| Objective: First, isolation and identification of the ADSCs from the inguinal of rats; Second, construction of lentiviral vector mediated HO-1 expressed with high level in ADSCs; Third, to investigate the viability 、 anti-apoptosis 、 osteogenic differentiation and migration effect of HO-1 overexpression in ADSCs. At last, the strategy of HO-1 delivery into bone tissue engineering was discussed.Methods: 1. ADSCs were isolated by the digestation of collagenase enzyme and centrifugation. Identification of the isolated ADSCs from the morphology 、 multipotential capability(the osteogenic and adipogenic differentiation) and cell surface marker. 2. Construction and transduction of expression vectors containing HO-1 gene, the result were demonstrated primarily by bacterial PCR. After enveloping, the virus collected at 48 and 72 h was used for transduction. ADSCs were transducted by lentivrus medium. The HO-1 protein expression levels in the ADSCs were assessed via western blot analysis. 3. Cell culture groups: We divided the cell cultures into four groups: ADSCs transduced with lentiviral-HO-1(A group); ADSCs transduced with lentiviral-GFP(B group); ADSCs-HO-1 treated with Sn-protoporphyrin(Sn PP, 5 μM)(D group), an inhibitor of HO activity; and un-transduced ADSCs without inhibitor treatment(C group). Cell viability was determined using the MTT assay method at different time points. The apoptosis assay in serum-deprive medium were investigated using a C6 flow cytometer with an apoptosis kit. Cell osteogenic differentiation assay was investigated through q RT-PCR after seven and fourteen days induction. After 21 days of osteogenic culture, the mineralization of the different groups was determined using 1% Alizarin red staining and confirmed by von Kossa staining kit. Cell scratch experiment and Transwell chamber were carried out to analyze cell migration assay.Results: 1. The adherent cells obtained from rat inguinal tissue were expanded in vitro and exhibited fibroblast-like morphology. The cells were positive for the rat MSC markers CD29(99.5%), CD90(52%), and CD44(70.7%) but were negative for CD31(6.58%) and CD45(3.73%). These results indicate that the expanded cells included a large population of ADSCs and were not contaminated with endothelialcells. We examined the adipogenic and osteogenic differentiation potentials of the isolated ADSCs. The results demonstrate that the expanded ADSCs used in this study have a multi-lineage differentiation potential. 2. Identification of the p Zs G-HO-1 and HO-1 amplification products The Puc57-HO-1 plasmid and p Zs G vector were double-digested with Not I and Bam H I simultaneously. The purified fragment of HO-1 and digested p Zs G vector were connected via in-fusion enzyme ligation. DH5α competent bacterial cells were transformed, and three positive bacterial clones containing the HO-1 coding sequence were identified via bacterial PCR. As predicted, the HO-1 product generated by the two primers ran approximately 870 bp. In addition, the primary expression of HO-1 was identified in ADSCs. 3. As indicated by MTT method, no difference was obtained on the first day between A and B groups. By the end of day 2, the percent cell viability(percentage) in the Lenti-HO-1 group was significantly increased compared with the C group(p < 0.05). We found that this protective effect could last for at least seven days. After the cells were stained with annexin V/PI, the percentages of early apoptotic cells were 31.87±3.15% and 28.67±2.43% in the B and C groups, respectively, whereas that in the A group was only 20.7±3.01%( p < 0.05). The percentages of late apoptotic/necrotic cells were 26.9±4.29% and 26.67±5.42% in the B and C groups, respectively, whereas that in A group was 20.6±1.93%( p < 0.05). The expression of the bone-related gene Runx2 and ALP in the A group on day 7 was markedly higher than those found in the B and C groups. At day 14, the expression levels of Runx2, ALP, OCN and Col I in the A group were elevated compared with the levels found in the B and C groups. No obvious in OCN and Col I gene expression was observed on day 7. However, at day 14, the expression of both of these genes was elevated compared with the levels detected on day 7. In addition, the calcium nodule formation in the A group was significantly promoted compared with the B and C groups. The results of scratch experiment was shown that the rate of migration for the A group was 69.3%±3.8%, which is significantly enhanced compared with B(31.5%±8.4%) and C groups(32.8%±8.6%). However, after the inhibition of HO-1activity by Sn PP, the rate of cell migration was only 23.2%±6.3%. In addition, the effect of HO-1 overexpression on migration was verified through a transwell chamber experiment. The conditioned medium from the A group significantly promoted the recruitment of non-transduced ADSCs, and the average number in each field was three-fold higher compared with that found in the C group.Conclusion: ADSCs were isolated successfully, which had multi-differentiation capacity and mesenchymal stem cell surface marker. The lentiviral vector containing HO-1 target gene was constructed, which could mediated HO-1 expression in ADSCs successfully. Without inhibitor of HO-1 activity, the overexpression of HO-1 increased cell viability of ADSCs, had an anti-apoptosis effect on ADSCs, stimulated the osteogenic differentiation of ADSCs; what’s more, the overexpression of HO-1 promotes the recruitment of non-transduced ADSCs. |
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