title: Inhibition of apoptosis by knockdown of caspase-3 with siRNA in rat bone marrow mesenchymal stem cells.
abstract: Transplantation of bone marrow mesenchymal stem cells is a promising new strategy for the repair of infarcted cardiac tissue. However, the majority of transplanted bone marrow mesenchymal stem cells (BMSCs) die soon after transplantation, due in part to oxidative stress in the ischemic region. Oxidative stress is known to induce apoptosis through the activation of caspase-3. The aim of this study is to determine whether small interfering RNA targeting caspase-3 can inhibit the apoptosis of rat BMSCs in vitro. Caspase-3 siRNA expression vectors were prepared and transfected into rat BMSCs in the presence of liposomes. Western blot assay and real-time polymerase chain reaction (RT-PCR) were performed to detect caspase-3 expression. A retrovirus packaging system was employed to package 293FT cells producing caspase-3 siRNA virus, which were transfected into rat BMSCs. Those stably expressing caspase-3 siRNA were screened by Western blot assay and RT-PCR to determine caspase-3 expression levels. Stable transfection of caspase-3 siRNA significantly decreased caspase-3 protein (0.26 ± 0.001 vs. 0.42 ± 0.004, P < 0.05) and mRNA expression (0.19 ± 0.002 vs. 1, P < 0.05) in BMSCs compared to non-transfected BMSCs. Cells were incubated in H2O2 to induce apoptosis, which was detected by TUNEL staining, and BMSC morphology was not altered by either transient or stable transfection of caspase-3 siRNA. H2O2-induced apoptosis of BMSCs stably transfected with caspase-3 siRNA was dramatically reduced compared to that of normal BMSCs (11.0 ± 3.2 vs. 25.8 ± 4.2, P < 0.05). Caspase-3 knockdown BMSCs are thus more resistant to apoptosis than normal BMSCs, potentially increasing their survival rates under conditions that cause oxidative stress.
authors.affiliation: Department of Cardio-Thoracic Surgery, The Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Hua
authors.forename: Li-Bao
authors.initials: LB
authors.lastname: Liu
authors.forename: Jia-Liang
authors.initials: JL
authors.lastname: Liu
authors.forename: Meng
authors.initials: M
authors.lastname: Wang
authors.forename: Hui-Qi
authors.initials: HQ
authors.lastname: Jiang
authors.forename: Kuan
authors.initials: K
authors.lastname: Zeng
authors.forename: Yan-Qi
authors.initials: YQ
authors.lastname: Yang
authors.forename: Song-Ran
authors.initials: SR
authors.lastname: Yang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D053148:Caspase 3 / Q000235:genetics*; D018929:Cell Culture Techniques; D055785:Gene Knockdown Techniques; D059630:Mesenchymal Stem Cells; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D034741:RNA, Small Interfering; D053148:Caspase 3
keywords: BMSC; Cell therapy; myocardial infarction; oxidative stress; stable transfection