| Objective:To judge the feasibility and the biochemical function integrity of Human umbilical cord MSCs separated by tissue explants method from the two aspects of morphology and immunophenotype. to investigate the improvement and therapeutical effect of Human umbilical cord MSCs transplantation on irreversible obstructive nephropathy from three different sides including biochemical indicator(serum creatinine and serum cystatin C), renal tissue sections HE staining, and the expression levels of VEGF protein and CTGF protein related with fibrosis in the renal tissues. To provide experimental and theoretical support for the further application of human umbilical cord MSCs in obstructive nephropathy.Methods: 1 MSCs were separated from fresh human umbilical cord by tissue explants method. Morphology of the cells separated was observed under a microscope. Cellular immunophenotype was detected by flow cytometry. 2 Ninety healthy rats were randomly divided into the model group( unilateral ureteral obstruction group, n=30, the rats were operated by ligation of right ureter and injected 1ml saline via tail vein 10 days after operation), the treatment group(unilateral ureteral obstruction + Human umbilical cord MSCs transplantation group, n=30, the rats were operated by ligation of right ureter and injected 1ml saline containing 1×107 Human umbilical cord MSCs via tail vein 10 days after operation) and the control group( sham surgery group, n=30, the rats were operated by sham surgery and injected 1ml saline via tail vein 10 days after operation). 10 rats of each group were killed and renal tissue was taken after haemospasia on the 1st, 2nd and 4th week after Human umbilical cord MSCs transplantation. At various time points,biochemical indicator(serum creatinine and serum cystatin C), renal tissue sections HE staining, and the expression levels of VEGF protein and CTGF protein related with fibrosis in the renal tissues were detected. 3 Experimental data was analysed by SPSS Statistics 19 software for statistical processing, in which measurement data was figured by( sx ±).(Test standard: P=0.05)Results: 1 Human umbilical cord MSCs separated by tissue explants method were feasible. Cellular immunophenotype of the separated cells detected by flow cytometry Flow indicated that these cells expressed CD73, CD90, CD105 but not expressed CD19, CD34, CD45, CD116 and HLA-DR, which conformed to Human mesenchymal stem cells characteristics. 2 At various time points after Human umbilical cord MSCs transplantation, there were significant differences in serum creatinine as well as serum cystatin C in the same group of the model group and the treatment group(P<0.05). Moreover, there were obvious differences in the two indexes respectively among the three groups at each time point(P<0.05). 3 Both the renal pathologic changes of the model group and the treatment group showed obvious vacuole-like dilations of renal tubular, infiltration of inflammatory cells and interstitial fibrosis at various time points after Human umbilical cord MSCs transplantation. The histopathological scores of the model group in the early stage after transplantation were relatively lower, which then continued to deteriorate since the second week after transplantation(P<0.05). Furthermore, the pathological injuries of the treatment group were significantly lighter than those of the model group at the same time point after transplantation, but they were deteriorating since the second week after transplantation as well(P<0.05). 4 The expression levels of VEGF protein in the model group increased in the beginning and then decreased as days progressed, and the expression levels at the three time points after transplantation were all higher than those in the control group(P<0.05). In the treatment group, the expression levels ofVEGF protein on the first week after transplantation were significantly higher than those in the control group, and reached the apex on the second week after transplantation. later the levels declined, which on the fourth week after transplantation were even lower when compared with those in the same group on the first week after transplantation(P<0.05). 5 The expression levels of CTGF protein in the control group were the lowest(P<0.05). The levels in the model group and the treatment group on the first week after transplantation were highest, then presented a decreasing trend as days go by, and respectively reached the lowest level on the fourth week after transplantation(P<0.05). However, no matter at which time points after transplantation, the expression levels of CTGF protein in the treatment group were lower than those of the model group(P<0.05).Conclusions: 1 Human umbilical cord MSCs separated by explant culture is proved to be feasible. The separated MSCs have obvious superiority in culture, subculture, differentiation as well as immune functions. 2 Human umbilical cord MSCs transplantation can help improve the renal function of irreversible obstructive nephropathy, reduce serum creatinine and serum cystatin C levels. Serum cystatin C level and serum creatinine level can all be used as the indexes to evaluate the development and improvement of obstructive nephropathy. The two indexes have almost the same change trend, while serum cystatin C level is more sensitive than serum creatinine level. 3 Human umbilical cord MSCs transplantation is effective treatment for irreversible obstructive nephropathy. It can relieve infiltration of inflammatory cells towards the peritubular interstitial, inhibit the apoptosis of renal tubular epithelial cells and help repair them. Besides, the therapeutical effect in the early stage of treatment(within about two weeks after transplantation) is the best, and the obvious effect after a single injection lasts about two weeks, which needs multiple injections to maintain curative effect. 4 The expression levels of CTGF and VEGF were correlated with the progression of interstitial fibrosis in obstructive nephropathy. The expressionlevels of VEGF have negative correlation with the degree of fibrosis, while the expression levels of CTGF have positive correlation with the degree of fibrosis. When obstructive nephropathy occurs, the kidney itself has the function of regulation, compensation and self repair, but still fails to stop the process of renal interstitial fibrosis. Studies had shown that Human umbilical cord MSCs can induce significant renal microvascular repair and reduce interstitial fibrosis, but the effect gradually declines as days progress, accompanied with the attenuation and apoptosis of human umbilical cord MSCs as well as the disappearance of immune regulation ability in the process of serial passage in vivo. 5 The specific frequency and dose standard of Human umbilical cord MSCs transplantation is still not confirmed at present, which will be the next research direction. |
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