阳虚质慢性失眠患者NK细胞和T细胞的特征研究

发布时间：2019-05-23 15:49

【摘要】：目的:通过探索阳虚质慢性失眠患者的淋巴细胞亚群、NK细胞亚群及所含颗粒酶、穿孔素、NK细胞杀伤功能及分泌功能、Th1、Th2、Th17细胞亚群变化,从NK细胞和T细胞的角度分析阳虚质慢性失眠患者的免疫紊乱状态,为阳虚质慢性失眠的微观化辨证提供理论依据。方法:本研究为横断面研究,纳入心理睡眠门诊失眠病人及健康体检人群,根据失眠病史的有无分为慢性失眠组和正常对照组,并进一步将慢性失眠组分别分为阳虚质组、非阳虚质组两个亚组及阳虚质组、非阳虚质组偏颇体质组、平和质组三个亚组,收集一般资料、中医体质调查问卷、匹兹堡睡眠量表,使用流式细胞术检测淋巴细胞亚群检、NK细胞杀伤功能、NK细胞亚群及所含颗粒酶、穿孔素、Th1/Th2/Th17细胞亚群,使用Elisa法检测NK细胞分泌的INFγ、TNF a水平。对研究结果进行统计分析,比较慢性失眠组与正常对照组间,慢性失眠组两个亚组间及三个亚组间的一般资料、PSQI量表及上述检测指标的差异,并分析阳虚程度与失眠程度及上述检测指标的相关性。结果:1.淋巴细胞亚群:慢性失眠组(n=59)的淋巴细胞总数、NK细胞数、总T淋巴细胞数、抑制性/细胞毒性T细胞数、总B淋巴细胞数较正常对照组(n=30)下降。慢性失眠组内,在阳虚质组与非阳虚质组两组间比较中,阳虚质组的NK细胞数、NK细胞百分比较非阳虚质组下降,而总T淋巴细胞数、总T淋巴细胞百分比、辅助性T细胞数则较非阳虚质组上升。慢性失眠组内,在阳虚质组、非阳虚质偏颇体质组、平和质组三组间的比较中,阳虚质组的NK细胞百分比较平和质组下降。慢性失眠组中总T淋巴细胞数(r=0.273)、总T淋巴细胞百分比(r=0.37)与阳虚程度呈正相关关系,NK细胞百分比与阳虚程度呈负相关关系(r=-0.273)。2.NK细胞亚群及所含颗粒酶、穿孔素:CD56+CD16-NK细胞亚群:慢性失眠组(n=32)的CD56+CD16-NK细胞亚群百分比较正常对照组(n=28)升高。慢性失眠组内,在阳虚质组与非阳虚质组两组间的比较中,阳虚质组CD56+CD16-NK细胞亚群的颗粒酶百分比、穿孔素百分比、颗粒酶荧光强度均较非阳虚质组下降。慢性失眠组内,在阳虚质组、非阳虚质偏颇体质组、平和体质组三组间的比较中,阳虚质组的CD56+CD16-NK细胞亚群的颗粒酶百分比较平和质组下降,而阳虚质组的CD56+CD16-NK细胞亚群的颗粒酶荧光强度较非阳虚质偏颇体质组、平和质组下降。CD56+CD16+NK细胞亚群:慢性失眠组(n=32)的CD56+CD16+NK细胞亚群百分比较正常对照组(n=28)下降,该亚群所含穿孔素百分比及穿孔素荧光强度较正常对照组上升。慢性失眠组内,在阳虚质组与非阳虚质组两组间的比较中,阳虚质组的CD56+CD16+NK细胞百分比、颗粒酶百分比、穿孔素百分比、穿孔素荧光强度均较非阳虚质组下降。慢性失眠组内,在阳虚质组、非阳虚质偏颇体质组、平和体质组三组间的比较中,阳虚质组CD56+CD16+NK细胞亚群颗粒酶百分比较非阳虚质偏颇体质组、平和质组下降。阳虚质组CD56+CD16+NK细胞亚群穿孔素荧光强度较平和质组下降。CD56-CD16+NK细胞:慢性失眠组(n=32)的CD56-CD16+NK细胞的颗粒酶百分比较正常对照组(n=28)减少,而颗粒酶荧光强度、穿孔素荧光强度均较正常对照组增加。慢性失眠组内,在阳虚质组、非阳虚质组两组间的比较中,阳虚质组的CD56-CD16+NK细胞亚群的颗粒酶百分比、穿孔素百分比、穿孔素荧光强度均较非阳虚质组下降。慢性失眠组内,在阳虚质组、非阳虚质偏颇体质组、平和体质组三组间的比较中,阳虚质组的CD56-CD16+NK细胞颗粒酶百分比、穿孔素百分比均较非阳虚质偏颇体质组减少,而阳虚质组的CD56-CD16+NK细胞穿孔素荧光强度则较平和质组减少。慢性失眠组中,阳虚程度与CD56+CD16+NK细胞亚群百分比(r=-0.446)、CD56+CD16+NK细胞亚群颗粒酶百分比(r=-0.647)、CD56+CD16+NK细胞亚群穿孔素荧光量(r=-0.465)、CD56+CD16-NK细胞亚群颗粒酶荧光量(r=-0.504)呈负相关关系。3.NK细胞杀伤功能及分泌功能:慢性失眠组(n=8)的NK细胞杀伤率、NK细胞分泌的TNFα、IFNy与正常对照组(n=7)无明显差异。4.Th1、Th2、Th17 细胞亚群:慢性失眠组(n=58)的Th1/Th2比值较正常对照组(n=22)下降。在慢性失眠组内,在阳虚质组、非阳虚质组两组间的比较中,阳虚质组的Th1细胞百分比、Th2细胞百分比、Th17细胞百分比均较非阳虚质组下降。在慢性失眠组内,在阳虚质组、非阳虚质偏颇体质组、平和体质组三组间的比较中,阳虚质组Th1细胞百分比、Th2细胞百分比均较非阳虚质偏颇体质组下降,而阳虚质组Th17细胞百分比则较非阳虚质偏颇体质组、平和质组下降。慢性失眠组中,阳虚程度与Th1细胞百分比(r=-0.452)、Th2细胞百分比(r=-0.39)、Th17细胞百分比(r=-0.387)细胞亚群呈负相关关系(P0.05)。结论:慢性失眠导致的免疫紊乱主要特征为NK细胞、T细胞数目的下降及相关功能亚群的偏移,其中的阳虚质慢性失眠还具有免疫激活能力下降的特征表现,且阳虚程度与上述指标变化程度存在相关关系,提示阳虚质慢性失眠是慢性失眠中免疫功能较易受失眠影响的一种类型。
[Abstract]:Objective: To study the lymphocyte subsets, NK cell subsets and the changes of the secretory function, Th1, Th2 and Th17 subsets in the patients with chronic insomnia with yang-yang deficiency. The immune disorder status of the patients with yang-deficiency and chronic insomnia is analyzed from the angle of NK cells and T-cells, and the theoretical basis for the micro-differentiation of the chronic insomnia with yang-deficiency is provided. Methods: The study was a cross-sectional study, which was included in the patients with insomnia and the healthy physical examination of the patients with insomnia. According to the history of insomnia, the patients were divided into the chronic insomnia group and the normal control group, and the chronic insomnia group was divided into two groups of Yang-Yang, non-Yang, and Yang-Yang, respectively. The three sub-groups of the non-yang-yang mass group, the partial constitution group and the mild mass group, the general information, the physical constitution questionnaire of the traditional Chinese medicine and the Pittsburgh sleep scale were collected, and the lymphocyte subgroup detection, the NK cell killing function, the NK cell subpopulation and the particle-containing enzyme and the perforin were detected by flow cytometry. The subsets of Th1/ Th2/ Th17 cells and the level of TNF a, which were secreted by NK cells, were detected by the Elisa method. The general data, PSQI scale and the difference between the two sub-groups and the three sub-groups in the chronic insomnia group and the normal control group, the PSQI scale and the above-mentioned detection index were compared, and the correlation between the degree of yang-yang and the level of insomnia and the above-mentioned detection index was analyzed. Results:1. The lymphocyte subpopulation: the total number of lymphocytes, the number of NK cells, the total T-lymphocyte number, the inhibitory/ cytotoxic T-cell number and the total B-lymphocyte number of the chronic insomnia group (n = 59) decreased with the normal control group (n = 30). In the chronic insomnia group, the number of NK cells and the percentage of NK cells in the Yang-Yang mass group were lower than that of the non-Yang-Yang mass group, while the total number of T-lymphocytes, the percentage of total T-lymphocytes and the number of helper T-cells were higher than that of the non-Yang-Yang mass group. In the chronic insomnia group, the percentage of NK cells in the Yang-Yang mass group was lower than that in the mass group. The total number of T lymphocytes in the chronic insomnia group (r = 0.273), the total T-lymphocyte percentage (r = 0.37) was positively related to the degree of yang-yang, and the percentage of NK cells was negatively correlated with the degree of yang-yang (r =-0.273). The percentage of CD56 + CD16-NK cells in the chronic insomnia group (n = 32) was higher than that in the normal control group (n = 28). In the chronic insomnia group, the percentage of the particles of CD56 + CD16-NK cell subpopulation, the percentage of perforin and the fluorescence intensity of the granular enzyme were lower than that of the non-Yang-Yang mass group. In the chronic insomnia group, the percentage of the granular enzyme of CD56 + CD16-NK cell subpopulation of the Yang-Yang mass group was lower than that in the mass group. However, the fluorescence intensity of CD56 + CD16-NK cell subsets in the Yang-Yang mass group was lower than that of the non-yang-deficient group. The percentage of CD56 + CD16 + NK cells in the chronic insomnia group (n = 32) was lower than that in the normal control group (n = 28). In the chronic insomnia group, the percentage of CD56 + CD16 + NK cells, the percentage of granular enzyme, the percentage of perforin and the fluorescence intensity of the perforin were lower than that of the non-Yang-Yang mass group. In the chronic insomnia group, the percentage of the granular enzyme of CD56 + CD16 + NK cell subpopulation in the Yang-Yang mass group was lower than that of the non-yang-deficient group and the quality group decreased in the comparison among the three groups. The fluorescence intensity of CD56 + CD16 + NK cell subpopulation in the Yang-Yang mass group was lower than that in the mass group. CD56-CD16 + NK cells: the percentage of the particles of CD56-CD16 + NK cells in the chronic insomnia group (n = 32) was lower than that in the normal control group (n = 28), while the fluorescence intensity of the granular enzyme and the fluorescence intensity of the perforin were higher than that of the normal control group. In the chronic insomnia group, the percentage of the particles of the CD56-CD16 + NK cell subpopulation of the Yang-Yang mass group, the percentage of the perforin and the fluorescent intensity of the perforin were lower than that of the non-Yang-Yang mass group in the comparison among the two groups of the Yang-Yang mass group and the non-Yang-Yang mass group. In the chronic insomnia group, the percentage of CD56-CD16 + NK cell particles and the percentage of perforin in the Yang-Yang mass group were lower than that of the non-yang-deficient group. The fluorescence intensity of CD56-CD16 + NK cells in the Yang-Yang mass group was lower than that of the mild group. The percentage of CD56 + CD16 + NK cell subpopulation (r =-0.446), CD56 + CD16 + NK cell subpopulation particle enzyme percentage (r =-0.465), CD56 + CD16 + NK cell subpopulation particle enzyme fluorescence (r =-0.465), CD56 + CD16-NK cell subpopulation particle enzyme fluorescence (r =-0.504) were negatively correlated with the chronic insomnia group. The NK cell killing rate of the chronic insomnia group (n = 8), the TNF level of NK cell secretion, IFNy and the normal control group (n = 7) had no significant difference.4. The ratio of Th1/ Th2 and Th17 cells in chronic insomnia group (n = 58) was lower than that of normal control group (n = 22). In the chronic insomnia group, the percentage of Th1 cells, the percentage of Th2 cells and the percentage of Th17 cells in the Yang-Yang mass group were lower than that of the non-Yang-Yang mass group. in the chronic insomnia group, the percentage of the Th1 cells and the percentage of the Th2 cells in the Yang-Yang mass group were lower than that of the non-yang-deficient group, The percentage of Th17 cells in the Yang-Yang mass group was lower than that of the non-yang-deficient group. The percentage (r =-0.452), the percentage of Th2 cells (r =-0.39) and the percentage of Th17 cells (r =-0.387) in the chronic insomnia group were negatively correlated with the percentage of Th1 cells (r =-0.452), the percentage of Th2 cells (r =-0.39), and the percentage of Th17 cells (r =-0.387) (P0.05). Conclusion: The immune disorder caused by chronic insomnia is mainly characterized by the decrease of the number of NK cells and T cells and the shift of related functional subpopulations. It is suggested that chronic insomnia with yang-deficiency can be a type of immune function in chronic insomnia which is more likely to be affected by insomnia.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R256.23

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