本文選題：NK細(xì)胞 + CIK細(xì)胞�。� 參考：《貴州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:研究體外擴(kuò)增的自然殺傷細(xì)胞(NK)及CIK細(xì)胞(CIK)在聯(lián)合西妥昔單抗(cetuximab)后對人EGFR陽性的結(jié)直腸癌細(xì)胞株DLD-1及Caco-2的殺傷效應(yīng),以及呼腸孤病毒(reovirus)能否進(jìn)一步增強(qiáng)CIK及NK細(xì)胞與cetuximab的聯(lián)合抗腫瘤效應(yīng),為臨床結(jié)直腸癌的治療提供新的治療策略。方法:從健康獻(xiàn)血者的外周血分離出單個核細(xì)胞(PBMC),分別常規(guī)誘導(dǎo)出CIK細(xì)胞及NK細(xì)胞,收集培養(yǎng)第14天的CIK細(xì)胞及NK細(xì)胞,ELISA實驗檢測CIK細(xì)胞培養(yǎng)上清中IFN-γ及TGF-β的水平,通過流式細(xì)胞分析儀分析CIK細(xì)胞及NK表型及結(jié)直腸癌細(xì)胞DLD-1和Caco-2的EGFR受體的表達(dá)水平。分別采用RTCA(RTCA)及乳酸脫氫酶(LDH)釋放實驗檢測NK細(xì)胞的細(xì)胞毒作用。利用TCID50法和甲基纖維素改良的噬斑實驗測定reovirus病毒的滴度,使用不同滴度的reovirus病毒感染CIK細(xì)胞及NK細(xì)胞。采用實時無標(biāo)記分析儀分別動態(tài)監(jiān)測CIK及NK細(xì)胞在不同滴度reovirus感染后聯(lián)合cetuximab對DLD-1及Caco-2細(xì)胞株的殺傷效應(yīng)。結(jié)果:流式細(xì)胞儀檢測CIK細(xì)胞亞群可見培養(yǎng)后CD8+T(67.5±16.5%)、CD3+CD56+NKT(20.1±13.7%)及CD16+CD56+NK細(xì)胞比例(7.0±6.6%)均較培養(yǎng)前CD8+T(26.7±9.5%)、CD3+CD56+NKT(3.1±1.8%)及CD16+CD56+NK(2.6±2.7%)顯著增高;CIK細(xì)胞培養(yǎng)培養(yǎng)上清中細(xì)胞因子濃度IFN-γ水平由34.8±35.4 ng/L增高到2041.8±697.9ng/L(P0.01),而同時TGF-β的水平由1293.8±633.9ng/L降為116.7±26.8ng/L(P0.01);經(jīng)過14天體外擴(kuò)增培養(yǎng)的NK細(xì)胞純度可達(dá)86.2%;DLD-1及Caco-2腫瘤細(xì)胞表面均高表達(dá)EGFR,分別為81.6%及82.9%;LDH釋放實驗結(jié)果與RTCA檢測所顯示的殺傷趨勢基本一致;TCID50法及噬斑實驗求得病毒滴度分別為2.53×109pfu/L及1.36×109 pfu/L。RTCA檢測發(fā)現(xiàn)NK細(xì)胞聯(lián)合西妥昔單抗對結(jié)直腸癌細(xì)胞株殺傷作用明顯高于單一的NK細(xì)胞組;CIK細(xì)胞對DLD-1及Caco-2細(xì)胞株的殺傷率分別為16.04±0.87%及27.67±3.10%,而當(dāng)聯(lián)合使用cetuximab后殺傷率分別增高到31.56±3.55%及35.96±1.73%,且用10pfu和100pfu滴度reovirus病毒感染CIK細(xì)胞及NK細(xì)胞后聯(lián)合cetuximab對腫瘤細(xì)胞殺傷效應(yīng)更明顯。結(jié)論:CIK細(xì)胞及NK細(xì)胞聯(lián)合cetuximab的殺傷效果明顯增強(qiáng),對KRAS野生型DLD-1細(xì)胞及KRAS突變型Caco-2細(xì)胞均有效,reovirus能進(jìn)一步增強(qiáng)CIK細(xì)胞和NK細(xì)胞與cetuximab的聯(lián)合抗腫瘤效應(yīng),為今后的臨床研究奠定實驗基礎(chǔ)。
[Abstract]:Objective: to study the killing effect of natural killer cells (NKK) and CIK cells (CIK) on EGFR positive colorectal cancer cell lines DLD-1 and Caco-2 after combined with cetuximab. And whether reovirus can further enhance the anti-tumor effect of CIK and NK cells combined with cetuximab, and provide a new treatment strategy for clinical colorectal cancer. Methods: mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy blood donors. CIK cells and NK cells were induced by routine method. The levels of IFN- 緯 and TGF- 尾 in the supernatant of CIK cell culture were detected by Elisa of CIK cells and NK cells on the 14th day of culture. Flow cytometry was used to analyze the expression of DLD-1 and Caco-2 EGFR receptors in CIK cells and NK cells. The cytotoxicity of NK cells was detected by RTCA and LDH release assay. The titer of reovirus virus was determined by TCID50 assay and modified plaque assay of methylcellulose. CIK cells and NK cells were infected with different titers of reovirus virus. The killing effects of CIK and NK cells combined with cetuximab on DLD-1 and Caco-2 cell lines after reovirus infection with different titers were dynamically monitored by a real-time unlabeled analyzer. Results: flow cytometry analysis of CIK cell subsets showed that the levels of IFN- 緯 in the supernatant of CD8 T cell culture were significantly higher than those of CD8 T _ (26.7 鹵9.5 鹵9.5T) and CD16 CD56 NK(2.6 鹵2.7 ~ (th) after culture. The ratio of CD3 CD56 NKT(20.1 鹵13.7T and CD16 CD56 NK cells were significantly higher than that of CD8 T _ (26.7 鹵9.5 鹵1.8T) and CD16 CD56 NK(2.6 鹵2.7T (P < 0.05). 34.8 鹵35.4 ng/L increased to 2041.8 鹵697.9 ng / L P 0.01g / L, while the level of TGF- 尾 decreased from 1293.8 鹵633.9ng/L to 116.7 鹵26.8ng / L P 0.01.The purity of NK cells cultured in vitro for 14 days was 86.2ng-1 and 82.9ng / L, respectively, which were 81.6% and 82.9%, respectively. The cytotoxic effect of NK cells combined with cetuximab on colorectal cancer cell line was significantly higher than that of single NK cell group. TCID50 assay and plaque assay showed that the titer of virus was 2.53 脳 109pfu/L and 1.36 脳 10 ~ 9 pfu/L.RTCA, respectively. The cytotoxicity of NK cells combined with cetuximab to DLD-1 was higher than that of single NK cell group. The killing rate of Caco-2 cell line was 16.04 鹵0.87% and 27.67 鹵3.10%, respectively, but the killing rate was increased to 31.56 鹵3.55% and 35.96 鹵1.73% respectively after combined use of cetuximab, and the killing effect of cetuximab combined with 10pfu and 100pfu titer of reovirus virus on CIK cells and NK cells was more obvious. Conclusion the killing effect of cetuximab combined with cetuximab was significantly enhanced by cetuximab, and both KRAS wild-type DLD-1 cells and KRAS mutant Caco-2 cells could be effectively treated with reovirus. The combined anti-tumor effect of CIK cells and NK cells with cetuximab was further enhanced. [WT5HZ] [WT5 "BZ] [WT5" BZ] [WT5 "BZ] [WT5BZ] To lay the experimental foundation for the clinical research in the future.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34

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