、1h、2h等时相点行MRI扫描,计算每个时相点的CNR和增强最明显时相点的相对信号强度,并比较各组的MRI增强特点。扫描结束后活杀动物,取材,行Endoglin免疫荧光;α-SMA、VEGF免疫组化染色。分别对Endoglin、α-SMA所标记的微血管计数(E-MVD、S-MVD),并计算二者之和(T-MVD)。同时用标记指数(LI)来反映VEGF阳性染色情况。分析各组增强最强时相点的相对信号强度与E-MVD、S-MVD、T-MVD及VEGF LI的相关性。
结果:(1)荧光法测定磁显葡胺含量的条件:激发波长275nm,发射波长312nm,pH范围6.0-8.0,线性范围5×10~(-5)mol/L—1mol/L。(2)所合成的IgG-SLs或者
Purpose: (1) To synthesize the sterically stabilized Gd-DTPA liposomes targeted to Endoglin and to evaluate its rudimental characteristics. (2) To detect glioma angiogenesis in vivo using MRI and sterically stabilized Gd-DTPA liposomes targeted to Endoglin and to establish a method in vivo to evaluate tumor angiogenesis.Materials and Methods: (1) Three steps were applied to synthesize thePDP-PEG-HEPE, which attaches antibody to the surface of liposome. Firstly,Boc-NH-PEG-HEPE was synthesized with Boc-NH-PEG-HEPE and HEPE. Thenselective deprotection of the tert-butoxycarbonyl (Boc) of Boc-NH-PEG-HEPEwas achieved by using hydrogen chloride (4M) in anhydrous dioxane solutionfor 30 min. Finally, PDP-PEG-HEPE was prepared after Boc-NH-PEG-HEPEreacted with SPDP. During the whole synthesise, TLC was done to prove theproduct.(2)Magneist solution with different concentration was confectedand its fluorescence was measured with HITACHI F-3000. The correlationbetween concentration and fluorescence was analyzed. And the stability offluorescence of magnevist was also observed. (3)IgG-SLs or Endoglin-SLswere prepared following the steps as following: Firstly, liposomes (PDP-SLs)containing HEPC, Choi, mPEG-DSPE, PDP-PEG-HEPE were parepared. Then thedithiol-bond of PDP in the PDP-SLs was reduced to - SH by dithiothreitol(DTT) and HS-SLs was thus obtained. Finally, the derivative anti-Endoglinantibody or IgG (malemidophenylbutyrate-Endoglin or IgG, MPB-Endoglin orMPB-IgG) was attached to the surface of PDP-SLs. The liposome diameter andsize distribution were determined by Nicomp 380 Particle Sizing System.Concentration of phospholipids was deterimined with molybdenum blue method.I25I radiolabelling tests was carried out to determined the coupling ratioof antibody to liposomes and the antibody density on the surface ofliposomes. ELISA studies was performed to confirmed theimmunoreacitivities of IgG-SLs and Endoglin-SLs. (4)Sterically stabilizedGd-labelled immunoliposomes targeted to Endoglin (Endoglin-Gd-SLs) wasprepared by the thin film hydration method and the freeze-thawing method.
The shape of Endoglin-Gd-SLs was observed by transmission electron microscope. The liposome diameter and size distribution were determined by Nicomp 380 Particle Sizing System. Gd-DTPA trapping efficency was determined by fluorescent method and the Gd-DTPA concentration in the liposome was also calculated. The effect of concentration, value of pH of Gd-DTPA solution, ratio of HEPC/Chol/mPEG-DSPE/PDP-PEG-HEPE, diameter of liposome on the Gd-DTPA trapping efficency and the Gd-DTPA concentration in the liposome were analyzed. The physical stability of liposome and the leakage ratio of Gd-DTPA were also observed. (5)Forty Fischer 344 rats implanted with F98 gliomas were randomized into four treatment groups and received either: (a)sterically stabilized Gd-labelled liposomes targeted to Endoglin (Endoglin-Gd-SLs);(b)sterically stabilized Gd-labelled liposomes covalently coupled with IgG (IgG-Gd-SLs) ;(c)sterically stabilized Gd-labelled liposomes (Gd-SLs); or (d)Gd-DTPA. MRI scan were performed before, immediately after, and 5, 30, 60, 120min after intravenous injection. MRI enhancement features of each group were observed. To quantify image enhancement over time, CNR at each time point and the relative intensity at time point of maximal enhancement were calculated. After imaging, tumors were resected for a -SMA, VEGF immunohistochemical staining and Endoglin immunofluorescence. E-MVD(microvessel density based on Endoglin immunofluorescence), S-MVD(microvessel density based on a -SMA immunofluorescence), T-MVD (T-MVD= E-MVD+S-MVD) were calculated. VEGF labeling index (VEGF LI) were measured to reflect VEGF expression. The correlation analysis between the relative signal intensity at time point of maximal enhancement and E-MVD, S-MVD, T-MVD and VEGF LI were performed. Results: (1)The results of TLC suggested that PDP-PEG-HEPE was successfully synthesized. (2) The optional conditions for quantitative determination of magnevist were as following: The excitated wavelength was 275nm, emission wavelength was 312nm. The fluorescence of magnevist was stable when pH value was 6.0-8.0, and it positively correlated well with concentration of magnevist when the concentration was 5× 10~(-5) mol/L-1mol/L. (3) Mean diameter of IgG-SLs and Endoglin-SLs was 143nm, the size distribution was homogeneous. Mean diameter of IgG-SLs and Endoglin-SLs was slightly larger than that of SLs and PDP-SLs. The concentration of phospholipids was 1. 5725mmol/L. The results of 125I radiolabeling tests showed the the coupling
ratio of antibody to liposomes was 54. 28%, the antibody density on the surface of liposomes was 78.13ug Ab/umol lipid, which met the needs of experiment. ELISA studies showed the immunoreacitivities of IgG-SLs and Endoglin-SLs was about 44.3% and 51%,respectively. (4)The liposome was round in shape, lipid-membrane and fingerprint-like structure were found. The trapping efficiency and concentration of Gd-DTPA was the highest when the pH value of Gd-DTPA was 6. 0-8. 0, but there was no difference between group pH6. 0 and group pH8.0, and there was also no difference between group pH6.0, pH8. 0 and group pH10.0. While there was significant difference between group pH6. 0, pH8. 0, pHlO. 0 and group 2.0, 4.0 (p<0. 05、 0.01) . The trapping efficiency and concentration of Gd-DTPA in the liposomes was 21. 8% 、 0. 067umol/umol respectively, when the concentration of Gd-DTPA solution was 0.67mol/L. While these was 13.4 % 、 0.022 umol/umol respectively, when concentration of Gd-DTPA solution was 0. 25mol/L. There was statistical significance between group 0. 5mol/L、 0. 67mol/L、 0. 72mol/L and group 0. 25mol/L (p<0. 05> 0.01) .As for the effect of the size of liposomes on the trapping efficiency and concentration of Gd-DTPA was concerned. The larger the size was, the more The trapping efficiency and concentration of Gd-DTPA was. Statistical significance was found between group lOOnm, 150nm, 200nm and group 50nm (p<0. 05> 0.01) , but there is no difference among the former three groups. Four groups (group A、 B、 C、 D) with different ratio of HEPC/Chol/mPEG-DSPE/PDP-PEG-HEPE were analyzed. The ratio was 3:2:0.1:0.02,4:1:0.3:0.05,2:1:0.1:0.01 , 11:8:0.1:0.01 respectively. The trapping efficiency was statistical different between the former two groups and the latter two groups.As for the concentration of Gd-DTPA in the liposomes was concerned, Statistical difference was found between group A and group C、 D (p<0.05), There was also difference between group B and group D (p<0. 05) . While there was no difference between group B and C, between group C and D. The stability of IgG-Gd-SLs stay between Gd-CLs and Gd-SLs during restoration. When IgG -Gd-SLs were suspended in either PBS or 1% human plasma for 10 hours, the leakage ratio of Gd-DTPA was 19. 8%, 11.2%, respectively. (5) Enhancement differences were observed among the four groups. Slight enhancement was found after 5 min and singnal intensity of enhancement region increased steadily within 1h in group Endoglin-Gd-SLs、 group IgG-Gd-SLs and group Gd-SLs. Difference of the three
groups lay in enhancement at 2h point. A more significant signal intensity increase was detected after 2h in group Endoglin-Gd-SLs. In group IgG-Gd-SLs, signal intensity of enhancement after 2h was less than that after 1h, but was more than that at baseline. While in group Gd-SLs, signal intensity of tumor almost return to baseline after 2h. In group Gd-DTPA, contrast enhancement was detected soon after Gd-DTPA injection and maximal enhancement was found after 5min. While singnal intensity decreased after 30min and returned to baseline after lh. In addition, the size of enhancement region over time was difference among the four groups. In group Endoglin-Gd-SLs, enhancement region was confined and did not expanded within 2h. While distended enhancement region from periphery to central within lh was found in group IgG-Gd-SLs and group Gd-SLs. In group Gd-DTPA, homogeneous enhancement was found soon after injection.Correlation analysis showed the relative intensity at time point of maximal enhancement correlated well with E-MVD, VEGF LI in the four groups (p<0.05, 0.01), it did not have correlation with S-MVD, T-MVD in group Endoglin-Gd-SLs(p>0.05) , while in other three groups, it correlated well with S-MVD, T-MVD (p<0. 05, 0.01).Conclusions:(1)The optional conditions for quantitative determination of magnevist were as following: The excitated wavelength was 275nm, emission wavelength was 312nm. The fluorescence of magnevist was stable when pH value was 6.0-8.0, and it positively correlated well with concentration of magnevist when the concentration was 5×10~(-5)mol/L-lmol/L. Fluorescent method is a simple and efficient method to quantitative determination of magnevist concentration. (2)The linkage method of antibodies to the terminal of PEG of liposome surface via thioether bond could retain their immunoreacitivities. The decrease of immunoreacitivities corrlate with two factors as following: the SMPB derivation of antibody, the steric hinderance effect of liposome. (3)Protocol of trapping Gd-DTPA was as following: 0. 67mol/L Gd-DTPA solution with pH 6. 0-8. 0, the 3/3/0. 1/0. 02 (mole ratio) blend of HEPC, Choline, mPEG-DSPE and PDP-PEG-HEPE, the mean diameter should be 100nm-150nm in order to meet the experiment. The stability ofIgG-Gd-SLs stay between Gd-CLs and Gd-SLs during restoration. (4) Enhancement of Endoglin-Gd-SL correlate well with glioma neovasculature.It is feasible and efficient to detect tumor angiogenesis in vivo using
材料与方法:(1)以叔丁氧碳基保护的双氨基聚乙二醇(Boc-NH-PEG-NHS)、氢化蛋黄磷酯酰乙醇胺(HEPE)为原材料,首先合成Boc-NH-PEG-HEPE,然后去除叔丁氧碳基(Boc基团),合成NH-PEG-HEPE。通过NH-PEG-HEPE和SPDP发生反应,得PDP-PEG-HEPE,用于桥接抗体和脂质体。(2)配制8个不同浓度的磁显葡胺溶液,用日立F-3000型荧光分光光度仪测定其荧光,建立标准曲线。并对其稳定性、回收率和精密度进行考察。(3)先制备含有PDP-PEG-HEPE的空间稳定脂质体(PDP-SLs),再通过二硫苏糖醇(DTT)将其还原为表面带有巯基的脂质体(HS-SLs),最后与SMPB衍生化后的IgG或抗Endoglin抗体相连接即可得空间稳定免疫脂质体(IgG-SLs或Endoglin-SLs),并用激光散射粒度分析仪行粒径分析。用钼蓝法测定磷脂浓度;用~(125)I标记法检测空间稳定免疫脂质体中抗体的连接效率、脂质体表面抗体密度;用酶联免疫吸附实验(ELISA)检测空间稳定免疫脂质体中抗体的免疫活性。(4)采用薄膜法和冻融法制备基于Endoglin靶的钆空间稳定型免疫脂质体对比剂(Endoglin-Gd-SLs),并用同样方法制备IgG-Gd-SLs、Gd-SLs。采用透射电镜观察脂质体的形态。并对其粒径大小及粒径分布进行分析。利用荧光法测定脂质体中Gd的包封率,并计算其载药量。分析不同HEPC/Chol/mPEG-DSPE/PDP-PEG-HEPE比例、Gd-DTPA浓度、pH值、脂质体的粒径等因素对Gd-DTPA包封率和载药量的影响。并对其粒径稳定性、药物泄漏率进行考察。(5)40只皮下接种F98胶质瘤的Fischer344大鼠随机分为四组,分别静脉注射本研究合成的Endoglin-Gd-SLs、IgG-Gd-SLs、Gd-SLs及Gd-DTPA。于注射前、注射后即刻、5min、30min、1h、2h等时相点行MRI扫描,计算每个时相点的CNR和增强最明显时相点的相对信号强度,并比较各组的MRI增强特点。扫描结束后活杀动物,取材,行Endoglin免疫荧光;α-SMA、VEGF免疫组化染色。分别对Endoglin、α-SMA所标记的微血管计数(E-MVD、S-MVD),并计算二者之和(T-MVD)。同时用标记指数(LI)来反映VEGF阳性染色情况。分析各组增强最强时相点的相对信号强度与E-MVD、S-MVD、T-MVD及VEGF LI的相关性。
结果:(1)荧光法测定磁显葡胺含量的条件:激发波长275nm,发射波长312nm,pH范围6.0-8.0,线性范围5×10~(-5)mol/L—1mol/L。(2)所合成的IgG-SLs或者
Purpose: (1) To synthesize the sterically stabilized Gd-DTPA liposomes targeted to Endoglin and to evaluate its rudimental characteristics. (2) To detect glioma angiogenesis in vivo using MRI and sterically stabilized Gd-DTPA liposomes targeted to Endoglin and to establish a method in vivo to evaluate tumor angiogenesis.Materials and Methods: (1) Three steps were applied to synthesize thePDP-PEG-HEPE, which attaches antibody to the surface of liposome. Firstly,Boc-NH-PEG-HEPE was synthesized with Boc-NH-PEG-HEPE and HEPE. Thenselective deprotection of the tert-butoxycarbonyl (Boc) of Boc-NH-PEG-HEPEwas achieved by using hydrogen chloride (4M) in anhydrous dioxane solutionfor 30 min. Finally, PDP-PEG-HEPE was prepared after Boc-NH-PEG-HEPEreacted with SPDP. During the whole synthesise, TLC was done to prove theproduct.(2)Magneist solution with different concentration was confectedand its fluorescence was measured with HITACHI F-3000. The correlationbetween concentration and fluorescence was analyzed. And the stability offluorescence of magnevist was also observed. (3)IgG-SLs or Endoglin-SLswere prepared following the steps as following: Firstly, liposomes (PDP-SLs)containing HEPC, Choi, mPEG-DSPE, PDP-PEG-HEPE were parepared. Then thedithiol-bond of PDP in the PDP-SLs was reduced to - SH by dithiothreitol(DTT) and HS-SLs was thus obtained. Finally, the derivative anti-Endoglinantibody or IgG (malemidophenylbutyrate-Endoglin or IgG, MPB-Endoglin orMPB-IgG) was attached to the surface of PDP-SLs. The liposome diameter andsize distribution were determined by Nicomp 380 Particle Sizing System.Concentration of phospholipids was deterimined with molybdenum blue method.I25I radiolabelling tests was carried out to determined the coupling ratioof antibody to liposomes and the antibody density on the surface ofliposomes. ELISA studies was performed to confirmed theimmunoreacitivities of IgG-SLs and Endoglin-SLs. (4)Sterically stabilizedGd-labelled immunoliposomes targeted to Endoglin (Endoglin-Gd-SLs) wasprepared by the thin film hydration method and the freeze-thawing method.
The shape of Endoglin-Gd-SLs was observed by transmission electron microscope. The liposome diameter and size distribution were determined by Nicomp 380 Particle Sizing System. Gd-DTPA trapping efficency was determined by fluorescent method and the Gd-DTPA concentration in the liposome was also calculated. The effect of concentration, value of pH of Gd-DTPA solution, ratio of HEPC/Chol/mPEG-DSPE/PDP-PEG-HEPE, diameter of liposome on the Gd-DTPA trapping efficency and the Gd-DTPA concentration in the liposome were analyzed. The physical stability of liposome and the leakage ratio of Gd-DTPA were also observed. (5)Forty Fischer 344 rats implanted with F98 gliomas were randomized into four treatment groups and received either: (a)sterically stabilized Gd-labelled liposomes targeted to Endoglin (Endoglin-Gd-SLs);(b)sterically stabilized Gd-labelled liposomes covalently coupled with IgG (IgG-Gd-SLs) ;(c)sterically stabilized Gd-labelled liposomes (Gd-SLs); or (d)Gd-DTPA. MRI scan were performed before, immediately after, and 5, 30, 60, 120min after intravenous injection. MRI enhancement features of each group were observed. To quantify image enhancement over time, CNR at each time point and the relative intensity at time point of maximal enhancement were calculated. After imaging, tumors were resected for a -SMA, VEGF immunohistochemical staining and Endoglin immunofluorescence. E-MVD(microvessel density based on Endoglin immunofluorescence), S-MVD(microvessel density based on a -SMA immunofluorescence), T-MVD (T-MVD= E-MVD+S-MVD) were calculated. VEGF labeling index (VEGF LI) were measured to reflect VEGF expression. The correlation analysis between the relative signal intensity at time point of maximal enhancement and E-MVD, S-MVD, T-MVD and VEGF LI were performed. Results: (1)The results of TLC suggested that PDP-PEG-HEPE was successfully synthesized. (2) The optional conditions for quantitative determination of magnevist were as following: The excitated wavelength was 275nm, emission wavelength was 312nm. The fluorescence of magnevist was stable when pH value was 6.0-8.0, and it positively correlated well with concentration of magnevist when the concentration was 5× 10~(-5) mol/L-1mol/L. (3) Mean diameter of IgG-SLs and Endoglin-SLs was 143nm, the size distribution was homogeneous. Mean diameter of IgG-SLs and Endoglin-SLs was slightly larger than that of SLs and PDP-SLs. The concentration of phospholipids was 1. 5725mmol/L. The results of 125I radiolabeling tests showed the the coupling
ratio of antibody to liposomes was 54. 28%, the antibody density on the surface of liposomes was 78.13ug Ab/umol lipid, which met the needs of experiment. ELISA studies showed the immunoreacitivities of IgG-SLs and Endoglin-SLs was about 44.3% and 51%,respectively. (4)The liposome was round in shape, lipid-membrane and fingerprint-like structure were found. The trapping efficiency and concentration of Gd-DTPA was the highest when the pH value of Gd-DTPA was 6. 0-8. 0, but there was no difference between group pH6. 0 and group pH8.0, and there was also no difference between group pH6.0, pH8. 0 and group pH10.0. While there was significant difference between group pH6. 0, pH8. 0, pHlO. 0 and group 2.0, 4.0 (p<0. 05、 0.01) . The trapping efficiency and concentration of Gd-DTPA in the liposomes was 21. 8% 、 0. 067umol/umol respectively, when the concentration of Gd-DTPA solution was 0.67mol/L. While these was 13.4 % 、 0.022 umol/umol respectively, when concentration of Gd-DTPA solution was 0. 25mol/L. There was statistical significance between group 0. 5mol/L、 0. 67mol/L、 0. 72mol/L and group 0. 25mol/L (p<0. 05> 0.01) .As for the effect of the size of liposomes on the trapping efficiency and concentration of Gd-DTPA was concerned. The larger the size was, the more The trapping efficiency and concentration of Gd-DTPA was. Statistical significance was found between group lOOnm, 150nm, 200nm and group 50nm (p<0. 05> 0.01) , but there is no difference among the former three groups. Four groups (group A、 B、 C、 D) with different ratio of HEPC/Chol/mPEG-DSPE/PDP-PEG-HEPE were analyzed. The ratio was 3:2:0.1:0.02,4:1:0.3:0.05,2:1:0.1:0.01 , 11:8:0.1:0.01 respectively. The trapping efficiency was statistical different between the former two groups and the latter two groups.As for the concentration of Gd-DTPA in the liposomes was concerned, Statistical difference was found between group A and group C、 D (p<0.05), There was also difference between group B and group D (p<0. 05) . While there was no difference between group B and C, between group C and D. The stability of IgG-Gd-SLs stay between Gd-CLs and Gd-SLs during restoration. When IgG -Gd-SLs were suspended in either PBS or 1% human plasma for 10 hours, the leakage ratio of Gd-DTPA was 19. 8%, 11.2%, respectively. (5) Enhancement differences were observed among the four groups. Slight enhancement was found after 5 min and singnal intensity of enhancement region increased steadily within 1h in group Endoglin-Gd-SLs、 group IgG-Gd-SLs and group Gd-SLs. Difference of the three
groups lay in enhancement at 2h point. A more significant signal intensity increase was detected after 2h in group Endoglin-Gd-SLs. In group IgG-Gd-SLs, signal intensity of enhancement after 2h was less than that after 1h, but was more than that at baseline. While in group Gd-SLs, signal intensity of tumor almost return to baseline after 2h. In group Gd-DTPA, contrast enhancement was detected soon after Gd-DTPA injection and maximal enhancement was found after 5min. While singnal intensity decreased after 30min and returned to baseline after lh. In addition, the size of enhancement region over time was difference among the four groups. In group Endoglin-Gd-SLs, enhancement region was confined and did not expanded within 2h. While distended enhancement region from periphery to central within lh was found in group IgG-Gd-SLs and group Gd-SLs. In group Gd-DTPA, homogeneous enhancement was found soon after injection.Correlation analysis showed the relative intensity at time point of maximal enhancement correlated well with E-MVD, VEGF LI in the four groups (p<0.05, 0.01), it did not have correlation with S-MVD, T-MVD in group Endoglin-Gd-SLs(p>0.05) , while in other three groups, it correlated well with S-MVD, T-MVD (p<0. 05, 0.01).Conclusions:(1)The optional conditions for quantitative determination of magnevist were as following: The excitated wavelength was 275nm, emission wavelength was 312nm. The fluorescence of magnevist was stable when pH value was 6.0-8.0, and it positively correlated well with concentration of magnevist when the concentration was 5×10~(-5)mol/L-lmol/L. Fluorescent method is a simple and efficient method to quantitative determination of magnevist concentration. (2)The linkage method of antibodies to the terminal of PEG of liposome surface via thioether bond could retain their immunoreacitivities. The decrease of immunoreacitivities corrlate with two factors as following: the SMPB derivation of antibody, the steric hinderance effect of liposome. (3)Protocol of trapping Gd-DTPA was as following: 0. 67mol/L Gd-DTPA solution with pH 6. 0-8. 0, the 3/3/0. 1/0. 02 (mole ratio) blend of HEPC, Choline, mPEG-DSPE and PDP-PEG-HEPE, the mean diameter should be 100nm-150nm in order to meet the experiment. The stability ofIgG-Gd-SLs stay between Gd-CLs and Gd-SLs during restoration. (4) Enhancement of Endoglin-Gd-SL correlate well with glioma neovasculature.It is feasible and efficient to detect tumor angiogenesis in vivo using
引文
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4. Rubenstein JL, Kim J, Ozawa T, et al. Anti-VEGF antibody treatment of glioblastoma prolongs survival but results in increased vascular cooption. Neoplasia, 2000;2:306～314
5. Kunkel P, Ulbricht U, Bohlen P, et al. Inhibition of glioma angiogenesis and growth in vivo by systemic treatment with a monoclone antibody againt vascular endothelial growth factor-2. Cancer Res, 2001,61;6624～6628
6.牛红梅。癌症患者循环中血管内皮生长因子的临床意义。国外医学临床生物化学与检验学分册,2002;23(3):166～167
7.张冬,邹利光,王文献,等。脑星形细胞瘤的MRI指标与MVD的关系。第三军医大学学报,2004;26(14):1308-1310
8. Brasch RC, Li KC,Husband JE, et al. In vivo monitoring of tumor angiogenesis with MR imaging. Academic Radiology,2000,7(10):812～821
9. Neeman M, Provenzale JM, Dewhirst MW. Magnetic resonance imaging applications in the evaluation of tumor angiogenesis.Seminars in Radiation Oncology,2001,11(1):70～78
10. Wikstrom P, Lissbrant IF, Stanttin P, et al. Endoglin (CD105) is expressed on immature blood vessel and is a marker for survival in prostate cancer. Prostate, 2002;51 (4):268-275
11.丁士刚,林三仁,吴国荣,等。胃癌组织中CD105标记的微血管密度及其临床意义。中华消化杂志,2004;24(7):431-432
12.陈国庭,尹浩然,朱正刚。Endoglin.肿瘤新生血管的标志物。国外医学生理、病理科学与临床分册,2002;22(1):7-8
13. Kumar S, Ghellal A, Li C, et al. Breast carcinoma: vascular density determined using CD105 anbody correlates with tumor prognosis. Cancer Res, 1999;59(4):856-861
14.牛国琴,刘敏,陆伟跃。空间稳定免疫脂质体体内过程及其对策的研究进展。国外医学 药学分册。2002,29(5):301-306
15. Han G, Tamaki M, Hruby VJ. Fast, efficient and selective deprotection of the tert-butoxycarbonyl (Boc) group using HC1/dioxane (4M). J Peptide Res, 2001; 58:338-341
16.唐尧,赵郁,饶桂香,等。复方氨基酸补铁液中氨基酸的薄层鉴别。华西药学杂志,1997:12(4):257-258
17. Carlsson J, Drevin H, et al. Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio) propionate, a new hetero bifunctional reagent. Biochem J, 1978;173:723-737.
18. Torchilin VP, Klibanov AL, et al. Targeted accumulation of polyethylene glycol-coatd immunoliposomes in infracted rabbit mycocardium. FASEB J, 1992;6:2716
19. Abou-Taleb SA, microdetermination of metals in organometallic compounds by the xoxide method after oxygen flask. Microchem J, 1986;33(1):29-33
20.中华人民共和国卫生部。WS-069(X-056)-96药品标准。北京:中华人民共和国卫生部药政局,1996
21.孙丽,游强等。RP-HPLC外标法测定钆喷酸葡胺原料及其注射液的含量。中国药事,2003,17(3):175-176
22.吴昭珲,王尊本,游文纬。应用二阶导数紫外分光光度法测定钆喷酸。分析化学,1997,25(2):243～245
23. Allen TM, Brandeis E, et al. A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells. Biochim Biophys Acta,1995; 1237:99-108
24. Schneider T, Sachse A, et al. Generaion of contrast-carrying liposomes of defined size woth a new continuous high pressure extrusion method. Int J Pharm, 1995;117:1-12
25. Mrsny RJ, Volwerk JJ,Griffith OH. A simplified procedure for lipid phosphorus analysis shows that digestion rates vary with phospholipid structure. Chem Phys Lipids,1986;39:185-191
26. Mercadal M, Domingo JC, et al. N-Palmitoylphosphatidylethanolamine stabilizes liposomes in the presence of human serum: effect of lipidic composition and system characterization. Biochim Biophys Acta, 1995; 1235:281-288
27. Sarma VR, Silverton EW. et al. The three-dimensional structure at 6A resolution of a human rGl immunoglobulin molecule. J Biol Chem,1971; 246:3752-3759
28. Fonseca M J, Van Winden ECA. et al. Doxorubicin induces aggregation of small negatively charged liposomes. Eur J Pharma Biopharm, 1997; 43 ;9-17
29. Allen TM, Brandeis E, et al. A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells. Biochim Biophys Acta,1995; 1237:99-108
30. Hansen CB, Kao GY, et al. Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of couling procedures. Biochim Biophys Acta, 1995,1239:133-144
31.张宇峰,谢蜀生。具有活性羧基末端的长循环脂质体的制备和分布。药学学报,2000;35(11):854-859。
32. Bangham AD, Standish MM, Watkins JC. Diffusion of univalent inos across the lamella of swollen phospholipids. J Mol Biol,1965; 13:238-252
33. Martin C, Woodle, Danilo D Lasic. Sterically stabilized liposomes. Biochimica et Biophysica Acta, 1992,1113:171-199
34.王闻珠,邓英杰。脂质体肺部给药研究进展。沈阳药科大学学报,2000;17(3):226-229。
35. Gregoriadis G. Liposome Technology. Boca Raton: CRC Press,1984.
36.陈忠斌,王升启,王弘,等。pH敏脂质体对反义寡核苷酸康流感病毒活性的影响。中国生物化学与分子生物学报,1999;15(4):553-557。
37.石丽萍,颜光涛,李英丽,等。酸敏脂质体的制备及其在肠缺血-再灌注小鼠体内重要脏器中的分布。中华危重病急救医学,2001;13(11):659-661。
38.邹一愚,顾学裘,崛越勇。肝动脉注射阿霉素温度敏感脂质体的制剂研究。药学学报,1991;26(8):622-626.
39. Bedu-Addo FK, Tang P, Xu Y, et al. Effects of polyethyleneglyeol chain length and phospholipids acyl chain composition and the interaction of polyethuleneglycol-phorpholipid conjugates with phospholipids: implications in liposomal drug delivery. Pharm Res, 1996;13(5):710
40. Mori A, Klibanov AL, Torchilin VP, et al. Influence of the steric barrier of amphiphathicpoly (etthlenegly-col) and ganglioside GM1 on the circulation time of liposomes and on the target binding of immunoliposomes in vivo. FEBS letter, 1991 ;284(2):263
41. Torchilin VP, Klibanov AL, et al. Targentd accumulation of polyethylene glycol-coated immunoliposomes in infracted rabbit myocardium. FASEB J,1992;6:2716
42. Torchilin VP, Papisov MI, et al. J Liposome Res, 1994。
43. Blume G, Cevc G, et al. Specific targeting with poly(ethylene glycol)-modified liposomes: coupling of homing devices to the ends of the polymeric chains combines effective target binding with long circulation times. Biochim Biophys Acta,1993;1149:180-184
44. Maruyama K, Takizaua T, et al. Targetability of novel immunoliposome modified with amphipathic poly (ethylene glycol) conjugated at their distal terminals to monoclonal antibodies. Biochim Biophys Acta, 1995; 1234:74-83
45. Torchilin VP. Poly (ethylene glycol)-coated anti-cardiac myosin immunoliposomes: factors influencing targeted accumulation in the infracted myocardium. Biochim Biophys Acta, 1996; 1279:75-83
46. DEL de Menezes. In vitro and in vivo targeting of immunoliposomal doxorubicin to human B-cell lymphoma. Cancer Res, 1998;58:3320-3330
47. Lundberg BB. Specific binding of sterically stabilized anti-B-cell immunoliposomes and cytotoxicity of entrapped doxorubicin. Int J Pharm, 2000;205:101-108
48. Moase EH. Anti-MUC-1 immunoliposomal doxorubicin in the treatment of murine models of metastatic breast cancer. Biochim Biophys Acta, 2001; 1510:43-55.
49. Park JW. Tumor targeting using anti-her2 immunoliposomes. J Control Release,2001 ;74:95-113
50. Klibanov AL, Maruyama K, Torchilin VP, et al. Amphipathic polyethleneglycols effectively prolong the circulation time of liposomes. FEBS Lett. 1990;268:235-237.
51. Matthay KK, Heath TD, et al. Specific enhancement of drug deliver to AKR lymphoma by antibody-targeted small unilamellar vesicles. Cancer Res,1984;44:1880-1886
52. Derksen JTP, Scherphof GL. An improved method for the covalent coupling of protein to liposomes. Biochim Biophys Acta, 1985 ;814:151-155
53.许乙凯,柴青芬,刘岘,等。生物素-亲合素预定位技术在肿瘤磁共振免疫成像中的初步应用研究。中国医学影像学杂志,2002,10(2):129-131
54. Ishida T, Iden DL, et al. A combinatorial approach to producing sterically stabilized immunoliposomal drugs. FEBS Lett, 1999;460:129-133
55. Torchilin VP, Levchenko TS. p-Nitrophenylcarbonyl-PEG-PE-liposomes fast and simple attachment of specific ligands including monoclonal antibodies to distal ends of PEG chains via p-nitrophenylcarbonyl groups. Biochim Biophys Acta, 2001;1511:397-411.
56. Sivakumar PA, Panduranga Rao K. Polymerized (ethylene glycol) dimetha crylate-cholesteryl methacrylate liposomes: preparation and stability studies. Reactive Functional Polymers,2001 ;49:179-187
57.王黎,侯宝光。氢化与非氢化卵磷脂对阿霉素脂质体体内外稳定性的影响。药学学报,2001;36(6):444-447
58. Mori A, Klibannov AL, Torchilin VP, et al. Influence of steric barrier activity of amphipathicpoly(ethyleneglycol) and ganglioside GM1 on the circulation time of liposomes and on the target binding of immunoliposomes in vivo. FEBS Lett, 1991;284:263-266.
59. Wang CY, Yughes KW, Huang L. Improved cytoplasmic delivery to plant protoplasts via pH sensitive liposome. Plant Physiol,1986; 82:179-186.
60. Tilcock CP, Ahkong QF, Parr M. An improved method for the preparation of liposomal Gadolinium-DTPA: Ionophore-mediated active entrapment of Gadolinium. Invest Radiol, 1991 ;26:242-247.
61. Kabalka GW, Davis MA, Moss TH, et al. Gadolinium-labeled liposomes containing various amphiphilic Gd-DTPA derivatives: targeted MRI contrast enhancement agents for the liver. Mag Reson Med, 1991; 19:406-415.
62. Barsky D, Putz B, Schulten K, et al. Theory of paramagnetic contrast agent in liposome systems. Mag Reson Med. 1992,24:1-13。
63. Litinger DC, Buiting AMJ, et al. Effect of liposome size on the circulation time and intraorgan distribution of amphipathic poly(ethylene glycol)-containing liposomes. Biochim Biophy Acta, 1994,1990:99-107
64. Nagayasu A, Uchiyama K, et al. Is control of distribution of liposomes between tumors and bone marrow possible? Biochim Biophy Acta, 1996; 1278:29-34
65. Bally MB, Naya R, et al. Studies on the myelosuppressive activity of doxorubicin entrapped in liposomes. Cancer Chemother Pharmacol, 1990;27:13-19.
66. Weidner N, Intratumor microvessel density as a prognostic factor in cancer. Am J Pathol, 1995, 147:9-19
67. Tanigawa N, Lu C, Mitsui T, et al. Quantitation of sinusoid like vessels in hepatocellular carcinoma: its clinical and prognostic significance. Hepatology, 1997, 26:1216～122
68. Winter PM, Caruthers SD, Kassner CA, et al. Molecular Imaging of angiogenesis in nascent Vx-2 rabbit tumors using a novel α v β 3-targeted nanoparticle and 1.5T magnetic resonance imaging. Cancer Res, 2003;63:5838-5843
69. Sipkins DA, Cheresh DA, Kazemi MR, et al. Detection of tumor angiogenesis in
2. Leenders WPJ, Kusters B, Waal RMW. Vessel co-option: how tumors obtain blood supply in the absence of sprouting angiogenesis. Endothelium,2002;9:83～8
3. Holash J, Maisonpierre PC, Compton D, et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science, 1999; 284:1994～1998
4. Rubenstein JL, Kim J, Ozawa T, et al. Anti-VEGF antibody treatment of glioblastoma prolongs survival but results in increased vascular cooption. Neoplasia, 2000;2:306～314
5. Kunkel P, Ulbricht U, Bohlen P, et al. Inhibition of glioma angiogenesis and growth in vivo by systemic treatment with a monoclone antibody againt vascular endothelial growth factor-2. Cancer Res, 2001,61;6624～6628
6.牛红梅。癌症患者循环中血管内皮生长因子的临床意义。国外医学临床生物化学与检验学分册,2002;23(3):166～167
7.张冬,邹利光,王文献,等。脑星形细胞瘤的MRI指标与MVD的关系。第三军医大学学报,2004;26(14):1308-1310
8. Brasch RC, Li KC,Husband JE, et al. In vivo monitoring of tumor angiogenesis with MR imaging. Academic Radiology,2000,7(10):812～821
9. Neeman M, Provenzale JM, Dewhirst MW. Magnetic resonance imaging applications in the evaluation of tumor angiogenesis.Seminars in Radiation Oncology,2001,11(1):70～78
10. Wikstrom P, Lissbrant IF, Stanttin P, et al. Endoglin (CD105) is expressed on immature blood vessel and is a marker for survival in prostate cancer. Prostate, 2002;51 (4):268-275
11.丁士刚,林三仁,吴国荣,等。胃癌组织中CD105标记的微血管密度及其临床意义。中华消化杂志,2004;24(7):431-432
12.陈国庭,尹浩然,朱正刚。Endoglin.肿瘤新生血管的标志物。国外医学生理、病理科学与临床分册,2002;22(1):7-8
13. Kumar S, Ghellal A, Li C, et al. Breast carcinoma: vascular density determined using CD105 anbody correlates with tumor prognosis. Cancer Res, 1999;59(4):856-861
14.牛国琴,刘敏,陆伟跃。空间稳定免疫脂质体体内过程及其对策的研究进展。国外医学 药学分册。2002,29(5):301-306
15. Han G, Tamaki M, Hruby VJ. Fast, efficient and selective deprotection of the tert-butoxycarbonyl (Boc) group using HC1/dioxane (4M). J Peptide Res, 2001; 58:338-341
16.唐尧,赵郁,饶桂香,等。复方氨基酸补铁液中氨基酸的薄层鉴别。华西药学杂志,1997:12(4):257-258
17. Carlsson J, Drevin H, et al. Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio) propionate, a new hetero bifunctional reagent. Biochem J, 1978;173:723-737.
18. Torchilin VP, Klibanov AL, et al. Targeted accumulation of polyethylene glycol-coatd immunoliposomes in infracted rabbit mycocardium. FASEB J, 1992;6:2716
19. Abou-Taleb SA, microdetermination of metals in organometallic compounds by the xoxide method after oxygen flask. Microchem J, 1986;33(1):29-33
20.中华人民共和国卫生部。WS-069(X-056)-96药品标准。北京:中华人民共和国卫生部药政局,1996
21.孙丽,游强等。RP-HPLC外标法测定钆喷酸葡胺原料及其注射液的含量。中国药事,2003,17(3):175-176
22.吴昭珲,王尊本,游文纬。应用二阶导数紫外分光光度法测定钆喷酸。分析化学,1997,25(2):243～245
23. Allen TM, Brandeis E, et al. A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells. Biochim Biophys Acta,1995; 1237:99-108
24. Schneider T, Sachse A, et al. Generaion of contrast-carrying liposomes of defined size woth a new continuous high pressure extrusion method. Int J Pharm, 1995;117:1-12
25. Mrsny RJ, Volwerk JJ,Griffith OH. A simplified procedure for lipid phosphorus analysis shows that digestion rates vary with phospholipid structure. Chem Phys Lipids,1986;39:185-191
26. Mercadal M, Domingo JC, et al. N-Palmitoylphosphatidylethanolamine stabilizes liposomes in the presence of human serum: effect of lipidic composition and system characterization. Biochim Biophys Acta, 1995; 1235:281-288
27. Sarma VR, Silverton EW. et al. The three-dimensional structure at 6A resolution of a human rGl immunoglobulin molecule. J Biol Chem,1971; 246:3752-3759
28. Fonseca M J, Van Winden ECA. et al. Doxorubicin induces aggregation of small negatively charged liposomes. Eur J Pharma Biopharm, 1997; 43 ;9-17
29. Allen TM, Brandeis E, et al. A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells. Biochim Biophys Acta,1995; 1237:99-108
30. Hansen CB, Kao GY, et al. Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of couling procedures. Biochim Biophys Acta, 1995,1239:133-144
31.张宇峰,谢蜀生。具有活性羧基末端的长循环脂质体的制备和分布。药学学报,2000;35(11):854-859。
32. Bangham AD, Standish MM, Watkins JC. Diffusion of univalent inos across the lamella of swollen phospholipids. J Mol Biol,1965; 13:238-252
33. Martin C, Woodle, Danilo D Lasic. Sterically stabilized liposomes. Biochimica et Biophysica Acta, 1992,1113:171-199
34.王闻珠,邓英杰。脂质体肺部给药研究进展。沈阳药科大学学报,2000;17(3):226-229。
35. Gregoriadis G. Liposome Technology. Boca Raton: CRC Press,1984.
36.陈忠斌,王升启,王弘,等。pH敏脂质体对反义寡核苷酸康流感病毒活性的影响。中国生物化学与分子生物学报,1999;15(4):553-557。
37.石丽萍,颜光涛,李英丽,等。酸敏脂质体的制备及其在肠缺血-再灌注小鼠体内重要脏器中的分布。中华危重病急救医学,2001;13(11):659-661。
38.邹一愚,顾学裘,崛越勇。肝动脉注射阿霉素温度敏感脂质体的制剂研究。药学学报,1991;26(8):622-626.
39. Bedu-Addo FK, Tang P, Xu Y, et al. Effects of polyethyleneglyeol chain length and phospholipids acyl chain composition and the interaction of polyethuleneglycol-phorpholipid conjugates with phospholipids: implications in liposomal drug delivery. Pharm Res, 1996;13(5):710
40. Mori A, Klibanov AL, Torchilin VP, et al. Influence of the steric barrier of amphiphathicpoly (etthlenegly-col) and ganglioside GM1 on the circulation time of liposomes and on the target binding of immunoliposomes in vivo. FEBS letter, 1991 ;284(2):263
41. Torchilin VP, Klibanov AL, et al. Targentd accumulation of polyethylene glycol-coated immunoliposomes in infracted rabbit myocardium. FASEB J,1992;6:2716
42. Torchilin VP, Papisov MI, et al. J Liposome Res, 1994。
43. Blume G, Cevc G, et al. Specific targeting with poly(ethylene glycol)-modified liposomes: coupling of homing devices to the ends of the polymeric chains combines effective target binding with long circulation times. Biochim Biophys Acta,1993;1149:180-184
44. Maruyama K, Takizaua T, et al. Targetability of novel immunoliposome modified with amphipathic poly (ethylene glycol) conjugated at their distal terminals to monoclonal antibodies. Biochim Biophys Acta, 1995; 1234:74-83
45. Torchilin VP. Poly (ethylene glycol)-coated anti-cardiac myosin immunoliposomes: factors influencing targeted accumulation in the infracted myocardium. Biochim Biophys Acta, 1996; 1279:75-83
46. DEL de Menezes. In vitro and in vivo targeting of immunoliposomal doxorubicin to human B-cell lymphoma. Cancer Res, 1998;58:3320-3330
47. Lundberg BB. Specific binding of sterically stabilized anti-B-cell immunoliposomes and cytotoxicity of entrapped doxorubicin. Int J Pharm, 2000;205:101-108
48. Moase EH. Anti-MUC-1 immunoliposomal doxorubicin in the treatment of murine models of metastatic breast cancer. Biochim Biophys Acta, 2001; 1510:43-55.
49. Park JW. Tumor targeting using anti-her2 immunoliposomes. J Control Release,2001 ;74:95-113
50. Klibanov AL, Maruyama K, Torchilin VP, et al. Amphipathic polyethleneglycols effectively prolong the circulation time of liposomes. FEBS Lett. 1990;268:235-237.
51. Matthay KK, Heath TD, et al. Specific enhancement of drug deliver to AKR lymphoma by antibody-targeted small unilamellar vesicles. Cancer Res,1984;44:1880-1886
52. Derksen JTP, Scherphof GL. An improved method for the covalent coupling of protein to liposomes. Biochim Biophys Acta, 1985 ;814:151-155
53.许乙凯,柴青芬,刘岘,等。生物素-亲合素预定位技术在肿瘤磁共振免疫成像中的初步应用研究。中国医学影像学杂志,2002,10(2):129-131
54. Ishida T, Iden DL, et al. A combinatorial approach to producing sterically stabilized immunoliposomal drugs. FEBS Lett, 1999;460:129-133
55. Torchilin VP, Levchenko TS. p-Nitrophenylcarbonyl-PEG-PE-liposomes fast and simple attachment of specific ligands including monoclonal antibodies to distal ends of PEG chains via p-nitrophenylcarbonyl groups. Biochim Biophys Acta, 2001;1511:397-411.
56. Sivakumar PA, Panduranga Rao K. Polymerized (ethylene glycol) dimetha crylate-cholesteryl methacrylate liposomes: preparation and stability studies. Reactive Functional Polymers,2001 ;49:179-187
57.王黎,侯宝光。氢化与非氢化卵磷脂对阿霉素脂质体体内外稳定性的影响。药学学报,2001;36(6):444-447
58. Mori A, Klibannov AL, Torchilin VP, et al. Influence of steric barrier activity of amphipathicpoly(ethyleneglycol) and ganglioside GM1 on the circulation time of liposomes and on the target binding of immunoliposomes in vivo. FEBS Lett, 1991;284:263-266.
59. Wang CY, Yughes KW, Huang L. Improved cytoplasmic delivery to plant protoplasts via pH sensitive liposome. Plant Physiol,1986; 82:179-186.
60. Tilcock CP, Ahkong QF, Parr M. An improved method for the preparation of liposomal Gadolinium-DTPA: Ionophore-mediated active entrapment of Gadolinium. Invest Radiol, 1991 ;26:242-247.
61. Kabalka GW, Davis MA, Moss TH, et al. Gadolinium-labeled liposomes containing various amphiphilic Gd-DTPA derivatives: targeted MRI contrast enhancement agents for the liver. Mag Reson Med, 1991; 19:406-415.
62. Barsky D, Putz B, Schulten K, et al. Theory of paramagnetic contrast agent in liposome systems. Mag Reson Med. 1992,24:1-13。
63. Litinger DC, Buiting AMJ, et al. Effect of liposome size on the circulation time and intraorgan distribution of amphipathic poly(ethylene glycol)-containing liposomes. Biochim Biophy Acta, 1994,1990:99-107
64. Nagayasu A, Uchiyama K, et al. Is control of distribution of liposomes between tumors and bone marrow possible? Biochim Biophy Acta, 1996; 1278:29-34
65. Bally MB, Naya R, et al. Studies on the myelosuppressive activity of doxorubicin entrapped in liposomes. Cancer Chemother Pharmacol, 1990;27:13-19.
66. Weidner N, Intratumor microvessel density as a prognostic factor in cancer. Am J Pathol, 1995, 147:9-19
67. Tanigawa N, Lu C, Mitsui T, et al. Quantitation of sinusoid like vessels in hepatocellular carcinoma: its clinical and prognostic significance. Hepatology, 1997, 26:1216～122
68. Winter PM, Caruthers SD, Kassner CA, et al. Molecular Imaging of angiogenesis in nascent Vx-2 rabbit tumors using a novel α v β 3-targeted nanoparticle and 1.5T magnetic resonance imaging. Cancer Res, 2003;63:5838-5843
69. Sipkins DA, Cheresh DA, Kazemi MR, et al. Detection of tumor angiogenesis in