| OBJECTIVE To study the isolation, culture and identification of human umbilical cord mesenchymal stem cells(UC-MSCs). To investigate the safety of heterogenous UC-MSCs transplantation via intravenous injection and the distribution of UC-MSCs in unilateral ureteral obstruction(UUO) rats with obstruction-induced renal fibrosis. To investigate the therapeutical effect and possible mechanism of human UC-MSCs transplantation in obstruction-induced renal fibrosis in rat.METHOD 1. Mesenchymal stem cells(MSCs) were isolated from umbilical cord by enzyme digestion and cultured in appropriate growth medium. The biological characteristics of adherent cells were investigated and growth curves of cells were drawn. The surface antigens were detected by flow cytometry and differentiation potentials were observed by inducing to osteoblasts and fat cells.2. UC-MSCs were recovered and enlarged after cryopreservation.84 healthy female SD rats aged 8 weeks were divided into four groups.?UUO group:These rats were operated by ligation of left ureter and injected 1ml PBS via the tail vein 7 days after operation.?UUO+MSCs group:These rats were operated by ligation of left ureter and injected 5×106UC-MSCs via the tail vein 7 days after operation.?Sham group: These rats were operated by sham surgery and injected lml PBS via the tail vein 7 days after operation.?Sham+MSCs group:These rats were operated by sham surgery and injected 5×106UC-MSCs via the tail vein 7 days after operation. Seven rats of esch group were killed on the 14th,21stand 28th day. The blood, heart, liver, lung, spleen and renal tissue were collected. At each time point, items were detected as follows:?Changes of body weight, blood routine, serum alanine aminotransferase and serum aspartate aminotransferase.?The kidneys of all rats were collected to have pathological analysis by HE, PAS and Masson stain. (3)The expression of MAB1281 (Labeled human UC-MSCs) in heart, liver, lung, spleen and renal tissue were observed by immunohistochemical staining.3.63 healthy female SD rats aged 8 weeks were divided into UUO group, UUO+MSCs group and Sham group. These three groups were treated as mentioned before. Seven rats of esch group were killed on the 14th,21st and 28th day. The urine, blood and renal tissue were collected. At each time point, items were detected as follows:?Changes of 24-hour urine protein, serum urea nitrogen and serum creatinine.?The kidney of all rats were collected to have renal pathological lesion scores.?The expression of?-SMA?Fn?TGF-?1 and BMP-7 in renal tissue were observed by immunohistochemical staining.?The quantity analysis of a-SMA?Fn?TGF-?1 and BMP-7 protein were further demonstrated with western blot.RESULT1. Adherent cells with fibroblastic morphology could be observed as early as 24-48 hours after the enzymatic digestion. The cell proliferation growth curve showed that doubing time of P4 UC-MSCs was 28h. No significant changes in cell morphology could been found after cryopreservation and recovery. The sample contained cells with an adipogenic phenotype when identified by oil red O staining after 14 days of adipogenic differentiation. The sample formed mineralized matrix when detected by the calcification of the matrix (von Kossa) after 14 days of osteogenic differentiation. Adhesive cells were all positive for MSC-related antigens, such as CD29, CD44, CD73, CD90, CD105, but negative for CD34, CD45, CD31 and HLA-DR. 2.?There were no significant difference in blood routine, changes of body weight, serum alanine aminotransferase and serum aspartate aminotransferase between Sham group and Sham+MSCs group in each time point (P>0.05).?Renal interstitial fibrosis(RIF) gradually increased with time in UUO group and UUO+MSCs group, but there were no obvious glomerular lesions.?There were no obvious abnormality observed by the naked eye in heart, liver, lung, spleen and renal of rats in four groups. There were no obvious inflammatory cell infiltration under the microscope in heart, liver, lung, spleen and renal of rats in Sham+MSCs groups.?Exogenous human UC-MSCs were found mainly in the lung and spleen, followed by kidney and heart, but no in liver. The UC-MSCs in spleen in UUO+MSCs group were significantly less than those in Sham+MSCs groups (P<0.01), but the UC-MSCs in kidney in UUO+MSCs group were more than those in Sham+MSCs groups. The UC-MSCs in left kidney in UUO+MSCs group were mainly in renal interstitial, while those in the kidney in Sham+MSCs group were mainly in glomerular.3.?There were no significant difference in 24-hour urine protein among these three groups in each time point (P>0.05). On the 14th day, the serum creatinine in UUO+MSCs group were less than those in UUO group (P<0.05).?On the 14th and 21st day, the renal pathological lesion scores, renal expression of Fn and a-SMA were lower in UUO+MSCs group than in UUO group (P<0.05), but there were no significant difference in these two groups on the 28th day(P>0.05).?On the 14th and 28st day, the renal expression of TGF-?1 were lower in UUO+MSCs group than in UUO group, but the renal expression of BMP-7 were higher than in UUO group (P<0.05).?A positive correlation was found between the renal expression of a-SMA and TGF-?1, while a negative correlation was found between the renal expression of a-SMA and BMP-7.CONCLUTION1. MSCs can be successfully isolated by enzyme digestion from human umbilical cord. The cultured cells, with the biological characteristics of MSCs, can be used for further research of cell transplantation.2. Heterogenous UC-MSCs transplantation is safe. The heterogenous UC-MSCs can migrate to the renal tissues of UUO rats via intravenous injection.3. The UC-MSCs transplantation contributed to decrease the fibrosis in obstruction-induced renal fibrosis in rat, but the mechanism of treatment needs further study. |
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