Characteristics of immune cell infiltration in inflamed mucosa of ulcerative colitis patients, hub gene candidates and key pathways in intestinal macrophage.

Tissue RNA was isolated from fresh mouse colon sample by Trizol reagent. Total RNA (1 µg) was reverse-transcribed using a reverse transcriptase kit (TakaRa, cat. RR036A-1, Tokyo, Japan). Hieff® qPCR SYBR Green Master Mix (Yeasen Biotechnolog, cat. 11201ES03, Shanghai, China) was used for Real-time qPCR. The comparative cycle threshold (Ct) method (2−ΔΔCT) was used to calculate relative expression, β-actin was used as the internal reference. We evaluated immunostaining by multiplying the intensity score and proportion score. Intensity score according to the intensity of cell staining: 0 for no positive staining (negative), 1 for light yellow (weakly positive), 2 for brown (positive) and 3 for dark brown (strongly positive). Proportion score according to the percentage of positive cells: 1 for ≤25%, 2 for 26%-50%, 3 for 51%-75% and 4 points for > 75%.

采用Trizol试剂从新鲜小鼠结肠组织样本中提取组织RNA。取总RNA 1 µg，使用反转录试剂盒（Takara，货号RR036A-1，日本东京）进行反转录反应。采用Hieff® qPCR SYBR Green Master Mix试剂盒（Yeasen Biotechnolog，货号11201ES03，中国上海）进行实时定量qPCR（Real-time qPCR）。采用比较循环阈值（Cycle threshold, Ct）法（2−ΔΔCT法）计算基因相对表达量，以β-肌动蛋白（β-actin）作为内参基因。本研究通过将染色强度评分与阳性细胞比例评分相乘，对免疫染色结果进行评估。染色强度评分依据细胞染色强度判定：0分代表无阳性染色（阴性），1分代表浅黄色（弱阳性），2分代表棕黄色（阳性），3分代表深棕褐色（强阳性）。阳性细胞比例评分依据阳性细胞占比判定：≤25%记1分，26%~50%记2分，51%~75%记3分，＞75%记4分。