肝癌靶向液态氟碳脂质微球造影剂的体外寻靶实验研究

目的 制备液态氟碳脂质微球造影剂并研究其体外基本特性,再引入抗人肝癌细胞单克隆抗体,采用生物素亲和素法,进行体外靶向结合的研究.方法 采用旋转蒸发法和高压均质法制备一种纳米液态氟碳脂质微球造影剂,观察其外观、形态、大小及分布情况,计算其浓度,并测定其粒径、电位;制备生物素的抗人肝癌细胞单克隆抗体HAb18,用HABA/Avidin试剂盒检测抗体的生物素化程度;制备NBD标记的生物素化的微球,采用生物素亲和素法研究其体外靶向作用.结果 所制备的液态氟碳脂质微球造影剂外观呈乳白色的混悬液,镜下观察,微球形态圆整,大小均一,性质稳定,平均粒径171.9 nm;采用生物素亲和素法制备的NBD标记的生物素化的微球可靶向结合到靶细胞上.结论 成功制备出稳定的纳米液态氟碳脂质微球造影剂,并引入抗人肝癌细胞单克隆抗体,通过生物素亲和素系统可将其特异结合到靶细胞上.     

携RGD肽的靶向相变光声/超声双模态高分子造影剂的制备及体外实验研究

目的制备一种携RGD肽的靶向相变型光声/超声双模态造影剂(RNP),并观察其体外光致相变以及细胞靶向效果。方法制备包裹ICG及液态氟碳(PFH)的RGD肽靶向高分子微球,在荧光显微镜下观察RGD肽与微球的结合情况,流式细胞术量化其结合率,脉冲激光辐照观察微球相变情况,用乳腺癌MD-MB231细胞验证其体外细胞靶向能力。结果制备的RNP平均粒径为(627±28.5)nm,微球与RGD肽结合率为98.9%,脉冲激光辐照后微球发生液气相变。体外靶向实验结果显示,与对照组相比,更多的靶向微球聚集在MD-MB231细胞的周围。结论成功制备出携RGD肽的靶向相变型光声/超声双模态造影剂,该造影剂对高表达整合素的乳腺癌MD-MB231细胞具有较高的靶向结合能力。     

叶酸受体靶向液态氟碳纳米粒造影剂的制备及体外寻靶

目的 制备叶酸受体靶向的被脂质包裹的液态氟碳纳米粒造影剂,鉴定其基本性质,观察其体外靶向性能.方法 以氯仿将偶联叶酸的磷脂[DSPE-PEG(2000)]按照一定比例溶解在成膜材料中,用旋转蒸发仪去除有机溶剂成膜,加入磷酸盐缓冲液重新水合后,在悬液中逐滴加入适量液态氟碳,均质乳化后得到叶酸受体靶向的纳米粒造影剂.在人卵巢癌SKOV3细胞中验证该造影剂的靶向性能(靶向造影剂组),并与不带叶酸的普通造影剂组和游离叶酸干预组相比较.结果 叶酸受体靶向液态氟碳纳米粒造影剂外观与普通造影剂无明显差别.体外靶向实验发现此纳米粒造影剂聚集且牢固地黏附于SKOV3细胞,而普通造影剂组和游离叶酸干预组则未见明显特异性结合.结论 成功制备了偶联叶酸的被脂质包裹的液态氟碳纳米粒造影剂,该造影剂对高表达叶酸受体的SKOV3细胞具有较强的亲和力,有望成为卵巢癌靶向显影与治疗的理想分子探针.     

去唾液酸糖蛋白受体介导的肝靶向纳米脂质超声造影剂的制备及体外实验

目的 以肝细胞膜特异性受体去唾液酸糖蛋白受体为靶向受体,利用共价结合的原理,制备出肝靶向性液态氟碳纳米超声造影剂,观察该造影剂的一般特性、与人肝细胞L02的靶向结合及体外聚集显像效果.方法 利用还原胺法制备去唾液酸糖蛋白特异性配体半乳糖化多聚赖氨酸(Gal-PLL);利用旋转蒸发及声振法制备液态氟碳纳米脂质超声造影剂;培养人肝细胞L02,于不同时间点观察与L02细胞的靶向结合;Philips iU 22超声诊断仪L12-5探头对比观察靶向液态氟碳纳米脂质超声造影剂的体外显像效果.结果 靶向液态氟碳纳米脂质超声造影剂粒径极小,分布均匀,形态呈圆球形,且能与L02有效结合;体外乳胶囊显示:脱气水侧呈现无回声,靶向液态氟碳纳米脂质超声造影剂侧呈现高回声.结论 以去唾液酸糖蛋白受体为靶向受体,自制的携半乳糖化多聚赖氨酸的靶向液态氟碳纳米脂质超声造影剂能有效与人肝细胞LO2靶向结合,该靶向超声造影剂能在体外聚集显影.该靶向超声造影剂粒径极小,是细胞水平上肝靶向性超声分子显像的一种理想的超声分子探针.

Abstract：

Objective To prepare the liver targeting nano-liquid perfluorocarbon ultrasound contrast agent and observe its general characteristics;to observe the targeting combined effects of the human liver cells L02 and the targeted ultrasound contrast agent ;to evaluate the gathering imaging effects of the targeted ultrasound contrast agent. Methods Amine method was used to prepared asialoglycoprotein Gal specific ligand polylysine (Gal-PLL), rotary evaporator and sonicated liquid fluorocarbon were used to prepare nano lipid ultrasound contrast agent. Human liver cell L02 were cultured, the combined effects were observed according to the reacting time of the cells and the targeted nano-lipid ultrasound contrast agent. The nanolipid ultrasound contrast agent and the degassed_ water_ were loaded into cysts and their ultrasound imaging effects were observed by ultrasound diagnostic apparatus Philips iU22. Results The particle size of the liquid fluorocarbon nano-targeted lipid ultrasound contrast agent was extremely small, uniform, cylindrical and spherical. The cysts in vitro showed that the side of the targeted liquid perfluorocarbon nano-lipid ultrasound contrast agent showed high echo. Conclusions The targeted liquid perfluorocarbon nano-lipid ultrasound contrast agent can be effectively targeted to the human liver cells L02 due to carrying home-made Gal-PLL. The targeted ultrasound contrast agents can be imaging by ultrasound and be confirmed in vitro.The size of the contrast agent was small, therefore, it can be considered an ideal ultrasonic molecular probe and achieve the ultrasound molecular imaging in cell level.     

载10-羟基喜树碱液态氟碳纳米粒制备及其表征、体外增强超声显像效果观察

目的 研制一种包裹液态氟碳（PFP）的新型载药纳米级超声造影剂,考察其基本特性并观察其体外增强超声显像效果。方法 采用旋转蒸发和探头超声法制备载10-羟基喜树碱（10-HCPT）的液态氟碳（PFP）纳米粒,在光学显微镜和透射电镜下观察纳米粒形态,采用马尔文仪测量纳米粒粒径和电荷。采用紫外分光光度计测定纳米粒包封率,并应用低强度聚焦超声（LIFU）辐照载药液态氟碳纳米粒溶液,观察纳米粒相变情况及其增强超声显像效果。结果 制备的载药脂质纳米粒外观为乳白色混悬液,在油镜及透射电镜下观察,载药纳米粒形态规则,分布均一,平均粒径约（500.82±25.97）nm,表面平均电位为（-47.77±3.09）mV;在体外经LIFU辐照后,可见纳米粒发生液气相变形成微泡,在基波模式和谐波模式下,均可显著增强超声显影。结论 成功研制了包裹液态氟碳的载药脂质纳米粒,可望成为一种新型的多功能超声造影剂。     

纳米级靶向超声造影剂的制备及对兔VX2肝肿瘤的显影试验

目的 制备一种具有靶向性的纳米级超声造影剂,测定其物理特性,并对兔VX2肝肿瘤的靶向性进行评估.方法 采用高速均质法制备纳米级靶向超声造影剂,测定粒径、Zeta电位、稳定性,观察体外显影、对体内肿瘤的显影,对肿瘤组织进行病理切片及荧光共聚焦观察.结果 纳米级靶向超声造影剂的粒径为(474.1±83.5)nm,Zeta电位为+(9.8±5.7)mV,溶液的性质稳定,其沉淀能够增强超声显影,体内能够增强肿瘤的显影,光镜下和荧光共聚焦显微镜下均能够观察到靶向造影剂聚集在肿瘤组织内.结论 自制的纳米级微球符合理想的靶向超声造影剂的要求,性质稳定,能够靶向肿瘤组织显影,有望成为一种新型的靶向超声造影剂及载基因或药物的靶向载体.     

载金纳米棒和液态氟碳纳米造影剂及其体外显影

目的制备一种载金纳米棒(Au)和液态氟碳全氟己烷(PFH)的纳米级造影剂,并对其进行体外双模态(光声/超声)显影研究,进一步实现体内显影。方法采用双乳化法制备包裹金纳米棒-液态氟碳的高分子聚合物(PLGA)纳米粒(GNPs)及只包裹双蒸水的纳米粒(WNPs)或只包裹金纳米棒的纳米粒(Au-NPs);MTT对不同浓度的GNPs行体外细胞毒性实验;分别对GNPs、W-NPs、A-NPs在激光辐照前后二维超声、造影强度和光声信号显影,并统计激光辐照前后二维超声灰度值、造影值及光声信号值。结果成功制备包裹金纳米棒-液态氟碳双模态纳米级造影剂,体外光声和造影效果好;GNPs对HUVECs的毒性,各个浓度抑制率较低,且各组间无统计学差异;在激光辐照前,GNPs、Au-NPs、W-NPs二维超声及造影模式下均为低回声;激光辐照后,GNPs超声灰度和造影强度明显增强,光声显影明显,W-NPs超声灰度和造影模式未见增强,光声未见显影,Au-NPs超声灰度造影强度未见明显增强,光声显影明显。结论成功制备包裹金纳米棒-液态氟碳的纳米级造影剂,实现了体外超声/光声双模态增强显影,为体内靶向显影提供实验基础。     

相变型液态氟碳纳米粒造影剂的制备及相变超声显影研究

目的制备一种相变型液态氟碳纳米粒超声造影剂(PC-PNPCA),研究其在体外相变及增强超声显影作用的能力。方法首先采用双步乳化法制备出以脂质为包壳、液态氟碳PFH为内核的纳米粒超声造影剂,检测该造影剂的粒径、电荷。然后分别用升高温度、低功率治疗超声及高强度聚焦超声辐照三种方法来激发纳米粒相变,用显微镜观察相变后产生的微泡,使用诊断超声仪在造影模式下观察造影剂相变后增强超声显影的情况。结果成功制备出脂质包裹PFH的PC-PNPCA,PC-PNPCA平均粒径为(206.87±21.29)nm。当温度升高至70℃以上,低功率治疗超声辐照10 s或高强度聚焦超声辐照2 s后,乳液内的纳米粒均可发生相变产生微泡,且能促进超声显影。结论制备的PC-PNPCA能在一定条件下发生相变并增强超声显影,为超声分子影像学提供了新型、可靠的研究平台。     

携Gelsolin单抗靶向相变型超声造影剂的制备及体外靶向实验

目的制备一种以凝溶胶蛋白(GSN)为靶点的包裹液态氟碳的纳米级PLGA高分子造影剂(GSN-PLGA-PFP),并评价其体外热致相变、靶向及显影能力。方法采用双乳化法及碳二亚胺法制备靶向相变型高分子超声造影剂。检测此造影剂的一般性质;显微镜加热板法进行热致相变;激光共聚焦显微镜体外观察GSN-PLGA-PFP被小鼠腹水型肝癌高淋巴道转移Hca-F细胞摄取的情况,初步评价其寻靶能力;体外低强度聚焦超声(LIFU)仪辐照后观察其超声显影效果。结果 GSN-PLGA-PFP的平均粒径为(328.59±3.82)nm,造影剂表面平均Zeta电位为(-10.36±1.34)mV,造影剂表面GSN单抗连接率高达98.31%,且于43.5℃时纳米粒开始发生相变;体外摄取实验显示Hca-F细胞可较多地摄取GSNPLGA-PFP,体外行LIFU辐照后可观察到显影效果明显增强。结论自制携Gelsolin单抗包载PFP的PLGA造影剂热致相变效果明显,可与Hca-F细胞特异度结合且体外显影效果较好。     

自制脂质纳米级超声造影剂体外基本特性和造影增强的实验研究

目的 研究自制脂质纳米级超声造影剂的体外基本特性和体内造影增强效果,为今后的靶向显像和载药治疗研究提供依据.方法 (1)造影剂复溶后观察微泡形态,检测其平均粒径,计数其浓度;(2)微泡造影剂常温放置1周、1个月和3个月后复溶,观察其基本特性改变情况;(3)观察此造影剂增强正常兔肝脏显影的情况.结果 自制脂质微泡镜下观察:形态好,粒径范围400～950 nm,平均粒径650 nm,分布均匀,微泡浓度为(3.81±0.33)×109/ml.造影剂放置1周、1个月和3个月后复溶,基本特性无明显改变;造影剂复溶后24 h内微泡能保持较高浓度.体内造影结果显示能显著增强兔肝脏血管及实质显像.结论 此脂质纳米级超声造影剂性质稳定,显像效果好,有极大的开发应用价值,值得进一步研究.     

肝移植在肝肿瘤治疗中的应用

自1963年Starzl施行首例人体原位肝移植(orthotopic liver transplantation，OLT)以后，20世纪70年代环孢霉素A(CSA)的出现，80年代UW保存液(University of Wiscosion Solution)和体外生物泵静脉转流技术的临床应用，90年代活体肝移植的开展均是肝移植发展史上重要的里程碑。     

非酒精性脂肪性肝病与肝癌

据世界卫生组织统计,我国肝癌发病人数占全球总发病人数的50%,因肝癌病死人数则占55%,实在是国人之大不幸。幸而我国已将乙肝疫苗接种列入儿童计划免疫,该计划免疫实施近20年来,估计至少减少了3000万例乙肝病毒感染者。已有报告表明,接种过疫苗之儿童肝癌发病率显著下降,相信三四十年后,我国肝癌高的发病率必然下降。     

脂肪肝背景下肝局灶性病变的超声造影诊断

目的 评价超声造影对脂肪肝背景下肝局灶性病变的诊断价值.方法 对常规超声不能确诊的脂肪肝背景下的67个肝局灶性病变进行了超声造影检查,将超声造影检查结果同增强CT、增强MRI及病理诊断进行对比,分析超声造影检查的准确性、敏感性和特异性.结果 超声造影检查后,2例病灶仍不能确诊.3例炎性假瘤和1例肝细胞肝癌被误诊.超声造影检查的准确性为91.O%(61/67),敏感性和特异性分别为91.7%(11/12)和90.9%(50/55).结论 超声造影可大大提高脂肪肝背景下肝局灶性病变的诊断.     

用于生物人工肝治疗的肝细胞研究进展

近年来以生物人工肝为主附加血或血浆灌流组成的体外组合型人工肝辅助装置 ,成为研究最多、最有希望的人工肝 ,常用的生物材料有肝酶类、肝细胞成分、肝组织片及培养肝细胞。其中以培养肝细胞最为理想。随着细胞培养技术和细胞工程技术的发展 ,肝细胞培养系统作为生物反应器提供肝功能支持的研究取得了重大进展 ,本文就肝细胞的来源、培养、储存等问题与前景做一综述。     

Liver fibrosis

Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation. Our knowledge of the cellular and molecular mechanisms of liver fibrosis has greatly advanced. Activated hepatic stellate cells, portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as major collagen-producing cells in the injured liver. These cells are activated by fibrogenic cytokines such as TGF-beta1, angiotensin II, and leptin. Reversibility of advanced liver fibrosis in patients has been recently documented, which has stimulated researchers to develop antifibrotic drugs. Emerging antifibrotic therapies are aimed at inhibiting the accumulation of fibrogenic cells and/or preventing the deposition of extracellular matrix proteins. Although many therapeutic interventions are effective in experimental models of liver fibrosis, their efficacy and safety in humans is unknown. This review summarizes recent progress in the study of the pathogenesis and diagnosis of liver fibrosis and discusses current antifibrotic strategies.     

Liver transplantation

Orthotopic liver transplantation (OLT) involves the substitution of a diseased native liver with a normal liver (or part of one) taken from a deceased or living donor. Considered an experimental procedure through the 1980s, OLT is now regarded as the treatment of choice for a number of otherwise irreversible forms of acute and chronic liver disease.The first human liver transplantation was performed in the United States in 1963 by Prof. T.E. Starzl of the University of Colorado. The first OLT to be performed in Italy was done in 1982 by Prof. R. Cortesini. The procedure was successfully performed at the Policlinico Umberto I of the University of Rome (La Sapienza).The paper reports the indications for liver transplantation, donor selection and organ allocation in our experience, surgical technique, immunosuppression, complications and results of liver transplantation in our center.     

Liver transplantation

Liver transplantation has become an established form of therapy for patients with almost any type of irreversible and severe liver disease. The remarkable success of liver transplantation has resulted from recent advances in immunosuppressive therapy, surgical techniques, and patient selection. Additional progress has been made in the management of the complex postoperative medical complications that may occur. Indeed, liver transplantation has contributed significantly to an improved quantity and quality of life for many patients with liver disease.