| Objectives:1.Isolate and culture bone marrow mesenchymal stem cells(BMSCs)of SD rats,and verify their osteogenic and adipogenic differentiation ability.2.The model of chronic kidney disease(CKD)in rats was established by injecting adriamycin(ADR)into the tail vein of SD rats.3.BMSCs were transplanted into renal tissue through renal artery of CKD rats to investigate whether they can improve renal function of CKD rats,prevent apoptosis of renal tissue in chronic kidney disease and inhibit inflammatory reaction in the process of chronic kidney disease.Methods:1.Separate and culture BMSCs,and detect the osteogenic and adipogenic differentiation ability Two SD rats were killed after neck cutting.The bilateral femurs and tibia were taken under sterile conditions.After soaking in sterile phosphate buffer,the bone marrow cavity was repeatedly washed with L-DMEM culture medium supplemented with 10% fetal bovine serum.After removing visible large tissue,the cells were repeatedly washed and blown until dispersed.After centrifugation,BMSCs were collected and subcultured.BMSCs were induced to differentiate into bone and fat,stained and observed with fluorescence and recorded.2.Establishment of CKD model in SD rats induced by adriamycin Randomly select30 rats and inject ADR into the tail vein according to the adriamycin solution(3.5mg/kg)corresponding to the weight of the rats to induce the modeling.After the modeling is successful,magnetic resonance examination and pathological section analysis are performed on the rats.Biochemical analysis was performed on serum creatinine(Scr),blood urea nitrogen(BUN)and 24 h urine protein.The non-modeling group also injected the same amount of physiological saline through the tail vein.The modeling group found that both Scr and BUN values were increased,and the above results verified the success of modeling.3.Grouping and BMSCs transplantation and testing the experimental results of rats Sixteen rats with successful CKD model were randomly selected and randomly divided into ADR and MSCs groups,in which ADR group was chronic kidney disease group(n=8)and MSCs group was BMSCs treatment group(n=8).Eight of the10 non-modeling rats were randomly selected into the control group,and the contro group was a sham operation control group(n=8).MSCs group rats were injected BMSCs suspension under renal artery.Rats in contro group and ADR group were injected with the same amount of culture medium into renal artery.Two weeks after transplantation of BMSCs,the blood and urine of rats in Control group,ADR group and MSCs group were collected.After the rats were killed,the renal tissues were selected for pathological examination,and the levels of Scr and BUN were detected.The renal tissues were analyzed by ELISA and Western blot.Results: 1.The adherent growth of BMSCs can occur after about 24 hours of primary culture.After fluid exchange,the cells showed a similar triangular shape change.With the continuous growth of the cells,the shape changed into a variety of shapes,mainly irregular strip shape and spindle shape.With the passage and culture of BMSCs cells,the morphology of cells gradually changed into long spindle or flat.After the slow fusion of the cell population,BMSCs cells adhered to the wall and grew in the culture medium,and the morphology was similar to that of fish.BMSCs were induced and cultured in osteogenic induction medium.After differentiation,they were stained with alizarin red,and the effect of osteogenic staining was observed under the microscope.After successful induction,alizarin red staining was positive,which proved that BMSCs successfully differentiated into osteoblasts.BMSCs were induced to differentiate and cultured by lipogenic induction medium.After differentiation,they were stained with oil red O,and the effect of lipogenic staining was observed under the microscope.After induction,oil red O staining was positive,which proved that BMSCs were successfully differentiated into lipogenic cells.2.Two weeks after the first injection of ADR solution,inject the same amount of ADR solution again,and then conduct modeling test.The magnetic resonance examination of rat kidney showed that the signal intensity of the renal cortex and medulla of rats in CKD group was lower than that in Sham group.The signal intensity of the renal cortex was decreased,and the cortex and medulla of rats in CKD group was unclear,while the cortex and medulla of rats in Sham group was clear.The renal function test results of rats showed that BUN and Scr in CKD group were significantly higher than those in Sham group.The pathological results showed that compared with the control group,the glomerular morphology of CKD group was atrophic,sclerotic,mesangial hyperplasia,and collagen deposition;Renal tubules atrophy,irregular morphology,inflammatory cell infiltration,interstitial fibrosis;The nucleus becomes smaller and condensed.To sum up,it shows that rat CKD modeling is successful.3.Scr and BUN tests were carried out on rats in three groups after one week of BMSCs transplantation.Compared with control group,the values of BUN,Scr,glomerular injury score and renal tubular injury score in ADR group were significantly higher(P<0.001);Compared with the ADR group,the BUN value of MSCs group decreased(P<0.05),the Scr value decreased(P<0.01),the glomerular injury score decreased(P<0.001),and the renal tubular injury score decreased(P<0.01).The results showed that BMSCs transplantation could alleviate the renal function damage of CKD rats.4.After two weeks of treatment,the renal tissue sections of rats in three groups were observed by HE staining and transmission electron microscopy.The pathological results showed that the morphology of glomerulus and renal tubules in Control group was normal;In ADR group,glomerulosclerosis,collagen deposition,mesangial hyperplasia,irregular,atrophic and broken renal tubules,renal interstitial fibrosis,and a large number of inflammatory cells infiltration;The degree of glomerulosclerosis and renal interstitial fibrosis in MSCs group was less than that in ADR group,and the degree of renal tubular atrophy was partially recovered than that in ADR group.The results of transmission electron microscope showed that the thickness of glomerular basement membrane in Control group was uniform,continuous and complete,the three-layer structure of the basement membrane was clear,the mitochondria were not swollen,the foot processes were arranged neatly,and covered the outside of the basement membrane,the structure was clear,and the foot processes were not fused;In ADR group,the basement membrane of glomerulus was irregularly thickened,the three-layer structure was fuzzy,the structure of podocytes was disorderly arranged,part of the pods were separated from the basement membrane,and the pods were severely fused,and the clear fenestra of the septum could hardly be seen,the cytoplasm of podocytes was significantly swollen,the mitochondria were not significantly swollen,part of the matrix was uneven,the crest was slightly broken,and a small amount of degranulation could be seen;Compared with ADR group,the thickening of glomerular basement membrane in MSCs group was reduced,the thickness was relatively uniform,the podocytes were not obviously separated from the basement membrane,the part of foot process was slightly fused,the gap diaphragm window was relatively obvious,the cytoplasm and mitochondria of podocytes were slightly swollen,the cristae were relatively regular,the arrangement of foot process was slightly disordered,and there was no obvious fusion.Through transmission electron microscope,it was found that MSCs treatment could improve the basement membrane(BM),mitochondria(M)and foot process(FP)of kidney in CKD rats.5.The statistical analysis of ELISA detection found that compared with the control group,the pro-inflammatory factor IL-1 in the renal tissue of ADR group β、The concentration of IL-6 was significantly increased,and the difference was statistically significant(P<0.001),while the concentration of anti-inflammatory factor IL-10 was decreased(P<0.01);After transplantation of BMSCs,compared with ADR group,the pro-inflammatory factor IL-1 in MSCs group β The concentration decreased(P<0.01),the concentration of IL-6 decreased(P<0.05),and the concentration of anti-inflammatory factor IL-10 increased(P<0.01).The results of ELISA showed that transplanted BMSCs could inhibit the inflammatory reaction in the process of CKD by reducing the concentration of pro-inflammatory factors and increasing the concentration of pro-inflammatory factors.6.Immunoblotting results of rat kidney tissue showed that compared with the control group,the protein expression of p53 was significantly up-regulated(P<0.01),the protein expression of m TOR was significantly up-regulated(P<0.05),the protein expression of Bax was significantly up-regulated(P<0.01),and the protein expression of Bcl-2 was significantly down-regulated(P<0.05).Compared with ADR group,the protein expression of P53,m TOR,Bax and Bcl-2 in MSCs group were significantly decreased(P.Compared with the control group,the protein expression of P53,m TOR,Bax and Bcl-2 in MSCs group did not change significantly,and the difference was not statistically significant.The above results showed that MSCs could significantly inhibit the apoptosis of renal cells in the process of adriamycin induced chronic kidney disease,which preliminarily confirmed the protective mechanism of MSCs on chronic kidney disease.Conclusions:1.Transplantation of BMSCs through renal artery improved the renal function of CKD rats to some extent.2.Transplantation of BMSCs via renal artery inhibits IL-1 in the process of chronic kidney disease to some extent β、 IL-6 inflammatory factor promotes the expression of anti-inflammatory factor IL-10.Through m TOR/p53 signal pathway,the process of cell apoptosis in chronic renal tissue was improved,and the injury of glomerulus and renal tubule and renal interstitial fibrosis were alleviated. |
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