家蚕微孢子虫感染对家蚕BmN细胞凋亡以及凋亡蛋白抑制因子IAPs表达的影响

Effect of Nosema bombycis on apoptosis of BmN cells and expression level of BmIAPs

摘要:

[目的] 在无任何外界凋亡因素诱导条件下，探究家蚕微孢子虫感染对家蚕卵巢细胞-BmN凋亡的影响，以及凋亡蛋白抑制因子IAPs实相表达的变化情况。[方法] 显微镜下观察家蚕微孢子虫感染BmN细胞后不同时间段宿主细胞的变化情况，以及利用荧光定量PCR方法检测家蚕促凋亡基因——细胞色素C（BmCyt c）表达水平的变化，随后检索家蚕基因组与蛋白质家族数据库搜寻家蚕凋亡蛋白抑制因子IAPs基因信息，并通过荧光定量PCR方法对这些基因的实相表达情况进行定量分析。[结果] 家蚕微孢子虫感染BmN细胞的前5 d，细胞状态未见明显变化。感染后7 d，BmN细胞的生长受到了一定程度的影响。第12天时，对照组中几乎所有细胞出现空泡化或细胞死亡的现象，而感染家蚕微孢子虫的BmN细胞未见空泡的出现，并且大量细胞形态完整，细胞核清晰可见。同时，BmCyt c基因的表达几乎一直处于被抑制状态，特别是感染后的第10天与第12天，该基因的表达量显著性降低（P<0.01）。通过数据库检索共得到4个家蚕凋亡蛋白抑制因子：BmIAP-1、BmIAP-2、BmSurvivin-1与BmSurvivin-2。荧光定量PCR结果表明：BmIAP-1和BmSurvivin-1基因在感染后期（10 d与12 d）表达量有上升趋势，尤其是感染后的12 d，表达量显著上升（P<0.01）。然而，BmIAP-2与BmSurvivin-2基因的表达在大多数时间段均处于下调状态。[结论] 当无任何外界凋亡因素诱导条件下，家蚕微孢子虫感染BmN细胞后可影响宿主细胞的生长，并可抑制细胞的正常生理凋亡。依据荧光定量PCR结果，我们推测在家蚕微孢子虫感染BmN细胞时，BmIAP-1和BmSurvivin-1蛋白可能在调节细胞凋亡的过程中起一定作用。

Abstract:

[Objective] To study the effect of Nosema bombycis on the apoptosis of BmN cells and the expression level of BmIAPs under the condition without external apoptosis-inducing factor.[Methods] We observed the change of BmN cells at different times by using microscope after inoculation with N. bombycis. The relative expression level of BmCyt c in the N. bombycis infected BmN cells was detected by using real-time PCR. We searched databases of silkworm genome and Pfam finding information of Bombyx mori inhibitor of apoptosis proteins (BmIAPs). Real-time PCR was used to demonstrate the relative expression level of BmIAPs in N. bombycis infected BmN cells.[Results] Compared with the normal BmN cells, BmN cells infected by N. bombycis had no obviously change at 5 d post-inoculation. The growth of BmN cells was influenced by N. bombycis at 7 d post-inoculation. The vacuolization and apoptosis were found in most BmN cells at 12 d. However, the phenomenon was not found in N. bombycis infected BmN cells. Moreover, the relative expression level of BmCyt c was suppressed in N. bombycis infected BmN cells at most of the time, except for 7 d. The expression of BmCyt c was lowest compared with control groups (P<0.01) at 10 d and 12 d post-inoculation. Through searching the databases, we obtained four BmIAPs, including BmIAP-1, BmIAP-2, BmSurvivin-1 and BmSurvivin-2. The results of real-time PCR indicated that the relative expression level of BmIAP-1 and BmSurvivin-1 had up-regulating tendency at 10 d and 12d post-inoculation, especially at 12 d post-inoculation. But, BmIAP-2 and BmSurvivin-2 had down-regulated expression at most times.[Conclusion] Under the condition without external apoptosis-inducing factor, growth and apoptosis of BmN cells can be influenced by N. bombycis. According to the results of real-time PCR, we speculated that BmIAP-1 and BmSurvivin-1 may play a certain role in regulating the apoptosis of BmN cells infected by N. bombycis.