本文選題：肺癌 + 腫瘤干細(xì)胞��； 參考：《第二軍醫(yī)大學(xué)》2012年博士論文

【摘要】：研究目的： 目前，隨著工業(yè)的迅速發(fā)展，不可避免的的環(huán)境污染的加重；大量使用農(nóng)藥造成糧食、蔬菜、水果的污染以及食品添加劑的使用；醫(yī)療水平提高、經(jīng)濟(jì)條件的改善；平均壽命的延長(zhǎng)；不良情緒和不良生活習(xí)慣等諸多因素使腫瘤發(fā)病率不斷升高，腫瘤已成為威脅人類健康的重要疾病。每年約有265萬(wàn)人最后失去生命就是因?yàn)轭净寄[瘤及腫瘤相關(guān)并發(fā)癥。近年來(lái)，，國(guó)內(nèi)外醫(yī)學(xué)相關(guān)工作者越來(lái)越重視對(duì)腫瘤及其并發(fā)癥的的研究，同時(shí)對(duì)腫瘤的診斷、治療包括內(nèi)外科的治療以及放化療等治療的結(jié)合取得了巨大的進(jìn)步，肺癌、肝癌、乳腺癌包括一些原發(fā)性惡性腫瘤的生存期及預(yù)后還是較差，對(duì)腫瘤發(fā)生發(fā)展轉(zhuǎn)移等機(jī)制的認(rèn)識(shí)不足是腫瘤治療預(yù)后不理想的主要原因，臨床上缺乏有效的治療手段。 我國(guó)衛(wèi)生部屬下腫瘤防治辦公室曾做過(guò)這樣的統(tǒng)計(jì)：我國(guó)腫瘤發(fā)病率排序中，肺癌、乳腺癌分別位居男、女性惡性腫瘤發(fā)病首位，而肺癌則是所有惡性腫瘤中死亡率最高的腫瘤。有學(xué)者在臨床中發(fā)現(xiàn)，對(duì)死亡的肺癌患者進(jìn)行尸檢，發(fā)現(xiàn)高達(dá)90%的肺癌死亡患者已經(jīng)有脊柱轉(zhuǎn)移。肺癌以及肺癌脊柱轉(zhuǎn)移瘤惡性程度非常高，生長(zhǎng)迅速，侵襲性非常強(qiáng)，發(fā)生脊柱轉(zhuǎn)移早，肺癌患者生存期預(yù)期差，生活質(zhì)量差，5年生存率低。國(guó)內(nèi)外對(duì)肺癌的治療是在外科治療的基礎(chǔ)上，結(jié)合化療、放療和免疫治療等措施，治療措施相對(duì)規(guī)范和統(tǒng)一。目前國(guó)內(nèi)外醫(yī)學(xué)界的一個(gè)挑戰(zhàn)性難題就是怎樣對(duì)肺癌轉(zhuǎn)移性脊柱腫瘤進(jìn)行有效的治療，現(xiàn)在國(guó)內(nèi)外對(duì)肺癌轉(zhuǎn)移性脊柱腫瘤患者的治療理念已由消極的對(duì)癥處理向以外科治療為主，結(jié)合放療、化療等綜合治療為輔的積極治療措施轉(zhuǎn)變，使患者的脊髓神經(jīng)功能得以改善，生活質(zhì)量有了顯著的提高。但仍很難阻止肺癌的復(fù)發(fā)和轉(zhuǎn)移。腫瘤的發(fā)生、轉(zhuǎn)移和侵襲的機(jī)制目前很難解釋，而腫瘤干細(xì)胞理論的提出則為其提供了很好的解決思路。腫瘤干細(xì)胞理論認(rèn)為僅有極少數(shù)腫瘤細(xì)胞（即腫瘤干細(xì)胞）而非全部腫瘤細(xì)胞具有致瘤性。腫瘤干細(xì)胞和正常干細(xì)胞一樣多處于靜止期，與大多數(shù)快速增殖的腫瘤細(xì)胞不同，常規(guī)放、化療都不易殺到它，即使殺死絕大多數(shù)腫瘤細(xì)胞，但只要有甚至只需要極少量的的腫瘤干細(xì)胞沒被殺死，腫瘤的復(fù)發(fā)則肯定存在。這一理論很好地解釋了眾多骨腫瘤醫(yī)師對(duì)肺癌脊柱轉(zhuǎn)移長(zhǎng)期療效較差的臨床困惑。 本研究針對(duì)肺癌脊柱轉(zhuǎn)移特性，選取肺癌細(xì)胞株A549分離獲得腫瘤干細(xì)胞，并進(jìn)行干細(xì)胞的鑒定，以此為基礎(chǔ)建立肺癌干細(xì)胞裸鼠脊柱轉(zhuǎn)移模型，為進(jìn)一步研究肺癌脊柱轉(zhuǎn)移機(jī)制、早期診斷及靶向治療提供依據(jù)。 研究方法： 選用肺癌細(xì)胞株A549，傳代培養(yǎng)采用添加生長(zhǎng)因子的無(wú)血清培養(yǎng)技術(shù)誘導(dǎo)肺癌球體細(xì)胞；應(yīng)用免疫熒光染色檢測(cè)腫瘤球體細(xì)胞中蛋白Sox2，Nanog及Oct4的表達(dá)，誘導(dǎo)肺癌細(xì)胞球；利用流式細(xì)胞術(shù)檢測(cè)體細(xì)胞中CD133標(biāo)志的表達(dá)；免疫磁珠法以CD133為標(biāo)志分選腫瘤干細(xì)胞。最后將分選所得腫瘤干細(xì)胞制成細(xì)胞懸液，采用左心室注射法，從而建立基于干細(xì)胞理論的肺癌干細(xì)胞裸鼠脊柱轉(zhuǎn)移模型。 結(jié)果： 1、肺癌細(xì)胞在無(wú)血清培養(yǎng)基中生長(zhǎng)，加入生長(zhǎng)因子，成功誘導(dǎo)腫瘤細(xì)胞球體形成； 2、應(yīng)用細(xì)胞免疫熒光檢測(cè)肺癌球體細(xì)胞中Sox2，Nanog及Oct4蛋白的表達(dá)，Sox2，Nanog及Oct4蛋白在多數(shù)細(xì)胞中陽(yáng)性表達(dá)，流式細(xì)胞術(shù)檢測(cè)肺癌脊柱轉(zhuǎn)移瘤球體細(xì)胞中CD133的表達(dá)。 3、細(xì)胞侵襲實(shí)驗(yàn)、細(xì)胞增殖實(shí)驗(yàn)及成瘤實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)CD133+細(xì)胞具有較強(qiáng)的侵襲性、增殖能力，能夠在小鼠體內(nèi)成瘤。 4、免疫磁珠法以CD133為標(biāo)志分選肺癌干細(xì)胞，經(jīng)左心室注射成功建立肺癌裸鼠脊柱轉(zhuǎn)移動(dòng)物模型并對(duì)其進(jìn)行相關(guān)研究。 結(jié)論： 利用普通細(xì)胞株細(xì)胞，在無(wú)血清培養(yǎng)基中添加細(xì)胞生長(zhǎng)因子EGF、TGF-β1、bFGF等能成功誘導(dǎo)肺癌細(xì)胞球體細(xì)胞，免疫熒光法檢測(cè)表明：Sox2，Nanog及Oct4蛋白在該細(xì)胞中多數(shù)細(xì)胞呈陽(yáng)性表達(dá)；流式細(xì)胞術(shù)檢測(cè)發(fā)現(xiàn)：CD133可以作為肺癌干細(xì)胞重要的表面標(biāo)志，基于以上原理，應(yīng)用免疫磁珠法分選出其中的CD133+細(xì)胞，細(xì)胞侵襲實(shí)驗(yàn)、細(xì)胞增殖實(shí)驗(yàn)及成瘤實(shí)驗(yàn)結(jié)果提示CD133+細(xì)胞具有很強(qiáng)的侵襲性、增殖能力，能夠在小鼠體內(nèi)成瘤。肺癌干細(xì)胞分離鑒定后配成懸液，經(jīng)左心室注射，成功建立肺癌脊柱轉(zhuǎn)移裸鼠模型，模型能模擬肺癌細(xì)胞經(jīng)動(dòng)脈途徑轉(zhuǎn)移至脊柱并形成轉(zhuǎn)移灶并發(fā)生發(fā)展的病理過(guò)程，而且模型復(fù)制成功率高，對(duì)于研究肺癌脊柱轉(zhuǎn)移的機(jī)制、探索肺癌脊柱轉(zhuǎn)移的治療意義重大。
[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錛

本文編號(hào)：1997253