肺癌干细胞脊柱转移裸鼠模型的建立及相关研究

发布时间：2018-06-08 21:41

  本文选题：肺癌 + 肿瘤干细胞　； 参考：《第二军医大学》2012年博士论文

【摘要】：研究目的： 目前，随着工业的迅速发展，不可避免的的环境污染的加重；大量使用农药造成粮食、蔬菜、水果的污染以及食品添加剂的使用；医疗水平提高、经济条件的改善；平均寿命的延长；不良情绪和不良生活习惯等诸多因素使肿瘤发病率不断升高，肿瘤已成为威胁人类健康的重要疾病。每年约有265万人最后失去生命就是因为罹患肿瘤及肿瘤相关并发症。近年来，，国内外医学相关工作者越来越重视对肿瘤及其并发症的的研究，同时对肿瘤的诊断、治疗包括内外科的治疗以及放化疗等治疗的结合取得了巨大的进步，肺癌、肝癌、乳腺癌包括一些原发性恶性肿瘤的生存期及预后还是较差，对肿瘤发生发展转移等机制的认识不足是肿瘤治疗预后不理想的主要原因，临床上缺乏有效的治疗手段。 我国卫生部属下肿瘤防治办公室曾做过这样的统计：我国肿瘤发病率排序中，肺癌、乳腺癌分别位居男、女性恶性肿瘤发病首位，而肺癌则是所有恶性肿瘤中死亡率最高的肿瘤。有学者在临床中发现，对死亡的肺癌患者进行尸检，发现高达90%的肺癌死亡患者已经有脊柱转移。肺癌以及肺癌脊柱转移瘤恶性程度非常高，生长迅速，侵袭性非常强，发生脊柱转移早，肺癌患者生存期预期差，生活质量差，5年生存率低。国内外对肺癌的治疗是在外科治疗的基础上，结合化疗、放疗和免疫治疗等措施，治疗措施相对规范和统一。目前国内外医学界的一个挑战性难题就是怎样对肺癌转移性脊柱肿瘤进行有效的治疗，现在国内外对肺癌转移性脊柱肿瘤患者的治疗理念已由消极的对症处理向以外科治疗为主，结合放疗、化疗等综合治疗为辅的积极治疗措施转变，使患者的脊髓神经功能得以改善，生活质量有了显著的提高。但仍很难阻止肺癌的复发和转移。肿瘤的发生、转移和侵袭的机制目前很难解释，而肿瘤干细胞理论的提出则为其提供了很好的解决思路。肿瘤干细胞理论认为仅有极少数肿瘤细胞（即肿瘤干细胞）而非全部肿瘤细胞具有致瘤性。肿瘤干细胞和正常干细胞一样多处于静止期，与大多数快速增殖的肿瘤细胞不同，常规放、化疗都不易杀到它，即使杀死绝大多数肿瘤细胞，但只要有甚至只需要极少量的的肿瘤干细胞没被杀死，肿瘤的复发则肯定存在。这一理论很好地解释了众多骨肿瘤医师对肺癌脊柱转移长期疗效较差的临床困惑。 本研究针对肺癌脊柱转移特性，选取肺癌细胞株A549分离获得肿瘤干细胞，并进行干细胞的鉴定，以此为基础建立肺癌干细胞裸鼠脊柱转移模型，为进一步研究肺癌脊柱转移机制、早期诊断及靶向治疗提供依据。 研究方法： 选用肺癌细胞株A549，传代培养采用添加生长因子的无血清培养技术诱导肺癌球体细胞；应用免疫荧光染色检测肿瘤球体细胞中蛋白Sox2，Nanog及Oct4的表达，诱导肺癌细胞球；利用流式细胞术检测体细胞中CD133标志的表达；免疫磁珠法以CD133为标志分选肿瘤干细胞。最后将分选所得肿瘤干细胞制成细胞悬液，采用左心室注射法，从而建立基于干细胞理论的肺癌干细胞裸鼠脊柱转移模型。 结果： 1、肺癌细胞在无血清培养基中生长，加入生长因子，成功诱导肿瘤细胞球体形成； 2、应用细胞免疫荧光检测肺癌球体细胞中Sox2，Nanog及Oct4蛋白的表达，Sox2，Nanog及Oct4蛋白在多数细胞中阳性表达，流式细胞术检测肺癌脊柱转移瘤球体细胞中CD133的表达。 3、细胞侵袭实验、细胞增殖实验及成瘤实验结果发现CD133+细胞具有较强的侵袭性、增殖能力，能够在小鼠体内成瘤。 4、免疫磁珠法以CD133为标志分选肺癌干细胞，经左心室注射成功建立肺癌裸鼠脊柱转移动物模型并对其进行相关研究。 结论： 利用普通细胞株细胞，在无血清培养基中添加细胞生长因子EGF、TGF-β1、bFGF等能成功诱导肺癌细胞球体细胞，免疫荧光法检测表明：Sox2，Nanog及Oct4蛋白在该细胞中多数细胞呈阳性表达；流式细胞术检测发现：CD133可以作为肺癌干细胞重要的表面标志，基于以上原理，应用免疫磁珠法分选出其中的CD133+细胞，细胞侵袭实验、细胞增殖实验及成瘤实验结果提示CD133+细胞具有很强的侵袭性、增殖能力，能够在小鼠体内成瘤。肺癌干细胞分离鉴定后配成悬液，经左心室注射，成功建立肺癌脊柱转移裸鼠模型，模型能模拟肺癌细胞经动脉途径转移至脊柱并形成转移灶并发生发展的病理过程，而且模型复制成功率高，对于研究肺癌脊柱转移的机制、探索肺癌脊柱转移的治疗意义重大。
[Abstract]:The purpose of the study is:
At present, with the rapid development of industry, the inevitable environmental pollution is aggravated; a large number of pesticides are used to cause food, vegetable, fruit pollution and the use of food additives; medical level is improved, economic conditions are improved, average life expectancy is prolonged; bad mood and bad habits and other factors make the incidence of cancer ill. The tumor has become an important disease that threatens human health. About 2 million 650 thousand people lose their lives at the end of the year because of cancer and tumor related complications. In recent years, medical workers both at home and abroad have paid more and more attention to the study of tumors and their complications. At the same time, the diagnosis of tumors, including the treatment of internal surgery, The combination of chemotherapy and chemotherapy has made great progress. Lung cancer, liver cancer, and breast cancer, including the survival and prognosis of some primary malignant tumors, are still poor. The lack of understanding of the mechanism of tumor development and metastasis is the main reason for the poor prognosis of the tumor treatment, and there is a lack of effective treatment on the bed.
The office of cancer prevention and control under the Ministry of health of our country has done such statistics: in the ranking of the incidence of cancer in China, lung cancer, breast cancer are in the first place, female malignant tumor is the first, and lung cancer is the highest death rate in all malignant tumors. 90% of the patients with lung cancer died of spinal metastasis. The malignancy of lung cancer and lung cancer is very high, rapid growth, strong invasiveness, early spinal metastasis, poor survival of lung cancer patients, poor living quality, low 5 year survival rate. The treatment of lung cancer at home and abroad is based on surgical treatment, combined with chemotherapy and radiotherapy The treatment measures, such as immunotherapy and other measures, are relatively standardized and unified. At present, a challenging problem in medical circles at home and abroad is how to treat metastatic spinal tumors of lung cancer effectively. Now, the treatment concept of metastatic spinal tumors in lung cancer patients at home and abroad has been treated from negative symptomatic treatment to surgical treatment. It is difficult to prevent the recurrence and metastasis of lung cancer. The mechanism of the occurrence, metastasis and invasion of the tumor is difficult to explain, and the theory of the stem cell of the tumor is very good. The tumor stem cell theory holds that only a few tumor cells (that is, cancer stem cells) and not all tumor cells have tumorigenicity. The tumor stem cells are at rest as many as normal stem cells. Unlike most rapidly proliferating tumor cells, conventional radiotherapy and chemotherapy are not easy to kill it, even if the vast majority of the tumors are killed. Cell, but as long as there is even a small amount of cancer stem cells that are not killed, the recurrence of the tumor is sure to exist. This theory is a good explanation for the clinical confusion of a large number of bone oncologists with a poor long-term effect on the metastasis of lung cancer.
This study aims at the characteristics of the spinal metastasis of lung cancer. The tumor stem cells were isolated from the lung cancer cell line A549, and the stem cells were identified. On this basis, the spinal metastasis model of nude mice was established to provide the basis for the further study of the mechanism of spinal metastasis, early diagnosis and target therapy.
Research methods:
The lung cancer cell lines were induced by A549, a serum-free culture technique with growth factors, and the expression of protein Sox2, Nanog and Oct4 in tumor cells were detected by immunofluorescence staining to induce lung cancer cell spheres; the expression of CD133 markers in somatic cells was detected by flow cytometry; the immunomagnetic bead method was used to detect the expression of CD133 markers. The tumor stem cells were selected as the marker of CD133. Finally, the cell suspension was made by the selected tumor stem cells, and the left ventricular injection method was used to establish the spinal metastasis model of nude mice in nude mice based on the stem cell theory.
Result锛

本文编号：1997253