柚皮素对人自然杀伤细胞杀伤肝癌细胞的影响及其机制

马利节; 于畅; 王芳; 唐亦非; 邬海龙; 孙学华; 高月求

doi:10.3969/j.issn.1001-5256.2022.08.019

柚皮素对人自然杀伤细胞杀伤肝癌细胞的影响及其机制

DOI: 10.3969/j.issn.1001-5256.2022.08.019

马利节^{1, 2a, 2b},
于畅^2b,
王芳^2b,
唐亦非^2a,
邬海龙³,
孙学华^2a,
高月求^2a, , ,

1.
上海中医药大学，上海 201203
2a.
上海中医药大学附属曙光医院肝病科，上海 201203
2b.
上海中医药大学附属曙光医院中心实验室，上海 201203
3.
上海健康医学院，上海 201203

基金项目:

国家自然科学基金面上项目 (81874436);

国家“艾滋病和病毒性肝炎等重大传染病防治”科技重大专项 (2018ZX10725504);

国家自然科学基金青年项目 (81904117)

伦理学声明：本研究于2018年11月20日通过上海中医药大学附属曙光医院伦理委员会审核批准，批号：2018-628-57-01。

利益冲突声明：本研究不存在研究者、伦理委员会成员以及与公开研究成果有关的利益冲突。

作者贡献声明：马利节、于畅负责课题设计，资料分析，撰写论文；王芳、邬海龙参与收集数据，修改论文；唐亦非参与修稿事宜；孙学华、高月求负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
高月求，gaoyueqiu@hotmail.com

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出版历程
- 收稿日期: 2021-11-09
- 录用日期: 2022-02-28
- 出版日期: 2022-08-20

Effect of naringenin on natural killer cell cytotoxicity against hepatocellular carcinoma cells in vitro and its mechanism

Lijie MA^{1, 2a, 2b},
Chang YU^2b,
Fang WANG^2b,
Yifei TANG^2a,
Hailong WU³,
Xuehua SUN^2a,
Yueqiu GAO^{2a
, ,
,}

1.
Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
2a.
Department of Hepatology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
2b.
Central Laboratory, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
3.
Shanghai University of Medicine & Health Sciences, Shanghai 201203, China

Research funding:

General Project of National Natural Science Foundation of China (81874436);

The State "AIDS and Viral Hepatitis and Other Major Infectious Diseases Prevention and treatment" Science and Technology Major Project (2018ZX10725504);

Youth Project of National Natural Science Foundation of China (81904117)

More Information

Corresponding author: GAO Yueqiu, gaoyueqiu@hotmail.com(ORCID: 0000-0001-7276-9810)

摘要

摘要: 目的将人外周血单个核细胞体外扩增为自然杀伤(NK)细胞，与肝癌细胞1∶ 1共培养。观察柚皮素对NK细胞杀伤率的影响并初步研究相关机制。方法淋巴细胞分离液分离人外周血单个核细胞，在体外经重组人白细胞介素2诱导培养NK细胞；0、3.125、6.25、12.5、25、50 μmol/L浓度柚皮素作用于人肝癌细胞0、24、48 h后CCK8检测肝癌细胞增殖情况；不同浓度柚皮素作用于人NK细胞24 h后，CCK8检测NK细胞增殖情况；柚皮素作用于NK-肝癌细胞1∶ 1共培养体系24 h后，CellTiter-LumiTM检测NK细胞杀伤率；实时荧光定量PCR法检测NK细胞中激活型受体NKG2D的基因表达，以及肝癌细胞中NKG2D配体的基因表达。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。结果经重组人白细胞介素2诱导培养后，NK细胞可扩增至人外周血单个核细胞的82.33%±0.70%；柚皮素作用24 h后，所有质量浓度组CLC5肝癌细胞的增殖率无明显差异(P值均＞0.05)，25及50 μmol/L质量浓度组柚皮素对NK细胞的增殖有较明显的促进作用(P值均＜0.000 1)；柚皮素作用NK-肝癌细胞1∶ 1共培养体系24 h后，25、50 μmol/L浓度组对NK细胞杀伤率均有明显提高(P值均＜0.000 1)；柚皮素作用于NK-肝癌细胞1∶ 1共培养体系24 h后，25、50 μmol/L浓度组柚皮素对NK细胞中NKG2D的表达均无影响，对肝癌细胞中MICB、ULBP2的表达亦无影响(P值均＞0.05)，对肝癌细胞中ULBP1、ULBP3等NKG2D配体的表达有明显上调作用(P值均＜0.001)。结论柚皮素可能通过上调肝癌细胞中NKG2D配体表达，从而增加NK细胞的杀伤活性。
- 癌, 肝细胞 /
- 柚皮素 /
- 自然杀伤T细胞 /
- 免疫疗法
Abstract: Objective To investigate the effect of naringenin on the killing rate of natural killer (NK) cells and related mechanism by amplification of human peripheral blood mononuclear cells into NK cells in vitro and co-culture with hepatocellular carcinoma (HCC) CLC5 cells at a ratio of 1∶ 1. Methods A lymphocyte separation medium was used to isolate human peripheral blood mononuclear cells, which were induced with recombinant human interleukin-2 in vitro to culture NK cells. CCK-8 assay was used to measure the proliferation of HCC cells after human HCC cells were treated with naringenin (0, 3.125, 6.25, 12.5, 25, and 50 μmol/L) for 0, 24, and 48 hours, and after human NK cells were treated with different concentrations of naringenin for 24 hours, CCK-8 assay was used to measure the proliferation of NK cells. CellTiter-LumiTM was used to measure the killing rate of NK cells after the NK-HCC cell co-culture system at the ratio of 1∶ 1 was treated with naringenin for 24 hours. Quantitative real-time PCR was used to measure the gene expression of the activating receptor NKG2D in NK cells and NKG2D ligands in HCC cells. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results After being induced and cultured by recombinant human interleukin-2, NK cells were amplified to 82.33%±0.70% of human peripheral blood mononuclear cells. After naringenin treatment for 24 hours, there was no significant difference in the proliferation rate of HCC CLC5 cells between all mass concentration groups (all P > 0.05), and in the 25 and 50 μmol/L mass concentration groups, naringenin significantly promoted the proliferation of NK cells (both P < 0.000 1). After the NK-HCC cell co-culture system at the ratio of 1∶ 1 was treated with naringenin for 24 hours, there was a significant increase in the killing rate of NK cells in the 25 and 50 μmol/L mass concentration groups (both P < 0.000 1). After the co-culture system was treated with naringenin for 24 hours, naringenin had no effect on the expression of NKG2D in NK cells in the 25 and 50 μmol/L mass concentration groups, and it also had no effect on the expression of MICB and ULBP2 in HCC cells (all P > 0.05); it significantly upregulated the expression of the NKG2D ligands such as ULBP1 and ULBP3 in HCC cells (all P < 0.001). Conclusion Naringenin may increase the killing activity of NK cells by upregulating the expression of NKG2D ligands in HCC cells.
- Carcinoma, Hepatocellular /
- Naringin /
- Natural Killer T-Cells /
- Immunotherapy

HTML全文

图 1 NK细胞培养及鉴定

注：a，NK细胞流式鉴定；b，显微镜下NK细胞形态。

Figure 1. NK cell culture and identification

下载: 全尺寸图片幻灯片

图 2 不同质量浓度柚皮素对肝癌细胞增殖率的影响

注：与0 μmol/L浓度组相比，P＜0.000 1。

Figure 2. Effects of different naringin concentrations on viability of hepatocellular carcinoma cells

下载: 全尺寸图片幻灯片

图 3 不同质量浓度柚皮素对NK细胞增殖率的影响

注：与0 μmol/L浓度组相比，P＜0.000 1。

Figure 3. Effects of different naringin concentrations on viability of NK cells

下载: 全尺寸图片幻灯片

图 4 不同质量浓度柚皮素对NK细胞杀伤率的影响

注：a, CellTiter-LumiTM检测原理；b, 柚皮素对NK细胞杀伤率的影响。与0 μmol/L浓度组相比，P＜0.000 1。

Figure 4. Effects of different naringin concentrations on lysis of NK cells

下载: 全尺寸图片幻灯片

图 5 柚皮素对NKG2D受体及配体的影响

注：a, 柚皮素对NKG2D受体的影响；b, 柚皮素对NKG2DL的影响。与对照组相比，P＜0.001，P＜0.000 1。

Figure 5. Effects of naringin on NKG2D receptor and ligand

下载: 全尺寸图片幻灯片

表 1 实时荧光定量PCR引物列表

Table 1. List of primers for fluorescence quantitative PCR

基因	正向(5′-3′)	反向(5′-3′)
β-actin	TGA CGA GGC CCA GAG CAA GA	ATG GGC ACA GTG TGG GTG AC
NKG2D	CCA CAG CAT CAT CAC ATG AAT T	CCA CCA CTA GTT TGT TCA ACA C
MICB	TCT TCG TTA CAA CCT CAT GGT G	TCC CAG GTC TTA GCT CCC AG
ULBP1	TAA GTC CAG ACC TGA ACC ACA	TCC ACC ACG TCT CTT AGT GTT
ULBP2	AGC AAC TGC GTG ACA TTC AG	GCC ATC CTA TAC AGT CTC CCA
ULBP3	TCT ATG GGT CAC CTA GAA GAG C	TCC ACT GGG TGT GAA ATC CTC

下载: 导出CSV

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