慢性再生障碍性贫血患者中医证型与外周血microRNA、淋巴细胞亚群及细胞因子的关系研究

Study on Correlation between TCM Syndromes and Peripheral Blood MicroRNA,Lymphocyte Subsets and Cytokines in Patients with Chronic Aplastic Anemia

目的:分析慢性再生障碍性贫血(CAA)患者外周血microRNA、淋巴细胞亚群、细胞因子水平与不同肾虚证型的关系。方法:纳入60例CAA患者,辨证分为肾阴虚组30例、肾阳虚组14例、肾阴阳两虚组16例,另选择20例健康志愿者作为正常对照组。采用实时荧光定量PCR方法检测受试者外周血单个核细胞miRNA-155-5p、miRNA-1260b表达情况,采用流式细胞术检测外周血淋巴细胞亚群[包括总T淋巴细胞(T)、辅助T淋巴细胞(Th)、细胞毒T淋巴细胞(Tc)、B淋巴细胞(B)、自然杀伤细胞(NK)],采用酶联免疫吸附法检测血浆干扰素-γ(IFN-γ)、白介素-12(IL-12)p70、肿瘤坏死因子-α(TNF-α)、转化生长因子-β1(TGF-β1)水平。结果:与正常对照组比较,肾阴虚组、肾阴阳两虚组、肾阳虚组患者的miRNA-155-5p、miRNA-1260b表达及Tc比例、IFN-γ、IL-12 p70、TNF-α水平均明显升高(P<0.01,P<0.05),NK细胞比例、Th比例、Th/Tc比值及TGF-β1水平均明显降低(P<0.05,P<0.01)。与肾阳虚组比较,肾阴虚组、肾阴阳两虚组患者的miRNA-155-5p、miRNA-1260b表达及Tc比例、IFN-γ、IL-12 p70、TNF-α水平均明显升高(P<0.01,P<0.05),Th比例、Th/Tc比值及TGF-β1水平均明显降低(P<0.05,P<0.01);肾阴虚组患者的Th比例低于肾阴阳两虚组(P<0.05),血浆IFN-γ、IL-12 p70、TNF-α水平高于肾阴阳两虚组(P<0.05)。结论:肾阴虚型、肾阴阳两虚型较肾阳虚型患者存在更严重的细胞免疫功能亢进及miRNA-155-5p、miRNA-1260b表达失调,而肾阴虚型较肾阴阳两虚型患者存在更严重的造血负调控因子紊乱。

Objective： To analyze the relationship between the levels of peripheral blood microRNA, lymphocyte subsets and cytokines and different types of kidney deficiency syndrome in patients with chronic aplastic anemia （CAA）. Methods： A total of 60 patients with CAA were included, who were divided into kidney yin deficiency group （ 30 cases）, kidney yang deficiency group （14 cases） and kidney yin and yang deficiency group （16 cases） by syndrome differentiation, meanwhile another 20 cases of healthy volunteers were included as normal control group. The expressions of miRNA-155-5p and miRNA- 1260b in mononuclear cells of peripheral blood were detected by real-time quantitative PCR. The proportion of peripheral blood lymphocyte subsets including total T lymphocyte （ T）, helper T lymphocyte （ Th ）, cytotoxic T lymphocyte （ Tc ）, B lymphocyte （B） and nature killer ceil （NK） were measured by flow cytometry. The plasma levels of IFN-γ, IL-12 p70, TNF- α and TGF-β1 were determined by enzyme-linked immunosorbent assay. Results： Compared with the normal control group, the expressions of miRNA-155-5p and miRNA-1260b, the proportion of Tc and the levels of IFN-γ, IL-12p70 and TNF-α were obviously increased in the kidney yin deficiency group, kidney yang deficiency group and kidney yin and yang deficiency group （P 〈 0.01, P 〈 0.05 ）, and the proportions of NK and Th, the Th/Tc ratio and the level of TGF-131 were significantly decreased （P 〈 0.05 ,P 〈 0.01 ）. Compared with the kidney yang deficiency group, the expressions of miRNA-155-5p and miRNA-1260b, the proportion of Tc and the levels of IFN-γ, IL-12p70 and TNF-α were obviously increased in the kidney yin deficiency group and kidney yin and yang deficiency group （P 〈0.01 ,P 〈0.05）, and the proportion of Th, the Th/Tc ratio and the level of TGF-β1 were markedly decreased （ P 〈 0.05, P 〈 0.01 ）. The proportion of Tit in the kidney yin deficiency group was lower than that in the kidney yin and yang deficiency group （ P 〈 0.05 ）, and the levels of IFN-γ,, IL- 12p70 and TNF-α were higher than those in the kidney yin and yang deficiency group （ P 〈 0. 05 ）. Conclusion： Compared with the patients of kidney yang deficiency type, the patients of kidney yin deficiency type and kidney yin and yang deficiency type show more serious cellular immune hyperfunction and dysregulation of miRNA-155-5p and miRNA-1260b, and compared with the patients of kidney yiu and yang deficiency type, the patients of kidney yin deficiency type show more serious disorder of hematopoietic negative regulatory factors.