【作者】 欧阳洋；

【导师】 李宜雄；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 研究背景生长抑素受体阳性细胞多见于神经内分泌肿瘤(neuroendocrine tumours, NETs),它是来源于神经内分泌细胞的一大类肿瘤,多发生于消化系统,但可散在发生于人体其它多个器官。其最主要来源部位是胃肠和胰腺(占75%),称为胃肠胰神经内分泌肿瘤(gastroenteropancreatic neuroendocrine tumours,GEP NETs)目前积极的外科手术治疗仍然是GEP NETs首选的方法。但由于病灶深藏在胃肠壁或腹膜后,早期诊断、精确定位困难,从而影响临床治疗效果。因此如何能对神经内分泌肿瘤进行早期诊断并精确定位成为国内外研究的重点。肿瘤学研究发现,NETs的细胞膜上均有生长抑素受体(somatostatin receptor, SSTR)的显著高密度分布。SSTR是一种G-蛋白偶联的细胞膜跨膜受体,有五种亚型(SSTR1-SSTR5), SSTR的配体是生长抑素(somatostatin,SST),目前引用较多的是生长抑素类似物(somatostatin analogues,SSTA)。SSTA可和SSTR特异性结合并进入细胞内。利用SSTA作为载体的功能,通过SSTR的内化机制将放射性同位素带到细胞内,造成肿瘤区域核素的浓聚,而被SPECT所检测,即生长抑素受体显像(somatostatin receptor scintigraphy,SRS)。SRS应用于NETs原发灶与转移灶的诊断,从上世纪90年代起,即已开始研究并应用于临床,已逐渐成为一种新的检查方法,目前在国外已广泛开展,但仅限于在同位素扫描方面。而在外科临床中,更需要了解病灶的精确空间定位以及其与周围脏器组织的关系,而这恰恰是MRI的优势所在。但MRI又不具有对NETs的特异性与敏感性。如何能将SRS跟MRI的优势互补,得到一种对NETs既具有特异性与敏感性,又具有高图像空间分辨力,能为外科医生提供清晰的三维空间图像,并能清楚的显示出病灶与周围组织器官的关系的这样一种检测方法成为我们所想要研究的方向。本研究旨在通过化学方法对MRI检测中常用的造影剂超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)进行修饰并实现和奥曲肽之间的耦联,得到一种SPIO-奥曲肽复合物,在奥曲肽与SSTR的特异性受体结合作用下实现Fe3O4颗粒选择性进入SSTR阳性肿瘤细胞内,形成细胞胞浆内Fe3O4颗粒的选择性聚集,局部驰豫时间发生极大的改变,行MRI扫描检测这种信号改变并获得图像,即生长抑素受体的磁共振分子成像(somatostatin receptor magnetic resonance molecular imaging, SRMRMI)。我们希望SRMRMI既能具有SRS的特异性与敏感性,又具有MRI的高图像空间分辨能力,从而为生长抑素受体阳性肿瘤的早期诊断及定位提供一个新的思路。全文共分为二个部分。目的：制备具有高分散性、稳定性、纳米层级的羧甲基葡聚糖改性磁性复合纳米Fe3O4粒子,并与奥曲肽偶联制成SPIO-奥曲肽偶联复合物。方法：采用超声预处理技术,在羧甲基葡聚糖.水分散体系中,通过化学共沉淀法制备羧甲基葡聚糖表面改性纳米粒子,偶联上奥曲肽后用红外光谱(IR)、原子力显微镜(AFM)、透射电子显微镜(TEM)、X射线衍射(XRD)、振荡样品磁强(VSM)等对产物进行了表征。结果：(1)以氯化铁和氯化亚铁为铁源,葡聚糖为修饰剂,将反应溶液超声预处理后,通过化学共沉淀法,成功制备了羧甲基葡聚糖磁性纳米粒子(CMD-MNPs)。并通过酰胺键将奥曲肽以共价键连接于纳米微粒之上,得到CMD-MNPs-Oc。(2)通过对CMD-MNPs-Oc粒子扫描电镜分析：其粒径为50nm左右,粒径分布窄,呈现很好的球形。超声预处理混合溶液,能够很好阻止Fe3O4纳米粒子的团聚。葡聚糖的修饰没有改变Fe3O4的晶体结构,而且大大提高磁性纳米粒子的水溶性和稳定性。(3)通过外加磁场时磁化曲线的检测,证明CMD-MNPs-Oc粒子具有超顺磁性,室温下饱和磁化强度为35emu·g-1。结论：1.经超声预处理制备的复合纳米粒子的分散性较好,且仍保持Fe3O4的晶相结构。2.磁性复合纳米粒子经羧甲基葡聚糖改性后,在水溶液中的稳定性显著提高,并仍具有超顺磁性。注射液,超顺磁性目的：检测胰腺癌BXPC-3细胞与结肠癌HCT-116细胞生长抑素受体各亚型的表达,确定生长抑素受体阳性及阴性细胞株；生长抑素受体阳性肿瘤细胞是否能特异性的摄取偶联奥曲肽的SPIO；检测与偶联奥曲肽的SPIO共培养后生长抑素受体阳性细胞与生长抑素受体阴性细胞MRI信号的差异。方法：分别培养BxPC-3胰腺癌细胞、HCT-116结肠癌细胞。使用RT-PCR分别检测两种细胞内SSTR1、SSTR2、SSTR3、SSTR4、SSTR5的mRNA表达,以确定生长抑素受体阳性及阴性肿瘤细胞株。两种细胞常规培养至对数生长期,将相同浓度CMD-MNPs-Oc溶液分别加入各自的培养基中,继续共同培养,即分为两组：A组为CMD-MNPs-Oc孵育后的BxPC-3细胞；B组为CMD-MNPs-Oc孵育后的HCT-116细胞。然后在相同的孵育时间分别取细胞混悬液获取细胞沉渣行电镜检查,观察Fe3O4颗粒是否被内摄进入肿瘤细胞内,并对比Fe3O4颗粒在细胞内分布部位及量的情况。使用HE染色及普鲁士蓝染色两种方法定量检测Fe3O4颗粒分别进入生长抑素阳性肿瘤细胞和生长抑素阴性肿瘤细胞内的量(分组同电镜检测)。用MRI机器对处理后的细胞混悬液进行扫描,检测Fe3O4颗粒在细胞内的选择性聚集及其驰豫性的改变(A、B分组同电镜检测,C组为无细胞的纯培养液作为空白对照)。结果：RT-PCR结果显示BxPC-3细胞中SSTR1、SSTR2、SSTR3、SSTR4、SSTR5均呈现阳性表达,相反HCT-116细胞中则表现为阴性表达。电镜结果显示,在A组中,进入细胞内的Fe3O4颗粒较多,且多位于胞浆而不是线粒体内,并可见有小吞饮泡形成；在B组中,进入细胞内的Fe3O4颗粒很少,且位于溶酶体内。HE染色结果显示A组CMD-MNPs-Oc孵育后的BxPC-3细胞的胞浆中见较多散在颗粒状物,而B组CMD-MNPs-Oc孵育后的HCT-116细胞内则未见颗粒状物。进一步普鲁士蓝染色结果显示A组BxPC-3细胞的胞浆内较多深蓝色或青色Fe3O4颗粒,其阳性细胞百分比为97%,而B组HCT-116细胞的胞浆内则极少见深蓝色或青色Fe3O4颗粒,阳性细胞百分比在6%。MRI检测结果A组细胞MRI扫描T2加权图像信号强度明显低于C组空白对照组的信号(P<0.001),其信号强度下降率为69.6%,其信号强度也明显低于B组,差异具有统计学意义(P<0.001)结论：1. BxPC-3细胞中生长抑素受体呈阳性表达,HCT-116细胞中生长抑素受体呈阴性表达。2. CMD-MNPs-Oc能通过受体结合作用将Fe3O4颗粒特异性、靶向性的被摄取到生长抑素受体阳性细胞胞浆内。3. CMD-MNPs-Oc通过特异性、靶向性的将Fe3O4颗粒被摄取到生长抑素受体阳性细胞胞浆内,从而实现在MRI检测下生长抑素受体阳性细胞与生长抑素受体阴性细胞信号的明显差异。更多还原

【Abstract】 Research background:Somatostatin receptor positive tumor cells are commonly seen in neuroendocrine tumours (NETs), they are originated from neuroendocrine cells and are often discovered in digestive system, however, they can also be sporadic in other body organs. This kind of tumour majorly originated in the stomach and intestine and pancreas, all of which account for 75% of the whole and are also known as gastroenteropancreatic neuroendocrine tumors (GEP NETs). The preferred therapy to GEP NETs mainly relies on aggressive surgical operation. However, since the lesion is usually hidden deeply within the gastrointestinal wall or retroperitoneum, it is hard for early diagnosis or accurate localization of the tumor, the effect of clinical treatment is therefore largely affected. Hence, proposing a method for early diagnosis and accurate localization of the neuroendocrine tumor has become a focus of international research.The recent researches in oncology have revealed the high density of somatostatin receptor (SSTR) on the cell membranes of NETS. As a G protein-coupled transmembrane receptor, SSTR has 5 subtypes:SSTR1 to SSTR5; and the subtypes of GEP NETs expression are primarily SSTR2 and SSTR3. The ligand of SSTR is somatostatin (SST),which has analogues called somatostatin analogues (SSTA). SSTA can be ligated to SSTR specifically and transited into cells. Therefore, We can take advantage of the endocytosis mechanism of SSTR, and use SSTA as a carrier protein to transport radioactive isotopes into cells. The enriched radioactive isotopes in tumor cells can be detected by SPECT, which is called somatostatin receptor scintigraphy(SRS). SRS had been studied and applied for clinical practice since 1990s to diagnose primary and metastatic lesions of NETs. As a new examine method, SRS has been widely used overseas but is limited in SPECT. In clinical surgery, it is essential to locate the exact position of the lesion and its relationship with the surrounding organs and tissues, and MRI has its advantage in revealing this. However, MRI doesn’t show any specificity and sensitivity to NETs. Thus it has become our goal to develop a method that combines both the advantages of SRS and MRI to reach a high specificity and sensitivity for NETs diagnosis and at the meantime presents high spatial resolutions to provide surgeons with a clearer 3-dementional imaging of the lesion and its relationship with the surrounding organs or tissues.The objective of our study is to develop a new early-dignosis and accurate positioning method for somatostatin receptor positive tumor with somatostatin receptor magnetic resonance molecular imaging (SRMRMI), which uses the coupling of chemically modified contrast medium superparamagnetic iron oxide(SPIO) usually applied in MRI and Octreotide, to form a SPIO-Octreotide complex, and selectively transport Fe3O4 particles into tumor cells by the specific combination of Octreotide with SSTR to form a enrichment of Fe3O4 particles in cytoplasm, which can be detected by MRI because of the change of relaxation time. We hope that SRMRMI has the potentials of both high specificity and sensitivity presented in SRS and high spatial resolutions presented in MRI, which provide us with a new way to early diagnose and locate the somatostatin receptor positive tumors. Our study can be divided into two parts.Objective:To prepare the carboxymethyldextran modified Fe3O4 magnetic nanoparticles (CMD-MNPs), which are of high dispersity and stability, and crosslink octreotide with CMD-MNPs. Methods:By means of chemical co-deposition method, modified magnetic nanoparticles were prepared in carboxymethyldextran-water dispersed phase by ultrasonic pretreatment. Crosslinked with octreotide, the product was characterized by IR spectrum, transmission electron microscopy, x-ray diffractometer, and vibrating sample magnetometer, and the T2 relaxativity in solution is measured.Results:(1) The carboxymethyldextran magnetic nanoparticles was prepared by chemical co-deposition method, and the octreotide was successfully linked to the CMD-MNPs with the amide linkage. The reaction solution which contained ferric chloride and ferrous chloride as the source of iron, and glucan as the modifying agent, was pretreated by ultrasonic equipment. (2) The scanning electron microscope shows that nanoparticles is globular and the radius is about 50nm, accompanied with concentrated distribution of the radius. The ultrasonic pretreatment could stop the gathering of the Fe3O4 nanoparticles. The glucan modifying agent has obviously reinforced the water-solubility of the nanoparticles with no change of the crystal construction of Fe3O4. (3) The composite nanoparticles show the property of superparamagetism. The saturated magnetizing strength is 35emu g-1 in the room temperature.Conclusions:1. The ultrasonic pretreatment can significantly improve the dispersibility of the composite nanoparticles. And it maintains the crystal construction which is similar to that of Fe3O4.2. The stability of the composite nanoparticles in the water is significantly improved. The composite nanoparticles also show the property of superparamagetism. Objective:To test the somatostatin receptor subtypes (SSTR1-5) mRNA expression in BxPC-3 cells and HCT-116cells to determine the somatostatin receptor positive and negative cell lines. To test the specific endocytosis of SSTR positive tumor cells to SPIO--Octreotide complex. To detect the difference of MRI signals in somatostatin receptor positive and negative tumor cells after CMD-MNPs-Oc co-cultivation.Methods:Both the BxPC-3 cells and the HCT-116 cells were cultured respectively, and the intracellular expression of somatostatin receptor subtypes (SSTR 1-5) mRNA was tested by reverse transcription polymerase chain reaction (RT-PCR) technique to determine the somatostatin receptor positive and negative cell lines. Both cells were cultured to logarithmic phase when CMD-MNPs-Oc solutions were added to the culture mediums respectively, which therefore comprises 2 groups of cells:Group A was BxPC-3 cells cultured with CMD-MNPs-Oc; Group B was HCT-116 cells cultured with CMD-MNPs-Oc. With the same incubation time, cell suspensions were collected from each group and after that centrifugated respectively to undergo electron microscopy in order to observe whether the Fe3O4 particles were uptaken into the tumor cells and compare the distribution and quantity of the Fe3O4 particles within the tumor cells. The cells underwent conventional HE staining and Prussian blue iron staining, and the ratio of the numbers of the positive iron containing cells to the numbers of the total cells were calculated for each group. The electron microscope inspection was applied to observe the selective uptake of high-density particles in the cytoplasm of cells in each group. The T2 relaxativity of resuspension in each group was detected by MR imaging. (Group A and B were calculated using Group C as control.)Results:RT-PCR showed positive expressions of SSTR1、SSTR2. SSTR3、SSTR4、SSTR5 in BxPC-3 cell line, and negative expressions of the above receptors in HCT-116 cell line. Electron microscope showed high intake of Fe3O4 particles in group A cells, with high concentration in cytoplasm rather than mitochondria and the formation of pinosome; While group B cells had lower Fe3O4 particles intake and the particles were mainly located in lysosome. HE staining after incubation of CMD-MNPs-Oc showed BxPC-3 cells in group A had many particles in cytoplasm while there is no particle in HCT-116 cell line. Prussian blue staining confirmed that cyan or dark blue particles was Fe3O4 particles, which had a high concentration in cytoplasm(positive rate 97%) in group A and a low concentration in cytoplasm(positive rate under 6%) in group B. MRI showed that T2 weighted spin echo sequence imaging of group A was lower than group C (P<0.001), and the calculated△SI% was 69.6%, lower than group B, the differences were of statistical significance (P<0.001)Conclusions:1. SSTR had positive expression in BxPC-3 cell line and negative expression in HCT-116 cell line.2. CMD-MNPs-Oc can specifically transport Fe3O4 particles into the cytoplasm of SSTR positive cells by receptor binding mechanism3. SSTR positive and negative cells showed different signals under the detection of MRI due to the specific uptake of Fe3O4 particles into the cytoplasm of SSTR positive cells by means of specific and targeted binding of CMD-MNPs-Oc to somatostatin receptor.更多还原