HA1077促进骨髓间充质干细胞修复皮肤创面的研究
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摘要
背景:
皮肤是人体面积最大的器官,是机体与外界的机械屏障,具有复杂的组织结构和多重生理功能。创(烧)伤后皮肤缺损或是糖尿病等慢性病所致顽固性皮肤溃疡是临床常见病症之一,及时有效的创面修复是此类患者救治的基础和核心问题。随着组织工程学的拓展,应用干细胞技术有望为创面修复提供新的手段。近年来,骨髓间充质干细胞在创伤修复方面的作用日益受到各国学者的广泛关注。
骨髓间充质干细胞具有高度自我更新能力以及多向分化潜能,多项体内外研究已证实其可以分化为成骨细胞、软骨细胞、成纤维细胞、脂肪细胞、心肌细胞、肝细胞、神经细胞等多种组织细胞。越来越多的学者倾向认同,骨髓间充质干细胞能够跨胚层分化为表皮细胞参与创面修复。然而,目前研究主要集中在骨髓间质干细胞的分化结果上,对于分化中的信号调控机制研究甚少,骨髓间充质干细胞向表皮细胞分化的机制尚不明确,缺乏有效调控手段。探索有效的骨髓间充质干细胞分化调控药物,提高骨髓间充质干细胞向表皮细胞分化效率,无疑对治疗大面积皮肤缺损或是慢性难愈性创面具有重要临床意义和广阔应用前景。
法舒地尔,英文名为Fasudil,别名为HA1077,化学名为六氢-1-(5-磺酰基异喹啉)-1(H)-1,4-氮杂,是日本旭化成株式会社和名古屋大学药理学研究室合作开发的一种新型异喹啉磺胺衍生物。盐酸法舒地尔为细胞内Ca2+拮抗剂,蛋白激酶抑制剂。其主要用途是用来改善和预防由多种原因引起的脑血管痉挛,选择性扩张痉挛的脑血管,改善脑缺血症状及伴随的神经元损伤。盐酸法舒地尔为Rho激酶特异性抑制剂,它主要是通过抑制Rho激酶活性而发挥其药理作用。Rho蛋白是细胞的转导通路的信号转换器或分子开关,Rho激活的信号传递与JNK、P38通路有关,而本实验室前期研究已证实ERK、p38两条信号通路与大鼠骨髓间充质干细胞诱导向表皮细胞分化相关。由此推测,法舒地尔极有可能参与骨髓间充质干细胞向表皮细胞分化的调控。基于以上认识,本研究深入探讨了骨髓间充质干细胞、盐酸法舒地尔与皮肤创面修复之间的关系。
目的:
1.探讨HA1077阻断Rho信号对骨髓间充质干细胞表达角蛋白的影响。
2.探讨HA1077对人骨髓间充质干细胞(hMSCs)向表皮细胞表型诱导中细胞周期的影响。
3.观察HA1077在大鼠皮肤创面愈合中的作用,探讨最佳HA1077促愈浓度。
4.观察HA1077联合骨髓间充质干细胞在难愈创面治疗中的作用
方法:
1.体外原代培养大鼠MSCs,纯化、鉴定、传代扩增。取第3代MSCs,分为正常对照组、单纯诱导组、Rho阻断组,于第1、3、5、7天应用流式细胞仪检测MSCs定向诱导中磷酸化P38和ERK表达;观察诱导后第7天各组细胞CK5/8、CK19的阳性表达率;观察2种浓度(10μmol/L,30μmol/L)HA1077阻断后,MSCs的CK5/8、CK19阳性表达率。
2.体外原代培养hMSCs,纯化、鉴定、传代扩增。取第3代hMSCs,体外诱导hMSCs向表皮细胞表型分化,分为正常对照组、单纯诱导组、Rho阻断组3组。应用流式细胞仪检测各组细胞诱导7天后hMSCs细胞周期和增殖细胞核抗原(PCNA)变化。
3.建立大鼠背部皮肤全层缺损模型,分为实验组和对照组,实验组分别用10μmol/L、20μmol/L、40μmol/L、80μmol/L、160μmol/L浓度梯度的盐酸法舒地尔注射液治疗大鼠皮肤缺损创面,对照组创面喷洒氯化钠溶液。观察各组动物创面愈合情况,并于创伤后第10天取创面组织进行组织学检查。
4.体外分离培养自体骨髓间充质干细胞,纯化、鉴定、传代扩增。临床病人慢性创面分为对照组、单纯HA1077组、HA1077联合细胞移植组,观察各组创面愈合情况。
结果:
1.磷酸化P38表达:正常对照组的磷酸化P38为0.02±0.009%;单纯诱导组诱导后第5天p38磷酸化水平较对照组增高,差异有统计学意义;Rho阻断组,磷酸化P38水平在第1天至第7天较对照组和单纯诱导组均呈升高,差异有统计学意义。
2.磷酸化ERK表达:正常对照组的磷酸化ERK水平为4.15±0.32%;单纯诱导组诱导后第3天至第5天磷酸化ERK表达均呈下降趋势低于对照组,差异有统计学意义,第7天时恢复至诱导前水平;Rho阻断组,ERK磷酸化水平第1天至第5天无明显变化,第7天时低于对照组和单纯诱导组,差异有统计学意义。
3.骨髓间充质干细胞角蛋白表达:诱导后第7天,对照组CK5/8阳性率为0.58±0.01%,CK19阳性率为2.04±0.13%;单纯诱导组CK5/8为1.81±0.05%,CK19为10.19±0.23%; Rho阻断1组(10μmol/L):CK5/8为21.65±0.75%,CK19为39.41±0.86%,与单纯诱导组相比,升高显著,P<0.01;Rho阻断2组(30μmol/L):CK5/8为21.07±0.57%,CK19为45.60±0.91%,较之单纯诱导组升高显著,P<0.01;两种HA1077 Rho阻断浓度对提高MSCs向表皮细胞分化效率差异不显著。
4.细胞周期检测结果显示,诱导第7天,对照组、诱导组和Rho阻断组各组处在G0/G1期的细胞分别为(92.32±0.32)%、(88.76±0.55)%、(79.60±1.99)%,各组间差异有统计学意义;各组处于S期的比例分别为:(8.10±0.54)%、(12.92±0.28)%、(19.22±0.89)%,各组间差异有统计学意义。
5.诱导第7天,对照组、诱导组、HA1077阻断组PCNA的阳性表达率分别为:( 4.28±0.96)%、(8.92±2.23)%、(11.23±0.89)%,其中HA1077阻断组PCNA的阳性表达率最高,各组间差异有统计学意义。
6. 20μmol/L HA1077组创面剩余面积明显小于同时间点其他各组,除创伤后第7天与80μmol/L组剩余创面面积比较差异无显著性外,其余各时间点差异均有显著性(P<0.05)。
7.创面组织学检查显示显示,创伤后第10天,创面肉芽组织明显增多,应用不同剂量HA1077治疗的各组均比对照组肉芽形成明显,且新生毛细血管丰富,创缘有新生表皮生成;应用20μmol/L HA1077组创面新生肉芽组织生长及新生表皮速度明显优于其他组别。
8.在治疗后第7天,对照组、单纯HA1077组、HA1077联合细胞移植组的创面愈合指数分别为5.22±5.26、6.86±5.38和9.33±8.23;在治疗第14天,各组创面愈合指数分别为10.29±8.75、13.35±10.57和36.32±9.92, HA1077联合细胞移植组创面愈合情况均优于单纯HA1077组和对照组,差异有统计学意义,而单纯应用HA1077组与对照组在各个观察时相点创面愈合情况差别不大。
结论:
1.在本实验诱导条件下,上游信号Rho对p38途径起反向调节。阻断Rho,可以提高p38的信号磷酸化水平。应用HA1077将上游信号Rho阻断后可增加p38途径激活,在一定程度上促进MSCs分化,表达角蛋白CK5/8、CK19阳性率增加。
2. HA1077在体外条件诱导下能促进hMSCs进入S期,PCNA表达增加,使hMSCs增殖能力提高,分化能力增强。PCNA的上调可能参与了MSCs的向表皮细胞的转分化过程,促进hMSCs向表皮细胞表型的分化。
3.研究表明HA1077可促进皮肤缺损创面愈合;20μmol/L HA1077为最佳创面促愈浓度。
4.结果提示HA1077可以促进MSCs细胞修复慢性皮肤创面。HA1077联合骨髓间充质干细胞治疗难愈性创面具有良好临床应用前景。
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 group
4. 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.
皮肤是人体面积最大的器官,是机体与外界的机械屏障,具有复杂的组织结构和多重生理功能。创(烧)伤后皮肤缺损或是糖尿病等慢性病所致顽固性皮肤溃疡是临床常见病症之一,及时有效的创面修复是此类患者救治的基础和核心问题。随着组织工程学的拓展,应用干细胞技术有望为创面修复提供新的手段。近年来,骨髓间充质干细胞在创伤修复方面的作用日益受到各国学者的广泛关注。
骨髓间充质干细胞具有高度自我更新能力以及多向分化潜能,多项体内外研究已证实其可以分化为成骨细胞、软骨细胞、成纤维细胞、脂肪细胞、心肌细胞、肝细胞、神经细胞等多种组织细胞。越来越多的学者倾向认同,骨髓间充质干细胞能够跨胚层分化为表皮细胞参与创面修复。然而,目前研究主要集中在骨髓间质干细胞的分化结果上,对于分化中的信号调控机制研究甚少,骨髓间充质干细胞向表皮细胞分化的机制尚不明确,缺乏有效调控手段。探索有效的骨髓间充质干细胞分化调控药物,提高骨髓间充质干细胞向表皮细胞分化效率,无疑对治疗大面积皮肤缺损或是慢性难愈性创面具有重要临床意义和广阔应用前景。
法舒地尔,英文名为Fasudil,别名为HA1077,化学名为六氢-1-(5-磺酰基异喹啉)-1(H)-1,4-氮杂,是日本旭化成株式会社和名古屋大学药理学研究室合作开发的一种新型异喹啉磺胺衍生物。盐酸法舒地尔为细胞内Ca2+拮抗剂,蛋白激酶抑制剂。其主要用途是用来改善和预防由多种原因引起的脑血管痉挛,选择性扩张痉挛的脑血管,改善脑缺血症状及伴随的神经元损伤。盐酸法舒地尔为Rho激酶特异性抑制剂,它主要是通过抑制Rho激酶活性而发挥其药理作用。Rho蛋白是细胞的转导通路的信号转换器或分子开关,Rho激活的信号传递与JNK、P38通路有关,而本实验室前期研究已证实ERK、p38两条信号通路与大鼠骨髓间充质干细胞诱导向表皮细胞分化相关。由此推测,法舒地尔极有可能参与骨髓间充质干细胞向表皮细胞分化的调控。基于以上认识,本研究深入探讨了骨髓间充质干细胞、盐酸法舒地尔与皮肤创面修复之间的关系。
目的:
1.探讨HA1077阻断Rho信号对骨髓间充质干细胞表达角蛋白的影响。
2.探讨HA1077对人骨髓间充质干细胞(hMSCs)向表皮细胞表型诱导中细胞周期的影响。
3.观察HA1077在大鼠皮肤创面愈合中的作用,探讨最佳HA1077促愈浓度。
4.观察HA1077联合骨髓间充质干细胞在难愈创面治疗中的作用
方法:
1.体外原代培养大鼠MSCs,纯化、鉴定、传代扩增。取第3代MSCs,分为正常对照组、单纯诱导组、Rho阻断组,于第1、3、5、7天应用流式细胞仪检测MSCs定向诱导中磷酸化P38和ERK表达;观察诱导后第7天各组细胞CK5/8、CK19的阳性表达率;观察2种浓度(10μmol/L,30μmol/L)HA1077阻断后,MSCs的CK5/8、CK19阳性表达率。
2.体外原代培养hMSCs,纯化、鉴定、传代扩增。取第3代hMSCs,体外诱导hMSCs向表皮细胞表型分化,分为正常对照组、单纯诱导组、Rho阻断组3组。应用流式细胞仪检测各组细胞诱导7天后hMSCs细胞周期和增殖细胞核抗原(PCNA)变化。
3.建立大鼠背部皮肤全层缺损模型,分为实验组和对照组,实验组分别用10μmol/L、20μmol/L、40μmol/L、80μmol/L、160μmol/L浓度梯度的盐酸法舒地尔注射液治疗大鼠皮肤缺损创面,对照组创面喷洒氯化钠溶液。观察各组动物创面愈合情况,并于创伤后第10天取创面组织进行组织学检查。
4.体外分离培养自体骨髓间充质干细胞,纯化、鉴定、传代扩增。临床病人慢性创面分为对照组、单纯HA1077组、HA1077联合细胞移植组,观察各组创面愈合情况。
结果:
1.磷酸化P38表达:正常对照组的磷酸化P38为0.02±0.009%;单纯诱导组诱导后第5天p38磷酸化水平较对照组增高,差异有统计学意义;Rho阻断组,磷酸化P38水平在第1天至第7天较对照组和单纯诱导组均呈升高,差异有统计学意义。
2.磷酸化ERK表达:正常对照组的磷酸化ERK水平为4.15±0.32%;单纯诱导组诱导后第3天至第5天磷酸化ERK表达均呈下降趋势低于对照组,差异有统计学意义,第7天时恢复至诱导前水平;Rho阻断组,ERK磷酸化水平第1天至第5天无明显变化,第7天时低于对照组和单纯诱导组,差异有统计学意义。
3.骨髓间充质干细胞角蛋白表达:诱导后第7天,对照组CK5/8阳性率为0.58±0.01%,CK19阳性率为2.04±0.13%;单纯诱导组CK5/8为1.81±0.05%,CK19为10.19±0.23%; Rho阻断1组(10μmol/L):CK5/8为21.65±0.75%,CK19为39.41±0.86%,与单纯诱导组相比,升高显著,P<0.01;Rho阻断2组(30μmol/L):CK5/8为21.07±0.57%,CK19为45.60±0.91%,较之单纯诱导组升高显著,P<0.01;两种HA1077 Rho阻断浓度对提高MSCs向表皮细胞分化效率差异不显著。
4.细胞周期检测结果显示,诱导第7天,对照组、诱导组和Rho阻断组各组处在G0/G1期的细胞分别为(92.32±0.32)%、(88.76±0.55)%、(79.60±1.99)%,各组间差异有统计学意义;各组处于S期的比例分别为:(8.10±0.54)%、(12.92±0.28)%、(19.22±0.89)%,各组间差异有统计学意义。
5.诱导第7天,对照组、诱导组、HA1077阻断组PCNA的阳性表达率分别为:( 4.28±0.96)%、(8.92±2.23)%、(11.23±0.89)%,其中HA1077阻断组PCNA的阳性表达率最高,各组间差异有统计学意义。
6. 20μmol/L HA1077组创面剩余面积明显小于同时间点其他各组,除创伤后第7天与80μmol/L组剩余创面面积比较差异无显著性外,其余各时间点差异均有显著性(P<0.05)。
7.创面组织学检查显示显示,创伤后第10天,创面肉芽组织明显增多,应用不同剂量HA1077治疗的各组均比对照组肉芽形成明显,且新生毛细血管丰富,创缘有新生表皮生成;应用20μmol/L HA1077组创面新生肉芽组织生长及新生表皮速度明显优于其他组别。
8.在治疗后第7天,对照组、单纯HA1077组、HA1077联合细胞移植组的创面愈合指数分别为5.22±5.26、6.86±5.38和9.33±8.23;在治疗第14天,各组创面愈合指数分别为10.29±8.75、13.35±10.57和36.32±9.92, HA1077联合细胞移植组创面愈合情况均优于单纯HA1077组和对照组,差异有统计学意义,而单纯应用HA1077组与对照组在各个观察时相点创面愈合情况差别不大。
结论:
1.在本实验诱导条件下,上游信号Rho对p38途径起反向调节。阻断Rho,可以提高p38的信号磷酸化水平。应用HA1077将上游信号Rho阻断后可增加p38途径激活,在一定程度上促进MSCs分化,表达角蛋白CK5/8、CK19阳性率增加。
2. HA1077在体外条件诱导下能促进hMSCs进入S期,PCNA表达增加,使hMSCs增殖能力提高,分化能力增强。PCNA的上调可能参与了MSCs的向表皮细胞的转分化过程,促进hMSCs向表皮细胞表型的分化。
3.研究表明HA1077可促进皮肤缺损创面愈合;20μmol/L HA1077为最佳创面促愈浓度。
4.结果提示HA1077可以促进MSCs细胞修复慢性皮肤创面。HA1077联合骨髓间充质干细胞治疗难愈性创面具有良好临床应用前景。
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 group
4. 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.
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10. Zuk PA,Zhu M,Mizuno H,et al.Multi-lineage cells from human adipose tissue:implications for cell based therapies. Tissue Eng,2001,7(2):211.
11. Shih-Chieh,Nien-jung C,Shie-Liang H et al.Isolation and characterization of Size-soieved stem cells from human bone marrow.Stem Cells,2002,20:249-258.
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13. Lennon D P, Haynesworth S E, Young R G et al. A chemically defined medium supports in vitro proliferation and maintains the osteochondral potential of rat marrow-derived mesenchymal stem cells. Exp Cell Res,19 95,219(1):211-222
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22.张奇、白晓东、杨洋等。法舒地尔对人骨髓间充质干细胞向表皮细胞分化的影响。武警医学,2005,16(9):647-650。
23.邹丽琴,苏踊跃,孙荣距等。大鼠骨髓间充质干细胞向表皮细胞分化的初步研究。第三军医大学学报,2006,28(6):584-586。
24. Yoon YS,Wecker A,Heyd L,et al.Clonally expanded novel multipotent stem cells from human bone marrow regenerate myoeardium after myocardial infarction. J Clin Invest. 2005, 115 (2):326-328.
25.景竹春、梁璐、韩忠朝。间充质干细胞在治疗皮肤缺损中的应用。国际生物医学工程杂志,2007,30(4):202-205。
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