目的 研究分泌白细胞介素17A（IL-17A）的γδ T细胞对日本血吸虫诱导的早期肝纤维化形成的影响。 方法 将10只6~8周龄C57BL/6野生型雌性小鼠随机分为健康对照组和野生感染组,每组5只。另5只6~8周龄C57BL/6背景的T细胞抗原受体δ链基因敲除（TCR δ KO）小鼠为TCR δ KO感染组。两个感染组每鼠经腹部贴片法感染日本血吸虫尾蚴（20 ± 2）条,健康对照组不感染。感染后6周,各组小鼠麻醉后脱颈处死,取肝组织研磨后裂解,沉淀部分经密度梯度离心收集肝非实质细胞,采用流式细胞术检测肝非实质细胞中γδ T细胞表达的IL-17A含量,采用ELISA检测裂解上清中IL-17A浓度。另取部分肝组织,4%多聚甲醛固定后,行苏木精-伊红（HE）和Masson染色,观察胶原纤维沉积情况。取各组小鼠外周血,ELISA检测血清中IL-17A浓度。体外培养的人肝星状细胞系（LX-2细胞）分为3组,实验组加入终浓度为10 ng/ml的IL-17A,阳性对照组和阴性对照组分别加入终浓度为10 ng/ml的IL-13和等量PBS,荧光定量PCR检测各组LX-2细胞α-平滑肌肌动蛋白（α-SMA）和Ⅰ型胶原（Col 1）mRNA的相对转录水平。 结果 流式细胞术检测结果显示,野生感染组小鼠表达IL-17A的Vγ2亚型γδ T细胞百分比为（48.0 ± 1.0）%,高于健康对照组的（17.0 ± 1.6）%（P < 0.01）。Masson染色结果显示,TCR δ KO感染组小鼠的胶原沉积面积为（3.5 ± 0.2）× 10⁴ μm²,小于野生感染组的（6.4 ± 0.5）× 10⁴ μm²（P < 0.01）。ELISA检测结果显示,TCR δ KO感染组小鼠血清和肝组织裂解液上清中的IL-17A浓度分别为（34.0 ± 22.3）和（101.6 ± 18.6）pg/ml,均低于野生感染组的（143.9 ± 33.8）（P < 0.05）和（217.2 ± 19.2）pg/ml（P < 0.01）。荧光定量PCR检测结果显示,经IL-17A刺激后,实验组LX-2细胞中α-SMA和Col 1 mRNA相对转录水平较阴性对照组分别上调（4.0 ± 0.7）和（8.7 ± 1.2）倍（P < 0.05或0.01）。 结论 分泌IL-17A的γδ T细胞可能参与日本血吸虫感染所致小鼠肝纤维化的进程中,其分泌的IL-17A激活肝星状细胞可能是其加据纤维化的途径。

Objective To investigate the effect of IL-17A-producing γδ T cells on the development of early liver fibrosis induced by Schistosoma japonicum infection. Methods Ten wild-type (WT) female C57BL/6 mice were randomly divided into the healthy control group and the WT infection group, five mice each. Another group of five C57BL/6-background T cell antigen receptor δ chain gene knockout (TCR δ KO) mice served as the TCR δ KO infection group. Mice in the two infection groups were infected with S. japonicum cercariae (20 ± 2 each) by using abdominal patches, while the healthy control group was not infected. Six weeks after infection, the mice were anaesthesized and sacrificed, and liver tissues were collected, homogenized, and lysed. The pellets after lysis underwent density gradient centrifugation to obtain liver nonparenchymal cells. The content of IL-17A expressed by γδ T cells among the liver nonparenchymal cells was determined by flow cytometry. The IL-17A concentration in the lysate supernatant was examined by ELISA. Another part of the liver tissue was fixed in 4% paraformaldehyde, followed by hematoxylin-eosin (HE) and Masson staining to observe the deposition of collagen fibers. Meanwhile, peripheral blood was collected the mice of all groups to determine the serum concentration of IL-17A. A human hepatic stellate cell line (LX-2 cells) was used to examine the relative transcription level of mRNA of α-smooth muscle actin (α-SMA) and collagen type I (Col 1) in the LX-2 cells by fluorescent quantitative PCR. The assay was carried out by assigning the cell culture into 3 groups, of which the test group was assayed by adding IL-17A to a final concentration of 10 ng/ml, while the positive group and negative control group was assayed by adding IL-13 to a final concentration of 10 ng/ml and equal volume of PBS. Results The results of flow cytometry showed that the percentage of IL-17A-secreting Vγ2 subtype γδ T cells in the WT infection group was (48.0 ± 1.0)%, which was significantly higher than that in the control group [(17.0 ± 1.6)%, P < 0.01]. Masson staining revealed that the collagen deposition area in the mice of the TCR δ KO group was (3.5 ± 0.2) × 10⁴ μm², which was smaller than that in the WT infection group [(6.4 ± 0.5) × 10⁴ μm², P < 0.01]. ELISA assay showed that the IL-17A concentration in the sera and liver lysate supernatant of the infected TCR δ KO mice was (34.0 ± 22.3) pg/ml and (101.6 ± 18.6) pg/ml, respectively, both lower than those in the WT infection group [(143.9 ± 33.8) pg/ml, P < 0.05; (217.2 ± 19.2) pg/ml, P < 0.01, respectively]. Fluorescent quantitative PCR showed that the mRNA expression of α-SMA and Col 1 in LX2 cells after IL-17A stimulation was up-regulated by (4.0 ± 0.7) and (8.7 ± 1.2) folds, respectively, compared to the control group (P < 0.05, P < 0.01). Conclusion The IL-17A secreted from γδ T cells may involve in the development of hepatic fibrosis induced by S. japonicum infection in mice probably via activating hepatic stellate cells.