Study On The HA1077's Enhancement Effect Of The Wound Repair By Mesenchymal Stem Cells

Background:Skin is the largest organ which has the complicated structure and multiple physical functions and is the barrier against the outside hazard. Skin defect and refectory wound of diabetes are the common disease in clinic, and the key of treatment is the repairing wound effectively. With the development of tissue work, stem cells provide the new approach to repair the wound. In recent years the role of mesenchymal stem cells (MSCs) on the wound repairing has been paid more and more attention.MSCs (mesenchymal stem cells) has the ability to self regeneration and multiple potential of differentiation, and be identified to different to osteoblas, chondrocyte, fibroblast, adipocyte, myocardial cell, hepatocyte and neurocyte. MSCs can be induced to be epidermal cell and take part in the wound repairing. At present plenty of study focuses on the results of MSCs differentiation while the fewer on the signal transduction during the differentiation. It is not clear the mechanism how the differentiation of MSCs to the epidermal cell and there is no effective signal modulation methods. It is very important in clinic to explore the effect medicine to modulate the differentiation of MSCs and improve the efficiency. Result of study might show the clinical significance on the treatment of major skin defect and refectory wound.Fasudil (HA1077), whose chemical name is hexa-hydr-1-5-sulfuryl isoquinoline-1-4-aza, is a new kind of isoquiline-sulfuryl compound. Fasudil is a Ca2+ intracellular antagonist and inhibitor of protein kinase. The main use of HA1077 is preventing and improving the cerebral angiospasm, selectively expanding the spastic cerebral vessels, improving the cerebral ischemia and nerve cell function. Fasudil is the specific blocker of Rho kinase. Rho is the molecular converter or switch and related to the JNK and P38 signal routes. In our previous study, the results show the tight relation among ERK, P38 routes and differentiation of MSCs to epidermal cell. We guess that Fasudil may take part in the modulation of differentiation to epidermal cell. So we investigate the relation among MSCs, Fasudil and wound repairing.Objective:1. Investigate the effect of blocking of Rho by HA1077 on the cytokeratin expression of MSCs.2. Investigate the effect of HA1077 on the cell cycle during the induction of MSCs to the cytokeratin expression.3. Observe the HA1077's effect on the wound healing of rat and investigate the best dose of HA1077.4. Observe the HA1077 and MSCs'effect on the clinical refractory wound.Methods:1. Bone marrow MSCs were separated from rats, then purified, identified and proliferated in culture medium. The passage 3 MSCs were randomly divided into control group, induction group, Rho inhibition group. At the 1d,3d,5d and 7d,Phosphorylation P38 and ERK levels were detected by flow cytometry. CK 5/8 and CK19 of MSCs at the 7d after induction were detected by flow cytometry. HA1077 effect on the CK5/8 and CK19 expression of MSCs in two dose(10μmol/L,30μmol/L) were observed.2. Isolation and identification of human bone mesenchymal stem cells. The cells of third passage were randomly divided into control group and induction group, HA1077 group. At the day 7 afetr induction, the MSCs cell cycle and PCNA (proliferating cell nuclear antigen were detected by FCM.3. The model of skin full thickness defect was set up on the rats'back. Then rats were divided into treatment group, control. In treatment group, different dose of HA1077 in 10μmol/L,20μmol/L,40μmol/L,80μmol/L,160μmol/L respectively were used to treated the rats wound ,while NaCl solution were used in the control group. The wound healings were observed and the tissue around the wound was examined by pathological methods at the 10d after injury.4. Patients themselves MSCs were separated in vitro, then purified, identified, proliferated in culture medium and identified. The chronic refractory wounds were divided into control group, HA1077 group, HA1077and MSCs group, the wound healing were observed. Results:1. Phosphorylation P38 expression: the Phosphorylation P38 was 0.02±0.009% in control group. At 5d in induction group, Phosphorylation P38 increased significantly. From 1d to 7d in Rho inhibition group, Phosphorylation P38 increased significantly comparing with control and inductiongroup.2. Phosphorylation ERK expression: the Phosphorylation ERK was 4.15±0.32% in control group. From 3d to the 5d, Phosphorylation ERK decreased significantly comparing with control group, then recovered to the normal at 7d. In Rho inhibition group,Phosphorylation ERK shown no change from 1d to 5d,while decreased significantly comparing with control group and induction group at 7d.3. Cytokeratin expression of MSCs: the CK5/8 positive rate was 0.58±0.01%, and CK19 positive rate was 2.04±0.13% in control group. At 7d after induction, CK5/8 was 1.81±0.05%,and CK19 was 10.19±0.23% in induction group. In Rho inhibition 1 group, CK5/8 was 21.65±0.75%,CK19 was 39.41±0.86%,which all were higher than induction group, P<0.01. In Rho inhibition 2 group, CK5/8 was 21.07±0.57% , CK19 was 45.60±0.91%,which all were higher than induction group, P<0.01. There was no difference about the results between the two kinds of HA1077 dose in Rho inhibition group4. Cell cycle shown that at 7d the rates of G0/G1 phase were (92.32±0.32)%,(88.76±0.55)%,(79.60±1.99)% in control, induction and HA1077 group respectively. There was significant difference among the groups. Rates of S phase were (8.10±0.54)%,(12.92±0.28)%,(19.22±0.89)% in control, induction and HA1077 group respectively. There was significant difference among the groups.5. PCNA rate of control group, induction group and HA1077 group were( 4.28±0.96)%,(8.92±2.23)%,(11.23±0.89) % respectively,the level of HA1077 group was highest among the groups. Result of cell cycle showed that S phase cell rate of HA1077 group was highest among the groups.6. Residue wound area of 20μmol/L HA1077 group was smaller than other groups obviously(P<0.05)at same time point , except at 7d when there was no difference comparing with 80μmol/L HA1077 group .7. Pathological examination show that the granulation tissue grew and epidermis regeneration among different dose HA1077 groups were better than in control group. 20μmol/L HA1077 group was better than other HA1077 groups in granulation tissue growth, epidermis regeneration.8. In the clinical observation, at 7d after treatment, WCI were 5.22±5.26,6.86±5.38 and 9.33±8.23 in control group,HA1077 group, HA1077 and MSCs group respectively. At 14d,WCI were 10.29±8.75, 13.35±10.57,and 36.32±9.92 in control group, HA1077 group, HA1077 and MSCs group respectively. The HA1077 and MSCs group was better than other two groups. There were no significant difference between HA1077 group and control group.Conclusion:1. During the induction of the study,the upper signal Rho had adverse effect on p38 route. Blocking of Rho could improve Phosphorylation P38 expression, then promote the MSCs differentiation and cytokeratin expression.2. In vitro, HA1077 promote hMSCs entering S phase and PCNA expression. The result show it can improve hMSCs proliferation and differentiation.The increasing of PCNA expression might take part in MSCs differentiation to the epidermis cell.3. Result show that HA1077 can enhance the wound healing. 20μmol/L of HA1077 is the best dose.4. Result show HA1077 can improve the MSCs'repairing the clinical wound. Use of HA1077 combined with MSCs to treat the refractory wound might have good future in clinic.