| Background: Our previous study has found that adipose tissue-derived stem cells(ADSCs) could engraft onto pulmonary arterioles and differentiated into pulmonary artery endothelial cells resulting in attenuation of pulmonary arterial remodeling, mean pulmonary arterial pressure(m PAP), and right ventricular hypertrophy(RVHI) in rats with monocrotaline(MCT)-induced pulmonary arterial hypertension(PAH). However, appropriate cell number, transplantation time and therapeutic window of ADSCs therapy have not been fully clarified. Objective: To investigate the therapeutic role of different number of ADSCs transplantation to different dose of MCT-induced PAH rats, and to explore the optimal cell number, intervention time and duration of therapy of ADSCs transplantation. Methods: ADSCs were isolated and cultured from subcutaneous adipose tissue of Sprague-Dawley rats’ groin by collagenase type I. The cell phenotype was identified by flow cytometry and the multiple differentiation potentials were confirmed by adipogenic and osteogenic differentiation respectively. m PAP and RVHI were determined at an appropriate time after ADSCs or normal saline(NS) was injected into rats through the left jugular vein as followed: ① Different number of ADSCs transplantation to PAH rats: 36 SD rats were divided into normal control group(NC group), PAH group and ADSCs group. PAH and ADSCs groups were administrated a single dose of 40mg/kg MCT by intraperitoneal injection. Different number of ADSCs [0.5×106(A1 group), 1.0×106(A2 group), 3.0×106(A3 group) and 5.0×106(A4 group)] were transplanted into rats at 7 days after MCT injection while NC and PAH group received equal volume of NS. The various parameters were detected at 4 weeks after MCT injection(n=6 for each group). ② Different number of ADSCs transplantation to normal rats: 25 SD rats were divided into NC and ADSCs group. Each group received an appropriate volume of NS through enterocoelia. Different number of ADSCs [0.5×106(N-A1 group), 1.0×106(N-A2 group), 3.0×106(N-A3 group) and 5.0×106(N-A4 group)] were transplanted into rats at 7 days after NS injection while NC and PAH group received equal volume of NS. The various parameters were detected at 3 weeks after ADSCs transplantation(n=5 for each group). ③ The transplantation of 1.0×106 ADSCs to different dose of MCT-induced PAH rats: 70 SD rats were divided into NC, PAH and ADSCs group. PAH and ADSCs groups were injected with 20mg/kg(P-20/A-20 group), 30mg/kg(P-30/A-30 group) and 40mg/kg(P-40/A-40 group) MCT through enterocoelia. ADSCs group were injected with 1.0×106ADSCs via left jugular vein at 7 days after MCT injection. The various parameters were detected at 4 and 8 weeks after MCT injection respectively(n=5 for each group at every time point). ④ 1.0×106 ADSCs were transplantated into PAH rats in different time point: 48 SD rats were divided into NC(NC-4w/NC-5w group), PAH(P-4w/P-5w group) and ADSCs group. PAH and ADSCs groups were injected with 40mg/kg MCT, then ADSCs group was injected with 1.0×106 ADSCs at day 1(A1d-4w group), day 7(A7d-4w group) and day 14(A14d-4w/A14d-5w group) via left jugular vein after MCT injection. Various parameters were detected at 4 weeks or 5 weeks after MCT injection respectively(n=6 for each group at every time point). Results: 1. ADSCs expressed mesenchymal stem cell markers CD29/CD90 without expression of the hematopoietic and endothelial cell markers CD31/CD34/CD45. Oil red O and alizarin red staining showed ADSCs could differentiate into adipocytes and osteocytes. 2. ①Compared with NC group, the m PAP and RVHI of PAH,A1,A3 and A4 group were increased, the RVHI of A2 group was increased while m PAP was no statistical difference; compared with PAH group, the m PAP of A2 group was decreased while RVHI was no statistical difference; there were no significant difference in m PAP and RVHI among PAH, A1, A3 and A4 group; there were also no significant difference in m PAP and RVHI between A1 and A2 group; compared with A2 group, the m PAP of A3 and A4 group was increased while RVHI was no statistical difference. ②Different number of ADSCs transplantation had no effect on m PAP and RVHI in normal rats. ③There were no obvious changes in m PAP and RVHI among NC, P-20 and A-20 group at 4 and 8 weeks after MCT injection. Compared with NC group, the m PAP and RVHI of P-30, A-30, P-40 and A-40 were increased at 4 weeks after MCT injection, while there were on statistical difference in m PAP and RVHI among NC, P-30 and A-30 group and all of rats in P-40 and A-40 group dead at 8 weeks after MCT injection. In addition, compared with P-40 group, the m PAP and RVHI of A-40 group were decreased at 4 weeks after MCT injection while there were no statistical difference in m PAP and RVHI between A-30 and P-30 group. ④Compared with NC group, the RVHI of P-4w, A1d-4w, A7d-4w, A14d-4w, P-5w and A14d-5w were increased; there were no significant difference in RVHI among P-4w, A1d-4w, A7d-4w and A14d-4w. Compared with NC group, the m PAP of P-4w, A1d-4w, P-5w and A14d-5w group were increased while it was no significant difference in A7d-4w and A14d-4w group; compared with P-4w group, the m PAP of A7d-4w and A14d-4w were decreased, while there were no significant difference in m PAP between these two groups as well as between P-4w and A1d-4w group; compared with A1d-4w group, the m PAP of A7d-4w was decreased while it was no significant difference in A14d-4w group. Compared with P-5w group, the m PAP and RVHI of A14d-5w was decreased. Conclusions: 1. A single dose of 40mg/kg MCT by intraperitoneal injection can establish a stable PAH rat model while lower dose of it can not. 2. 7 or 14 days after intraperitoneal injection of 40mg/kg MCT, transplantation of 1×106 ADSCs for 2-3 weeks could effectively reduce m PAP in rats. 3. RVHI is closely related to m PAP in PAH rats. |
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