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

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.

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

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.