| Acute pulmonary thromboembolism (APTE) is a common clinical condition associated with significant morbidity and mortality. At present, the pulmonary embolism treatment with anticoagulation and thrombolytic therapy. But the drug treatment has poor specificity, short half-life, side effects and other disadvantages. Although promising, bone marrow-derived mesenchymal stem cell (BMSC) treatment for thrombus resolution remains controversy in previous studies. It has not been evaluated if BMSC is effective against APTE. In this study, using the external jugular vein injection of thrombus to replicate animal model of acute pulmonary embolism, and bone marrow-derived mesenchymal stem cell for therapy. We developed a new analysis method with serial-sectioning of the whole lung and calculating the size of each thrombus in volume and compared this new method with the conventional non-serial sectioning method previously reported. We found the employment of serial-sectioning of the whole lung is necessary for quantitative evaluation of pulmonary thrombi.Many studies have reported that cell have a sex, and found that female stem cells have more advantages than males in the treatment of many diseases.Treatment of thrombosis by stem cells have been reported in many studies, but did not explore the gender differences. This study was the first to explore the gender of the bone marrow mesenchymal stem cells on acute pulmonary embolism treatment, found that female bone marrow mesenchymal stem cells therapy was better than the male. GAPDH as a housekeeping protein has a variety of biological functions involved in addition to glycolysis, including DNA repair, tRNA transporter, membrane fusion and transport, cytoskeleton dynamics and cell death. Some scholars found gender differences in the expression of GAPDH on the study of rat brain. We hypothesized that the expression of GAPDH differences between male and female stem cell caused the acute pulmonary embolism treatment differences, our experiments also demonstrated that GAPDH played a critical role in the superior function of female BMSCs, possibly by regulating the expression of urokinase plasminogen activator. ObjectTo observe the effects of female or male BMSCs on treatment of PTEMethodFemale and male C57BL/6J mice,20-23g,6-8-week old, received intravenous injection of autologous thrombi via right jugular vein to produce PTE model. Then the mice were either untreated or treated with female or male BMSCs (2×106cells in0.5ml PBS via tail vein) in a sex-matched or a sex-mismatched way at4h,8h,or16h separately after thrombi injection. The experimental groups studied in either gender mice and each time point were as follows:1) PTE mice treated with PBS (control group);2) PTE mice treated with female BMSCs (F-BMSCs group); and3) PTE mice treated with male BMSCs (M-BMSCs group). One day after thrombi injection, mice were sacrificed, and the lungs were collected for further analysis.Result1. PTE model was successfully pruducedThe model group after thrombosis reproduction, through the stained sections can see very clear thrombus. The analysis of all emboli in the model group and treatment group, found the volume of most emboli was0.1-5×106μm3by a new analysis method with serial-sectioning of the whole lung and calculating the size of each thrombus in volume. The area of most emboli was1-10×103μm2by the conventional non-serial sectioning method previously reported. Pulmonary embolus distribution showed that more emboli were in the right lung than left lung, most emboli were located in the small blood vessels with the diameter0-30μm by the two ways which were generally consistent with clinical thrombosis and pulmonary distribution.2. It is more scientific and more reasonable to analyse the treatment effect by the total volume of thrombosis than by the total area; male and female stem cells stem cells were effective for treatment of acute pulmonary embolism, and the female stem cells have more advantages.About54%thrombus were missed by the total area as the literature commonly used. It is more accurate and objective to evaluate the treatment effect through the volume of all thrombus by serial sectioning.4h after autologous thrombi injection, both female BMSCs and male BMSCs had the effect, and the treatment effect of female BMSCs treatment effect was better, either in male mouse or in female mouse;8h after autologous thrombi injection, in male mouse we found that both female BMSCs and male BMSCs had the effect, and the treatment effect of female BMSCs treatment effect was better, in female mouse both female BMSCs and male BMSCs had the effect, but there was no difference between them;16h after autologous thrombi injection, in male mouse we found that only female BMSCs had the effect, and in female mouse both female BMSCs and male BMSCs had the effect, but there was no difference between them.ConclusionThe external jugular vein injection of thrombus can be successfully established an animal model of acute pulmonary embolism model of reproduction, distribution and clinical data of pulmonary thromboembolism is basically the same, this method of replication thrombosis of pulmonary embolism is feasible and reliable. Compared with the common area relative to the literature analysis and calculation method for thrombosis, we created whole lung sections thrombus volume analysis, more accurate and objective. Male and female stem cells are effective in the early treatment of thrombosis, and the female stem cell treatment effect better; and in the late thrombosis, only the females of stem cells. ObjectTo determine the role of GAPDH in the regulation of male and female BMSCs for the treatment of pulmonary embolism, to further explore the mechanism of stem cells in the treatment of acute pulmonary embolism.Method1. Female BMSCs were stably transfected with GAPDH interference plasmid or empty plasmid, male BMSCs were stably transfected with GAPDH overexpression plasmid or empty plasmid, and then western blotting to observe GAPDH expression2. Female and male C57BL/6J mice,20-23g,6-8-week old, received intravenous injection of autologous thrombi via right jugular vein to produce PTE model. Then the mice were either untreated or treated with BMSCs treatment with manipulated GAPDH expressions (2x106cells in0.5ml PBS via tail vein) in a sex-matched or a sex-mismatched way at4h after thrombi injection. The experimental groups studied in either gender mice were as follows:1) PTE mice treated with PBS (control group);2) PTE mice treated with female BMSCs with manipulated GAPDH expressions (F-BMSCs-siRNA-GAPDH group or F-BMSCs-siRNA vector group); and3) PTE mice treated with male BMSCs with manipulated GAPDH expressions (M-MSCs-OE-GAPDH group or M-MSCs-OE vector group). One day after thrombi injection, mice were sacrificed, and the lungs were collected for further analysis.3. Seria sections of whole lung, all odd pieces for HE staining, to find that continuous sections appeared thrombosis, then even section corresponding for immunofluorescence to test the localization of stem cells; western blotting to test the expression of uPA.Result 1. Stable transfection of GAPDHUnder normal circumstances, the expression of GAPDH was higher in female BMSCs than in male BMSCs; female BMSCs were transfected with GAPDH interference plasmid, GAPDH expression were significantly decreased, compared with the pure female BMSCs; male BMSCs were transfected with GAPDH overexpression plasmid, expression of GAPDH was significantly increased, compared with the pure male BMSCs.2. Reverse test in male and female acute PTE mice after manipulation of GAPDH expression in BMSCsStem cell therapy initiated4hours after embolization, the thrombus volume increased significantly in F-BMSCs-SiRNA-GAPDH group compared with F-BMSCs group in either male mice or female mice; while the thrombus volume reduced significantly in M-MSCs-OE-GAPDH group compared with M-BMSCs group in either male mice or female mice.3. Stem cell tracking and uPA expressionImmunofluorescence experiments showed that green fluorescence labeling stem cells located in the intravascular emboli edge attachment. The uPA expression was higher in female BMSCs than male BMSCs, while male BMSCs were transfected with GAPDH overexpression plasmid, expression of uPA was significantly increased, compared with the pure male BMSCs.ConclusionThe first part shows that female BMSCs had better treatment effect than male BMSCs on PTE. This part of the study shows that maybe the reason was that GADPH expression in female BMSCs was higher than in male BMSCs. Reverse test in male and female acute PTE mice after manipulation of GAPDH expression in BMSCs confirmed that treatment effect differences was caused by the different GAPDH expressions between the two BMSCs.uPA Changes in the BMSCs regulated by GAPDH and stem cell attachment in the rim of thrombus, showd that BMSCs can promote the dissolution of thrombi in the early period of acute PTE mainly through the secretion of some thrombolytic factors. |
PDF Full Text Request