磁共振功能成像用于超声微泡—纳米脂质体复合体介导肝细胞生长因子基因治疗肝纤维化的研究
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摘要
肝纤维化是肝脏各种慢性损伤导致纤维素沉积形成，进而改变肝脏组织结构和功能，甚至引起肝硬化和肝衰竭。基因治疗肝纤维化是一种新的有效治疗手段。目前许多生长因子已成功用于肝纤维化基因治疗。肝细胞生长因子（HGF）能促进成熟细胞有丝分裂，是有潜力的抗纤维化因子，能够防止许多器官纤维化，包括肝脏。可是缺乏一种安全有效的基因传递载体一直是临床应用的障碍。微泡是临床医学影像超声的显像剂，能在超声波引导下产生空化效应，利用这种特征传递基因可提高转染效率。但微泡在体内通常不稳定，并且直径大约1-6um，无法渗透出静脉，而且靶向分子很难在微泡表面修饰。脂质体作为药物和基因传递载体都很有优势，但转染效率低，并且没有靶向性。因此，我们应用生物素-亲和素系统结合超声微泡和阳离子纳米脂质体形成生物素化超声微泡和生物素化阳离子纳米脂质体复合体（Bio-MB＋Bio-CNLP）载体。有效克服两者的不足，明显提高转染效率，经体内及体外实验证实Bio-MB＋Bio-CNLP能携带HGF基因靶向治疗肝纤维化，是一种新型有效的基因载体。
肝活组织检查一直是肝纤维化诊断的金标准，但具有有创性、少数可见并发症及样本误差等缺点限制了临床使用。急需研究一种无创、有效评估肝纤维化程度的诊断技术。这种技术必须没有活组织检查的风险,能简单、方便有效评估抗纤维化程度及治疗效果。本实验我们通过胆总管结扎术建立大鼠肝纤维化模型,尾静脉注射Bio-MB+Bio-CNLP-pCDH-HGF载体,治疗后分别用磁共振扩散加权成像(diffusion-weighted imaging，DWI)、磁共振灌注成像（Perfusionimaging，PWI）和磁共振波普分析（magnetic aesonance spectroscopy，MRS）三种MRI功能成像评估治疗效果。结果表明磁共振功能成像能准确监测肝纤维化发展过程，评估肝纤维化治疗效果。本研究显示三种MRI功能成像参数能定量分析肝纤维化,与肝纤维化分期具有显著相关性，可以为临床诊断和评估肝纤维化治疗提供依据。
第一部分Bio-MB+Bio-CNLP介导HGF基因治疗肝纤维化生物学效应的体外初步研究
目的：构建一个新型的生物素化超声微泡基因载体，评估载体介导HGF基因的靶向性和安全性以及对肝脏细胞的作用
方法：运用生物素-亲和素系统构建Bio-MB+Bio-CNLP载体并且和HGF基因重组；分别转染肝星状细胞(HSC-T6)和肝细胞(L02),分为4组：（1） Bio-MB+Bio-CNLP+HGF无超声辐照；（2）Bio-MB+Bio-CNLP+HGF超声辐照；（3）脂质体+HGF;（4）Bio-MB+Bio-CNLP超声辐照。转染成功后分别用流式细胞仪检测转染率和凋亡率；细胞免疫荧光染色、ELISA和Western blot检测蛋白表达；MTT检测细胞活性。
结果：超声辐照组转染成功，而无超声辐照组则无转染；与单独脂质体转染比较，Bio-MB+Bio-CNLP介导HGF明显提高转染率；HGF基因转染HSC-T6细胞后72小时蛋白表达量最高，细胞凋亡率明显增加；而转染L02细胞活性明显增加。
结论：Bio-MB+Bio-CNLP能靶向传递HGF基因并明显提高转染率，促进HSC细胞凋亡，增加正常干细胞活性，表明我们成功制备了一种新型的具有靶向性、安全性和有效性Bio-MB+Bio-CNLP载体。
第二部分磁共振功能成像对大鼠肝纤维化诊断、定量分析研究
目的：探索一种新型靶向超声微泡载体介导HGF基因体内表达治疗肝纤维化，并且通过MRI功能成像检测疗效。
方法：通过胆总管结扎构建大鼠肝纤维化模型，尾静脉注射Bio-MB+Bio-CNLP+HGF悬液，肝脏超声辐照定位转递HGF基因治疗。分别运用DWI、PWI和MRS三种功能成像检测治疗效果，并与病理检查结果对照。
结果：我们靶向传递HGF基因并且产生抗纤维化效果；MRI功能成像各参数能准确区分肝纤维化分期。
结论：Bio-MB+Bio-CNLP在体内能靶向传递HGF基因，有效治疗肝纤维化，MRI功能成像参数能用于评估肝纤维化程度和疗效检测。
Liver fibrosis is the wound-healing response to various liver damage，in the form of a progressive accumulation of fibrosis．It alters the tissuestructure and function of liver，leading to cirrhosis and liver failure, Genetherapy may provide novel treatment strategies for a wide range of liverfibrosis. HGF was originally characterized as a potent mitogen for maturehepatocytes, has emerged as a potent anti-fibrotic cytokine that preventstissue fibrosis in various organs, including the liver.[，However, the lack of asafe and effective method for gene delivery to a specific organ or cellremains an obstacle to clinical application. Microbubbles are contrast agentsfor medical ultrasound imaging, improve transfection efficiency afterultrasound-induced cavitation, However, microbubbles are generallyunstable and their mean diameter of around1–6μm is too large forintravascular applications. Moreover, functional particles such as targetingmolecules are difficult to modify on the surface of microbubbles., Theirsurface should be easily modified with functional molecules for targeting after injection into the blood and ultimately， Liposomes have someadvantages as drug, antigen and gene delivery carriers, Their size can beeasily controlled and they can be modified to add a targeting function.but thegene expression was very low Therefore, we developed novelBio-MB+Bio-CNLP, Here, we assessed the feasibility ofBio-MB+Bio-CNLP for gene delivery after cavitation induced byultrasound.
Chronic diffuse liver disease is a worldwide problem.Whereashistology remains the gold standard by which to diagnose hepatic fibrosis,Several clinical limitations are associated with the use of liver biopsy.It isan invasive and costly procedure prone to complications, sampling error isinherent in the technique, Alternative techniques for assessingthe severityof diffuse liver disease are urgently needed. and such a tool would reducebiopsy related risks and costs, could be useful for guiding anti-fibrotictreatments, monitoring treatment efficacy, and helpful in the clinicalevaluation of new anti-fibrotic drugs. Furthermore, the identification ofoccult advanced fibrosis, may direct further management, and has essentialprognostic implications.
We established a bile duct ligation (BDL) rat model of hepatic fibrosis.Bio-MB+Bio-CNLP-pCDH-HGF carrier was administered through tail-veininjections. The gene therapy efficacy was evaluated in vivo. Before and aftertreatment, the rats were examined with diffusion-weighted imaging （DWI）、 Perfusion imaging (PWI) and magnetic aesonancespectroscopy(MRS) respectively. Magnetic resonance functional imagingcould potentially provide a more accurate representation of the diseaseprocess. Recently, in vivo Magnetic resonance functional imaging hasbeen suggested for the evaluation of chronic liver disease and discuss thevalue of MR functional imaging in early diagnosis、quantization analysisand staging of hepatic fibrosis by analyzing the changes of perfusionparameters of MR functional imaging of different degree of hepaticfibrosis
Part I Enhanced effect of HGF on liver cell growth with specialemphasis on hepatic stellate cells by a novel modified ultrasoundtargeted microbubble gene delivery system
Purpose: To evaluate the efficacy and safetyof human hepatocytegrowth factor (HGF) delivery by a novel ultrasound targeted microbubblesystemusing avidin to crosslink biotinylated ultrasound microbubbles(Bio-MB) and biotinylated cationic nano-liposomes (Bio-CNLP), which isreferred to as Bio-MB+Bio-CNLP.
Methods: HGF was linked to Bio-MB+Bio-CNLP, and transfected tohepatic stellate cells (HSC-T6) and normal liver cell line L02according tothe following four groups:(1) Bio-MB+Bio-CNLP+HGF, no ultrasonicirradiation group;(2) Bio-MB+Bio-CNLP+HGF+ultrasonic irradiation group;(3) liposomes+HGF;(4) Bio-MB+Bio-CNLP+ultrasonicirradiation group. Protein expression, L02cell viability, and HSC-T6cellapoptosis rate were determined respectively.
Results: Compared with liposome mediated transfection, Bio-MB+Bio-CNLP mediated transfection rate increased markedly, with the highestprotein expression in HGF transfected HSC-T6cells at72hours aftertransfection. Compared with mock transfection control group, the apoptosisrate was32.8%and7.5%respectively. Cell viability significantly increasedin HGF transfected L02compared with normal L02.
Conclusion: Introducing HGF using an ultrasound-mediated Bio-MB+Bio-CNLP system improved gene transfection rate and promoted normalliver cell growth. This appears to be a promising approach for targetedtherapy.
Part Ⅱ Efficacy of HGF for treating hepatic fibrosis and itsrelationship with MRI functional imaging parameters
Object: The aims of the present study were: i) to explore the interest oftreating liver fibrosisusing human hepatocyte growth factor (HGF)expression vector carried by a novel ultrasound targeted microbubbledelivery system.; b) to explore the diagnostic interest of MR functionalimaging parameters in evaluating liver fibrosis
Method: We established a rat model of hepatic fibrosis by bile duct ligation. The rats were administered HGF linked to biotynilated ultrasoundmicrobubbles and biotynilated cationic nano-liposomes, and compared tosham-operated animals. The impact on hepatic fibrosis was evaluated byhisto-pathology, hydroxyproline content, with special focus on MRfunctional imaging parameters.
Result: Our targeted gene therapy produced a significant anti-fibrosiseffect, as shown by liver histology and decreased hepatic collagen content.Moreover, proved of interest as indicated by the respective evolution of MRfunctional imaging parameters.
Conclusion: This is the first in vivo report of using ultrasoundmicrobubble-cationic nanoliposomes complex for gene delivery. The dataindicate that, in our experimental model, this approach is efficient tocounteract the fibrosis process. MRI functional imaging parameters appearsa promising imaging technique for reflecting liver fibrosis..
肝活组织检查一直是肝纤维化诊断的金标准，但具有有创性、少数可见并发症及样本误差等缺点限制了临床使用。急需研究一种无创、有效评估肝纤维化程度的诊断技术。这种技术必须没有活组织检查的风险,能简单、方便有效评估抗纤维化程度及治疗效果。本实验我们通过胆总管结扎术建立大鼠肝纤维化模型,尾静脉注射Bio-MB+Bio-CNLP-pCDH-HGF载体,治疗后分别用磁共振扩散加权成像(diffusion-weighted imaging，DWI)、磁共振灌注成像（Perfusionimaging，PWI）和磁共振波普分析（magnetic aesonance spectroscopy，MRS）三种MRI功能成像评估治疗效果。结果表明磁共振功能成像能准确监测肝纤维化发展过程，评估肝纤维化治疗效果。本研究显示三种MRI功能成像参数能定量分析肝纤维化,与肝纤维化分期具有显著相关性，可以为临床诊断和评估肝纤维化治疗提供依据。
第一部分Bio-MB+Bio-CNLP介导HGF基因治疗肝纤维化生物学效应的体外初步研究
目的：构建一个新型的生物素化超声微泡基因载体，评估载体介导HGF基因的靶向性和安全性以及对肝脏细胞的作用
方法：运用生物素-亲和素系统构建Bio-MB+Bio-CNLP载体并且和HGF基因重组；分别转染肝星状细胞(HSC-T6)和肝细胞(L02),分为4组：（1） Bio-MB+Bio-CNLP+HGF无超声辐照；（2）Bio-MB+Bio-CNLP+HGF超声辐照；（3）脂质体+HGF;（4）Bio-MB+Bio-CNLP超声辐照。转染成功后分别用流式细胞仪检测转染率和凋亡率；细胞免疫荧光染色、ELISA和Western blot检测蛋白表达；MTT检测细胞活性。
结果：超声辐照组转染成功，而无超声辐照组则无转染；与单独脂质体转染比较，Bio-MB+Bio-CNLP介导HGF明显提高转染率；HGF基因转染HSC-T6细胞后72小时蛋白表达量最高，细胞凋亡率明显增加；而转染L02细胞活性明显增加。
结论：Bio-MB+Bio-CNLP能靶向传递HGF基因并明显提高转染率，促进HSC细胞凋亡，增加正常干细胞活性，表明我们成功制备了一种新型的具有靶向性、安全性和有效性Bio-MB+Bio-CNLP载体。
第二部分磁共振功能成像对大鼠肝纤维化诊断、定量分析研究
目的：探索一种新型靶向超声微泡载体介导HGF基因体内表达治疗肝纤维化，并且通过MRI功能成像检测疗效。
方法：通过胆总管结扎构建大鼠肝纤维化模型，尾静脉注射Bio-MB+Bio-CNLP+HGF悬液，肝脏超声辐照定位转递HGF基因治疗。分别运用DWI、PWI和MRS三种功能成像检测治疗效果，并与病理检查结果对照。
结果：我们靶向传递HGF基因并且产生抗纤维化效果；MRI功能成像各参数能准确区分肝纤维化分期。
结论：Bio-MB+Bio-CNLP在体内能靶向传递HGF基因，有效治疗肝纤维化，MRI功能成像参数能用于评估肝纤维化程度和疗效检测。
Liver fibrosis is the wound-healing response to various liver damage，in the form of a progressive accumulation of fibrosis．It alters the tissuestructure and function of liver，leading to cirrhosis and liver failure, Genetherapy may provide novel treatment strategies for a wide range of liverfibrosis. HGF was originally characterized as a potent mitogen for maturehepatocytes, has emerged as a potent anti-fibrotic cytokine that preventstissue fibrosis in various organs, including the liver.[，However, the lack of asafe and effective method for gene delivery to a specific organ or cellremains an obstacle to clinical application. Microbubbles are contrast agentsfor medical ultrasound imaging, improve transfection efficiency afterultrasound-induced cavitation, However, microbubbles are generallyunstable and their mean diameter of around1–6μm is too large forintravascular applications. Moreover, functional particles such as targetingmolecules are difficult to modify on the surface of microbubbles., Theirsurface should be easily modified with functional molecules for targeting after injection into the blood and ultimately， Liposomes have someadvantages as drug, antigen and gene delivery carriers, Their size can beeasily controlled and they can be modified to add a targeting function.but thegene expression was very low Therefore, we developed novelBio-MB+Bio-CNLP, Here, we assessed the feasibility ofBio-MB+Bio-CNLP for gene delivery after cavitation induced byultrasound.
Chronic diffuse liver disease is a worldwide problem.Whereashistology remains the gold standard by which to diagnose hepatic fibrosis,Several clinical limitations are associated with the use of liver biopsy.It isan invasive and costly procedure prone to complications, sampling error isinherent in the technique, Alternative techniques for assessingthe severityof diffuse liver disease are urgently needed. and such a tool would reducebiopsy related risks and costs, could be useful for guiding anti-fibrotictreatments, monitoring treatment efficacy, and helpful in the clinicalevaluation of new anti-fibrotic drugs. Furthermore, the identification ofoccult advanced fibrosis, may direct further management, and has essentialprognostic implications.
We established a bile duct ligation (BDL) rat model of hepatic fibrosis.Bio-MB+Bio-CNLP-pCDH-HGF carrier was administered through tail-veininjections. The gene therapy efficacy was evaluated in vivo. Before and aftertreatment, the rats were examined with diffusion-weighted imaging （DWI）、 Perfusion imaging (PWI) and magnetic aesonancespectroscopy(MRS) respectively. Magnetic resonance functional imagingcould potentially provide a more accurate representation of the diseaseprocess. Recently, in vivo Magnetic resonance functional imaging hasbeen suggested for the evaluation of chronic liver disease and discuss thevalue of MR functional imaging in early diagnosis、quantization analysisand staging of hepatic fibrosis by analyzing the changes of perfusionparameters of MR functional imaging of different degree of hepaticfibrosis
Part I Enhanced effect of HGF on liver cell growth with specialemphasis on hepatic stellate cells by a novel modified ultrasoundtargeted microbubble gene delivery system
Purpose: To evaluate the efficacy and safetyof human hepatocytegrowth factor (HGF) delivery by a novel ultrasound targeted microbubblesystemusing avidin to crosslink biotinylated ultrasound microbubbles(Bio-MB) and biotinylated cationic nano-liposomes (Bio-CNLP), which isreferred to as Bio-MB+Bio-CNLP.
Methods: HGF was linked to Bio-MB+Bio-CNLP, and transfected tohepatic stellate cells (HSC-T6) and normal liver cell line L02according tothe following four groups:(1) Bio-MB+Bio-CNLP+HGF, no ultrasonicirradiation group;(2) Bio-MB+Bio-CNLP+HGF+ultrasonic irradiation group;(3) liposomes+HGF;(4) Bio-MB+Bio-CNLP+ultrasonicirradiation group. Protein expression, L02cell viability, and HSC-T6cellapoptosis rate were determined respectively.
Results: Compared with liposome mediated transfection, Bio-MB+Bio-CNLP mediated transfection rate increased markedly, with the highestprotein expression in HGF transfected HSC-T6cells at72hours aftertransfection. Compared with mock transfection control group, the apoptosisrate was32.8%and7.5%respectively. Cell viability significantly increasedin HGF transfected L02compared with normal L02.
Conclusion: Introducing HGF using an ultrasound-mediated Bio-MB+Bio-CNLP system improved gene transfection rate and promoted normalliver cell growth. This appears to be a promising approach for targetedtherapy.
Part Ⅱ Efficacy of HGF for treating hepatic fibrosis and itsrelationship with MRI functional imaging parameters
Object: The aims of the present study were: i) to explore the interest oftreating liver fibrosisusing human hepatocyte growth factor (HGF)expression vector carried by a novel ultrasound targeted microbubbledelivery system.; b) to explore the diagnostic interest of MR functionalimaging parameters in evaluating liver fibrosis
Method: We established a rat model of hepatic fibrosis by bile duct ligation. The rats were administered HGF linked to biotynilated ultrasoundmicrobubbles and biotynilated cationic nano-liposomes, and compared tosham-operated animals. The impact on hepatic fibrosis was evaluated byhisto-pathology, hydroxyproline content, with special focus on MRfunctional imaging parameters.
Result: Our targeted gene therapy produced a significant anti-fibrosiseffect, as shown by liver histology and decreased hepatic collagen content.Moreover, proved of interest as indicated by the respective evolution of MRfunctional imaging parameters.
Conclusion: This is the first in vivo report of using ultrasoundmicrobubble-cationic nanoliposomes complex for gene delivery. The dataindicate that, in our experimental model, this approach is efficient tocounteract the fibrosis process. MRI functional imaging parameters appearsa promising imaging technique for reflecting liver fibrosis..
引文
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