纳米炭淋巴结示踪在直肠癌中的运用及新型药物纳米载体的制备与作用研究
本文选题:纳米炭 + 直肠癌 ; 参考:《南方医科大学》2017年博士论文
【摘要】:直肠癌是目前人类最高发的恶性肿瘤之一,手术治疗一直是最有效的治疗手段。根据目前的TNM肿瘤分期标准,术后准确的区域淋巴结转移情况对于患者病理分期及后期治疗和随访计划指定有着重要的重要指导意义。为了得到准确的区域淋巴结评价,目前美国的NCCN指南要求在结直肠癌术后标本中必须获取12-14枚淋巴结。还有一些研究认为从手术标本中获取更多的淋巴结对于术后准确的区域淋巴结转移及预后评估更有利。新辅助放化疗后进行根治性直肠前切除是目前NCCN指南推荐的局部进展期直肠癌的标准治疗方案。大量的研究表明这种方案可以增加术中肿瘤切除率及保留肛门括约肌的比例,也可以降低术后局部复发率。然而目前大量的研究证明新辅助放化疗后直肠癌手术标本中获取的淋巴结术后会显著减少。虽然针对新辅助放化疗后这一类患者来说,从标本中获取淋巴结数目减少对于患者预后评价的影响还有争议,但也有一些观察性研究表明,在这一类患者中,区域淋巴结阳性患者的标本淋巴结获取数量明显多于区域淋巴结阴性患者。这就预示着提高这一类患者术后标本中淋巴结检出数目,有可能可以提高癌转移淋巴结的发现率,从而获得更加准确的区域淋巴结转移情况,以指导术后治疗及随访计划的制定,为患者带来更大的获益。在之前的研究中,我们发现肿瘤周围粘膜下注射纳米炭混选注射液可以提高胃肠癌手术标本中淋巴结获取数目,而不增加手术时间和标本处理时间。为了进一步评价新辅助放化疗后直肠癌患者术后标本中淋巴结获取数目和阳性癌转移淋巴结发现率之间的关系,并且评价纳米炭混悬注射液在提高该类患者淋巴结检出数目中的作用,我们从2013年8月开始对新辅助放化疗直肠癌患者运用了这项技术。虽然纳米炭等淋巴结示踪剂可以特异性识别淋巴结,显著术者术中、术后标本淋巴结的识别能力。但目前临床上使用所有淋巴结示踪剂均不具备肿瘤特异性,无法再术中实时辨别转移淋巴结。这使得淋巴结示踪剂的使用受到了很大的限制。因此,我们计划合成新型的纳米材料,预期使其既要有特异性的淋巴结示踪功能,也要可以卸载可特异性识别肿瘤细胞的荧光材料,以达到术中实时辨认癌转移淋巴结的作用。为达到此目的,我们设计了结合氧化铁纳米簇和葡聚糖的新型可携载纳米淋巴结示踪材料(DIONs)。该纳米材料的大小限制在100-200nm之间,这使得它可以像纳米炭一样通过淋巴管内皮细胞之间的间隙进入淋巴管而特异性进入淋巴结中,使淋巴结显现出铁离子的黑褐色。并且因为在纳米簇上修饰合成了葡聚糖,而使纳米簇具有大量的羟基结构。这些羟基结构可以通过共轭效应链接可以识别肿瘤细胞的荧光药物。在特异性进入淋巴结后,在癌转移淋巴结的酸性环境下大量释放药物,产生远大于正常的自体荧光,以达到鉴别癌转移淋巴结的作用。本研究就在于研究合成的DIONs的方法及其淋巴结示踪效果和生物安全性,为后期链接肿瘤特异性荧光药物做基础。第一部分术前粘膜下注射纳米炭对于提高新辅助直肠癌患者淋巴结病理分期的作用研究目的分析新辅助放化疗后直肠癌患者淋巴结检出数目和淋巴结病理分期之间的关系。并评价纳米炭混选注射液在提高新辅助放化疗后直肠癌患者淋巴结检出数目及准确淋巴结病理分期的作用。方法 从2008年9月到2014年7月,152名连续的局部进展期直肠癌患者接受新辅助放化疗后根治性切除手术,其中45例患者术前经肛行纳米炭混悬液肿瘤周围粘膜下注射。分析所有患者术后淋巴结检出数目和癌转移淋巴结发现率之间的关系,并分析注射纳米炭患者和未注射纳米炭患者之间淋巴结检出数目和淋巴结病理分期之间的关系。结果 在淋巴结检出1-5枚的患者中,术后淋巴结阳性比例8.6%,明显低于淋巴结检出数目17-44枚的37.8%。多因素分析显示淋巴结检出数目是淋巴结癌转移发现率的独立危险因素(p=0.036)。纳米炭组患者术后淋巴结检出数目明显高于对照组患者(21.1 vs 8.0,p = 0.000)。对照组术后淋巴结阳性患者比率明显少于纳米炭组(19.6%vs 35.6%,p = 0.037)。在纳米炭组中发现的58枚癌转移淋巴结中,45枚(77.6%)被黑染。在37枚小于5mm的癌转移淋巴结中33枚(89.2%)被黑染。结论 使用纳米炭等淋巴结示踪剂可以提高新辅助放化疗后直肠癌患者术后淋巴结检出数目,并且得到更加准确的淋巴结病理分期。第二部分葡聚糖修饰纳米氧化铁分散液的制备及淋巴结示踪的作用和生物安全性研究目的本研究的目的在于制备和表征可携载药物的葡聚糖修饰纳米氧化铁分散液(DIONs),评价其淋巴结示踪功能及生物安全性。方法我们利用三价铁、二价铁与右旋糖干经化学共沉淀得到DIONs并表征其理化性质。将合成的DIONs于大鼠胃壁浆膜下注射,观察DIONs的区域淋巴结示踪功能。并通过DIONs与人淋巴细胞共培养及CCK-8法评价其体外细胞生物安全性。通过观察大鼠注射后三个月内不同时期肝肾功能及腹腔情况,了解DIONs的体内生物安全性。通过DIONs携载Cy5荧光以及对示踪大鼠的解剖,了解DIONs在心、脑、肝、肺、肾等重要器官的代谢情况。结果我们合成的DIONs流体动力学直径为165nm,可将氧化铁纳米簇离子包裹起来,形成稳定纳米结构;有很好的水溶性、磁性和稳定性,而且本身为深棕色。其合适的大小、稳定性及黑褐色使得经过胃壁浆膜下注射后淋巴结特异性好,6小时后区域淋巴结开始染色,24小时染色时间最佳,48小时后逐渐褪色但仍可清晰辨认,得到良好的区域淋巴结示踪效果。通过DIONs与人淋巴细胞共培养及CCK-8法检测,可见其有良好的体外细胞生物安全性。通过长时间(浆膜下注射4天至3个月)对大鼠血液学及组织病理学观察,DIONs具有良好的生物安全性及相容性。通过DIONs携载Cy5荧光以及对示踪大鼠的解剖,了解DIONs仅存在于注射胃壁局部及淋巴结中,并不进入心、脑、肝、肺、肾等重要器官,具有良好的淋巴结特异性,不会对重要器官造成损伤。结论我们成功合成了一种新型的可携载的具有淋巴结示踪功能的纳米材料DIONs。它具有良好的生物安全性及相容性,并且淋巴结示踪效果明显。为下一步利用该纳米材料携载药物进行淋巴结肿瘤特异性示踪及癌转移淋巴结的药物治疗奠定了基础。
[Abstract]:Rectal cancer is one of the highest malignant tumors in human being. Surgical treatment has always been the most effective treatment. According to the current standard of TNM tumor staging, accurate regional lymph node metastasis after operation has important guiding significance for the patient's pathological staging, later treatment and follow-up plan. The NCCN guidelines in the United States now require 12-14 lymph nodes in the specimens after colorectal surgery. Some studies suggest that obtaining more lymph nodes from the surgical specimen is more favorable for accurate postoperative regional lymph node metastasis and prognostic evaluation. A standard treatment for locally advanced rectal cancer recommended by the NCCN guide is currently recommended. A large number of studies have shown that this scheme can increase the rate of intraoperative tumor resection and the retention of the anal sphincter, and also reduce the local recurrence rate. However, a large number of studies have proved that the new adjuvant chemoradiotherapy has been proved to be a new type of rectal cancer. There is a significant decrease in postoperatively. Although there is a dispute over the impact of the reduction of the number of lymph nodes on the prognosis of the patients after neoadjuvant chemoradiotherapy, there are some observational studies showing that in this type of patients, the number of lymph nodes in the regional lymph node positive patients is significantly more than that in the area. This is an indication that the number of lymph nodes detected in the specimen after surgery in this class may improve the detection rate of metastatic lymph nodes, and thus obtain more accurate regional lymph node metastases to guide postoperative treatment and follow-up planning and to bring greater benefits to patients. In the study, we found that the number of lymph nodes in the surgical specimens of gastric cancer could be improved by the injection of nano carbon mixed injection by Submucous injection of the tumor around the tumor, without increasing the time of operation and the time of treatment. In order to evaluate the number of lymph node acquisition and the positive metastatic lymph node in the postoperative specimens of rectal cancer after neoadjuvant radiotherapy and chemotherapy The relationship between the present rate and the evaluation of Carbon Nanoparticles Suspension Injection's role in improving the number of lymph node detection in this type of patients, we have been using this technique since August 2013 for neoadjuvant radiotherapy and chemotherapy for rectal cancer patients. Although nano carbon and other lymph node tracers can specifically identify lymph nodes, significant surgical procedures, and postoperative specimens have been used. But the use of all lymph node tracers in clinical use is not specific and can not identify the metastatic lymph nodes in real time. This makes the use of lymph node tracers very limited. Therefore, we plan to synthesize new nanomaterials, which are expected to have specific lymph node tracers. In order to achieve this goal, we designed a new portable carrying nano lymph node tracer (DIONs) with iron oxide nanoclusters and dextran. The size of the nanomaterial is limited to 100-200nm. This allows it to enter the lymph nodes through the space between the lymphatic endothelial cells, like the nano carbon, and specifically enter the lymph nodes, making the lymph nodes appear to be dark brown in the iron ions. And because the dextran is synthesized on the nanoclusters, and the nanoclusters have a large number of hydroxyl structures. These hydroxyl structures can be conjugated by conjugation. The effect links can identify the fluorescent drugs of the tumor cells. After the specific entry of the lymph nodes, a large number of drugs are released in the acid environment of the metastatic lymph nodes, producing far greater than normal autofluorescence to identify the role of the metastatic lymph nodes. This study is based on the study of the combined DIONs method and its lymph node tracing effect. Biological safety, which is the basis for late linking of tumor specific fluorescent drugs. Part 1 the role of submucosal injection of carbon nanoscale before operation to improve lymph node pathological staging of neoadjuvant rectal cancer patients: to analyze the relationship between the number of lymph node detection and the pathological staging of lymph nodes after neoadjuvant radiotherapy and chemotherapy. The effect of the valence nano carbon mixed injection on the number of lymph nodes and the accurate lymph node pathological staging of rectal cancer patients after neoadjuvant chemoradiotherapy. Methods from September 2008 to July 2014, 152 consecutive patients with locally advanced rectal cancer underwent radical resection after neoadjuvant radiotherapy and chemotherapy, of which 45 patients were treated by anus preoperatively. The relationship between the number of lymph nodes detected after operation and the rate of metastatic lymph node discovery in all patients was analyzed, and the relationship between the number of lymph node detection and the pathological staging of lymph nodes was analyzed. The results were found in 1-5 patients with lymph nodes. In the case of postoperative lymph node positive ratio of 8.6%, significantly lower than the number of lymph nodes detected by 17-44 37.8%. multivariate analysis, the number of lymph nodes detected is an independent risk factor for lymph node metastasis (p=0.036). The number of lymph nodes detected in the nanoscale group was significantly higher than that of the control group (21.1 vs 8, P = 0). The rate of posterior lymph node positive patients was significantly less than that in the nanoscale group (19.6%vs 35.6%, P = 0.037). Of the 58 metastatic lymph nodes found in the nanoscale group, 45 (77.6%) were black. 33 (89.2%) in the 37 lymph nodes less than 5mm were black. Conclusion the use of nano carbon and other lymph node tracers can improve the new adjuvant radiotherapy and chemotherapy. The number of lymph nodes detected in colorectal cancer patients and more accurate lymph node pathological staging. The preparation of second partial glucan modified iron oxide dispersions and the role of lymph node tracer and biosafety study aim to prepare and characterize the dispersions of dextran modified nano iron oxide, which can carry drugs. Liquid (DIONs), evaluation of its lymph node tracer function and biological safety. Methods we used trivalent iron, two iron and dextran to obtain DIONs and characterize its physicochemical properties. The synthetic DIONs was injected under the serous of the rat gastric wall, and the regional lymph node tracing function of DIONs was observed. And the co culture of human lymphocytes was co cultured with DIONs. CCK-8 method was used to evaluate the biological safety of the cells in vitro. By observing the liver and kidney function and the abdominal condition in different periods of three months after injection, the biological safety of DIONs was understood. The metabolism of the important organs such as the heart, brain, liver, lung and kidney was understood by carrying the Cy5 fluorescence and the anatomy of the traced rats by DIONs. The result was that we combined with the results of the metabolism of the important organs of the heart, brain, liver, lung and kidney. The DIONs fluid dynamic diameter is 165nm, which can wrap the iron oxide nanocluster ions and form a stable nanostructure. It has good water solubility, magnetism and stability, and itself is dark brown. The appropriate size, stability and black brown make the lymph node specificity better after subserous injection of the gastric wall and the regional lymph nodes 6 hours later. The 24 hour dyeing time was the best, the color fading gradually after 48 hours, but it could be discoloration gradually, but still can be clearly identified, and a good regional lymph node tracer effect was obtained. Through DIONs and human lymphocyte co culture and CCK-8 method, it can be found that it has a good biological safety in vitro. The hematology of rats through the long time interval (4 days to 3 months under the serous injection) And histopathological observation, DIONs has good biological safety and compatibility. Through carrying the Cy5 fluorescence with DIONs and the anatomy of the traced rats, it is understood that DIONs exists only in the local and lymph nodes of the gastric wall, and does not enter the important organs such as heart, brain, liver, lung and kidney. It has a good lymph node specificity and does not cause damage to important organs. Conclusion we have successfully synthesized a new portable DIONs. nanomaterial with lymph node tracer function. It has good biological safety and compatibility, and the lymph node tracer effect is obvious. The next step is to use the nano material to carry the drugs for lymph node tumor specific tracer and cancer metastasis lymph node. The treatment laid the foundation.
【学位授予单位】:南方医科大学
【学位级别】:博士
【学位授予年份】:2017
【分类号】:R735.37
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