| Objective: The co-transplantation of Bone Marrow Mesenchymal Stem Cell(BMSC)and islet cells can improve the survival rate,promote the proliferation of grafts and increase the insulin release,therefore,co-transplantation of BMSC with islets may be a feasible solution to the problem of islet transplantation alone,but this solution requires a large number of BMSC,so we look for BMSC that can stimulate BMSC proliferation,and can regulate its secretion function of the drug has a certain practical significance.In order to investigate the mechanism of the proliferation of rat BMSC induced by the euonymus fortunei drug-containing serum,the model of inflammatory injury of rat islets was established by using the INS-1 islet cells and BMSC,to observe the protective effect of BMSC on INS-1 cells in co-culture with INS-1 cells.Methods: Rat BMSC were isolated,cultured and purified by whole-bone marrow adherent method.The morphology of BMSC was observed under microscope.The surface markers CD44,CD45 and CD90 of BMSC were identified by Flow cytometry The SD rats were given intragastric administration of euonymus fortunei extract for 2 times per day,and the euonymus fortunei drug-containing serum was extracted from abdominal aorta after 1 week.The rat BMSC was treated with serum containing different concentrations(1%、2%、4%、8%、16%).The cell growth was compared by MTT assay Then the rat BMSC were divided into blank control group,euonymus fortunei drug-containing serum group,Notch1 siRNA group and Notch1 siRNA + euonymus fortunei drug-containing serum group,the proliferation rate of BMSC was measured by MTT assay after the serum containing euonymus fortunei was cultured for 24 hours,the contents of Jagged1,NICD,DLL1 mRNA and Jagged1,Notch1,DLL1,Hes1,Hey1 protein were measured by totalRNA and protein of each group.MTT assay was used to detect the viability of INS-1 islet cells induced by LPS in different time and concentration.The model of inflammatory injury of islets of INS-1 was established with LPS and BMSC was pretreated with euonymus fortunei drug-containing serum.The contents of TNF-α、IL-6 and insulin were detected by ELISA kit.Results:1.The BMSC of SD rats were observed with an inverted microscope.When cultured for 96 hours,the colony area increased continuously,and gradually fused with the surrounding colonies.When transferred to the first generation,the cells were passivated,simple in shape and grew in a long shuttle shape,mainly in parallel arrangement or in a whirlpool shape.2.The results showed that the positive rate of CD90 and CD44 were98.22% and 86.95%,respectively,while the negative rate of CD45 was 9.9%.The positive rate of CD90 and CD44 was 98.22% and 86.95%,respectively.3.The Rat BMSC was treated with different concentration of euonymus fortunei and its absorbance was measured by MTT method.The results showed that the concentration of euonymus fortunei was between 1% and16%,which could promote the proliferation of BMSC,when the concentration of serum was 8%,the effect on cell proliferation was the most significant(p < 0.01).As the concentration continued to increase,the proliferation rate showed a downward trend,therefore,the concentration of euonymus fortunei 8% serum is the best drug intervention concentration.4.After 24 hours of siRNA silencing Notch1,the expression of Notch1 mRNA in Notch1 siRNA group was significantly lower than that in NC siRNA group(p < 0.05).5.The proliferation rate of BMSC in the euonymus fortunei drug-containing serum was measured by MTT method.The results showed that the proliferation rate of BMSC in the euonymus fortunei drug-containing serum group and Notch1 siRNA + euonymus fortunei drug-containing serum group BMSC were higher than that in the control group,compared with Notch1 siRNA group,the proliferation rate of BMSC in Notch1 siRNA + euonymus fortunei drug-containing serum group was higher than that in Notch1 siRNA group(p < 0.05).6.After 24 hours of intervention with drug-containing serum,the expression of Notch1 signal pathway related genes Jagged1,NICD,DLL1 mRNA in blank control group,euonymus fortunei drug-containing serum group,Notch1 siRNA Group,Notch1 siRNA + euonymus fortunei z containing serum group were detected by RT-q PCR.The results showed that Jagged1,NICD,DLL1 mRNA levels in the serum group with euonymus fortunei were significantly higher than those in the blank control group(p <0.05),the expression of Jagged1,NICD and DLL1 mRNA in Notch1 siRNA group decreased significantly(p < 0.05),compared with Notch1 siRNA group,the expression of Jagged1,NICD and DLL1 mRNA in Notch1 siRNA +euonymus fortunei serum group increased significantly(p < 0.05).7.After 24 hours of intervention with drug-containing serum,Western Blot was used to detect the expression of Notch1 signal pathway related proteins Jagged1,Notch1,DLL1,Hes1,Hey1 in blank control group,euonymus fortunei drug-containing serum group,Notch1 siRNA Group,Notch1 + euonymus fortunei drug-containing serum group.The results showed that the contents of Jagged1,Notch1,DLL1,Hes1 and Hey1 in the medicated serum group were significantly higher than those in the control group(p < 0.05).The contents of Jagged1,Notch1,DLL1,Hes1 and Hey1 decreased significantly in Notch1 siRNA group(p < 0.05).The levels of Jagged1,Notch1,DLL1,Hes1 and Hey1 in Notch1 siRNA + euonymus fortunei serum group were significantly higher than those in Notch1 siRNA group(p < 0.05).8.The results showed that with the increase of LPS concentration,the viability of INS-1 islet cells decreased continuously.After 6 hoof LPS treatment,INS-1 islet cells were injured by LPS,the half lethal dose(IC 50)of LPS to INS-1 islets was 1.93 μg/m L,the IC 50 of LPS to INS-1 islets was1.76 μg/m L at 12 h,and LPS to INS-1 islets was 24 h,the IC50 of INS-1islets was 1.21 μg/m L.The effect time was 24 hoand the intervention concentration was 1.21 μg/m L.9.The contents of TNF-α and IL-6 in the culture supernatant of INS-1islet cells in the model group were significantly higher than those in the control group(p < 0.01).Compared with the model group,the contents of TNF-α and IL-6 in the supernatant of INS-1 islet cells in the control group and the experimental group were significantly decreased(p < 0.01),the contents of TNF-α and IL-6 in the supernatant of INS-1 islet cells were significantly decreased(p < 0.01).10.Compared with the blank group,the Insulin content in the culture supernatant of INS-1 islet cells in the model group decreased significantly(p< 0.01),and the Insulin content in the model group decreased significantly(p< 0.01),the Insulin content in the culture supernatant of INS-1 islet cells was significantly increased in the control group and the experimental group(p <0.01),the Insulin content in the culture supernatant of INS-1 islet cells in the experimental group was significantly increased(p < 0.01).Conclusions:1.Euonymus fortunei medicated serum can promote BMSC proliferation in rats.2.The mechanism of promoting BMSC proliferation by euonymus fortunei drug-containing serum may be related to the activation of Notch1 signal pathway.3.BMSC after intervention with medicated of euonymus fortunei drug-containing serum,can promote the release of insulin from islet cells and improve the impairment of insulin release induced by inflammatory injury. |
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