| ObjectiveThe green fluorescent protein-labeled bone marrow mesenchymal stem cells were cultured in vitro and transplanted into osteoporotic rats.To explore the distribution of bone marrow mesenchymal stem cells in rats and the therapeutic effects on bone mineral density and bone microstructure on osteoporosis.Methods1.Green fluorescent protein(GFP)-labeled rat bone marrow stromal stem cells(GFP-rBMSCs)were cultured and their surface specific antigens CD34,CD44,CD45 and CD90 were determined.2.Forty-eight female SD rats aged 3 months were randomly divided into 2 groups,the operation group(n=24)and the sham operation group(n=24).The operation group underwent ovariectomized(OVX)surgery.The osteoporosis model was established successfully after 24 weeks.The rats in the operation group were randomly divided into 2 groups:control group(n=12)and stem cell transplantation group(n=12).3.Before the transplantation of GFP-rBMSCs,cell counting and cell cycle were detected by flow cytometry.Stem cell transplantation group:Rats were reinfused with 1 ml of bone marrow mesenchymal stem cells(1 × 106cells/ml,resuspended in saline),and repeated every 2 weeks.Control group:Rats were reinfused with 1 ml of normal saline through the tail vein and repeated every 2 weeks.4.Six rats in each group were randomly selected-After the rats were anesthetized and shaved,they were placed in a small animal living imager.The migration and distribution of GFP-rBMSCs in rats after transplantation were carried out at Oh,48h and 72h.5.Serum,lumbar vertebrae and femur were extracted 4 weeks after transplantation.Flow cytometry was used to detect the expression of GFP cells in the femur bone marrow cavity.After rinsing the cells in the femur bone marrow cavity of rats,the expression of GFP fluorescence was observed under an inverted fluorescence microscope and photographed.6.Micro-CT was used to observe the lumbar spine microstructure and HE staining to evaluate the femoral trabecular structure of the bone.7.The contents of serum urinary N-telopeptide of type I collagen(PINP)were detected by ELISA kit.8.Rat bone tissue was extracted after grinding,the expression levels of alkaline phosphatase(ALP)and tartrate resistant acid phosphatase(TRAP)were measured.9.SPSS 22.0 was adopted for statistical analysis,data were expressed as mean(x±s)standard deviation.Two independent samples were compared by t test,and P<0.05 was considered statistically significant.Result1.Under the fluorescence microscope,the observed characterizations of cultured GFP-rBMSCs mainly showed as follows:fusiform,spindle-shaped,a few polygons,arranged radially,having bright GFP green fluorescence.Flow cytometry indicated that CD44 and CD90 were positive,while CD45 and CD34 were negative respectively.2.The small animal live imager showed that there was no GFP fluorescence expression signal at Oh in the stem cell transplantation group,and GFP fluorescence expression signals were obvious collected at 48h and 72h.The intensity of GFP fluorescence signal collected at 72 h was greater than 48 h.In the vicinity of the lumbar vertebrae of the rats,the liver and spleen had strong fluorescence signals,but the distribution of GFP-rBMSCs in the bone marrow cavity of rat bone tissue could not be accurately observed.No GFP fluorescence expression was detected in the control group at Oh,48h,and 72h.3.After 4 weeks of transplantation of GFP-rBMSCs,flow cytometry indicated that the bone marrow cavity GFP expression of the stem cell transplantation group was stronger than that of the control group,and there was a statistical difference(P<0.05).4.Fluorescence microscope showed that the fluorescence signal of GFP expression was found in the stem cell transplantation group,but no fluorescence expression was found in the control group,and there was a statistical difference(P<0.05).5.Mirco-CT gave the parameters of bone mineral density(BMD),trabecular number(Tb.N),bone volume(BV),tissue volume(TV),bone volume fraction(BV/TV)of the 2 groups were increased and had statistical difference(P<0.05).The trabecular separation(Tb.Sp)of the experimental group was lower than that of the control group,and there was statistical difference(P<0.05).Although the trabecular thickness(Tb.Th)was numerically high,the difference was not significant(P>0.05).6.Hematoxylin-eosin staining(HE)staining showed that the trabecular bone of the control group was thinner,thinner and smaller than that of the stem cell transplantation group.The spacing between the trabeculae was widened,continuously decreased,and the free end increased.7.the content of P1NP in serum was detected.The content of PINP in stem cell transplantation group was higher than that of the control group(P<0.05).8.The results showed that the expression level of ALP activity in stem cell transplantation group was significantly higher than that of the control group(P<0.05).The expression level of TRAP activity in the stem cell transplantation group was lower than that of the control group showing a statistical difference(P<0.05).Conclusion1.Bone marrow mesenchymal stem cell transplantation has a therapeutic effect on rat osteoporosis.2.The distribution of the GFP-labeled bone marrow mesenchymal stem cells after transplantation in rats was observed by an in vivo small animal imaging system and the GFP-labeled bone marrow mesenchymal stem cells can enter the femoral bone marrow cavity of rats.. |
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