钙调神经磷酸酶对前B淋巴细胞及其转化的白血病细胞凋亡影响的研究

本研究比较钙调神经磷酸酶（calcineurin,PP2B,PP3）在小鼠前B淋巴细胞系（S9）及其转化的白血病细胞系（S4C2）中的差异表达,并探讨其参与白血病细胞凋亡的可能机制.用实时定量PCR技术检测H2AX磷酸化酶ATM、ATR、DNA-PKs、JNK1、P38和γ-H2AX磷酸酶PP1、PP2A、calcineurin、PP4、PP6、PP5 γ-PP2c、WIP-1在S9细胞和S4C2细胞之间的表达差异.用CCK-8法和流式细胞术检测伊马替尼（IM）和环孢霉素A（CsA）对细胞的毒性作用和凋亡的影响.利用Western blot技术检测药物对S9细胞和S4C2细胞凋亡的影响.结果发现,钙调神经磷酸酶基因在白血病细胞S4C2中的表达约为S9细胞的3.5倍,而其余基因的表达在两种细胞间无明显差异.IM和CsA对S4C2细胞的促凋亡作用明显强于S9细胞,同时两种药物对S4C2细胞毒性作用高于对S9细胞的毒性作用.钙调神经磷酸酶蛋白在S4C2细胞中的表达高于S9细胞.CsA特异性抑制钙调神经磷酸酶活性后,DNA损伤标记物γ-H2AX在S9细胞的表达明显低于S4C2细胞,而伊马替尼对其表达的影响在两细胞系之间无明显差异,联合应用两种药物时γ-H2AX在S9细胞的表达也低于S4C2细胞.结论：钙调神经磷酸酶在B淋巴细胞白血病细胞中发挥一定的抗凋亡作用,环孢素A抑制钙调神经磷酸酶活性促进了白血病细胞的凋亡.

Influence of Calcineurin on Apoptosis of pre-B Lymphocytes and the Leukemia Cells Derived from pre-B lymphocytes

This study was aimed to compare the differential expressions of calcineurin （PP2B,PP3） in the mouse Pre-B cell lines （S9） and the tumor cell lines （S4C2） derived from pre-B lymphocytes,and to clarify its possible mechanism involving in the leukemia cell apoptosis.The quantitative real-time PCR was used to detect the differential expressions of H2AX-associated phosphakinase ATM,ATR,DNA-PKs,JNKI,P38 and the γ-H2AX-related phosphatase PP1,PP2A,calcineurin,PP4,PP6,PP5 between S9 and S4C2 cell lines.CCK-8 assay and flow cytometry were used to detect the effect of imatinib（IM） and cyclosporine A（CsA） on cytotoxicity and apoptosis of 2 cell lines.The Western blot was used to detect the effects of 2 drugs on apoptosis of S9 and S4C2 cell lines.The results showed that the expression level of calcineurin gene in the leukemia cell S4C2 was about 3.5 times of that in S9 cells,while the expression of other genes in these 2 kinds of cells was not significantly different.The apoptosis and toxicity of IM and CsA on S4C2 cells was significantly stronger than that on S9 cells.The expression level of calcineurin in S4C2 cells was higher than that in S9 cells.When CsA inhibited the calcineurin activity,the expression of DNA damage marker γ-H2AX in S9 cells was significantly lower than that in S4C2 cells,while the expression level of γ-H2AX between the two cell lines was no significantly different after treatment with imatinib,the expression level of γ-H2AX in S9 cells was lower than that in S4C2 cells when the two drugs were combined.It is concluded that the calcineurin plays a role of antiapoptosis in B leukemic cells,cyclosporine A can promote the leukemia cell apoptosis.