【作者】 纪振钢；

【导师】 范清宇； 裘秀春； 张殿忠；

【作者基本信息】 第四军医大学 ， 外科学， 2010， 博士

【摘要】 研究背景:药物耐药是限制肿瘤的化疗疗效的主要问题。肿瘤可能是在治疗前就对化学药物耐受,而耐药也能通过最初对化疗敏感的肿瘤的治疗过程中获得。这些继发耐药的一个令人沮丧的特征是这些肿瘤不但对最初用于治疗它们的药物耐药,而且对其他不同作用机制的药物产生交叉耐药。耐药,不管是原发还是继发,被认为将对超过90%的转移癌患者治疗失败,而且耐药的微转移肿瘤细胞也可能降低在后续处理中的化疗的作用。显然,如果肿瘤耐药能被克服,那么肿瘤患者存活率的提高将是非常显著的。许多因素将影响化疗药物的敏感性。这些包括限制到达肿瘤的药物数量,影响肿瘤微环境的机制等。肿瘤细胞耐药可以在多个水平发生,包括增加药物排出,减少药物摄入,药物失活,药物靶点的改变,药物造成的损害作用,凋亡的逃逸。脊索瘤比较少见,是具有局部侵袭的骨原发恶性肿瘤,来源于脊索残存物。肿瘤主要发生在中轴骨,好发于颅底区域的蝶骨和骶尾部。脊索瘤通常生长缓慢,低度恶性,对原发肿瘤的控制仍是治疗上的主要挑战。脊索瘤的治疗方法主要依赖于外科手术,手术切除加术后放疗是目前最好的治疗方法。化学治疗对脊索瘤不是主要选择,多因为对化疗药物的细胞毒性抵抗所致,而新近发现的靶向制剂可能提供新的选择。骨肉瘤是一种常见的骨原发恶性肿瘤,其化疗后常有多药耐药现象发生,严重影响患者的治疗效果和预后,因此对其耐药性的检测、耐药机制及逆转的研究一直是众多学者研究的热点。在体外建立多药耐药的细胞模型已逐渐成为研究骨肉瘤多药耐药性的有利工具。紫杉醇作为一种化疗药物,在本中心开展了大量的临床、基础研究并作为骨肉瘤化疗的基础药物,其在临床初始化疗缓解后常会产生耐药性,严重影响患者治疗效果,因此采用紫杉醇诱导建立骨肉瘤多药耐药细胞模型,对探讨骨肉瘤细胞多药耐药机制和耐药逆转治疗具有重要意义。研究目的:探讨多药耐药基因1(MDR1)、缺氧诱导因子1α(HIF-1α)蛋白、多药耐药相关蛋白(MRP1)在脊索瘤中的表达与脊索瘤病理特征及放化疗抵抗之间的关系。研究脊索瘤CM-319细胞的放化疗敏感性。建立骨肉瘤耐紫杉醇细胞系,探讨骨肉瘤多药耐药的发生机制。研究内容:1、脊索瘤细胞系CM-319的生物学鉴定:通过免疫细胞化学染色、细胞周期分析、透射电镜、糖原染色等分析CM-319的生物学特性。2、脊索瘤细胞系中放化疗相关基因的检测:采用RT-PCR、间接免疫荧光、免疫细胞化学Envision法及western blott法对脊索瘤细胞系CM-319中HIF-1α和MDR1、MRP1基因及蛋白的表达进行检测,探讨HIF-1α和MRP1与脊索瘤化疗耐药、放疗抵抗的关系。3、脊索瘤组织MDR1、HIF-1α和MRP1的表达及临床意义:选取2000年1月至2008年12月接受手术治疗的50例脊索瘤患者的组织石蜡标本,用免疫组化Envision法检测MDR1、HIF-1α、MRP1蛋白的表达情况,并分析其与放化疗抵抗的关系。4、脊索瘤细胞系对放化疗的敏感性研究:通过细胞系体外照射及不同化疗药物对细胞系的敏感性实验,观察脊索瘤细胞系对放疗及化学药物的敏感性。5、人骨肉瘤耐药细胞系MG-63/PTX、SOSP-9607/PTX、OS-9901/PTX的建立及多药耐药机制研究:采用紫杉醇大剂量间断冲击培养法,诱导建立三株紫杉醇耐药骨肉瘤细胞系,并运用MTT法检测6种药物敏感性变化及细胞生长规律变化,流式细胞仪分析亲本细胞系和耐药细胞系的细胞膜表面P-gp蛋白表达率、罗丹明123排出情况,RT-PCR检测细胞内耐药基因的变化,免疫细胞化学检测细胞内P-gp蛋白的表达,western blot法检测细胞内P-gp蛋白的表达。研究结果:1、脊索瘤细胞系CM-319呈上皮样生长,CK、EMA、S-100、Vimentin阳性,富含糖原,染色体为亚三倍体核型,侵袭能力低于骨肉瘤,异种成瘤能力100%。2、脊索瘤细胞系中能检测到HIF-1α及MRP1mRNA的表达,但未检测到MDR1mRNA的表达(P<0.01)。间接免疫荧光染色及免疫细胞化学染色结果提示脊索瘤存在HIF-1α及MRP1的表达。western blot法提示CM-319中存在HIF-1α和MRP1的表达,而不表达MDR1。3、50例脊索瘤组织MDR1的阳性表达率为10%,与髓核的表达(20%)、骨软骨瘤的表达(13.3%)无统计学差异。HIF-1α、MRP1的阳性表达率分别为80%和74%,均明显高于髓核表达的20%、26.7%,骨软骨瘤的表达的20 %和26.7%,具有统计学意义(P<0.01),同时相关性分析显示肿瘤组织中HIF-1α与MRP1两者之间具有正相关性(r=0.8,P<0.01),其两者与MDR1相比呈负相关性。结合临床资料分析,脊索瘤肿瘤组织中MDR1、HIF-1α、MRP1的表达与患者性别、年龄、发病次数相比无统计学意义。4、紫杉醇相对于其他化疗药物对CM-319有较高的抑制率,高浓度紫杉醇与卡铂联合用药对CM-319的抑制有协同作用。紫杉醇作用细胞后电镜下观察到细胞的凋亡现象,细胞周期检测发现细胞阻滞在S期和G2/M期,抑制了细胞的生长。小剂量射线能对紫杉醇起到化疗增敏作用,低浓度紫杉醇能对射线起到放疗增敏作用。5、历时12个月建成人骨肉瘤耐药细胞系MG-63/PTX、SOSP-9607/PTX、OS-9901/PTX能在含PTX 1μg/mL的培养基中稳定生长并传代,其对PTX的耐药指数为69.8、24.4、26.88,并与卡铂、顺铂、表柔比星、氨甲喋呤、阿霉素等多种化疗药物存在不同程度的交叉耐药性;罗丹明排出实验显示耐药细胞内罗丹明含量明显低于亲本细胞;RT-PCR结果显示在耐药细胞系中存在MDR1基因的表达,而在亲本细胞系中无MDR1基因的表达。免疫细胞化学、流式细胞仪及western blot实验均显示在耐药细胞中存在P-gp的表达。研究结论:1、CM-319作为稳定的脊索瘤细胞系,具有上皮组织和间叶组织的特征,性质稳定,为继续研究脊索瘤的放化疗及多药耐药提供基础。CM-319表达肿瘤放化疗抵抗相关的基因HIF-1α和MRP1,而不表达MDR1。脊索瘤组织中同样表达HIF-1α和MRP1,而不表达MDR1。HIF-1α和MRP1的表达呈正相关,与脊索瘤的放化疗抵抗相关。2、CM-319细胞对多种化疗药物不敏感,紫杉醇相对于其他药物有较高的杀伤率,紫杉醇与放射线对脊索瘤的治疗能起到增敏作用。3、MDR1/P-gp在多药耐药的特性上起着至关重要的作用,三株骨肉瘤多药耐药细胞亚系的成功建立为进一步研究骨肉瘤耐药特征及逆转方法打下基础。更多还原

【Abstract】 BackgroundDrug resistance is a major problem that limits the effectiveness of chemotherapies used to treat cancer. Tumors may be intrinsically resistant to chemotherapy prior to treatment. However, drug resistance can also be acquired during treatment by tumors that are initially sensitive to chemotherapy. A frustrating property of such acquired resistance is that the tumors not only become resistant to the drugs originally used to treat them, but may also become cross-resistant to other drugs with different action mechanisms. Drug resistance, whether intrinsic or acquired, is believed to cause treatment failure in over 90% of patients with metastatic cancer, and resistant micrometastic tumor cells may also reduce the effectiveness of chemotherapy in the followed treatment. Obviously, if drug resistance could be overcome, the impact on survival rate would be highly significant. There are many factors that affect drug sensitivity. These include mechanisms such as those that limit the amount of drug reaching the tumor and those affecting the tumor micro-environment. Here we will provide an overview of cancer cell-specific mechanisms of drug resistance and highlight examples that have clinical relevance. Cancer cell resistance to chemotherapy can occur at many levels, including increased drug efflux and decreased drug influx; drug inactivation; alteration in drug target; processing of drug-induced damage; and evasion of apoptosis.Chordomas are rare, slowly growing, locally aggressive neoplasms of bone that arise from embryonic remnants of the notochord. These tumors typically occur in the axial skeleton and have a proclivity for the spheno-occipital region of the skull base and sacral regions. Chordomas are usually relatively slow-growing, lowgrade malignancies. Control of primary disease remains the major challenge. The therapeutic approach to chordoma has traditionally relied mainly on surgical control. More recently, particularly with the advent of charged particle radiotherapy, radiation therapy has been demonstrated to be a valuable modality for local control. Medical therapy continues to be suboptimal in chordomas, which is relatively refractory to cytotoxic chemotherapy. However, new targeted agents may offer therapeutic alternatives.Osteosarcomas are the most frequent malignant bone tumors. Further studies have shown that multidrug resistance (MDR) to chemotherapeutic agents is a major barrier to the successful treatment of osteosarcomas. As the basic drug of chemotherapy, paclitaxel will create drug resistance after initial effective chemotherapy. ObjectiveThe expression of HIF-1αand MRP1 and their relationship with clinicopathological features and resistance to chemotherapy and radiotherapy were investigated tin chordoma. The sensitivity of chordoma cell line to chemoradiation in vitro was studied. To establish drug-resistant human osteosarcoma cell lines to plataxel based on three osteosarcoma cell lines and to explore the molecular mechanism of osteosarcoma multidrug resistance.Methods1. Characteristics of chordoma cell line CM-319 were analysed through immunocytochemical technique, cell cycle, transmission electron microscope and glycogen staining.2. The expression of HIF-1α, MDR1 and MRP1 was detected with reverse transcription polymerase chain reaction (RT-PCR), indirect immunofluorescence staining, immunocytochemical technique (ChemMateTM Envision) and western blot. The relationship between HIF-1α, MRP1 and resistance to chemotherapy and radiotherapy was statistically analyzed.3. The integrated clinical data were retrospectively analyzed through 50 patients who had undergone radical operation from Jan. of 2000 to Dec. of 2008 in Orthopedics Oncology Institute of Chinese PLA, and the paraffin-embedded tissues of the chordoma samples were collected. The expression of HIF-1α, MRP1 and MDR1 was detected with immunohistochemical technique (ChemMateTM Envision).The relationship between HIF-1α, MRP1 and resistance to chemotherapy and radiotherapy, clinicopathological features were statistically analyzed. 4. The sensitivity of chordoma cell lines to chemoradiation: through chemoradiation in vitro irradiation, and different chemical drugs, the sensitivity of the cell was observed by radiotherapy and chemotherapy.5. Resistant cell lines MG-63/PTX, SOSP-9607/PTX and OS-9901/PTX were developed by pulse drug exposure to paclitaxel. The growth curves and drug resistance in drug resistant cell lines to anticancer agents were detected by MTT assay. Flow cytometry assay was used to evaluate P-gp expression and Rhodamine 123 accumulation assay of both drug resistant cell lines and their parental cell lines. Multidrug resistant genes in cells were investigated with RT-PCR and the characteristics were determined by light microscopy, electron microscopy, and flow cytometry. P-gp was detected by western blot.Results1. Under phase contrast microscope, chordoma cells showed epithelial arrangement, simple layer strapping growth, and overlapping phenomenon occurred. Cells were CK, EMA, S-100 and Vimentin positive, rich in glycogen, hypo-triploid karyotype. Invasion ability in vitro was lower than osteosarcoma, and the cell line had a 100% ability of translated tumor formation.2. The positive expression of HIF-1αand MRP1 was detected in CM-319 in normal oxygen conditions, while MDR1 was not detected by RT-PCR(P<0.01) and western blot. The positive expression of HIF-1αprotein was detected in cytoplasm and nucleus, and MRP1 mostly in cytoplasm with indirect immunofluorescence staining and immunocytochemical technique.3. The positive expression rate of MDR1 in 50 patients who suffered from chordoma was 10%, while 13.3% in osteochondroma and 20% in nucleus pulposus. There is no significant difference between chordoma and nucleus pulposus or osteochondroma (P>0.05).The positive expression rate of HIF-1αand MRP1 in 50 patients was 80% and 74%, respectively, and significantly higher than that in the patients with osteochondroma (20% and 26.7 %, P<0.01) and in nucleus pulposus (20% and 26.7 %, P<0.01). There was positive correlation between the expression of HIF-1αand MRP1 (r=0.8, P < 0.01). In 50 patients, a negative correlation trend was found between the expression of HIF-1α, MRP1 and tumor size, ages of patients as well as recurrence rate.4. Paclitaxel had much higher inhibition rate in chordoma than other chemical drugs. High concentration of paclitaxel combined with carboplatin showed synergism to the inhibition of CM-319. Cell apoptosis phenomenon was observed under electron microscope. Block of cell cycle which inhibited cell growth was detected in S period and the G2/M period through FCM, inhibiting cell growth. Paclitaxel showed higher chemotherapy sensitivity under the condition of low-dose X-rays, and also X-ray radiation showed radiation sensitivity under the low concentration of paclitaxel.5. Three human osteosarcoma multidrug resistant cell lines MG-63/PTX, SOSP-9607/PTX and OS-9901/PTX were successfully established during a 12-month period with stable resistance to paclitaxel (1μg /mL). The resistance index was respectively 69.8, 24.4 and 26.88 to paclitaxel and drug resistant cells also exhibited cross-resistance to many other chemotherapeutic agents (Carboplatin, Cisplatin, Epirubicin, Methotrexate and Adriamycin, etc). FCM showed the concerntration of Rhodamine 123 in drug resistant cell lines was far lower than parental cell lines. MDR1, MRP1, LRP and ABCG2 genes were expressed in drug resistant cell lines, while in parental cell lines MDR1 was not expressed. P-gp expression was found in drug resistant cell lines with immunohistochemical method, FCM and western blot method.Conclusions1. CM-319,as a stable chordoma cell line, has the characteristics of epithelium and mesenchymal tissue, which provides a basis for the mechanism of chemoradiation and drug resistance. The overexpression of HIF-1α, MRP1 plays an important role in the resistance to chemotherapy and radiotherapy of chordoma cell line. And the expression of MDR1 was not detected. The expression of HIF-1αis positively correlated with the expression of MRP1. Overexpression of MRP1 and HIF-1αmay play a vital role in resistance to chemotherapy and radiotherapy and better predicting the prognosis in patients suffered from chordoma.2. CM-319 was not sensitive to many anticancer drugs, but paclitaxel was an exception. Paclitaxel and radiation together could sensitize the chemoradiation effect.3. MDR1/P-gp may play a crucial role in the characteristic of multidrug resistance. Human osteosarcoma multidrug resistant cell lines can be used for the study of drug resistance, reversal and MDR mechanisms.更多还原