本文選題：樹突狀細(xì)胞 + 高強(qiáng)度聚焦超聲　； 參考：《泰山醫(yī)學(xué)院》2005年碩士論文

【摘要】：目的：建立體外大量擴(kuò)增小鼠樹突狀細(xì)胞(dendritic cell, DC)的方法；探討用高強(qiáng)度聚焦超聲(high intensity focused ultrasound, HIFU)輻照腫瘤細(xì)胞制備腫瘤抗原致敏DC和制備DC腫瘤疫苗的可行性,為HIFU開辟新的應(yīng)用領(lǐng)域。 方法：①應(yīng)用1ng/ml IL-4和lOng/ml GM-CSF聯(lián)合誘導(dǎo)培養(yǎng)小鼠骨髓細(xì)胞,光鏡及電子顯微鏡觀察DC發(fā)育過(guò)程中形態(tài)學(xué)變化；流式細(xì)胞術(shù)檢測(cè)DC表面分子CD80、CD86、H-2Kd及I-Ad的表達(dá)情況；②固定輻照時(shí)間,應(yīng)用不同功率的HIFU輻照CT26細(xì)胞株后,用臺(tái)盼蘭染色、MTT法測(cè)定活腫瘤細(xì)胞數(shù),同時(shí)用臺(tái)盼蘭染色觀察腫瘤細(xì)胞的形態(tài)學(xué)變化,分析HIFU劑量與腫瘤細(xì)胞存活率的關(guān)系,了解劑量-效應(yīng)關(guān)系；③固定輻照時(shí)間和超聲強(qiáng)度,輻照CT26細(xì)胞懸液,制備腫瘤細(xì)胞抗原并致敏DC,致敏DC與T細(xì)胞功孵育48小時(shí)后提取上清,通過(guò)ELISA法檢測(cè)上清中IL-12、IFN-γ的含量,并與凍融組、單純CT26、對(duì)照組進(jìn)行比較。 結(jié)果：①用IL-4和GM-CSF聯(lián)合培養(yǎng)小鼠骨髓細(xì)胞3天后,可見細(xì)胞形態(tài)發(fā)生改變,細(xì)胞形態(tài)不規(guī)則。培養(yǎng)第6～8天,細(xì)胞表面出現(xiàn)較多毛刺樣突起,拉長(zhǎng),為典型的DC特征。流式細(xì)胞儀檢測(cè)DC表面分子有CD80、CD86、H-2Kd及I-Ad表達(dá)；②隨著HIFU輻照劑量的增加腫瘤細(xì)胞的存活率迅速減少,腫瘤細(xì)胞的碎片逐漸增多。超聲劑量為1000W/cm2×30秒時(shí),無(wú)細(xì)胞存活,腫瘤細(xì)胞完全失去細(xì)胞形態(tài),全部被撕裂成碎片;③通過(guò)ELISA法檢測(cè)HIFU、凍融、單純CT26三組上清中IL-12、IFN-γ的含量顯著高于對(duì)照組(p均0.05),HIFU、凍融兩組上清中IL-12、IFN-γ的含量顯著高于單純CT26組(p均0.05),但H1FU、凍融組之間比較在統(tǒng)計(jì)學(xué)上無(wú)顯著性意義(p0.05)。 結(jié)論：應(yīng)用IL-4聯(lián)合GM-CSF培養(yǎng)小鼠骨髓細(xì)胞,可大量擴(kuò)增成熟DC,培養(yǎng)的DC符合其自身的特性;HIFU能滅活腫瘤細(xì)胞且能破碎腫瘤細(xì)胞,HIFU制備的腫瘤抗原可體外致敏DC,并可使DC成熟分泌大量IL-12,同時(shí)能誘導(dǎo)T細(xì)胞分泌大量IFN-γ。
[Abstract]:Objective: to establish a method to amplify dendritic cells (DCs) in vitro, and to investigate the feasibility of preparing tumor antigen-sensitized DC and DC tumor vaccine by high intensity focused ultrasound (HIFU) irradiation. To open up a new application field for HIFU. Methods 1ng/ml IL-4 and lOng / ml GM-CSF were used to induce and culture mouse bone marrow cells. The morphological changes of DC were observed by light microscope and electron microscope, and the expression of CD80 CD86 + H-2Kd and I-Ad on DC surface was detected by flow cytometry. CT26 cells were irradiated with HIFU of different power. The number of live tumor cells was determined by Trypan blue colorimetric assay. The morphological changes of tumor cells were observed by trypan blue staining, and the relationship between HIFU dose and survival rate of tumor cells was analyzed. To understand the dose-effect relationship between irradiation time and ultrasound intensity, CT26 cell suspension was irradiated to prepare tumor cell antigen and sensitize DC. the supernatant was extracted after incubation with T cells for 48 hours. The content of IL-12 IFN- 緯 in the supernatant was detected by Elisa. And compared with freeze-thaw group, CT26 and control group. Results the bone marrow cells of mice were cultured with IL-4 and GM-CSF for 3 days. On the 6th day of culture, there were many burrs on the surface of the cells, which were typical DC characteristics. Flow cytometry was used to detect CD80, CD86, H-2Kd and I-Ad expression on DC surface. With the increase of HIFU irradiation dose, the survival rate of tumor cells decreased rapidly, and the fragments of tumor cells increased gradually. When the ultrasound dose was 1000 W / cm 2 脳 30 seconds, no cells survived, the tumor cells completely lost the shape of the cells, all of them were torn into fragments, HIFU3 was detected by Elisa, freeze-thaw, freezing and thawing. The level of IL-12 IFN- 緯 in supernatant of CT26 group was significantly higher than that in control group (P < 0.05), and the content of IL-12 and IFN- 緯 in supernatant of freeze-thaw group was significantly higher than that in CT26 group (P < 0.05). Conclusion: bone marrow cells of mice were cultured with IL-4 combined with GM-CSF. The cultured DC could inactivate tumor cells, and the tumor antigens prepared by HIFU could sensitize DCin vitro, and make DC mature and secrete a large amount of IL-12, and induce T cells to secrete a large amount of IFN- 緯.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 葉欣,劉愛華,宋文剛,楊憲勇,李毅;人臍血清代替胎牛血清體外培養(yǎng)臍血樹突狀細(xì)胞[J];中國(guó)輸血雜志;2004年05期

2 趙洪,袁永熙,陸昌宜,王怡,蔡端,孫福成,蔣繼偉;高強(qiáng)度聚焦超聲治療乳腺癌后機(jī)體免疫功能的變化[J];中國(guó)超聲診斷雜志;2003年06期

3 王文見;超聲治療腫瘤與免疫[J];國(guó)外醫(yī)學(xué)(腫瘤學(xué)分冊(cè));1998年06期

4 張奕,劉炳亞;以樹突狀細(xì)胞為基礎(chǔ)的腫瘤疫苗研究進(jìn)展[J];國(guó)外醫(yī)學(xué)(腫瘤學(xué)分冊(cè));2000年01期

5 葉欣,費(fèi)興波;高強(qiáng)度聚焦超聲治療腫瘤[J];國(guó)外醫(yī)學(xué)(腫瘤學(xué)分冊(cè));2004年01期

6 高雅娟;熱休克蛋白與腫瘤免疫[J];國(guó)外醫(yī)學(xué)(免疫學(xué)分冊(cè));2000年05期

7 馬志方;樹突狀細(xì)胞治療泌尿系統(tǒng)腫瘤的進(jìn)展[J];國(guó)外醫(yī)學(xué).泌尿系統(tǒng)分冊(cè);2001年05期

8 高尚梅,賴春冬,周顰,王芷龍,白晉,鄒建中,王志剛,王智彪;高強(qiáng)度聚焦超聲致培養(yǎng)腫瘤細(xì)胞死亡的閾值量研究[J];臨床超聲醫(yī)學(xué)雜志;1999年01期

9 周顰,王懷碧,付敏,賴春冬,伍烽,白晉,鄒建中,王智彪;高強(qiáng)度聚焦超聲對(duì)離體腫瘤細(xì)胞的急性生物學(xué)效應(yīng)[J];臨床超聲醫(yī)學(xué)雜志;1999年03期

10 張唯力,喬天愚,劉川,涂剛,王智彪,伍烽,陳文直;高強(qiáng)度聚焦超聲對(duì)膀胱癌細(xì)胞及兔移植性腎腫瘤的實(shí)驗(yàn)研究[J];臨床超聲醫(yī)學(xué)雜志;2000年04期

，

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