原发性胆汁性肝硬化患者外周血NKT细胞体外活化及相关细胞因子表达分析

Analysis of NKT cells and related cytokines in peripheral blood of the patients with primary biliary cirrhosis

目的 分析与评价PBC患者外周血TCRVα24＋Vβ11＋NKT数量和功能状态及其与PBC发生的关系.方法 制备60例PBC患者（PBC组）和60名年龄及性别匹配健康人（健康对照组）PBMC,以FACS检测TCRVα24＋Vβ11＋NKT数量;以α半乳糖酰基鞘胺醇（α-galcer）和IL-2诱导PBMC中NKT的扩增活化,用ICS-FC检测细胞内表达IL-4和IFN-γ的NKT比值;用ELISpot和ELISA定量检测细胞内及上清液IL4和IFN-γ的表达量.结果 FACS检测PBC组和健康对照组TCRVα24＋Vβ11＋NKT扩增前后的比率分别为[0.16（0.11～0.26）]%、[0.82（0.61～0.89）]%、[0.33（0.27～0.38）]%、[27.40（23.52～33.87）]%,前组显著低于后组（Z值分别为6.563、7.707,P＜均0.01）.扩增活化后PBC组和健康对照组TCRVα24＋Vβ11＋NKT的扩增倍数分别为扩增活化前的[96.05（80.50～100.27）]倍和[134.65（121.60～142.13）]倍,PBC组明显低于健康对照组（Z=6.462,P＜0.01）.同时于活化后用ICS-FC检测细胞内细胞因子,PBC组和健康对照组IFN-γ＋与TCRVα24＋Vβ11＋NKT比分别为[38.98（36.73～42.98）]%、[34.56（30.12～36.78）]%,前组显著高于后组（Z=3.158,P＜0.05）;PBC组和健康对照组IL-4＋与TCRVα24＋Vβ11＋NKT细胞比分别为[0.193（0.179～0.218）]%和[34.36（30.93～38.77）]%,前组显著低于后组（Z=6.476,P＜0.01）.ELISpot和ELISA定量检测扩增活化后PBC组分泌IFN-γ的斑点形成细胞数（SFC）和上清液IFN-γ分泌量分别为[410（380～500）]SFC/1×106 PBMC、（67.21±11.27）ng/L,健康对照组[340（280～390）]SFC/1×106PBMC、（31.45±8.17）ng/L,前组的IFN-γ斑点形成细胞数及分泌量显著高于后组（Z=4.312,P＜0.05、t=27.25,P＜0.01）;PBC组的IL-4斑点形成细胞数和分泌量分别为[73（60～100）]SFC/1×106PBMC、（12.65±4.17）μg/L,健康对照组[245（230～280）]SFC/1×106PBMC、（28.31±6.31）μg/L,PBC组IL-4却显著低于健康对照组（Z=5.112,P＜0.01、t=25.34,P＜0.01）.结论 PBC组外周血TCRVα24＋Vβ11＋NKT数量较健康对照组明显减少,经α-galcer及IL-2体外活化后扩增倍数及分泌细胞因子IL-4的功能较健康对照组显著降低,而分泌IFN-γ能力却显著高于健康对照组,NKT数量的减少及免疫调节功能的紊乱可能是PBC发病的重要因素之一.

Objective To analyze and evaluate the changes of quantity and function of TCBVα24＋ Vβ11＋ NKT in PBMC of the patients with PBC and its relationship with the occurrence of PBC. Methods Flow cytometry was utilized to count TCRVα24＋ Vβ11＋ NKT cells in PBMC in 60 cases of PBC and 60 cases of age-matched and gender-matched controls. NKT cells were activated and expanded by α-galcer and IL-2 in vitro. The percentages of positive NKT cells expressing IL-4 and IFN-γ were determined by ICS-FC. The levels of serum IL-4 and IFN-γ were tested by ELSIA. The numbers of cells secreting IFN-γ and IL-4 were detected by ELISpot. Results The ratios of NKT cells in PBC group were [0. 16（0. 11-0. 26） ]% before expansion and [0. 82 （0. 61-0. 89） ]% after expansion, significantly lower than control group [（0.33 （0.27-0.38） ]% and [27.40 （23.52-33.87） ]%, respectively, （Z=6.563, 7.707, P〈0. 01 ）. Seven days after expansion by α-galcer and IL-2, the expansion folds of NKT cells were 96. 05 （80.50-100.27） in PBC group and 134.65 （121.60-142. 13） in control group, respectively （Z =6.462,P 〈 0. 01 ）. At the same time, the ratio of IFN-γ＋ and TCRVα24＋ Vβ11＋ NKT detected by ICS-FC in PBC group was significantly higher than that in control group [ 38. 98 （ 36.73-42. 98 ） ]% vs [ 34. 56 （ 30. 12-36. 78 ） ] %, Z = 3. 158, P 〈 0. 05, while the ratio of IL-4＋ and TCRVα24 ＋ Vβ11＋ NKT cells in PBC group was significantly lower than that in control group[0. 193（0. 179-0. 218） ]% vs [34. 36 （30. 93-38. 77） ]%,Z =6. 476, P 〈0. 01. The number of IFN-γ SFC detected by ELISpot were [410（380 ~500） ] SFC/1 × 106 PBMC in PBC group and [ 340（280 ~ 390）] SFC/1 × 106 PBMC in control group, respectively （Z = 4. 312, P 〈0. 05）. The levels of serum IFN-γ in PBC group and control group were （67.21 ± 11.27） ng/L and （31.45 ± 8. 17） ng/L, respectively （ t = 27.25, P 〈 0. 01 ）. The level of IFN-γ in PBC group was higher than that of control group. The number of IL-4 SFC were [73（60 ~ 100） ]SFC/1 × 106 PBMC in PBC group vs [245（230 -280） ] SFC/1 × 106 PBMC in control group, Z=5. 112,P 〈0. 01. The levels of serum IL-4 in PBC group and control group were （12.65 ±4. 17） μ/L and （28.31 ±6.31） μg/L, respectively （t =25.34,P 〈 0. 01 ）. The level of IL-4 in PBC group was lower than that of control group. Conclusions The quantity of TCRVα2.4＋ Vβ11＋ NKT in PBC group is lower than that in control group. After in vitro activation, the capacity of expansion and producing IL-4 of NKT is decreased in PBC group, while the capacity of producing IFN-γ of NKT is increased in PBC group. The reduction of NKT cells and the immune dysfunction may be one of the important factors in the pathogenesis of PBC.