The Gender Of The Bone Marrow Mesenchymal Stem Cells In The Treatment Effect And Its Mechanisms Of Cell Therapy Research To Pulmonary Hypertension

Part I The gender of the bone marrow mesenchymal stem cells in the treatment effect to pulmonary hypertensionObjective:For specific tracer stem cells into the body positioning and change, male stem cells transplant into female animals,in vivo by a Y chromosome-specific tracer, it is inevitable to ignore gender differences in stem cell treatment effect. In this study, bone marrow mesenchymal stem cells (BMSCs) separation from the same viviparous female and male mice of different genders observed BMSCs gender differences in the effect of treatment of pulmonary hypertension.Methods:In this study,8-week-old weight about22g C57mice, intravenous injection60mg/kg MCT to induced pulmonary hypertension, after three days intravenous give2×106different gender BMSCs treatment, after28days to evaluate treatment effects, used right heart hypertrophy index RV/(V+S) and lung tissue biopsy shows small pulmonary vascular muscle degree (muscularization) as indicators.Results: (1) Optical microscope visible a single BMSC show spindle-shaped, polygonal cell fusion was cobblestone; flow identification of cell surface markers: CD29-PE+(99.96%±0.03%);CD34-PE(0.34%±0.13%); D45-APC-Cy7(1.97%±0.38%).(2) In the treatment of pulmonary hypertension induced by MCT, female mice right heart hypertrophy index after the model significantly increased (0.278±0.009vs0.357±0.019,p<0.05), female BMSCs reduce right ventricular hypertrophy index exhibit significantly better than male BMSCs (0.275±0.026vs0.311±0.022, p<0.05); in male mice, female BMSCs also showed significantly better than male BMSCs (0.2835±0.028vs0.326±0.045, p<0.05).(3) In the treatment of pulmonary hypertension induced by MCT, pulmonary muscle of female mice (muscularization) after the model significantly increased (38.3%±2.9%vs82.3%±7.8%, p<0.05), but female BMSCs reduce pulmonary muscularization significantly better than male BMSCs (41.7%±5.8%vs55.3%±5.0%,p<0.05); and in male mice, female BMSCs also also showed significantly better than male BMSCs (40.0%±4.8%vs54.0%±6.9%, p<0.05).Conclusion:MCT-induced pulmonary hypertension in mice models, male and female BMSCs can effectively slow down right ventricular hypertrophy and pulmonary muscle degree of the mouse model, which play the role of effective pulmonary hypertension, female BMSCs exhibiting greater than the therapeutic effect of male BMSCs. Part Ⅱ The gender differences of bone marrow mesenchymal stem cells in the biological mechanisms in vivoObjective:Bone marrow mesenchymal stem cells(BMSCs) is introduced into the occurrence of pulmonary hypertension in the human and animal body, inevitable exposure to different pathological environment, the more common, including oxidative stress, inflammatory response, and so on. BMSCs must be able to adapt to these micro-environment to play a role in endothelial repair and treatment of pulmonary hypertension. Observation and comparison in oxidative stress, inflammatory response, female, male BMSCs biology behavior charge including the calcium signal, proliferation and migration, connexin43expression and other aspects of the differences, in order to clarify BMSCs in the treatment of pulmonary hypertension manifested in gender differences in mechanism.Methods:With different concentrations of histamine (1μmol/L,10μmol/L,100μmol/L) and different concentrations (100μmol/L,250μmol/L,500μmol/L) of hydrogen peroxide(H2O2) to stimulate BMSCs, ion imaging system with real-time fluorescence detection [Ca2+] i changes, sure the concentration of histamine and hydrogen peroxide you can make BMSCs exhibit distinct calcium signaling differences. Determine the concentration of histamine and hydrogen peroxide to stimulate BMSCs, detected cell proliferation by MTS, migration with the transwell chamber, using Western blot method compare male and female BMSCs connexin43and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) protein level.Results:(1) Histamine-induced female and male BMSCs calcium reaction:1μM histamine stimulation female BMSCs (F-BMSCs) approximately78%of cells showing typical calcium oscillation, male BMSCs (M-BMSCs)71.4%of the cells showed typical calcium peak and sustained elevated calcium; lOμM histamine stimulation F-BMSCs about78%of the cells showed typical calcium oscillation,74%of the M-BMSCs showed typical calcium peak and sustained elevated calcium;100μM histamine stimulation F-BMSCs approximately61%of the cells showed typical calcium peak and sustained calcium increase,67.4%the M-BMSCs showing typical calcium peak and sustained calcium increase.(2) H2O2-induced female and male BMSCs calcium reaction:100μM H2O2stimulate female BMSCs (F-BMSCs) approximately81%of cells showing typical calcium oscillation, male BMSCs (M-BMSCs)79.5%of the cells showed typical calcium peak and sustained elevated calcium;250μM H2O2stimulation F-BMSCs about73.3%of the cells showed typical calcium oscillation,75%of M-BMSCs showed typical calcium peak and sustained calcium increase;500μM H2O2to stimulate F-BMSCs about73%of the cells showed typical calcium oscillation,79%M-BMSCs showed typical calcium peak and sustained calcium increase.(3) BMSCs proliferation curve show that female BMSCs compared to male BMSCs is faster; in cell growth lag phase, with lOμM histamine and100μM H2O2stimulate cells after24h proliferation. Female, male BMSCs proliferation in the absence of stimulation did not differ significantly, but10μM histamine stimulation proliferation of female BMSCs accelerate and male BMSCs proliferation did not change significantly (1.42±0.08vs1.01±0.05, p<0.05),100μM H2O2stimulate proliferation of female BMSCs accelerate and male BMSCs proliferation did not change significantly (1.34±0.11vs0.94±0.06, P<0.05).(4)10μM histamine and100μM H2O2stimulate cell migration compared, without stimulation female BMSCs migrate each field the number of cells than male BMSCs (32.6±5.8vs9.8±4.9,p<0.05),10μM histamine stimulate female BMSCs migration-cells increased and male BMSCs did not change significantly (40.8±7.7vs30.4±4.0, p<0.05);100μM H2O2stimulation female BMSCs migrated cells increase and male BMSCs did not change significantly (42.3±8.4vs32.4±5.4, p<0.05).(5) Histamine and H2O2stimulated cells Connexin43expression:female, male BMSCs in the absence of stimulation Connexin43phosphorylated and non-phosphorylated expression did not differ significantly, histamine stimulation female BMSCs phosphorylation Connexin43(pCx43) increased and male BMSCs reduced (1.83±0.44vs0.57±0.14,p<0.05), female BMSCs dephosphorylated Cx43(dpCx43) reduced and male BMSCs increase (0.66±0.33vs1.46±0.19, p<0.05); H2O2stimulation female BMSCs pCx43increased and male BMSCs decrease (1.65±0.27vs0.60±0.03, p<0.05), female BMSCs dpCx43decreased and male BMSCs increased (0.89±0.19vs 1.31±0.14,p<0.05).(6) Histamine and H2O2stimulation BMSCs GAPDH protein levels:in the absence of any stimulus male BMSCs GAPDH basal levels equivalent to0.626±0.07in female BMSCs, there are significant differences (p<0.05). Female BMSCs after histamine stimulation is an increase of1.38±0.15(p<0.05), after H2O2stimulation female BMSCs GAPDH protein levels increased to1.36±0.17(p<0.05), while male BMSCs did not change significantly (p>0.05).Conclusion:H2O2and/or histamine stimulated analog oxidative stress, inflammation status in vitro tests, the different genders BMSCs in cell proliferation, migration, and [Ca2+]i dynamics and other aspects were significant difference. Surprisingly, compared with male BMSCs, female BMSCs have a higher GAPDH (glyceraldehyde-3-phosphate dehydrogenase) protein expression levels. GAPDH has recently been shown to be a multifunctional protein, have different functions. We propose that GAPDH may be a key factor for BMSCs gender differences. Part Ⅲ Affect therapeutic effect mechanism of gender differences bone marrow mesenchymal stem cells in vivoObjective:In order to explore GAPDH as a key role in bone marrow mesenchymal stem cells(BMSCs) treat pulmonary hypertension of mice, as well as in vivo tracking BMSCs treat pulmonary hypertension possible mechanisms.Methods:Separation and purification BMSCs with viviparous different gender C57mice, using man-regulation GAPDH over-expression (overexpression-GAPDH) or interfere with plasmid (siRNA-GAPDH) between male and female BMSCs GAPDH expression, through the detection GAPDH expression and evaluation on the biological behavior of BMSCs in calcium signaling, proliferation and migration.In vivo MCT-induced pulmonary hypertension mouse model, the male and female mouse model given human intervention GAPDH expression of the male and female BMSCs therapy, and use plasmid carrying the GFP tracer to stem cells, using tissue immunofluorescence technical observations BMSCs on endothelial repair.Results:(1) Westernblot detection transfected BMSCs GAPDH expression:F-BMSCs after the interference level of GAPDH and no significant difference between the base levels of the M-BMSCs, M-BMSCs after overexpression of GAPDH levels were significantly increased and the F-the BMSCs basic levels no significant difference.(2) Histamine and H2O2h-induced transfected BMSCs calcium reaction:10μM histamine stimulation F-BMSCs+siRNA vector about65%cells showed typical calcium oscillation, approximately57%F-BMSCs+siRNA-GAPDH showed typical calcium peak and sustained calcium increase, approximately68%of M-BMSCs+overexpression vector showed typical calcium peak and sustained calcium increase, approximately64%of M-BMSCs+overexpression GAPDH showed typical calcium oscillations;100μM H2O2stimulate F-BMSCs+siRNA vector about64%of the cells showed typical calcium oscillation. F-BMSCs+siRNA-GAPDH approximately71%cells showed typical calcium peak and sustained calcium increase, M-BMSCs+overexpression vector approximately79%cells showed typical calcium peak and sustained calcium increase, M-BMSCs+overexpression-GAPDH approximately60%cells showed typical calcium oscillation.(3) Histamine and H2O2-induced transfected cell proliferation and migration:histamine and H2O2-induced interference GAPDH of female BMSCs proliferation did not change significantly, whereas overexpression of GAPDH male BMSCs proliferation accelerated (*p<0.05), effectively reversing BMSCs proliferation change. Histamine and H2O2-induced interference or overexpression cell in cell migration change. Migration did not change significantly after histamine and H2O2-induced interference GAPDH female BMSCs, while the over-expression of GAPDH of male BMSCs migration accelerate (*p<0.05), effectively reversing the changes in BMSCs migration ability.(4) Cells after transfection efficacy of treatment of pulmonary hypertension in mice: overexpression of GAPDH of male BMSCs for the mitigation right ventricular hypertrophy of MCT-induced rat model of pulmonary hypertension and right ventricular hypertrophy (RV/(LV+S)) is better than the interfere GAPDH of female BMSCs (*p<0.05). Overexpression of GAPDH of male BMSCs for ease pulmonary vascular muscle degree of MCT-induced rat model of pulmonary hypertension is better than interference GAPDH of female BMSCs (*p<0.05).(5) In vivo, the cells were observed after transfection efficacy of the treatment of pulmonary hypertension in mice comparison:either female rats or male mice, over-expression GAPDH of male BMSCs involved in the repair of vascular endothelial share of the total number of blood vessels was significantly increased (*p<0.05), the interference GAPDH of female BMSCs participation repair of vascular endothelial proportion of the total number of blood vessels attributable to lower (*p<0.05).Conclusions:GAPDH has recently been shown to be a multifunctional protein, have different functions. Human control BMSCs’ GAPDH protein levels by molecular biology techniques, effectively reversing male and female BMSCs in the biological behavior and the treatment effect of pulmonary hypertension. We implanted BMSCs can be directly involved in the repair of vascular endothelium in vivo tracer technology.