骨髓基质干细胞与受损PC12细胞的共育体系减少PC12的凋亡

The co-culture system of MSCs and injured PC12 in vitro could inhibit the apoptosis of PC12

目的建立体外骨髓基质干细胞(MSCs)与Aβ1-40损伤PC12的共育体系,探讨共育体系抑制Aβ1-40致PC12凋亡的效应与可能机制。方法分别培养MSCs与PC12,以Aβ1-40刺激PC12后,用转移筛网转移PC12。实验分A组:正常培养的PC12+MSCs共育;B组:Aβ1-40刺激的PC12+ MSCs共育;C组:正常PC12的培养上清+MSCs;D组:受损PC12上清+MSCs;E组:普通1640培养的MSCs。用PI和Annexin-V进行细胞的双染凋亡检测及电镜检测PCI2凋亡,以ELISA方法检测各组中上清液的TGF-β、NGF、BDNF、bFGF含量。结果骨髓基质干细胞与Aβ1-40损伤PC12共育组即B组凋亡细胞数最少(B组46.17%±8.28%,对照组86.39%±9.34%,F=61.637,P<0.01),ELISA结果显示各组均能检测到bFGF,B组上清中bFGF分泌最高[B组(598.76±41.32) pg/ml,对照组(296.43±47.86) pg/ml,F=24.15,P<0.01)],各组均检测到TGF-β、NGF和BDNF分泌,但差异无统计学意义。结论MSCs与Aβ1-40刺激的PC12的共育体系能减少受损PC12的凋亡。

Objective To establish the co-culture system of marrow stromal cells （MSCs） and Aβ1-40injured PC12 in vitro and to evaluate the effect and mechanisms of the system inhibiting apoptosis of PC12 induced by Aβ1-40 Methods MSCs and PC12 were cultured in vitro and identified by CD44 immunofluorescent staining; PC12 were damaged by Aβ1-40, and transferred by transwell followed by the classification into 5 groups. PI and Annexin-V co-fluorescent staining was performed, then PC12 apeptosis were detected by flow cytometry and EM; Supernatant was analyzed by enzyme-linked immunoadsordent assay （ELISA） to detected TGF-β,NGF,BDNF, and bFGF. Results About 96% MSCs showed CD44 positive cells. Co-culture group had the lowest rate of PC12 apeptosis （46. 17% ± 8. 28% ,F = 61. 637 ,P 〈 0. 01）. ELISA identified bFGF in all the groups, but only in experimental group, there was a significant difference compared with control group （ （598.76±41.32） pg/ml,F =24. 15, P〈0.01） ; TGF-β,NGF, BDNF could be detected without a significant difference compared with control group （ P 〉 0. 05 ） . Conclusion The co-culture system of MSCs and Aβ1-40 injured PC12 in vitro could inhibit apoptosis of PC12 induced by Aβ1-40 Thus grafted MSCs have the possibility to inhibit neuronal apeptosis by Aβ in the diseased brain.