Objective HIF-1a, SDF-1a and VEGF gene expression affected by HIF-1a siRNA in MSCs.
Methods Bone marrow mesenchymal stem cells were cultured in vitro (3–5 generation,), inverted microscope was used to observed morphology, flow cytometry was used to detective of surface markers CD11b /c, CD34, CD44 and CD90. MSCs were divided into five groups, hypoxia group (only hypoxia), liposome group (MSCs transfected with empty liposomes), siRNA609 (MSCs transfected with siRNA609 sequence), siRNA658 (MSCs transfected with siRNA658 sequence), siRNA2070 (MSCs transfected with siRNA2070 sequence). The cells were cultured under hypoxia for 24 h, HIF-1a gene expression was dectived by RT-PCR. MSCs were divided into four groups, control group (without any treatment), hypoxia group (hypoxia 24 h), liposome control group (MSCs transfected with liposome then hypoxic 24 h), RNA interference (MSCs transfected with RNA interference sequences then hypoxic 24 h). MSCs mRNA expression RT-PCR, MSCs supernatant protein levels was detected by ELISA, MSCs hypoxic supernatant stimulate rat smooth muscle cell proliferation was detected by CCK8.
Results 1. Flow cytometry detective CD11b /c negative, CD34 negative, CD44 positive, CD90 positive cells respective reached 84.2%±0.2%, 97.91%±0.7%, 99.8%±0.9%, 97.7%±0.4% and CD44+/CD34—cell number reached 99.4%±0.4%. 2. SiRNA transfected cells could be detected the green fluorescent signal, lipsome group and the control group could not be detected the green fluorescence signal. 3. RT-PCR results showed that siRNA609, siRNA658, siRNA2070 group compared with hypoxia group HIF-1a gene expression was lower (p<0.05), the siRNA658 group is the least (p<0.05). 4. RT-PCR results revealed that compared with the normal control group hypoxia group HIF-1 a, SDF-1 a and VEGF gene expression increased (p<0.05), and compared with the lipsome control group RNA interference group HIF-1 a, SDF-1 a and VEGF gene expression reduced (p<0.05). 5. ELISA results revealed that compared with the normal control group hypoxia group HIF-1 a, SDF-1 a and VEGF content were increased (p<0.05), compared with the liposome control group RNA interference group HIF-1 a, SDF-1 a, VEGF content was decreased (p<0.05). 6. CCK8 results revealed that compared with the normal group control group hypoxia can promote smooth muscle cell proliferation (p<0.05), compared with the liposome control group RNA interference group proliferation is weak (p<0.05).
Conclusion 1. HIF-1a, SDF-1a and VEGF gene expression can be affected by HIF-1a siRNA in MSCs. 2. Hypoxia can made HIF-1 a, SDF-1 a and VEGF gene expression increased. 3. SDF-1 a and VEGF gene expression may be controlled by HIF-1 a in MSC. 4. Cell culture medium stimulate SMC profliction can be reduced by RNA interfence.
- Hypoxia-inducible factor
- RNA interference
- cell factor
- Stromal Derived Factor 1 a
- Vascular endothelial growth factor
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