SPIO结构,功能及应用浓度研究新进展

超顺磁性氧化铁（SPIO）具有典型的晶体结构,有效成分是Fe3O4晶体核心,其作为纳米粒子,常包被葡聚糖右旋糖酐或其他物质而具有一定的水溶性或生物活性。Fe3O4具有极强铁磁性和超顺磁性,能缩短周围氢质子的弛豫时间,降低正常组织的信号强度,使T2加权图像信号明显下降,SPIO因能被网状内皮系统所摄取而已被用作临床磁共振成像（MRI）T2加权造影剂;同时,SPIO也可被组织中不同类型细胞所摄取的浓度的不同而显示出不同的影像学差异,因此SPIO的应用浓度对细胞的活性及MRI效应有着重要的影响。本文就相关进展做一综述。     

超顺磁性氧化铁的合成及其对细胞标记相关研究进展

细胞移植在修复损伤组织等方面具有极大的前景.应用细胞治疗常规治疗效果不佳的疾病或建立更为优化的治疗方案,移植细胞的在体连续动态追踪显得尤为重要.超顺磁性氧化铁(SPIO)的应用,改善了以往只能依靠免疫组织化学、电镜等侵袭性方法的不便.研究者建立了多种制备该颗粒的方法,并摸索其标记细胞的最佳条件,通过动物实验,证实了SPIO活体示踪细胞的可行性及其优势,为细胞移植运用于临床治疗及疗效评价提供了可靠手段.     

SPIO标记猪脂肪干细胞的生物学特性与多向分化潜能及体外3.0T MR成像

背景：不同种类细胞的最佳化标记方案需要大量实验证明,而每种细胞对应标记策略的安全性检测至关重要。 目的：应用超顺磁性氧化铁联合多聚左旋赖氨酸标记猪脂肪干细胞,探讨磁标记对细胞生物学特性和多向分化潜能的影响以及标记细胞体外3.0T MR成像特性。 方法：五指山小型猪皮下脂肪分离培养脂肪干细胞;超顺磁性氧化铁-多聚左旋赖氨酸复合物标记液标记脂肪干细胞;应用3.0T MR对不同浓度标记细胞进行T1WI、T2WI及T2*WI序列体外成像。 结果与结论：普鲁士蓝染色显示标记细胞胞质内含有多少不等的蓝染铁颗粒,细胞标记率近100%;标记细胞向心肌、骨、脂肪方向诱导分化成功;不同浓度标记细胞MR扫描显示,随细胞浓度升高,3种序列信号变化率均增加;相同浓度细胞T2*WI信号变化率最大,T1WI最小,同一浓度3种MR序列间信号变化率差异均有显著性意义(P < 0.01);3.0T MR成像能检测到至少1×10⁶ L^-1标记细胞。结果显示应用超顺磁性氧化铁联合多聚左旋赖氨酸标记方案可有效标记脂肪干细胞,不影响细胞活力、增殖及多向分化能力;T2*WI序列检测标记细胞最敏感。     

磁共振对超顺磁性氧化铁标记的大鼠间充质干细胞成像的研究

目的:探讨磁共振对超顺磁性氧化铁(SPIO)纳米颗粒体外标记大鼠间充质干细胞进行成像的可行性.方法:多聚赖氨酸修饰的Fe3O4纳米颗粒.分别培养大鼠间充质干细胞至对数生长期,更换为分散多聚赖氨酸修饰的Fe3O4纳米颗粒的培养液,取所培养的细胞进行电镜超微结构观察.磁共振T1WI、FSE T2WI、FIESTA T2*WI三个序列对培养6 h的细胞群成像.结果:同一条件下培养6 h.见有多聚赖氨酸修饰的Fe3O4纳米颗粒进入大鼠间充质干细胞的细胞质内.对培养6h的细胞群磁共振成像中.标记后的大鼠间充质干细胞在FSE T1WI、FSET2WI、FIESTA T2WT三个序列中均可显示SPIO信号.其中在FSE T2WI上各标记细胞的EP管信号均较T1WI信号改变明显.结论:超顺磁性氧化铁纳米颗粒可以标记大鼠间充质干细胞,应用磁共振可以对其进行监测.     

SPIO标记骨髓间充质干细胞移植入心肌梗死大鼠的示踪研究

目的探讨超顺磁性氧化铁微粒（SPIO）体外标记骨髓间充质干细胞（BMSCs）及体内示踪移植入大鼠心肌梗死心脏的BMSCs的能力。方法使用左旋多聚赖氨酸-SPIO共培养方式标记BMSCs。采用普鲁士蓝染色观察细胞内铁颗粒,流式细胞术检测细胞活力,将经过SPIO标记的干细胞移植入心肌梗死大鼠心脏,应用1.5TMRI系统行磁标记干细胞成像。超声心动图检测各组心脏的左室射血分数（EF）,左室舒张末期内径（LVIDd）,左室收缩末期内径（LVIDs）及短轴缩短率（FS）。结果普鲁士蓝染色显示,SPIO标记的BMSCs细胞胞质内出现细小的蓝色铁颗粒,标记效率为（99.81±1.57）%;与正常未标记细胞相比较,细胞的活力差异无统计学意义（P〉0.05）。标记了SPIO的BMSCs体内MRI成像时显示,细胞移植区域信号缺失,对应区域病理切片普鲁士蓝染色可见胞浆内染色阳性的细胞。超声心动图显示,PBS组FS移植前后没有明显变化,BMSC组FS从移植前的（23.1±1.88）%上升到第1周的（31.28±4.15）%。BMSC组EF在移植前是（51.13±5.07）%,第1周时上升到（60.12±8.40）%。结论 SPIO能成功地标记BMSCs,且对BMSCs的活力无明显影响。BMSCs移植后能改善心梗大鼠的心功能。SPIO标记的BMSCs移植后在大鼠体内的分布、迁移过程可用MRI进行检测评价。     

超顺磁性氧化铁增强的磁共振成像在肝占位病变诊断中的价值

肝脏占位病变的检出与定性一直是影像学研究的重点与难点之一。CT、磁共振(MRI)的问世,使这一领域有了突飞猛进的发展。但某些占位病变,在常规CT、MRI平扫和增强扫描中与正常肝组织之间密度／信号差较小而易漏诊及误诊或定性困难。为弥补这一不足,各种用于肝脏的特异性对比剂应运而生,超顺磁性氧化铁(SPIO)就是其中之一。自     

超顺磁性氧化铁微粒磁标记骨髓间充质干细胞效率实验研究

目的探讨不同浓度超微超顺磁性氧化铁微粒（USPIO）与多聚左旋赖氨酸（PLL）标记大鼠骨髓问充质十细胞（BMSCs）的磁标记效率及对细胞生长活力的影响,寻找最佳配比浓度。方法实验采用6周龄Wister近交系大鼠,150g芹右．雄性。用贴壁法分离培养BMSCs。使用6nmUSPIO—PLL复合物标记BMSCs。实验分6组,对照组为未标记的BMSCs（A组）。按照PLL的有无及PLL质量浓度梯度（0、0．25、0．50、0．75、1．00μg／mL）分为5个实验组（B～F组）;其中每个实验组再根据不同浓度铁离子与PLL结合（USPIO的终质量浓度分别为25、50、100、150μg/mL）。使用电子显微镜及光学显微镜观察标记后的BMSCs及BMSCs内的USPIO微粒。BMSCs活力测定采用台盼蓝排除实验。BMSCs生长曲线绘制采用MTT法。MRI观察体外标记后BMSCs的显影。火焰法测量BMSCs内铁含量,验证铁含量与MRI信号的关系。统计学分析采用方差分析。,结果台盼蓝染色证实90％以上标记后BMSCs均拒染台盼蓝。根据BMSCs生长曲线判断单纯使用铁离子质量浓度达到200μg／mL时或PLL用量达1．00μg／mL会对BMSCs的生长产生一定的抑制作用。火焰法测得单独USPIO标记BMSCs与USPIO—PLL标记BMSCs铁含量差异有统计学意义（P〈0．05）。T2WI及SWI序列图像从B组至F组信号强度逐级下降,反映出铁离子的变化情况。结论使用USPIO（100μg／mL）结合PLL（0．75μg／mL）标记BMSCs即对细胞活性和生长无不利影响．且标记BMSCs的信号强度较强。     

SPIO标记脂肪干细胞移植退变椎间盘内的MRI活体示踪

背景：国内外动物实验多是荧光标记骨髓间充质干细胞的移植,以SPIO标记脂肪干细胞移植后活体示踪对退变椎间盘修复作用的研究较少。 目的：活体监测SPIO标记的脂肪干细胞在退变椎间盘内的存活、迁移和转归,以及脂肪干细胞对退变椎间盘的修复及延缓退变作用。 方法：新西兰大白兔20只,兔椎间盘被分为4组,即正常对照组(L1/2),脂肪干细胞组(L2/3),PBS组(L3/4),SPIO-脂肪干细胞组(L4/5)。透视引导下用18G穿刺制作退变模型后2周行SPIO标记的脂肪干细胞移植。 结果与结论：SPIO-脂肪干细胞移植后即刻T2WI/FFE序列上可见椎间盘内明显低信号,8周后仍可检测到低信号。脂肪干细胞移植组与同时间点PBS组比较,椎间盘退变程度轻。提示SPIO-脂肪干细胞移植至椎间盘后,可通过MRI进行监测;脂肪干细胞椎间盘内移植有助于修复退变椎间盘和延缓椎间盘退变。     

超微超顺磁性氧化铁的制备及其对家兔毒性作用的研究

目的：制备超微超顺磁性氧化铁（USPIO）,观察其对家兔的毒性作用,并研究其物理、磁学性质,探讨其作为磁共振阴性对比剂的可能性。方法：采用化学共沉淀法制备四氧化三铁（Fe3O4）纳米粒,采用X射线粉末衍射仪、透射电镜、磁强计及1．5T超导型磁共振仪等测定其相关理化指标。选取家兔20只,随机分为实验组（10只）和对照组（10只）,实验组按1．25ml／kg耳缘静脉注射样品、对照组按1．25ml／kg耳缘静脉给予生理盐水。给药后于饲养2周末,检测其血清主要生化指标、观察主要脏器的病理学改变。并行MR检查观察实验组肝、脾的增强效果。结果：成功制备USPIO,核心粒径小于10nm,饱和磁化强度为47．2emu／g。给药后饲养2周,家兔无死亡。给药2周末予MR检查,实验组肝、脾在T2WI信号降低,对照组肝、脾在T2WI信号未见降低;两组家兔各项血清生化指标比较,差异均无统计学意义（P〉0．05）;其肝、脾组织进行普鲁士蓝染色,实验组家兔肝、脾内分布少许铁蓝色颗粒,而对照组均无铁蓝色颗粒。结论：采用化学共沉淀法制备的USPIO符合作为磁共振成像阴性对比剂的要求,通过给药家兔血清学、病理组织学及磁共振成像检查,说明USPIO生物相容性较好且毒性低,可作为磁共振的阴性对比剂用于肝、脾等部位磁共振成像。     

Immunohistochemical staining of hepatocellular carcinoma with monoclonal antibody against inhibin

Inhibin is a peptide hormone produced by ovarian granulosa cells. During a recent study investigating the immunohistochemical staining of ovarian granulosa cell tumours and other neoplasms with an anti-inhibin monoclonal antibody, we identified strong cytoplasmic staining of hepatocytes. In the present study we investigated the immunostaining of hepatocellular carcinoma and other neoplasms involving the liver with anti-inhibin to determine whether the antibody may be of value in the differential diagnosis of hepatic neoplasms. Immunostaining for α-fetoprotein was also performed. With anti-inhibin there was positive, generally strong, cytoplasmic staining of 17 of 19 cases of hepatocellular carcinoma, including the pleomorphic and glandular variants. There was positive staining of six of 20 cases of adenocarcinoma. In these, positive staining was generally focal, of weak intensity and involved the luminal surface of neoplastic glands. There was no staining of five cases of neuroendocrine tumour. There was positive staining for α-fetoprotein in 13 of 19 cases of hepatocellular carcinoma and in two of 20 cases of adenocarcinoma but no staining of neuroendocrine tumours. Immunostaining with anti-inhibin antibody may be of value in the differentiation of hepatocellular carcinoma from other neoplasms involving the liver. The antibody is a more sensitive, but less specific, immunohistochemical marker for hepatocellular carcinoma than is α-fetoprotein.     

Immunohistochemical staining of hepatocellular carcinoma with monoclonal antibody against inhibin

Inhibin is a peptide hormone produced by ovarian granulosa cells. During a recent study investigating the immunohistochemical staining of ovarian granulosa cell tumours and other neoplasms with an anti-inhibin monoclonal antibody, we identified strong cytoplasmic staining of hepatocytes. In the present study we investigated the immunostaining of hepatocellular carcinoma and other neoplasms involving the liver with anti-inhibin to determine whether the antibody may be of value in the differential diagnosis of hepatic neoplasms. Immunostaining for α-fetoprotein was also performed. With anti-inhibin there was positive, generally strong, cytoplasmic staining of 17 of 19 cases of hepatocellular carcinoma, including the pleomorphic and glandular variants. There was positive staining of six of 20 cases of adenocarcinoma. In these, positive staining was generally focal, of weak intensity and involved the luminal surface of neoplastic glands. There was no staining of five cases of neuroendocrine tumour. There was positive staining for α-fetoprotein in 13 of 19 cases of hepatocellular carcinoma and in two of 20 cases of adenocarcinoma but no staining of neuroendocrine tumours. Immunostaining with anti-inhibin antibody may be of value in the differentiation of hepatocellular carcinoma from other neoplasms involving the liver. The antibody is a more sensitive, but less specific, immunohistochemical marker for hepatocellular carcinoma than is α-fetoprotein.     

Diffusion-weighted MR imaging before and after contrast enhancement with superparamagnetic iron oxide for assessment of hepatic metastasis

Purpose

The purpose of our study was to validate diffusion-weighted MRI (DWI) before and after superparamagnetic iron oxide (SPIO) injection for assessment of hepatic metastases.

Materials and Methods

Eighty-six hepatic metastases (size range, 0.3-4.7 cm; mean, 1.5 cm) verified pathologically or by follow-up imaging studies in 22 consecutive patients (17 men and 5 women; 44-83 years; mean age, 60 years) during a 13-month period were enrolled. Hepatic MRI, including DWI (b-factors=50, 400, 800 s/mm²) with breath-holding technique of single-shot spin-echo echo-planar imaging (TR/TE=1000/69 ms, average=2) before and after SPIO administration, were retrospectively reviewed by two independent radiologists with a 5-point scale confidence score for each hepatic lesion on pre-contrast DWI (pre-DWI), SPIO-enhanced DWI (SPIO-DWI), and SPIO-enhanced T2^*-weighted imaging (SPIO-T2^*wI).

Results

For all lesions, SPIO-T2^*wI showed significantly higher confidence score in the diagnosis of hepatic metastases than pre-contrast or SPIO-DWI regardless of the size of b-factors (p<0.05) with only one exception; using b-factor=50 s/mm², the score of SPIO-T2^*wI was still higher than SPIO-DWI but there was no statistical significance given by observer 1 (p=0.730). For the subcentimeter lesions (n=37), SPIO-T2^*wI showed the highest score, and using b-factor=50 or 400 s/mm² SPIO-DWI showed similar confidence scores to SPIO-T2^*wI by both observers (p>0.05). Pre-DWI using b-factor=50 sec/mm² was also comparable with SPIO-T2^*wI by observer 1 (p=0.060).

Conclusion

Pre-DWI has a limited value for the assessment of hepatic metastases, however, the repetition of DWI after SPIO injection using small b-factors could complement SPIO-T2^*wI, especially for subcentimeter lesions.     

裸鼠人肝癌模型模拟临床化疗实验研究

目的 探讨人肝癌组织裸鼠原位种植模型体内筛选供瘤病人敏感的化疗药物可行性。方法 荷瘤鼠随机分为对照组和表阿霉素（E—ADM）、5氟脲嘧啶（5-FU）、丝裂霉素（MMC）四组。腹腔化疗每周1次，共4次。B超观测移植瘤大小，化疗结束1wk后处死荷瘤鼠．分别用免疫组化（SP）、RT—PCR、Western blot方法检测移植瘤MDR1和LRP蛋白及其mRNA表达情况。选择敏感和低致多药耐药的化疗药物用于供瘤病人。结果 3个化疗组肿瘤体积均逐渐缩小，与对照组相比差异均有显著性（P〈0．05）。各检测方法均示E—ADM组MDR1和LRP蛋白及其mRNA表达升高，差异有显著性（P〈0．05）。5-Fu组LRP表达升高，差异有显著性（P〈0．05）。MMC和5-Fu对供瘤病人化疗一个疗程，病人骨痛症状消失或明显减轻，SPECT示个别骨转移灶消失，多个骨转移灶缩小。结论 E—ADM化疗可以诱导肿瘤组织MDR1和LRP表达升高；5-Fu化疗则可诱导LRP表达升高。裸鼠原位种植瘤敏感的化疗药物同样供瘤病人也敏感。     

Difference in the three-dimensional structure of the sinusoids between hepatocellular carcinoma and cirrhotic liver

The purpose of the present study was to estimate the difference in three-dimensional (3-D) structure of sinusoids between hepatocellular carcinoma (HCC) and cirrhotic liver, by the use of topology. Ten surgically resected lesions of HCC and 10 lesions of liver cirrhosis (LC) were used. Computer-alded reconstruction models of HCC sinusoids and LC sinusoids were developed from 20 4μm thick serial tissue sections from each specimen. A topologlcal invariant, called the first Bettl numberp., was used to estimate the complexity degree of the 3-D sinusoidal structure. The mean p, of the sinusoidal network in the examined tissue, 200X200X80 μm³ in size, was 46.5X33.0 In 10 HCC and 84.9 ±19.1 in 10 cirrhotic livers. There was a statistically significant difference between the two values (P<0.01), while there was no significant difference in the sinusoidal volume of the same size tissue between the HCC and the cirrhotic liver. It was found, therefore, that the sinusoidal network of HCC was more sparsely and coarsely knit in 3-D space than that of the cirrhotic liver.     

Combined hepatocellular carcinoma and osteoclast-like giant cell tumor of the liver: Possible clue to histogenesis

Hepatic giant cell tumor is extremely rare, and only five cases have been reported of overt hepatocellular carcinoma, thus its histogenesis is controversial. Herein is reported a case of simultaneous hepatocellular carcinoma and osteoclast-like giant cell tumor in a single tumor. A liver tumor was found in a 74-year-old woman. Histologically the tumor consisted of two distinct components: mononuclear and multinuclear giant cells with osteoclastic giant cells, and a conventional hepatocellular carcinoma. The boundary between the two components showed transitional features. Immunohistochemistry showed that the osteoclast-like giant cells were CD68 and vimentin positive, but cytokeratin and AFP negative, while spindle-shaped cells were positive only for vimentin. In a portion of the hepatocellular carcinoma the cells were cytokeratin-8 and AFP positive. Ki-67 positivity was 10% for the hepatocellular carcinoma, 60% for the spindle-shaped cells, and 0% for the giant cells. It is possible that the tumor might have had a hepatocellular carcinoma origin, given the more highly proliferative sarcomatous changes and reactive osteoclast-like cells. This case provides a clue to the histogenesis of hepatic giant cell tumors.     

运用MRI评估超顺磁性氧化铁纳米粒在肝硬化肝癌组织中分布变化的实验研究

目的研究超顺磁性氧化铁纳米粒(SPIO)作为MRI对比剂在肝硬化肝癌组织中分布变化情况,探讨其用于分子靶向成像的可行性。方法 SPF级雄性SD大鼠35只,体质量(200±20)g,鼠龄3个月。按完全随机分组的方法分成实验组25只和对照组10只,实验组予质量浓度0.1mg/mL的二乙基亚硝胺(DENA)溶液自由饮用,对照组饮用灭菌0.9%氯化钠溶液。于给药后20周先进行MRI平扫,再注入SPIO后1、24、48、72、96h分别行MRI增强扫描,每一时段5只。同理,空白对照组每一时段取2只。分析MRI图像,取血液标本进行肝功能测定,取肝脏标本进行苏木精-伊红和普鲁士蓝染色病理学检查。结果实验组大鼠肝功能指标丙氨酸氨基转移酶及天冬氨酸转氨酶[分别为(130.43±8.83)、(415.00±55.44)U/L]较对照组[分别为(39.57±7.25)、(132.93±39.03)U/L]显著升高(P<0.001)。正常肝组织及肝硬化组织,注入SPIO后1 h,各序列上肝脏组织信号明显下降,24 h肝脏组织信号强度下降百分比(PSIL)达到最大,48、72、96 h后均下降,各时段PSIL差异有统计学意义(P<0.001);而20周肝癌组织,注入SPIO各时段其信号无明显变化(P>0.05)。普鲁士蓝染色显示,正常肝组织及肝硬化组织蓝染的Kupffer细胞数在24 h达到最多,后逐步减少;部分高分化肝癌组织散在少许蓝染的Kupffer细胞,低分化肝癌组织内无蓝染细胞。不同肝脏组织SPIO增强后MRI各序列信号变化与组织中蓝染的Kupffer细胞数呈线性相关趋势,具有显著统计学意义(r正常=0.927,r肝硬化=0.912,P<0.01)。结论通过MRI扫描动态观察SPIO分布变化情况,不仅可用于肝脏恶性病变的检出,而且具有将SPIO作为MRI对比剂用于分子靶向成像的潜能。     

Utility of pancreatic digestive enzyme immunohistochemistry in the differential diagnosis of hepatocellular carcinoma, cholangiocarcinoma and metastatic adenocarcinoma of the liver

To test the diagnostic utility of pancreatic digestive enzyme immunohistochemistry in liver cancers, the expression of three pancreatic digestive enzymes (trypsinogen, chymotrypsinogen and pancreatic lipase) was investigated in cholangiocarcinoma (CC) (n = 42), hepatocellular carcinoma (HCC) (n = 35), combined HCC-CC (n = 11) and metastatic adenocarcinoma (MA) of the liver (n = 34; 4 gastric cancer, 5 pancreatic cancer and 25 colon cancer). In CC, 15 (36%) expressed one or more of these enzymes, while the remaining 27 (64%) did not express any enzymes. In MA, 13 (38%) expressed one or more of these enzymes, while the remaining 21 (62%) did not express any enzymes. Expression of trypsinogen, chymotrypsinogen and lipase was noted in 15 CC (36%), 11 CC (25%) and 15 CC (36%), respectively, and in 9 MA (26%), 6 MA (18%) and 13 MA (38%), respectively. There was no significant difference in the positive ratio of each enzyme between CC and MA. In positive cases, the enzymes were expressed with a cytoplasmic granular pattern. In MA, there was no significant difference in the positive ratio of the enzymes among the primary sites. In contrast to CC and MA, these enzymes were not expressed in any cases of HCC and combined HCC-CC. These data suggest that pancreatic digestive enzyme immunohistochemistry may be useful for differential diagnosis between HCC and CC or MA as well as between combined HCC-CC and CC or MA, but it is not useful for differential diagnosis between CC and MA. A positive reaction for these enzymes is indicative of CC or MA and is against the diagnosis of HCC or combined HCC-CC, and a negative reaction is noncontributory to the differential diagnosis.