| Objective:To investigate the effect of exocrine derived from adipose stem cells during osteoinduction on the proliferation and mineralization of osteoblasts.Methods:(1)Adipose stem cells were extracted from SD rats and identified by microscope and flow cytometry.(2)Adipose stem cells were divided into two groups: one group was cultured in osteogenic induction medium,the other group was cultured in common medium;After 14 days of culture,the exosomes in the two groups of culture media were separated by ultradifferential centrifugation,and the morphology of exosomes was observed by transmission electron microscope.(3)The miRNA sequence of the two secretions was performed,and the miRNA expression differences in the two secretions were quantified and compared by miRNA microarray analysis.(4)Adipose stem cells were transfected with miR-34a-5p silencing lentivirus.After 48 hours of transfection,the success of miR-34a-5p silencing was confirmed by PCR;At the same time,whether miR-34a-5p produced by adipose stem cells silenced by miR-34a-5p in the exocrine body during osteogenesis induction is also silenced.(5)The osteoblasts were divided into four groups: the control group was given normal medium culture without any intervention;In observation group A,the exocrine secreted by normal adipose stem cells during osteogenesis induction was given intervention;Observation group B was treated with miR-34a-5p to silence the secretion of adipose stem cells during osteogenesis induction;Observation group C,given exocrine secretion+Wnt secreted by normal adipose stem cells during osteogenesis induction/ β-RPRD1 A,a blocker of the catenin pathway,intervened.(7)To construct the rat model of femoral bone defect,the rats were randomly divided into three groups: normal saline group,0.2 ml of normal saline was injected into the bone defect;In the normal exocrine group,0.2 ml of exocrine saline produced during osteogenesis induction with normal adipose stem cells was injected into the bone defect;In the miR-34a-5p silencing exocrine group,0.2 ml of exocrine saline containing miR-34a-5p silencing adipose stem cells produced during osteoinduction was injected into the bone defect;After 4 weeks of treatment,the femoral modeling area of rats was extracted for HE staining.Results:(1)The adipose stem cells obtained by the standard method are closely arranged under the microscope,with complete morphology and good growth status;Flow cytometry showed that more than 99% of the cells expressed CD29 and CD90 on the surface,and the results showed that the obtained cells were adipose stem cells.(2)Through transmission electron microscope,it was observed that the exocrine body was a round membranous vesicle with a diameter of about 100 nm.(3)Through miRNA microarray analysis,it was found that there was a significant difference in miRNA in the secretion of adipose stem cells after osteogenesis induction and without osteogenesis induction.Compared with the miRNA in the secretion of adipose stem cells without osteogenesis induction,eight miRNAs,including miR-302b-3p,miR-302d-3p,and miR-34a-5p,were significantly overexpressed in the secretion of adipose stem cells induced by osteogenesis,of which miR-34a-5p was most significantly overexpressed(P<0.01).(4)PCR confirmed that miR-34a-5p silencing virus successfully silenced the miR-34a-5p gene of adipose stem cells,and the expression of miR-34a-5p in the secretions secreted by the corresponding cells also decreased.(5)CCK-8experiment showed that the number of osteoblasts in observation group A was significantly higher than that in control group and observation group B(P<0.05);There was no significant difference between control group and observation group B(P>0.05).The alkaline phosphatase staining experiment showed that the alkaline phosphatase staining of observation group A was deeper than that of control group and observation group B(P<0.05);There was no significant difference between control group and observation group B(P>0.05).Through the alizarin red staining experiment,it was found that the alizarin red staining of observation group A was deeper than that of control group and observation group B(P<0.05);There was no significant difference between control group and observation group B(P>0.05).The results of PCR showed that the expression of osteogenic differentiation gene in observation group A was higher than that in control group and observation group B(P<0.05);There was no significant difference between control group and observation group B(P>0.05).(6)The results of PCR showed that RPRD1 A blocked the secretion of adipose stem cells induced by osteogenesis and promoted the high expression of osteoblastic marker genes(P<0.05);Mi R-34a-5p silencing blocked the secretion of adipose-derived stem cells induced by osteogenesis to promote osteoblast Wnt/ β-The activation marker gene of the catenin pathway was highly expressed(P<0.05).(7)Through animal experiment HE staining,it was found that the bone mass of mice treated with exocrine secretion induced by adipose stem cells was significantly increased and the continuity of bone trabeculae was more complete.Conclusion:The secretion derived from adipose stem cells during osteoinduction activates Wnt via miR-34a-5p/ β-Catenin signaling pathway promotes osteoblast proliferation and mineralization. |
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