新型慢性同种异体肾移植排斥反应小鼠模型的建立及单细胞深层表型分析

doi:10.3877/cma.j.issn.1674-3903.2025.06.003

目的

构建一种新的慢性同种异体肾移植排斥反应小鼠模型。

方法

选取C57BL/6和Balb/c小鼠作为实验动物，设置对照移植组、普通移植组和新型移植组。术后定期检测小鼠血清肌酐和血尿素氮。移植后16周获取小鼠移植肾组织进行病理和单细胞深层表型分析。使用Banff评分进行小鼠移植肾病理评估。使用CellRanger 3.1软件处理原始测序数据。分析基因表达结果并将其与公开可用的细胞类型特异性基因数据库进行比较，确定主要细胞类型并进行注释。使用Seurat软件包中CellCycleScoring函数进行细胞周期评分。使用R语言ClusterProfiler包进行基因本体(GO)功能富集分析。正态分布计量资料组间比较采用单因素方差分析，非正态分布计量资料组间比较采用Kruskal-Wallis H检验，计数资料组间比较采用卡方检验。采用Kaplan-Meier法绘制生存曲线，并采用Log-rank检验进行比较。P<0.05为差异具有统计学意义。

结果

术后不同时间点新型移植组小鼠术后血清肌酐(除术后8周外)和血尿素氮水平均高于对照移植组和普通移植组(P均<0.05)。病理分析结果示对照移植组、新型移植组和普通移植组小鼠移植肾间质纤维化、肾小管萎缩、动脉纤维内膜增厚和慢性移植肾肾小球病Banff评分差异均有统计学意义(H＝24.385、24.120、23.550和26.840，P均<0.05)。新型移植组、普通移植组和对照移植组移植肾纤维化区域和淋巴细胞浸润比例差异均有统计学意义(F＝129.071和9.543，P均<0.05)。免疫组织化学染色结果半定量分析示新型移植组、普通移植组和对照移植组Ⅲ型胶原蛋白、α-平滑肌肌动蛋白、CD3和CD19阳性区域差异均有统计学意义(F＝740.142、223.663、106.611和1 026.355，P均<0.05)。免疫荧光染色结果示，新型移植组小鼠移植肾组织单核细胞及巨噬细胞浸润较对照移植组更强。细胞类型分析和注释结果示对照移植组上皮细胞占主导地位，新型移植组免疫细胞比例更高。单细胞测序分析表明，新型移植组中免疫细胞比例显著增加；细胞周期分析示该组细胞多处于增殖活跃的G2M期和S期。基因集变异分析及GO分析示，新型移植组中与免疫激活相关的Hallmark基因集表达显著上调。

结论

新型慢性同种异体肾移植排斥反应小鼠模型可较好地反映慢性同种异体肾移植排斥反应和同种异体移植肾间质纤维化过程。

关键词: 肾移植, 慢性同种免疫损伤, 同种异体肾间质纤维化, 单细胞测序

Objective

To establish a novel mouse model of chronic renal allograft rejection.

Methods

C57BL/6 and Balb/c mice were selected as experimental animals to establish three groups: syngeneic control group, conventional allograft group and novel allograft group. Post-operative serum creatinine and blood urea nitrogen levels were monitored regularly. At 16 weeks post-transplantation, graft tissues were harvested for pathological assessment and deep phenotypic analysis. The Banff scoring system was used to evaluate renal allograft pathology. Raw sequencing data were processed using CellRanger 3.1 software. Major cell types were identified and annotated by comparing gene expression profiles with publicly available cell-type-specific gene databases. Cell cycle scoring was performed using the CellCycleScoring function in the Seurat package. Gene Ontology (GO) functional enrichment analysis was conducted using the ClusterProfiler package in R. Comparisons of normally distributed quantitative data were performed using one-way analysis of variance, while non-normally distributed data were analyzed using the Kruskal-Wallis H test. Categorical data were compared using the Chi-square test. Survival curve was generated using the Kaplan-Meier method and compared using the Log-rank test. A P-value <0.05 was considered statistically significant.

Results

Serum creatinine (except at 8 weeks post-transplantation) and blood urea nitrogen levels in the novel allograft group were significantly higher than those in the syngeneic control and conventional allograft group at various post-operative time points (all P<0.05). Pathological analysis showed statistically significant differences in Banff scores for interstitial fibrosis, tubular atrophy, vascular fibrous intimal thickening, and transplant glomerulopathy among the syngeneic control, novel allograft, and conventional allograft groups (H=24.385, 24.120, 23.550 and 26.840, respectively, all P<0.05). Significant differences were observed in the fibrosis area and lymphocyte infiltration rate among the novel allograft, conventional allograft, and syngeneic control group (F=129.071 and 9.543, respectively, all P<0.05). Semi-quantitative analysis of immunohistochemical staining revealed statistically significant differences in the positive areas of Collagen-Ⅲ, alpha-smooth muscle actin, CD3 and CD19 among the three groups (F=740.142, 223.663, 106.611 and 1 026.355, respectively, all P<0.05). Immunofluorescence staining results indicated stronger infiltration of monocytes and macrophages in the graft tissues of the novel allograft group compared to the syngeneic control group. Cell type identification and annotation showed that epithelial cells dominated in the syngeneic control group, whereas the novel allograft group exhibited a higher proportion of immune cells. Single-cell sequencing analysis demonstrated a significant increase in the proportion of immune cells in the novel allograft group; cell cycle analysis suggested that cells in this group were predominantly in the active proliferative G2M and S phases. Gene set variation analysis and GO analysis showed significant upregulation of Hallmark gene sets related to immune activation in the novel allograft group.

Conclusion

The novel chronic renal allograft rejection mouse model effectively recapitulates the processes of chronic allograft rejection and renal interstitial fibrosis.

Key words: Kidney transplantation, Chronic homologous immune injury, Allogeneic renal interstitial fibrosis, Single-cell sequencing

图1 对照移植组、新型移植组和普通移植组小鼠肾移植术后生存曲线

表1 对照移植组、新型移植组和普通移植组小鼠术后血清肌酐和尿素氮比较(μmol/L，

±s)

组别	例数	术后血清肌酐
组别	例数	4周	8周	12周	14周	16周
对照移植组	10	40.64±1.49	47.40±1.56	41.66±1.19	41.77±0.67	42.63±1.67
新型移植组	10	72.84±6.51^ab	53.87±5.50	67.63±5.51^ab	84.41±5.24^ab	91.50±7.48^ab
普通移植组	10	55.33±4.51^a	51.33±6.66	60.50±8.41^a	58.67±3.82^a	59.83±3.79^a
F值	－	36.021	1.240	15.803	97.768	75.682
P值	－	<0.05	>0.05	<0.05	<0.05	<0.05
组别	倒数	术后血尿素氮
组别	倒数	4周	8周	12周	14周	16周
对照移植组	10	13.89±2.40	11.75±1.30	11.36±1.93	12.27±2.90	12.99±2.34
新型移植组	10	25.12±2.02^ab	20.26±1.91^ab	19.94±3.48^ab	24.27±1.93^ab	30.99±3.48^ab
普通移植组	10	19.75±1.05^a	17.55±1.65^a	18.50±1.89^a	19.52±2.40^a	21.41±3.21^a
F值	－	25.886	21.141	9.789	18.359	26.206
P值	－	<0.05	<0.05	<0.05	<0.05	<0.05

表1 对照移植组、新型移植组和普通移植组小鼠术后血清肌酐和尿素氮比较(μmol/L，

±s)

组别	例数	术后血清肌酐
组别	例数	4周	8周	12周	14周	16周
对照移植组	10	40.64±1.49	47.40±1.56	41.66±1.19	41.77±0.67	42.63±1.67
新型移植组	10	72.84±6.51^ab	53.87±5.50	67.63±5.51^ab	84.41±5.24^ab	91.50±7.48^ab
普通移植组	10	55.33±4.51^a	51.33±6.66	60.50±8.41^a	58.67±3.82^a	59.83±3.79^a
F值	－	36.021	1.240	15.803	97.768	75.682
P值	－	<0.05	>0.05	<0.05	<0.05	<0.05
组别	倒数	术后血尿素氮
组别	倒数	4周	8周	12周	14周	16周
对照移植组	10	13.89±2.40	11.75±1.30	11.36±1.93	12.27±2.90	12.99±2.34
新型移植组	10	25.12±2.02^ab	20.26±1.91^ab	19.94±3.48^ab	24.27±1.93^ab	30.99±3.48^ab
普通移植组	10	19.75±1.05^a	17.55±1.65^a	18.50±1.89^a	19.52±2.40^a	21.41±3.21^a
F值	－	25.886	21.141	9.789	18.359	26.206
P值	－	<0.05	<0.05	<0.05	<0.05	<0.05

表2 对照移植组、新型移植组和普通移植组小鼠移植肾病理Banff评分指标比较[分，M (P₂₅，P₇₅)]

组别	例数	肾间质纤维化	肾小管萎缩	动脉纤维内膜增厚	慢性移植肾肾小球病
对照移植组	10	0.80(0.70，1.00)	0.75(0.60，0.90)	0.00(0.00，0.50)	0.00(0.00，0.00)
普通移植组	10	1.25(1.00，1.50)^a	1.15(1.00，1.40)^a	0.80(0.50，1.00)^a	0.50(0.00，0.80)^a
新型移植组	10	2.25(2.00，2.50)^ab	2.05(1.90，2.30)^ab	1.90(1.70，2.20)^ab	1.65(1.50，1.80)^ab
H值	-	24.385	24.120	23.550	26.840
P值	-	<0.05	<0.05	<0.05	<0.05

表2 对照移植组、新型移植组和普通移植组小鼠移植肾病理Banff评分指标比较[分，M (P₂₅，P₇₅)]

组别	例数	肾间质纤维化	肾小管萎缩	动脉纤维内膜增厚	慢性移植肾肾小球病
对照移植组	10	0.80(0.70，1.00)	0.75(0.60，0.90)	0.00(0.00，0.50)	0.00(0.00，0.00)
普通移植组	10	1.25(1.00，1.50)^a	1.15(1.00，1.40)^a	0.80(0.50，1.00)^a	0.50(0.00，0.80)^a
新型移植组	10	2.25(2.00，2.50)^ab	2.05(1.90，2.30)^ab	1.90(1.70，2.20)^ab	1.65(1.50，1.80)^ab
H值	-	24.385	24.120	23.550	26.840
P值	-	<0.05	<0.05	<0.05	<0.05

图2 对照移植组、新型移植组和普通移植组小鼠移植肾组织HE、Masson和PAS染色结果(×400)

图3 对照移植组、新型移植组和普通移植组小鼠移植肾组织免疫组织化学染色结果(×200)注：Collagen-Ⅲ. Ⅲ型胶原蛋白；α-SMA.α-平滑肌肌动蛋白

图4 对照移植组、新型移植组和普通移植组小鼠移植肾组织免疫荧光染色结果(×100)注：蓝色．DAPI(4′，6-二脒基-2-苯基吲哚);绿色．F4/80;红色．Ly6c;粉色．CD11b

图5 新型移植组和对照移植组小鼠移植肾细胞单细胞测序结果注：t-SNE. t分布随机邻近嵌入；a．基于样本分组的t-SNE可视化图；b．经无监督聚类鉴定出的16个细胞亚群的t-SNE可视化图；c．基于经典标记物注释后的7种主要细胞类型的t-SNE可视化图；d．堆叠柱状图展示新型移植组与对照移植组中各细胞类型相对比例分布

图6 新型移植组与对照移植组单细胞测序分析与细胞群体功能分析结果注：a．细胞周期分析及各类型细胞占比图；b．GSVA分析结果；c．GO分析结果；SYN.对照移植组；Allo.新型移植组

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The establishment and deep phenotyping of a novel chronic renal allograft rejection mouse model

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The establishment and deep phenotyping of a novel chronic renal allograft rejection mouse model