Effect Of Invigorating Kidney And Nourishing Essence On MSCs Proliferation In Vitro And Its Mechanism

Research purpose and senseAccording to "essence and blood have the same source" and "stem cells have the same properties as congenital essence", we use these methods of nourishing essence and promoting blood, nourishing essence and fostering yin, nourishing essence and enriching yang, invigorating qi and manufacturing marrow to transforming blood, to study the effect of invigorating kidney and nourishing essence on mesenchymal stem cells (MSCs) proliferation in vitro. Combining traditional chinese medicine and stem cell research can provide new ideas for culture and amplification of MSCs in vitro, and provide strong experimental evidence for assisting clinical stem cell transplantation for treatment of diseases by Chinese medicine.On the basis of drug screening, we observed the influence of traditional Chinese medicine monomer, active ingredients and medicated serum which is useful for the promotion of MSCs on cytokine expression, to analyze their possible pathway. It can provide experimental basis for explaining its mechanism of promoting proliferation of MSCs, furnish more adequate scientific basis for the "essence" of the stem cell theory, and this study will be improved to the level of molecular.Research methods1. MSCs was isolated and purified by whole bone marrow adherent method; MSCs was identified by morphologic characteristics, surface antigens detected with flow cytometer.2. The effect of nourishing essence herbs-induced proliferation on MSCs was detected by MTT,it help us to judge which traditional Chinese medicine can promote proliferation of MSCs and to study its best time, concentration for promoting proliferation. 3. To cultivate MSCs 72h by medium of APS and its medicated serum, medium of THSG and polygonum multiflorum medicated serum, different cytokine mRNA expression were detected by RT-PCR, SCF mRNA expression was detected by Real-time PCR;SCF protein expression was detected by western blot;SCF content of supernatant was detected by ELISA.4. To cultivate MSCs 72h by polygonatum medicated serum, different cytokine mRNA expression were detected by RT-PCR, G-CSF mRNA expression was detected by Real-time PCR;G-CSF protein expression was detected by western blot.5. To cultivate MSCs 72h by dodder medicated serum,different cytokine mRNA expression were detected by RT-PCR, BMP-2 mRNA expression was detected by Real-time PCR;BMP-2 protein expression was detected by western blot.6. To cultivate MSCs 72h by medium of morinda officinalis polysaccharide, different cytokine mRNA expression were detected by RT-PCR, LIF,GM-CSF mRNA expression were detected by Real-time PCR;LIF,GM-CSF protein expression were detected by western blot.7. The weak kidney animal model was established by ovariectomy on female Sprague-Dawley (SD) rats aged 3 month;some model animal were fed with zuo gui pill.8. To investigate MSCs proliferation capacity of normal group, model group, and zuo gui pill group by comparing results of morphologic characteristics, surface antigens andβ-galactosidase staining senescent cells of differernt groups;different cytokine mRNA expression in the course of MSCs proliferation of differernt groups were detected by RT-PCR.9. To investigate differentiation capacity into osteoblasts of normal group, model group, and zuo gui pill group by compairing the expression of alkaline phosphatase (ALP) activity and the number of ALP stained positive cells.10. To investigate differentiation capacity into steatoblast of normal group, model group, and zuo gui pill group by compairing the expression of triglyeride (TG) and the number of oil red stained positive cells.Results1. We established a mature separation, purification, amplification technology platform of SD rat MSCs. The results of the surface antigens of MSCs detected by immunofluorescence and flow cytometer showed the positive expressions of CD29,CD44 and the negative expressions of CD34,CD45.2.10-1mol/L THSG,10% polygonum multiflorum medicated serum can significantly promote the proliferation of MSCs, and significantly promote SCF mRNA expression.10-1mol/L THSG significantly promote membrane binding and soluble SCF protein expression,10% polygonum multiflorum medicated serum significantly promote soluble SCF protein secretion.3.10% polygonatum medicated serum can significantly promote the proliferation of MSCs, and significantly promote G-CSF mRNA and G-CSF protein expression.4.1mg/mL morinda officinalis polysaccharide and 10% dodder medicated serum can significantly promote the proliferation of MSCs. 1mg/mL Morinda officinalis polysaccharide significantly promote LIF,GM-CSF mRNA and LIF protein expression.10% dodder medicated serum significantly promote BMP-2 mRNA expression.5.1mg/mL APS,10% APS medicated serum can significantly promote the proliferation of MSCs, and significantly promote SCF mRNA expression. 1mg/mL APS significantly promote membrane binding and soluble SCF protein expression, 10% APS medicated serum significantly promote soluble SCF protein secretion.6. Unit volume of primary bone marrow mononuclear cells, the number of CD34 negative cells in model group was significantly higher than normal group, and there was a trend that the number of CD105 positive cells in model group lower than normal group. From growth curve of P3,P5 MSCs, the proliferation capacity of MSCs in model group was significantly lower than normal group and Zuo gui pill group;β-galactosidase staining showed that the model group had the highest positive rate of aging cells, significantly higher than the normal group.7. In the proliferation process of MSCs, GM-CSF, LIF, CSF, BMP-2 mRNA expression of model group was significantly lower than normal group.8. On 18,21d of differentiating into osteoblasts, MSCs ALP expression of normal group was significantly higher than model group,Zuo gui pill group. From 14d, the rate of ALP stained positive cells of model group and Zuo gui pill group MSCs was significantly lower than normal group. From 14d of differentiating into steatoblast, the rate of oil red stained positive cells of model group and Zuo gui pill group MSCs was significantly higher than normal group.Conclusion1. The stable and uniformal MSCs could be obtained by whole bone marrow adherent method.2. 10-4mol/L THSG can promote the proliferation of MSCs, and it may be related to the promotion of SCF mRNA,membrane binding and soluble SCF protein expression.10% polygonum multiflorum medicated serum can promote the proliferation of MSCs, and it may be related to the promotion of SCF mRNA and soluble SCF protein expression.3.10% polygonatum medicated serum can promote the proliferation of MSCs, and it may be related to the promotion of G-CSF mRNA and G-CSF protein expression.4.10% dodder medicated serum can promote the proliferation of MSCs, and it may be related to the promotion of BMP-2 mRNA expression. lmg/mL morinda officinalis polysaccharide can promote the proliferation of MSCs may be related to the promotion of LIF,GM-CSF mRNA and LIF protein expression.5. lmg/mL APS can promote the proliferation of MSCs, and it may be related to the promotion of SCF mRNA,membrane binding and soluble SCF protein expression.10% APS medicated serum can promote the proliferation of MSCs, and it may be related to the promotion of SCF mRNA and soluble SCF protein expression.6. The decreased proliferation capacity of MSCs from ovariectomized rats might be associated with the decreased expression of GM-CSF,LIF,CSF,BMP-2 mRNA. To some extent, Zuo gui pill can improve proliferation capacity of weak kidney rats MSCs.7. Compared with normal rats MSCs, the ability of differentiation into osteoblast of MSCs from ovariectomized rats increased, and the ability of differentiation into adipocytes of MSCs reduced.