【摘要】：目的:優(yōu)化NK細(xì)胞培養(yǎng)體系,提高NK細(xì)胞在體外培養(yǎng)的效率,檢測(cè)不同培養(yǎng)體系NK細(xì)胞對(duì)乳腺癌細(xì)胞T47D體外殺傷效果。本文為NK細(xì)胞在腫瘤過(guò)繼細(xì)胞免疫治療的臨床應(yīng)用提供了理論與實(shí)踐依據(jù)。方法:采集健康人靜脈全血,分離出外周血單個(gè)核細(xì)胞,通過(guò)添加不同的細(xì)胞因子與添加ErbB2抗體的培養(yǎng)體系進(jìn)行NK細(xì)胞培養(yǎng),實(shí)驗(yàn)分為NK1組(培養(yǎng)基中含IL-2)、NK2組(培養(yǎng)基中含IL-2,IL-15和IL-21)、和NK3組(ErbB2抗體包被后培養(yǎng)基中含IL-2)三組。分別將培養(yǎng)第0天、7天和第14天的NK細(xì)胞進(jìn)行流式分析檢測(cè)CD3、CD4、CD8和CD56分子表型,檢測(cè)不同培養(yǎng)時(shí)期NK細(xì)胞的比率。將培養(yǎng)14天后的三組培養(yǎng)體系的NK細(xì)胞分別以效靶比5:1、10:1、20:1分別和T47D細(xì)胞共同培養(yǎng),用CCK-8法進(jìn)行體外腫瘤細(xì)胞殺傷實(shí)驗(yàn)。結(jié)果:第7天時(shí)流式結(jié)果顯示:NK3組與NK1及NK2組細(xì)胞相比,NK細(xì)胞比率開(kāi)始有顯著升高(P0.05),NK1與NK2組相比無(wú)顯著性差異(P0.05);第14天時(shí)三組細(xì)胞相比,NK3組中NK細(xì)胞比率與NK1及NK2相比升高,有顯著性差異(P0.05)。經(jīng)離心收集NK細(xì)胞并調(diào)整細(xì)胞濃度,分別以效靶比5:1、10:1、20:1與T47D共培養(yǎng)24小時(shí)后,以CCK-8法檢測(cè)細(xì)胞存活率,NK3組NK細(xì)胞對(duì)T47D的細(xì)胞的殺傷活性最高,與NK1及NK2組相比均有顯著性差異(P0.05)。結(jié)論:經(jīng)不同的方案誘導(dǎo)培養(yǎng)NK細(xì)胞,結(jié)果顯示添加了ErbB2抗體的培養(yǎng)組,NK細(xì)胞的比率顯著高于其它培養(yǎng)體系培養(yǎng)的細(xì)胞;并且添加了ErbB2抗體誘導(dǎo)培養(yǎng)體系培養(yǎng)出的NK細(xì)胞對(duì)乳腺癌細(xì)胞T47D的體外殺傷效果顯著優(yōu)于其它各組。
[Abstract]:Aim: to optimize the NK cell culture system, improve the efficiency of NK cell culture in vitro, and detect the killing effect of NK cells on breast cancer cell line T47D in vitro. This study provides a theoretical and practical basis for the clinical application of NK cells in tumor adoptive cellular immunotherapy. Methods: peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy people. NK cells were cultured by adding different cytokines and ErbB2 antibody. The experiment was divided into NK1 group (containing IL-2 in culture medium). NK2 group (containing IL-2,IL-15 and IL-21 in culture medium) and NK3 group (containing IL-2 in ErbB2 antibody coated medium). NK cells were cultured on day 0, day 7 and day 14 respectively. Flow cytometry was used to detect the phenotypes of CD3,CD4,CD8 and CD56, and the ratio of NK cells at different culture stages was detected. After 14 days of culture, NK cells were co-cultured with T47D cells with the effect-target ratio of 5, 10 and 20, respectively. The killing effect of T47D cells in vitro was tested by CCK- 8 method. Results: the results of flow cytometry on the 7th day showed that the ratio of NK cells in NK3 group was significantly higher than that in NK1 and NK2 groups (P0.05), but there was no significant difference between NK1 group and NK2 group (P0.05). On the 14th day, the ratio of NK cells in NK3 group was significantly higher than that in NK1 and NK2 groups (P0.05). After centrifugation, NK cells were collected and the cell concentration was adjusted. After 24 hours of co-culture with T47D with the effect-target ratio of 5, 10, 20, respectively, the survival rate of T47D cells was measured by CCK-8 assay. The cytotoxicity of NK cells to T47D cells in NK3 group was the highest. Compared with NK1 and NK2 group, there was significant difference (P0.05). Conclusion: NK cells were induced by different methods. The results showed that the ratio of NK cells in the culture group added with ErbB2 antibody was significantly higher than that in other culture systems. In addition, the killing effect of NK cells induced by ErbB2 antibody on breast cancer cell line T47D in vitro was significantly better than that of other groups.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q813.11;R730.51

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