| Objective:1.The properties of rat adipose mesenchymal stem cells(ADSCs)transfected with granulocyte chemotactic protein 2(GCP-2)gene were determined by cell experiments.2.To establish a rat model of pulmonary hypertension(PAH)and observe the relieving effect of adipose mesenchymal stem cells(ADSCs)transfected with GCP-2gene on pulmonary hypertension.Methods:1.GCP-2-ADSCs were prepared,and the expression of GCP-2 and eNOS proteins in ADSCs transfected with GCP-2 gene was detected by Western blotting.In addition,real-time quantitative PCR(QPCR)was used to detect the expression changes of GCP-2 and eNOS factors in ADSCs before and after transfection of GCP-2.The changes of cell migration and tubule formation ability of ADSCs before and after transfection of GCP-2 gene were compared.2.Twenty-four healthy male 6-week-old SD rats were randomly divided into normal control group(Control group),monocrotaline(MCT)-induced pulmonary hypertension group(PAH group),untransfected rat adipose mesenchymal stem cell treatment group(ADSCs group)and GCP-2 transfected rat adipose mesenchymal stem cell treatment group(GCP-2-ADSCs group),with 6 rats in each group.Preparation of PAH model: PAH group,ADSCs group and GCP-2-ADSCs group were respectively given intraperitoneal injection of 1% monocrotaline(MCT)50mg/Kg,while Control group was given the same amount of normal saline intraperitoneal injection.After 3 weeks of modeling,the mean pulmonary artery pressure(m PAP)of the model of pulmonary hypertension induced by MCT was measured to verify the effect of the model.After the successful establishment of the model,the corresponding cell therapy was carried out.The rats in the simple adipose mesenchymal stem cell treatment group(ADSCs group)and the GCP-2 transfected rat adipose mesenchymal stem cell treatment group(GCP-2-ADSCs)were injected with1 × 106 corresponding cells from the tail vein and diluted with 0.5ml saline for 2weeks,once a week.At the same time,the same amount of normal saline was injected into the tail vein in the Control group and the MCT-induced PAH group.After the corresponding cells were treated for 2 weeks,m PAP was measured,and then the corresponding heart tissue was weighed to calculate the right ventricular hypertrophy index(RVHI).The lung tissue was stained with HE to observe the changes of lumen size and vascular wall thickness of pulmonary arterioles.Westernblotting was used to detect the expression of GCP-2,eNOS and other proteins in local lung tissue,and QPCR to detect the expression of GCP-2,α-actinmRNA and other cytokines in lung tissue of rats in each group.ELISA was used to detect the expression of IL-17 and other inflammatory factors and anti-inflammatory factor IL-10 in the lung tissue of rats in each group.Results:1.Cell assay results(1)FACS analysis showed that ADSCs and GCP-2-ADSCs showed MSCspecific characteristics and expressed CD29 positively,with expression rates of99.46% and 99.62%,respectively.CD45 was negative and the expression rates were0.03% and 0.02%,respectively.(2)Cell migration and tubule formation experiments showed that the cell migration ability of GCP-2-ADSCs was significantly enhanced than that of simple ADSCs after 24 h cell culture.And compared with the pure ADSCs,GCP-2-ADSCs significantly improve the ability of cell of blood vessel formation,and this difference was found to be statistically significant(P <0.01).(3)Western blotting detected the expression levels of GCP-2 and eNOS proteins: Compared with the simple ADSCs,the expression levels of GCP-2 and eNOS proteins detected in GCP-2 and ADSCs increased,and this difference was found to be statistically significant(P <0.01).(4)The mRNA expression of anti-inflammatory vasodilator factor was detected by QPCR,and the expression of inflammatory factors was detected by Elisa.Compared with ADSCs alone,GCP-2-ADSCs increased the expression of antiinflammatory vasodilator factor GCP-2 and eNOS.At the same time,the expression of inflammatory factor TNF-α was inhibited,and this difference was found to be statistically significant(P <0.01).2.Therapeutic effect of GCP-2-ADSCs on cardiopulmonary tissue in rats with pulmonary arterial hypertension(1)Compared with PAH group and ADSCs group,m PAP and RV/(LV+S)in GCP-2-ADSCs group were significantly decreased,and this difference was found to be statistically significant(P <0.01).(2)HE staining of lung tissue specimens: Under the optical microscope(×200),inflammatory cell infiltration of lung tissue in the GCP-2-ADSCs group was significantly reduced,and pulmonary artery stenosis was significantly improved,relative to the PAH group and the ADSCs group.(3)ELISA was used to detect the levels of inflammatory and antiinflammatory factors in the lung tissue of rats in each group: compared to the PAH group and the ADSCs group,although the levels of the inflammatory factors IL-17,TNF α,IL-6,and IL-23 in the GCP-2-ADSCs group were significantly lower than those in the PAH and ADSCs groups,the level of expression of the anti-inflammatory factor IL-10 was significantly increased(P <0.01).(4)QPCR was used to detect the mRNA expression of GCP-2,VEGF and α-actin in the local lung tissue of the rats in each group: compared with Control group,the expression of GCP-2mRNA in PAH group decreased significantly,while the expression of VEGF and α-actin increased significantly,and the difference was statistically significant(P<0.01).In comparison to the PAH group,the mRNA expression of GCP-2 in GCP-2-ADSCs group increased,while the expression of VEGF and α-actin decreased significantly,and this difference was found to be statistically significant(P <0.01).(5)Western Blotting was used to detect the protein expressions of GCP-2,VEGF,α-actin,Apelin,and MCP-1 in the local lung tissue of the rats in each of the groups: in comparison to the PAH group,the expression of GCP-2 which had antiinflammatory effect and Apelin protein that improved endothelial function in GCP-2-ADSCs group was significantly higher than that in GCP-2-ADSCs group,but the expression of VEGF,α-actin and MCP-1protein which could promote vascular proliferation decreased significantly,and this difference was found to be statistically significant(P <0.01).Conclusions:1.ADSCs transfected with GCP-2 gene can interfere with MCT-induced PAH rats,reduce mean pulmonary artery pressure,improve pulmonary vascular remodeling and right ventricular hypertrophy,which may be a new target for the treatment of pulmonary hypertension.2.ADSCs transfected with GCP-2 gene upregulated the expression of GCP-2,eNOS and Apelin in lung tissue,which may relieve pulmonary artery pressure in PAH rats by activating Apelin/APJ signal transduction and targeting regulation of downstream eNOS. |
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