VNN1对胰腺癌癌旁胰岛的影响及USPIO-VNN1对胰腺癌细胞示踪作用的研究

发布时间：2018-07-09 20:06

本文选题：胰腺癌的早期诊断 + 新发糖尿病　；参考：《浙江大学》2016年博士论文

【摘要】：背景:胰腺癌恶性程度高,预后极差。由于缺乏早期症状及预测的标记物,致使胰腺癌的早期诊断十分困难。手术治疗是胰腺癌仅有的根治手段,但由于该病在确诊时通常已是晚期,导致可行手术治疗的患者不足20%。因此,早期诊断已成为提高胰腺癌手术切除率、进而有效改善其预后的关键。许多研究表明新发糖尿病可以作为胰腺癌的早期并发症,此型糖尿病又被称为胰腺癌相关性新发糖尿病。因此,新发糖尿病可以用于胰腺癌的早期筛查。然而,我国新发糖尿病的发病率要远高于胰腺癌,如何将少量的胰腺癌相关性新发糖尿病患者从众多的普通新发糖尿病患者中筛选出来仍是个难题。我们课题组在前期研究中发现,重组人血管非炎性因子1(Vanin-1,VNN1)在胰腺癌相关性新发糖尿病患者的癌组织及外周血细胞内呈特异性高表达,其可用于普通新发糖尿病与胰腺癌相关性新发糖尿病之间的鉴别,进而可用于胰腺癌的早期筛查。然而,VNN1引起胰腺癌相关性新发糖尿病发生的具体机制仍不十分明确。在此研究中,我们探究了胰腺癌细胞内VNN1的高表达对癌旁原代胰岛的影响及其分子机制。同时,我们还使用临床标本检测了 VNN1的下游产物是否也可用于胰腺癌的早期诊断。此外,我们在本课题组前期研究的基础上合成了 VNN1功能化的超顺磁性氧化铁纳米颗粒(USPIO-VNN1),还使用磁共振成像技术观察了 USPIO-VNN1对VNN1高表达胰腺癌细胞系的靶向示踪作用,并进一步探讨了USPIO-VNN1是否可对早期胰腺癌进行特异性成像。方法:使用VNN1基因过表达载体转染PANC-1和CFPAC-1两株胰腺癌细胞系,建立VNN1高表达的稳转胰腺癌细胞系PANC-VNN1和CFPAC-VNN1。从C57BL/6J(B6)小鼠胰腺内分离出原代胰岛,并使用6孔板专用的transwell小室(0.4μm径)构建了胰腺癌细胞与小鼠原代胰岛的共培养体系。小鼠原代胰岛与胰腺癌细胞共培养24小时后,使用台盼蓝染色检测原代胰岛的活性;使用流式技术检测原代胰岛的活性及其内部活性氧簇(reactiveoxygenspecies,ROS)的含量;使用胰岛素释放实验检测原代胰岛细胞的功能;将200或400胰岛当量(isletequivalent,IEQ)的原代胰岛移植到B6rag1-/-(免疫缺陷)1型糖尿病小鼠肾包膜下,采用尾静脉取血法连续监测小鼠血糖水平变化;使用免疫组织化学染色法检测原代胰岛内胰岛素(insulin)的含量及胰腺癌患者组织标本内VNN1的含量;使用谷胱甘肽(glutathione,GSH)测定试剂盒检测原代胰岛细胞内GSH的含量;使用western blot法检测原代胰岛内促凋亡蛋白cleaved caspase-3、cleaved caspase-9及抗氧化蛋白过氧化物酶体增殖物激活受体 γ(peroxisome proliferator-activated receptor ganma,PPARy)的含量。我们还使用高效液相色谱(high performance liquid chromatography,HPLC)法检测了胰腺癌细胞条件培养基内、原代胰岛细胞内及胰腺癌患者血清标本内半胱胺(cysteamine)的含量。我们采用三乙酰丙酮铁作为前体制备超小超顺磁性氧化铁纳米颗粒(USPIO)的铁核心,然后使用高分子聚合物—聚乙二醇(polyethylene glycol,PEG)对USPIO进行包被后合成水溶性良好的PEG-USPIO,并使用透射电子显微镜、纳米激光粒度仪、低温磁场测试和试样制备系统、傅里叶变换红外光谱仪等设备检测PEG-USPIO的理化性质。使用N-羟基丁二酰亚胺(N-hydroxysuccinimide,NHS)和二乙基二乙氨丙酯(ethyldiethylaminopropylcarbodiimide,EDC)将PEG-USPIO 和VNN1单克隆抗体连接后合成USPIO-VNN1。将PANC-VNN1及对照细胞分别与含有PEG-USPIO(铁浓度为40 ug/ml)或USPIO-VNN1(铁浓度为40 ug/ml)的培养基共孵育24h后,使用普鲁士蓝染色及3.0 T临床磁共振成像系统分别检测PEG-USPIO、USPIO-VNN1对胰腺癌细胞的标记效率。使用PANC-VNN1及对照细胞建立裸鼠皮下瘤模型,并通过尾静脉分别注射PEG-USPIO溶液(铁浓度为20 mg/kg)或USPIO-VNN1溶液(铁浓度为20 mg/kg)24小时后,行磁共振扫描检测USPIO-VNN1对VNN1高表达胰腺癌细胞系的靶向示踪作用。结果:台盼蓝染色和流式分析显示,与未处理组(未与胰腺癌细胞共培养的胰岛)和对照细胞组(与对照细胞共培养的胰岛)相比,经PANC-VNN1和CFPAC-VNN1细胞共培养24小时后的原代胰岛(PANC-VNN1和CFPAC-VNN1细胞组)的存活率明显下降。Western blot结果显示PANC-VNN1和CFPAC-VNN1细胞组的原代胰岛内促凋亡蛋白cleaved caspase-3及cleaved caspase-9的含量明显增加,免疫组化染色显示该组原代胰岛结构的完整性也受到了明显的破坏。与未处理组和对照细胞组相比,PANC-VNN1和CFPAC-VNN1细胞组的原代胰岛的胰岛素基础释放量显著降低。分别将未处理组、对照细胞组及PANC-VNN1细胞组的原代胰岛移植到B6 rag1-/-(免疫缺陷)1型糖尿病小鼠肾包膜下。移植200或400 IEQ原代胰岛后,与未经胰腺癌细胞共培养组及对照细胞共培养组的糖尿病小鼠相比,PANC-VNN1细胞共培养组的糖尿病小鼠更晚达到正常血糖水平。HPLC法分析显示,使用完全培养基培养胰腺癌细胞24小时后,PANC-VNN1和CFPAC-VNN1细胞分泌至胞外条件培养基内cysteamine(VNN1的下游产物)的量明显高于对照细胞。与未处理组及对照组相比,PANC-VNN1和CFPAC-VNN1细胞组的胰岛细胞内cysteamine和ROS的浓度显著增高、GSH和PPARγ的表达量明显下降。分别使用浓度为10μM的抗氧化剂GSH和1OμM的PPARγ激动剂噻唑烷二酮(thiazolidinedione,TZD)预孵育原代胰岛2小时,再将这些原代胰岛与胰腺癌细胞共培养24小时,结果显示PANC-VNN1细胞组的原代胰岛内ROS的含量明显下降,此外,该组原代胰岛的存活率和胰岛素释放能力均显著升高.我们使用临床组织标本再次发现胰腺癌相关性新发糖尿病患者癌组织内的VNN1蛋白呈特异性高表达。此外,我们还使用HPLC法检测了临床患者及健康人群血清内cysteamine的含量,结果发现与不伴有新发糖尿病的胰腺癌患者、新发2型糖尿病患者及健康人群相比,胰腺癌相关性新发糖尿病患者血清内cysteamine的浓度明显增高。经 ROC 曲线(receiver operating characteristic curve)分析后,我们发现检测血清内cysteamine的浓度从新发2型糖尿病患者中鉴别胰腺癌相关性新发糖尿病患者的曲线下面积(Area Under Curve,AUC)为0.828 ± 0.039(P0.05)。电子显微镜扫描显示USPIO的氧化铁核心直径约为3-7nm,分布均匀。USPIO经高分子聚合物PEG包被后合成PEG-USPIO,纳米激光粒度仪分析结果显示PEG-USPIO在水溶液中的平均粒径约为20nm。采用震动样品磁强计测试后发现,PEG-USPIO在室温下的饱和磁力为35 emu/g。使用NHS/EDC法将PEG-USPIO和VNN1单克隆抗体连接后合成USPIO-VNN1。将PANC-VNN1和对照细胞分别与铁浓度为40 ug/ml的PEG-USPIO及USPIO-VNN1共孵育24h后,普鲁士蓝染色显示经 USPIO-VNN1 共培养的 PANC-VNN1 细胞(USPIO-VNN1 +PANC-VNN1细胞组)内出现细小的蓝色铁颗粒,而其它3组(PEG-USPIO +对照细胞组、PEG-USPIO + PANC-VNN1细胞组、USPIO-VNN1 +对照细胞组)细胞内未见明显的蓝色铁颗粒。MRI扫描显示,USPIO-VNN1+PANC-VNN1细胞组可见T2信号(横向弛豫时间加权信号)减低,而其它3组未见明显信号改变。通过尾静脉将PEG-USPIO溶液(铁浓度为20 mg/kg)或USPIO-VNN1溶液(铁浓度为20 mg/kg)分别注射入PANC-VNN1或对照细胞荷瘤鼠体内24小时后,MRI扫描显示尾静脉注射USPIO-VNN1溶液的PANC-VNN1细胞荷瘤鼠瘤块内可见信号减低,而其它3组荷瘤鼠瘤块内未见明显信号改变。将瘤块的组织切片进行普鲁士蓝染色后发现,在USPIO-VNN1 + PANC-VNN1细胞组瘤块内可见散在的铁颗粒沉积,而其它3组则未见此现象。此外,我们还发现USPIO-VNN1在小鼠体内具有良好的生物相容性,未见明显细胞毒性。结论:胰腺癌细胞内VNN1高表达可以通过旁分泌cysteamine的方式加剧癌旁胰岛的氧化应激反应,进而抑制胰岛的活性及功能。VNN1的下游产物cysteamine在胰腺癌相关性新发糖尿病患者血清内呈特异性高表达,检测血清内cysteamine的浓度可以用于胰腺癌相关性新发糖尿病和单纯新发糖尿病之间的鉴别,进而可能用于胰腺癌的早期筛查。此外,我们制备的MRI造影剂USPIO-VNN1可以对VNN1高表达的胰腺癌细胞进行活体靶向示踪,结合我们前期的研究说明USPIO-VNN1可能对继发有新发糖尿病的早期胰腺癌组织进行特异性成像,进而有可能提高胰腺癌的早期诊断率。
[Abstract]:Background: pancreatic cancer has a high malignancy and a poor prognosis. Early diagnosis of pancreatic cancer is difficult due to the lack of early symptoms and predicted markers. Surgical treatment is the only radical cure for pancreatic cancer. However, the disease is usually advanced at the time of diagnosis, resulting in less than 20%. of patients who have been operated on. Therefore, early diagnosis has been made. Many studies have shown that new onset diabetes can be an early complication of pancreatic cancer. This type of diabetes is also known as a new type of diabetes associated with pancreatic cancer. Therefore, new onset diabetes can be used for early screening of pancreatic cancer. However, the incidence of new onset diabetes in China The rate is far higher than that of pancreatic cancer. How to screen a small number of pancreatic cancer related new diabetic patients from a large number of common new diabetic patients is still a difficult problem. In our previous study, we found that recombinant human vascular non inflammatory factor 1 (Vanin-1, VNN1) is in the cancer tissue and outside of the new diabetic patients with pancreatic adenocarcinoma. There is a specific high expression in the peripheral blood cells, which can be used to differentiate between new diabetes and pancreatic cancer related new onset diabetes, and can be used for early screening of pancreatic cancer. However, the specific mechanism of VNN1 induced pancreatic cancer related new onset diabetes is still not clear. In this study, we explored pancreatic cancer cells. The effect of high expression of internal VNN1 on the paracancerous islets of the pancreas and its molecular mechanism. We also used clinical specimens to detect whether the downstream products of VNN1 can also be used for the early diagnosis of pancreatic cancer. In addition, we have synthesized the VNN1 functionalized superparamagnetic iron oxide nanoparticles (USPIO-VNN1) on the basis of our previous study. Magnetic resonance imaging (MRI) was used to observe the target tracer effect of USPIO-VNN1 on VNN1 high expression of pancreatic cancer cell lines, and further explore whether USPIO-VNN1 could be specific imaging of early pancreatic cancer. Method: transfection of VNN1 gene overexpression vector to two pancreatic cancer cell lines of PANC-1 and CFPAC-1, and to establish the stability of high expression of VNN1. The pancreatic cancer cell lines PANC-VNN1 and CFPAC-VNN1. isolated the original islets from the pancreas of C57BL/6J (B6) mice. The co culture system of pancreatic cancer cells and the primary islets of mice was constructed by using the special Transwell chamber of 6 orifice plates (0.4 micron m). The primary pancreatic islets and pancreatic cancer cells were cultured for 24 hours, and trypan blue staining was used to detect the original generation. The activity of islets, the activity of the original islets and the content of reactiveoxygenspecies (ROS), the function of the primary islet cells using insulin release, and the original islet of the 200 or 400 islet equivalent (isletequivalent, IEQ) to B6rag1-/- (immunodeficiency) type 1 diabetic mice The content of insulin (insulin) in the original islet and the content of VNN1 in the tissue specimens of pancreatic cancer were detected by immunohistochemistry staining method, and the content of GSH in the primary islet cells was detected by glutathione (glutathione, GSH), and West was used. Ern blot method was used to detect the content of cleaved Caspase-3, cleaved caspase-9 and antioxidant protein peroxisome proliferator activated receptor gamma (peroxisome proliferator-activated receptor Ganma, PPARy) in the original islet. The content of cysteamine (cysteamine) in the serum samples of pancreatic cancer patients in the condition of pancreatic cancer cell condition. We use three acetyl acetone iron as the precursor to prepare the iron core of super small superparamagnetic iron oxide nanoparticles (USPIO), and then use the high molecular polymer polyethylene glycol (polyethylene glycol, PEG). USPIO was used to synthesize PEG-USPIO with good water solubility, and the physical and chemical properties of PEG-USPIO were detected by transmission electron microscope, nano laser particle size analyzer, low temperature magnetic field test and sample preparation system, Fourier transform infrared spectrometer and so on. N- hydroxy butyl two imide (N-hydroxysuccinimide, NHS) and two ethyl two ethyl propyl ester were used. (ethyldiethylaminopropylcarbodiimide, EDC) combined PEG-USPIO and VNN1 monoclonal antibodies to synthesize USPIO-VNN1. to incubate PANC-VNN1 and control cells with PEG-USPIO (iron concentration 40 ug/ml) or USPIO-VNN1 (iron concentration 40 ug/ml) for incubating 24h, using Prussian blue staining and 3 T clinical magnetic resonance imaging system The labeling efficiency of PEG-USPIO, USPIO-VNN1 for pancreatic cancer cells was detected. PANC-VNN1 and control cells were used to establish nude mice model of subcutaneous tumor, and PEG-USPIO solution was injected through the tail vein (iron concentration was 20 mg/kg) or USPIO-VNN1 solution (iron concentration was 20 mg/kg) for 24 hours, and the high expression of USPIO-VNN1 to VNN1 high expression pancreas was detected by magnetic resonance scanning. Results: trypan blue staining and flow analysis showed that the primary islets (PANC-VNN1 and CFPAC-VNN1 cells) were cultured for 24 hours after PANC-VNN1 and CFPAC-VNN1 cells, compared with the untreated group (the islets not co cultured with pancreatic cancer cells) and the control cell group (the islets co cultured with the control cells). The survival rate of.Western blot showed a significant increase in the contents of cleaved caspase-3 and cleaved caspase-9 in the primary islets of PANC-VNN1 and CFPAC-VNN1 cells. Immunohistochemistry showed that the integrity of the original islet structure of the group was also obviously broken. The insulin base release of the primary islets of the NC-VNN1 and CFPAC-VNN1 cells decreased significantly. The original islets of the untreated group, the control cell group and the PANC-VNN1 cell group were transplanted under the renal capsule of the B6 rag1-/- (immunodeficiency) type 1 diabetic mice. After the transplantation of the 200 or 400 IEQ original islets, the co culture group and the non pancreatic cancer cell co culture group and the other group were compared. Compared with the diabetic mice of the cell co culture group, the diabetic mice in the PANC-VNN1 cell co culture group were more late to reach the normal blood glucose level by.HPLC assay. After 24 hours of complete culture medium, PANC-VNN1 and CFPAC-VNN1 cells secreted to the quantity of cysteamine (downstream products of VNN1) in the extracellular conditioned culture. Compared with the control group, the concentration of Cysteamine and ROS in the islet cells of PANC-VNN1 and CFPAC-VNN1 cells increased significantly compared with those in the untreated and control groups, and the expression of GSH and PPAR gamma decreased significantly. The pre incubation of the antioxidant GSH and 1O micron M of the PPAR gamma agonist, thiazolidane two ketones, respectively, was preincubated. The original islets were cultured for 2 hours, and then the original islets and pancreatic cancer cells were co cultured for 24 hours. The results showed that the content of ROS in the original islet of PANC-VNN1 cell group decreased obviously. In addition, the survival rate and insulin release ability of the original islets of the group were significantly increased. The VNN1 protein in the cancerous tissues of the patients with urine is highly specific. In addition, we also use HPLC to detect the content of cysteamine in the serum of the patients and healthy people. The results showed that the pancreatic cancer patients with non new diabetes mellitus, new type 2 diabetes and healthy people, were associated with new diabetic patients with pancreatic cancer. The concentration of cysteamine in serum increased significantly. After the analysis of the ROC curve (receiver operating characteristic curve), we found that the concentration of cysteamine in the serum was detected in the new onset type 2 diabetic patients with the area under the curve (Area Under Curve, AUC) of 0.828 + 0.039 (P0.05). The microscope scan showed that the core diameter of the iron oxide core of USPIO was about 3-7nm, and the distributed uniform.USPIO was synthesized by the high molecular polymer PEG package. The results of the nano laser particle size analyzer showed that the average particle size of PEG-USPIO in the aqueous solution was about 20nm., and the saturated magnetic force of PEG-USPIO at room temperature was found at room temperature. After connecting PEG-USPIO and VNN1 monoclonal antibodies to 35 emu/g. to synthesize USPIO-VNN1., PANC-VNN1 and control cells were incubated with PEG-USPIO and USPIO-VNN1 respectively with iron concentration of 40 ug/ml, respectively, and Prussian blue staining showed that USPIO-VNN1 co cultured PANC-VNN1 cells appeared to be fine. Small blue iron particles, and other 3 groups (PEG-USPIO + control cell group, PEG-USPIO + PANC-VNN1 cell group, USPIO-VNN1 + control cell group) no obvious blue iron particles.MRI scan showed that the USPIO-VNN1+PANC-VNN1 cell group showed T2 signal (transverse relaxation time weighted signal) decreased, but the other 3 groups did not have obvious signal change. The PEG-USPIO solution (iron concentration 20 mg/kg) or USPIO-VNN1 solution (iron concentration 20 mg/kg) was injected into the tail vein for 24 hours in PANC-VNN1 or the control cells of the tumor mice respectively. The MRI scan showed that the PANC-VNN1 cells in the PANC-VNN1 cell of the tail vein injected with USPIO-VNN1 solution were reduced in the tumor block, while the other 3 groups of tumor bearing mice were not in the tumor block. Visible signal change was seen. After staining the tissue section of the tumor block with Prussian blue, the scattered iron particles were found in the USPIO-VNN1 + PANC-VNN1 cell group, while the other 3 groups did not see this phenomenon. In addition, we also found that USPIO-VNN1 had good biocompatibility in mice and no obvious cytotoxicity. Conclusion: pancreas The high expression of VNN1 in adenocarcinoma cells can increase the oxidative stress response of the islets paracancerous by paracrine cysteamine, and then inhibit the activity of the islet and the downstream product cysteamine of the functional.VNN1 in the serum of the new diabetic patients with pancreatic cancer. The concentration of cysteamine in the serum can be detected in the pancreas. The identification of cancer related new onset diabetes and simple new onset diabetes may be used for early screening of pancreatic cancer. In addition, our MRI contrast agent USPIO-VNN1 can target VNN1 highly expressed pancreatic cancer cells in vivo. Combined with our previous study, we suggest that USPIO-VNN1 may be secondary to new onset diabetes. Specific imaging of early pancreatic cancer tissue may improve the early diagnosis rate of pancreatic cancer.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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