Analysis of sinnalling pathway of apoptosis degradation using PKCδ gene-targeting mice

已有研究表明，蛋白激酶Cδ(PKCδ)是蛋白激酶C家族成员的一种亚型，在细胞凋亡时被半胱氨酸天冬氨酸氨基转移酶(Caspase)切割，转化为活化的蛋白激酶Cδ。本研究的目的是通过建立PKCδ基因缺陷的小鼠来阐明PKCδ的生理功能，构建靶向载体并将其导入胚胎干细胞，获得同源重组的ES细胞的PKCδ基因。按照基因消融小鼠的标准方法，用突变的ES细胞产生PKCδ缺陷小鼠。PKCδ缺陷小鼠存活、健康且不易发生癌症。组织学检查显示，PKCδ缺陷小鼠脾肿大是由于T和B淋巴细胞的异常聚集。在淋巴结中，B细胞数量的增加比T细胞数量的增加更为明显。体外培养实验表明，PKCδ缺陷的B细胞生长速度快于野生型B细胞。我们认为，由于缺乏发育异常，PKCδ对发育过程中的细胞凋亡不是必不可少的。此外，PKCδ缺陷小鼠的胸腺细胞和胚胎成纤维细胞的凋亡似乎没有受到影响。但B细胞的凋亡程度有所减轻，提示PKCδ可能在B细胞凋亡的信号通路中发挥重要作用。

It has been reported that protein kinase Cδ (PKCδ) a subtype of PKC family members, is cleaved by caspase resulting in conversion to activated PKCδ upon apoptosis. The aim of this research project is to elucidate the physiological function of PKCδ by generating mice deficient in PKCδ gene.We constructed the targeting vector and transfect it into embryonic stem (ES) cells to obtain the homologous recombinant of ES cells for PKCδ locus. According to the standard method to generate gene-ablated mice, PKCδ-deficient mice were produced with the mutant ES cells. The PKCδ-deficient mice were viable, healthy, and not prone to cancer development.Histlogical examinations of the PKCδ-deficient mice revealed splenomegaly due to abnormal accumulation of T and B lymphocytes. In lymph nodes, increase in the number of B cells was more apparent than that of T cells. In vitro culture experiments showed that PKCδ-deficient B cells grew faster than wild-type B cells.We concluded that PKCδ is not essential for the apoptosis during development, because of the lack of developmental abnormalities. Furthermore, apoptosis of thymovytes and embryonic fibroblasts from PKCδ-deficient mice seems unaffected. However, the extent of apoptosis in B cells appeared to be reduced, suggesting that PKCδ may play an important role in the signaling pathway of apoptosis in B cells.