| AIM:To observe the effects of bone marrow mesenchymal stem cells (MSCs) on the restoration of acute liver injury induced by carbon tetrachloride (CCL4) in rats via RhoA-ROCK signaling pathway.METHODS:MSCs were isolated from male Sprague-Dawley (SD) rats, cultured and purified in a MSCs culture system in vitro using their adherent characteristics, MSCs were identified using flow cytometry. MSCs were induced and differentiated with hepatocyte growth factor (HGF). Differentiating cells were identified by periodic acid-schiff (PAS) staining. Rats were divided into 3 groups: normal control (N,n=10), CCL4 (C,n=10) and CCL4 plus MSCs (T,n=10). All the rats were sampled at the appointed times and various target markers were determined as follows: hematoxylin and eosin (HE) staining, as well as the index changes of liver's enzymology, were applied for evaluating the improvement of hepatic histological damage. The expression of RhoA mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR), and the expression of RhoA protein was assayed by Western blotting.RESULTS:1.MSCs primary cultured in vitro adhered to plastic surface within 24h and reached 90% confluence within 7d. Then the cells were expanded by subculture and passaged into fourth generation. 80% of the MSCs in forth generation grew into G0/G1stage, testing phase, while few cells lied in active proliferation stage, which according to the characteristics of adult stem cells. MSCs induced with HGF were found morphologically from shuttle to ellipse- and rotundity-like shape. On day 14, the cells grew into hepatocyte-like cells and were positive for PAS staining.2.In the T group, Liver function of CCL4-induced acute hepatic injury after MSCs transplantation was improved markedly as compared with the C group (on day 1, ALT 89.70±3.09 vs 147.59±6.83, P<0.05; AST 263.67±17.05 vs 472.68±19.04, P<0.01; on day 7, ALT 42.38±14.31 vs 92.75±6.70, P<0.05; AST 173.85±16.80 vs 260.41±25.35, P<0.05), consistent with the improvement of hepatic histology.3.Little expression of RhoA was detected in the normal control. In the C group, both mRNA and protein of RhoA increased significantly as compared with normal control (1.39±0.046 vs 0.57±0.010, P<0.01; 1.23±0.020 vs 0.35±0.036, P<0.01), then decreased slowly (on day 7, 0.76±0.019 vs 0.57±0.011, P<0.01; 0.87±0.042 vs 0.34±0.018, P<0.01). In the T groups, the expression mRNA and protein of RhoA were obviously decreased , in concomitant with the restoration of liver histology and function,as compared with the C group (on day 1, 1.08±0.012 vs 1.39±0.046, P<0.01; 1.08±0.017 vs 1.23±0.020, P<0.01. on day 7, 0.56±0.018 vs 0.76±0.019, P<0.01; 0.36±0.014 vs 0.87±0.042, P<0.01).CONCLUSIONS:1.Direct anchoring method is simple, feasible and less influential on the cells activity, when used to separate MSCs. MSCs can be easily separated and cultured to provide a good source for research in vivo and in vitro; MSCs can be induced and differentiated into hepatocyte-like cells in vitro with HGF, and the differentiated cells contained glycogen granules.2 . MSCs transplantation can repair the injured liver function and histological structure induced by CCL4 to some extent.3.Rho-ROCK signaling pathways are involved in the process of acute hepatic injury in rats induced by CCL4. MSCs transplantation may accelerate the restoration of acute liver injury by inhibition of RhoA-ROCK signaling pathway.
|
PDF Full Text Request