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摘要：
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摘要：目的 研究Glt25d1基因在刀豆蛋白A（concanavalin A，Con A）诱导的自身免疫性肝炎中的
作用及其对巨噬细胞和中性粒细胞的影响。方法 随机抽取6～8周无特定病原体雌性Glt25d1 +/- 小鼠
及与其同窝出生的野生型（wild type，WT）小鼠，体质量（20 ± 2）g，分为WT对照组、Glt25d1 +/- 对照
组、WT造模组和Glt25d1 +/- 造模组，每组8只。造模组通过内眦静脉注射Con A，剂量为10 mg/kg，对照
组给予相同剂量生理盐水注射。造模12 h后处死小鼠。检测小鼠血浆中丙氨酸氨基转移酶（alanine
aminotransferase，ALT）和天门冬氨酸氨基转移酶（aspartate aminotransferase，AST）水平，观
察肝组织病理。采用流式细胞术检测小鼠外周血、脾脏和肝脏中巨噬细胞和中性粒细胞的比例。
采用定量逆转录PCR（quantitative reverse transcriptase-mediated PCR，qRT-PCR）检测肝脏中单
核-巨噬细胞趋化蛋白-1（monocyte/macrophage chemotaxis protein Ⅰ，MCP-Ⅰ）、巨噬细胞炎
性蛋白1α（macrophage inflammatory protein Ⅰα，MIP-Ⅰα）、巨噬细胞炎性蛋白2（macrophage
inflammatory protein Ⅱ，MIP-Ⅱ）及淋巴细胞抗原6G（lymphocyte antigen 6 complex locus G，
Ly6G）等mRNA的相对表达量。结果 WT对照组和Glt25d1 +/- 对照组小鼠ALT [（52.00 ± 18.19）U/L
vs （45.00 ± 6.85）U/L]和AST [（168.17 ± 21.33）U/L vs（276.17 ± 83.37）U/L]水平差异无统计学意义
（t值分别为0.360、1.255，P值分别为0.7263、0.2589）。Con A造模12 h后，WT造模组小鼠ALT
和AST水平分别为（3089.67 ± 663.92）U/L、（3099.50 ± 519.15）U/L，Glt25d1 +/- 造模组小鼠ALT和
AST水平分别（11565.17 ± 1381.38）U/L、（10875.17 ± 1558.68）U/L，均显著高于未造模组（P均＜
0.05），Glt25d1 +/- 造模组小鼠ALT和AST水平均显著高于WT造模组（t值分别为5.530、4.733，P值分
别为0.0003、0.0032）。肝组织HE染色示：WT对照组和Glt25d1 +/- 对照组小鼠肝组织未见明显损伤，
Con A诱导12 h后，与WT Con A造模组小鼠相比，Glt25d1 +/- 造模组小鼠肝脏坏死面积更广、汇管区炎
性细胞浸润更显著。Glt25d1 +/- 对照组小鼠脾脏中巨噬细胞比例显著高于WT对照组[（5.04 ± 0.32）%
vs（3.48 ± 0.31）%]，差异有统计学意义（t = 2.954，P = 0.0131），外周血[（0.26 ± 0.09）% vs（0.63 ±
0.16）%]和肝脏[（1.58 ± 0.11）% vs（2.83 ± 0.43）%]中差异无统计学意义（t值分别为1.456、1.626，
P值分别为0.1733、0.1351）。Con A诱导12 h后，与WT造模组相比，Glt25d1 +/- 造模组小鼠外周血中
巨噬细胞比例显著降低[（0.12 ± 0.21）% vs （0.43 ± 0.10）%，t = 2.955，P = 0.0144]，肝脏中巨噬
细胞比例显著升高[（8.34 ± 0.30）% vs （1.03 ± 0.53）%，t = 6.992，P = 0.0002]，脾脏中巨噬细胞
比例无显著差异[（3.98 ± 0.39）% vs （3.82 ± 0.21）%，t = 0.372，P = 0.7173]。与WT对照组小鼠相
比，Glt25d1 +/- 对照组小鼠脾脏中中性粒细胞比例显著升高[（5.20 ± 0.76）% vs （2.46 ± 0.35）%，
t = 3.836，P = 0.0028]，外周血[（0.67 ± 0.26）% vs （1.96 ± 0.77）%]和肝脏[（3.68 ± 1.33）% vs （3.76 ±
1.12）%]中中性粒细胞比例无显著差异（t值分别为1.078、0.039，P值分别为0.3040、0.9698）。Con A诱
导12 h后，与WT造模组小鼠相比，Glt25d1 +/- 造模组小鼠外周血中性粒细胞比例显著降低[（1.37 ±
0.22）% vs （14.40 ± 2.72）%，t = 4.782，P = 0.0088]，肝脏中巨噬细胞比例显著升高[（8.08 ± 1.93）%
vs （1.64 ± 0.71）%，t = 3.788，P = 0.0043]，脾脏中差异无统计学意义[（5.81 ± 0.84）% vs （5.96 ±
0.42）%，t = 0.176，P = 0.8621]。WT对照组小鼠MCP-Ⅰ、MIP-Ⅰα、MIP-Ⅱ及Ly6G mRNA相对表
达量分别为0.66 ± 0.31、1.08 ± 0.23、1.04 ± 0.17、1.05 ± 0.19，Glt25d1 +/- 对照组小鼠上述指标分别为
0.60 ± 0.13、1.56 ± 0.11、0.77 ± 0.07、0.92 ± 0.16，差异均无统计学意义（P均＞ 0.05）。WT造模组小
鼠MCP-Ⅰ、MIP-Ⅰα、MIP-Ⅱ及Ly6G mRNA相对表达量分别为1.29 ± 0.41、1.11 ± 0.18、1.16 ± 0.19、
0.90 ± 0.20，Glt25d1 +/- 造模组小鼠上述指标分别为2.51 ± 0.26、4.72 ± 0.45、2.62 ± 0.36、2.36 ± 0.59，
Glt25d1 +/- 造模组均显著高于WT造模组，差异有统计学意义（P均＜ 0.05）。结论 Glt25d1基因敲低可
加重小鼠Con A诱导的自身免疫性肝炎，降低外周血巨噬细胞和中性粒细胞比例，升高肝脏中巨噬细
胞和中性粒细胞比例，并诱导肝脏细胞因子如MCP-Ⅰ、MIP-Ⅰα、MIP-Ⅱ及Ly6G转录水平升高。
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Abstract: Objective To investigate the role of Glt25d1 in concanavalin A (Con A) induced autoimmune
hepatitis, and its effects on macrophages and neutrophils in this progression. Methods The specific pathogen
free Glt25d1 +/- mice and wild type (WT) mice born in the same litter were randomly selected, the mice were
all female and 6～8 weeks, the body mass were (20 ± 2) g. The mice were divided into WT control group,
Glt25d1 +/- control group, WT Con A administration group and Glt25d1 +/- Con A administration group, 8
mice in each group. Mice in administered group were given Con A via the internal iliac venous plexus with
a dose of 10 mg/kg body mass, while mice in control group were injected with the same dose of saline. Mice
were sacrificed after Con A challenged for 12 h. The levels of alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) were detected and the liver histopathology were observed. Flow cytometry were used
to detect the percentage of macrophages and neutrophils in blood, spleen and liver single-cell suspension of
mice. Quantitative reverse transcriptase-mediated PCR (qRT-PCR) were used to detect the relative expression
of monocyte/macrophage chemotaxis protein Ⅰ (MCP-Ⅰ)、macrophage inflammatory protein Ⅰα (MIP-
Ⅰα)、macrophage inflammatory protein Ⅱ (MIP-Ⅱ) and lymphocyte antigen 6 complex locus G (Ly6G)
mRNA. Results There were no statistically significant differences of ALT [ (52.00 ± 18.19) U/L vs (45.00 ±
6.85) U/L ] and AST [ (52.00 ± 18.19) U/L vs (45.00 ± 6.85) U/L ] in mice between WT control group and
Glt25d1 +/- control group (t = 0.360, 1.255, P = 0.7263, 0.2589). After Con A administrated for 12 h, the ALT
and AST levels of mice in WT Con A administration group were (3089.67 ± 663.92) U/L and (3099.50 ±
519.15) U/L, respectively, and in Glt25d1 +/- Con A administration group were (11565.17 ± 1381.38) U/L and
(10875.17 ± 1558.68) U/L, respectively, the levels of ALT and AST were significantly higher than those in
control group (all P ＜ 0.05). In addition, the ALT and AST levels of Glt25d1 +/- Con A administration group
were significantly increased compared with those in WT Con A administration group (t = 5.530, 4.733; P =
0.0003, 0.0032). HE staining showed that there was no significant liver injury of mice in both of WT control
group and Glt25d1 +/- control group. After Con A administrated for 12 h, compared with mice of WT Con
A administration group, liver pathological section of mice in Glt25d1 +/- Con A administration group had
wider area of necrosis and showed more significant infiltration of inflammatory cells in the portal area. The
percentage of macrophage in spleen of mice in Glt25d1 +/- control group were significantly higher than that in
WT control group [(5.04 ± 0.32)% vs (3.48 ± 0.31)%], the difference was statistically significant (t = 2.954, P = 0.0131).
There were no statistically significant differences of macrophage percentage in peripheral blood [(0.26 ± 0.09)%
vs (0.63 ± 0.16)%] and liver [(1.58 ± 0.11)% vs (2.83 ± 0.43)%] between Glt25d1 +/- control group and WT control
group (t = 1.456, 1.626; P = 0.1733, 0.1351). After Con A administrated for 12 h, in comparison with WT Con
A administration group, the percentage of macrophage in Glt25d1 +/- Con A administration group decreased
significantly in peripheral blood [(0.12 ± 0.21)% vs (0.43 ± 0.10)%; t = 2.955, P = 0.0144] and increased in
liver [(8.34 ± 0.30)% vs (1.03 ± 0.53)%; t = 6.992, P = 0.0002], and there was no significant difference in
spleen [(3.98 ± 0.39)% vs (3.82 ± 0.21)%; t = 0.372, P = 0.7173]. Compared with WT control group, the
percentage of neutrophil of spleen in Glt25d1 +/- control group increased significantly [(5.20 ± 0.76)%
vs (2.46 ± 0.35)%; t = 3.836, P = 0.0028], while there were no significant differences in peripheral blood [(0.67 ±
0.26)% vs (1.96 ± 0.77)%] and liver [(3.68 ± 1.33)% vs (3.76 ± 1.12)%; t = 1.078, 0.039, P = 0.3040, 0.9698].
After Con A administrated for 12 h, in comparison with WT Con A administration group, the percentage
of neutrophil in peripheral blood of Glt25d1 +/- Con A administration group decreased significantly [(1.37 ±
0.22)% vs (14.40 ± 2.72)%; t = 4.782, P = 0.0088], and in liver increased significantly [(8.08 ± 1.93)% vs
(1.64 ± 0.71)%; t = 3.788, P = 0.0043], there was no statistical difference of neutrophil in spleen [(5.81 ±
0.84)% vs (5.96 ± 0.42)%; t = 0.176, P = 0.8621]. The relative expression of MCP-Ⅰ, MIP-Ⅰα, MIP-Ⅱ and
Ly6G mRNA in WT control group were 0.66 ± 0.31, 1.08 ± 0.23, 1.04 ± 0.17 and 1.05 ± 0.19, respectively,
and in Glt25d1 +/- control group were 0.60 ± 0.13, 1.56 ± 0.11, 0.77 ± 0.07 and 0.92 ± 0.16, respectively, the
differences were not statistically significant (all P ＞ 0.05). The relative expression of MCP-Ⅰ, MIP-Ⅰα,
MIP-Ⅱ and Ly6G mRNA in WT control group were 1.29 ± 0.41, 1.11 ± 0.18, 1.16 ± 0.19 and 0.90 ± 0.20,
respectively, and in Glt25d1 +/- control group were 2.51 ± 0.26, 4.72 ± 0.45, 2.62 ± 0.36 and 2.36 ± 0.59 in
Glt25d1 +/- Con A administration group, respectively. The above indicators in Glt25d1 +/- Con A administration
group were higher than those in WT Con A administration group, the differences were statistically significant
(all P ＜ 0.05). Conclusions Glt25d1 gene-knockdown aggravated Con A-induced autoimmune hepatitis
in mice. Deficiency of Glt25d1 reduced the proportion of macrophages and neutrophils in peripheral blood,
while increased the proportion of macrophages and neutrophils in liver, and also induced the increasing
transcription levels of MCP-Ⅰ, MIP-Ⅰα, MIP-Ⅱ, and Ly6G mRNA.
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