本文關(guān)鍵詞： 乳腺癌 放射治療 循環(huán)腫瘤細(xì)胞 侵襲轉(zhuǎn)移 基質(zhì)金屬蛋白酶-2　出處：《上海交通大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：背景和目的放射治療可降低乳腺癌患者保乳和乳房切除后的局部復(fù)發(fā)風(fēng)險,提高生存率,在早期和局部進(jìn)展期乳腺癌治療中發(fā)揮重要作用。然而,近年來研究發(fā)現(xiàn)放射治療還可能通過改變腫瘤微環(huán)境,增加腫瘤細(xì)胞的侵襲轉(zhuǎn)移潛能。這里我們探討放射后乳腺癌細(xì)胞侵襲及轉(zhuǎn)移潛能的變化及其機制。材料和方法1、在體外以不同劑量(0、4、8Gy)X線照射人乳腺癌MDA-MB-435細(xì)胞。明膠酶譜法檢測放射后24h、48h細(xì)胞培養(yǎng)上清液中基質(zhì)金屬蛋白酶MMP-2活性,Western bloting檢測放射后24h、48h細(xì)胞內(nèi)MMP-2蛋白表達(dá)水平,Transwell法測定放射后24h、48h細(xì)胞的侵襲能力,評價放射治療對乳腺癌侵襲潛能的影響及其機制。2、構(gòu)建同時表達(dá)增強型綠色熒光蛋白(Enhanced Green Fluorecence Protein,e GFP)和熒光素酶(Luciferase,Luc)的慢病毒載體,將GFP-Luciferase融合蛋白載體轉(zhuǎn)染到人乳腺癌MDA-MB-435細(xì)胞株。將穩(wěn)定表達(dá)GPF和1uciferase的MDA-MB-435細(xì)胞(MDA-MB-435-e GFP-Luc)接種裸鼠,建立原位乳腺癌轉(zhuǎn)移動物模型并在接種后第二周分組,放射治療組采用8Gy單次劑量照射原位腫瘤,采用在體流式細(xì)胞儀(In Vivo Flow Cytometry,IVFC)每周檢測裸鼠耳根部動脈循環(huán)腫瘤細(xì)胞(Circulating tumour cells,CTCs),同步采用小動物活體成像系統(tǒng)(In Vivo Image System,IVIS)監(jiān)測腫瘤生長及全身轉(zhuǎn)移情況。接種后第6周處死兩組裸鼠,取出肺組織進(jìn)行活組織成像及病理組織切片HE染色,比較兩組裸鼠肺組織轉(zhuǎn)移灶數(shù)目,并采用免疫組織化學(xué)染色檢測原發(fā)腫瘤組織內(nèi)MMP-2表達(dá),評價放射治療對乳腺癌轉(zhuǎn)移潛能的影響及其機制。結(jié)果1、放射后乳腺癌MDA-MB-435細(xì)胞侵襲能力、MMP-2表達(dá)及活性顯著提高并均呈現(xiàn)時間及劑量依賴性,單次8Gy照射48h達(dá)到最高。2、成功建立了穩(wěn)定表達(dá)GFP/Luciferase的乳腺癌細(xì)胞株:綠色熒光表達(dá)強,GFP轉(zhuǎn)染效率達(dá)到99.58%,體外發(fā)光能力與細(xì)胞數(shù)目呈正相關(guān)(R2=0.998)。腫瘤細(xì)胞接種后第二周IVFC探測到體內(nèi)CTCs信號峰,IVIS成像檢查檢測到肺內(nèi)微小轉(zhuǎn)移灶。腫瘤細(xì)胞的形態(tài)學(xué)特征、生長速度、侵襲及遷移特性與轉(zhuǎn)染前無明顯變化。3、照射組接受放射后腫瘤體積、CTCs數(shù)目、腫瘤部位熒光強度呈現(xiàn)先減小后增加趨勢。放療后第4周(即腫瘤接種后第6周)對照組和實驗組腫瘤體積分別為519.66±32.89mm3,497.28±45.86mm3(p0.05)無統(tǒng)計學(xué)差異,每小時CTCs數(shù)目分別為7.8±1.9,11.8±2.3(p0.05)有統(tǒng)計學(xué)差異,提示放療后第4周腫瘤細(xì)胞侵襲轉(zhuǎn)移能力獲得進(jìn)一步增強。放射治療組腫瘤組織MMP-2表達(dá)及肺轉(zhuǎn)移灶明顯多于對照組。結(jié)論1、結(jié)合IVFC和IVIS技術(shù)可以實現(xiàn)對MDA-MB-435-GFP-Luc細(xì)胞系原位乳腺癌模型的CTCs、腫瘤生長及微小轉(zhuǎn)移灶進(jìn)行早期、實時動態(tài)監(jiān)測。2、放射線照射能夠通過增強乳腺癌細(xì)胞MMP-2的表達(dá)或活性而增強其侵襲轉(zhuǎn)移潛能。
[Abstract]:Background and objective radiotherapy can reduce the risk of breast conserving and local recurrence after mastectomy in breast cancer patients, improve survival rate and play an important role in early and locally advanced breast cancer treatment. In recent years, studies have found that radiotherapy may also change the tumor's microenvironment, To investigate the changes and mechanisms of invasion and metastasis potential of breast cancer cells after radiation. Materials and methods 1. In vitro, human breast cancer MDA-MB-435 cells were irradiated with different doses of 0, 4 and 8 Gy ~ (-1) rays. The activity of matrix metalloproteinase (MMP-2) in the supernatant of cell culture at 24 h after radiation was detected by gel enzyme assay and the expression level of MMP-2 protein in the cell at 24 h or 48 h after radiation was detected by Western bloting. The invasion ability of cells was measured by Transwell method at 24 h or 48 h after irradiation. To evaluate the effect of radiotherapy on the invasive potential of breast cancer and its mechanism. 2. To construct a lentivirus vector expressing enhanced Green Fluorecence protein (GFP) and luciferase luciferase (Lucc) simultaneously. GFP-Luciferase fusion protein vector was transfected into human breast cancer MDA-MB-435 cell line. MDA-MB-435-e GFP-Lucc cells, which expressed GPF and 1uciferase stably, were inoculated into nude mice to establish the animal model of breast cancer metastasis in situ and divided into groups at the second week after inoculation. In the radiotherapy group, the tumor was irradiated with a single dose of 8 Gy. In vivo flow cytometry (in Vivo Flow Cytometry) was used to detect the circulating tumour tumour cells CTCs in the ear root artery of nude mice every week. The tumor growth and metastasis were monitored by the small animal in vivo imaging system (in Vivo Image system IVISs). The nude mice were killed 6 weeks after inoculation, and the two groups were killed at 6 weeks after inoculation. Lung tissue was taken out for biopsy and HE staining. The number of lung metastatic foci in the two groups was compared, and the expression of MMP-2 in the primary tumor tissue was detected by immunohistochemical staining. To evaluate the effect of radiotherapy on the metastatic potential of breast cancer and its mechanism. Results 1. The expression and activity of MMP-2 in MDA-MB-435 cells were significantly increased in a time and dose-dependent manner. The breast cancer cell line expressing GFP/Luciferase stably was successfully established. The transfection efficiency of GFP was 99.58, and the luminescence ability in vitro was positively correlated with the number of cells. The tumor cells were inoculated at the second week after inoculation. IVFC detects in vivo CTCs signal peaks and IVIS imaging detects tiny metastases in the lung. Morphological features of tumor cells, The growth rate, invasion and migration characteristics were not significantly different from those before transfection. The fluorescence intensity of tumor site decreased first and then increased. There was no significant difference in tumor volume between the control group and the experimental group at the 4th week after radiotherapy (that is, the 6th week after tumor inoculation), the tumor volume of the control group and the experimental group were 519.66 鹵32.89mm3n3c497.28 鹵45.86mm3p0.05, respectively, and the number of CTCs per hour was 7.8 鹵1.9nil 鹵2.3p0.05, respectively. The results suggest that the ability of invasion and metastasis of tumor cells is further enhanced at the 4th week after radiotherapy. The expression of MMP-2 and lung metastasis in the radiotherapy group is significantly higher than that in the control group. Conclusion 1. Combined with IVFC and IVIS technique, the tumor cell line of MDA-MB-435-GFP-Luc can be achieved. In situ breast cancer model, CTCs, tumor growth and micrometastasis were performed early. Real-time dynamic monitoring. 2. Radiation irradiation can enhance the invasion and metastasis potential of breast cancer cells by enhancing the expression or activity of MMP-2.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.9;R730.55
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本文編號：1507158