芦可替尼对 JAK2 V617F 阳性细胞免疫检查点分子表达的影响。

Effects of Ruxolitinib on Immune Checkpoint Molecule Expression in JAK2 V617F-Positive Cells.

出版信息

Clin Lab. 2024 Oct 1;70(10). doi: 10.7754/Clin.Lab.2024.240340.

DOI:10.7754/Clin.Lab.2024.240340

Abstract

BACKGROUND

This study aimed to explore the clinical significance of ruxolitinib and its effects on the proliferation and apoptosis of human erythroleukemia (HEL) cells and the expression of immune checkpoint molecules programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), and regulatory T cells (Tregs) in HEL cells and JAK2 V617F-positive patients with myeloproliferative neoplasms (MPNs).

METHODS

JAK2 V617F-positive patients with MPNs admitted to the Baoding No. 1 Hospital from January 2016 to September 2023 were recruited, including 30 patients for the newly diagnosed group and 10 for the treatment group. Additionally, 15 healthy volunteers were selected as the control group. JAK2 V617F mutation was detected by using fluorescence quantitative PCR, and the expression levels of phosphorylated JAK2 (p-JAK2), PD-1, and PD-L1 in fresh bone marrow were examined by immunohistochemistry. HEL cells were treated with ruxolitinib at different concentrations (0, 50, 100, 250, 500, and 1,000 nmol/L). Cell viability was detected by CCK-8 assay. The mRNA expression levels of JAK2, PD-1, and PD-L1 were determined by using fluorescence quantitative PCR. The protein expression of p-JAK2 was detected by Western blot and those of PD-1 and PD-L1 were evaluated by flow cytometry. The expression of PD-1, PD-L1, and Tregs after the 48-hour co-culture of primary bone marrow cells and HEL cells were also analyzed by flow cytometry.

RESULTS

In the newly diagnosed group, the bone marrow myeloid cells highly expressed p-JAK2, PD-1, and PD-L1. The Tregs expression in their peripheral blood increased and was significantly higher than those in the treatment and control groups (all p < 0.05). Ruxolitinib at different concentrations could inhibit the proliferation of HEL cells and was positively correlated with treatment time and dose. Additionally, ruxolitinib could reduce p-JAK2, PD-1, and PD-L1 expression in HEL cells and Tregs expression.

CONCLUSIONS

Ruxolitinib reduces the expression of p-JAK2, PD-1, and PD-L1 in JAK2 V617F-positive cells by specifically inhibiting the JAK2 signaling pathway, thereby suppressing the progression of MPNs.

摘要

背景

本研究旨在探讨鲁索替尼的临床意义及其对人红白血病（HEL）细胞增殖和凋亡以及免疫检查点分子程序性死亡受体-1（PD-1）、程序性死亡配体 1（PD-L1）和调节性 T 细胞（Tregs）在 HEL 细胞和 JAK2 V617F 阳性骨髓增殖性肿瘤（MPN）患者中的表达的影响。

方法

选取 2016 年 1 月至 2023 年 9 月保定第一医院收治的 JAK2 V617F 阳性 MPN 患者 30 例作为新诊断组，10 例作为治疗组，另选取 15 例健康志愿者作为对照组。采用荧光定量 PCR 检测 JAK2 V617F 突变，免疫组化法检测新鲜骨髓中磷酸化 JAK2（p-JAK2）、PD-1 和 PD-L1 的表达水平。用不同浓度（0、50、100、250、500 和 1000 nmol/L）的鲁索替尼处理 HEL 细胞，用 CCK-8 法检测细胞活力，用荧光定量 PCR 法检测 JAK2、PD-1 和 PD-L1 的 mRNA 表达水平，用 Western blot 法检测 p-JAK2 蛋白表达，用流式细胞术检测 PD-1 和 PD-L1 蛋白表达。用流式细胞术分析原代骨髓细胞与 HEL 细胞共培养 48 小时后 PD-1、PD-L1 和 Tregs 的表达。

结果

在新诊断组中，骨髓髓系细胞高度表达 p-JAK2、PD-1 和 PD-L1。其外周血 Tregs 表达增加，明显高于治疗组和对照组（均 P<0.05）。不同浓度的鲁索替尼可抑制 HEL 细胞的增殖，且与治疗时间和剂量呈正相关。此外，鲁索替尼可降低 HEL 细胞中 p-JAK2、PD-1 和 PD-L1 的表达以及 Tregs 的表达。