Zhanjiang Institute of Clinical Medicine, Central People's Hospital of Zhanjiang, Guangdong Medical University, Zhanjiang, Guangdong, China.
Department of Hematology, Central People's Hospital of Zhanjiang, Guangdong Medical University, Zhanjiang, Guangdong, China.
PeerJ. 2024 Apr 24;12:e17261. doi: 10.7717/peerj.17261. eCollection 2024.
Protein C (PC) is an anticoagulant that is encoded by the PROC gene. Validation for the function of PC was carried out in mouse models.
In this study, autosomal recessive PC deficiency (PCD) was selected as the target, and the specific mutation site was chromosome 2 2q13-q14, PROC c.1198G>A (p.Gly400Ser) which targets G399S (GGT to AGC) in mouse models. To investigate the role of hereditary PC in mice models, we used CRISPR/Cas9 gene editing technology to create a mouse model with a genetic PCD mutation.
The two F0 generation positive mice produced using the CRISPR/Cas9 gene editing technique were chimeras, and the mice in F1 and F2 generations were heterozygous. There was no phenotype of spontaneous bleeding or thrombosis in the heterozygous mice, but some of them were blind. Blood routine results showed no significant difference between the heterozygous mice and wild-type mice ( > 0.05). Prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) were prolonged in the heterozygous mice, while the level of fibrinogen content (FIB) decreased, suggesting secondary consumptive coagulation disease. The protein C activity of heterozygous mice was significantly lower than that of wild-type mice ( < 0.001), but there was no significant difference in protein C antigen levels ( > 0.05). H&E staining showed steatosis and hydrodegeneration in the liver of heterozygous mice. Necrosis and exfoliated epithelial cells could be observed in renal tubule lumen, forming cell or granular tubules. Hemosiderin deposition was found in the spleen along with splenic hemorrhage. Immunohistochemistry demonstrated significant fibrin deposition in the liver, spleen, and kidney of heterozygous mice.
In this study, heterozygotes of the mouse model with a PC mutation were obtained. The function of PC was then validated in a mouse model through genotype, phenotype, and PC function analysis.
蛋白 C（PC）是一种抗凝剂，由 PROC 基因编码。PC 的功能验证是在小鼠模型中进行的。
本研究选择常染色体隐性 PC 缺乏症（PCD）为研究对象，其特定突变位点位于染色体 2 2q13-q14，PROC c.1198G>A（p.Gly400Ser），在小鼠模型中靶向 G399S（GGT 突变为 AGC）。为了研究遗传性 PC 在小鼠模型中的作用，我们使用 CRISPR/Cas9 基因编辑技术构建了一个具有遗传 PCD 突变的小鼠模型。
使用 CRISPR/Cas9 基因编辑技术产生的 2 只 F0 代阳性小鼠均为嵌合体，F1 和 F2 代的小鼠均为杂合子。杂合子小鼠无自发性出血或血栓形成的表型，但部分小鼠失明。血常规结果显示杂合子小鼠与野生型小鼠无显著差异（>0.05）。PT、APTT 和 TT 延长，FIB 水平降低，提示继发性消耗性凝血病。杂合子小鼠的 PC 活性显著低于野生型小鼠（<0.001），但 PC 抗原水平无显著差异（>0.05）。H&E 染色显示杂合子小鼠肝脏有脂肪变性和水样变性。肾小管腔中可见坏死和脱落的上皮细胞，形成细胞或颗粒管型。脾内可见含铁血黄素沉积，伴有脾出血。免疫组化显示杂合子小鼠肝、脾、肾均有明显的纤维蛋白沉积。
本研究获得了 PC 基因突变的小鼠杂合子。通过基因型、表型和 PC 功能分析，在小鼠模型中验证了 PC 的功能。
