全反式维甲酸调控Kruppel因子5在预防移植静脉术后狭窄的作用及其机制

All trans retinoic acid mediated kruppel like factor 5 during the prevention of post transplant vein stenosis

目的探讨全反式维甲酸(ATRA)调控Kruppel因子5(Klf5)在预防移植静脉术后狭窄的作用及机制。方法建立新西兰大白兔(海军军医大学动物实验中心提供)静脉动脉化动物模型,按完全随机分为造模组(A、B、C组分别为饲养2、4、8周)和ATRA组(D、E、F组分别为饲养2、4、8周,鼻饲ATRA10 mg/d)、空白对照组(G组),各6只。分别获取移植静脉标本,行苏木精-伊红染色及免疫组化检验。建立人脐静脉平滑肌细胞(SMC)KLF5过表达细胞模型,以ATRA干预,划痕实验、细胞增殖实验(CCK-8)明确SMC增殖及迁移情况;免疫共沉淀明确ATRA对Klf5-RARa结合的阻断作用。两组间计量资料采用两独立样本t检验,多组间计量资料采用单因素方差分析,组间两两比较采用LSD-t检验方法。结果各组管径:A组(107.58±18.11)μm,B组(144.65±26.10)μm,C组(160.28±28.06)μm,D组(78.42±9.00)μm,E组(102.75±16.47)μm,F组(117.47±38.06)μm,G组(35.73±6.04)μm。组间比较,建模组各组(A、B、C各组)明显高于空白对照组(G组)(t=5.81、8.81、10.08,P<0.01),同期建模组各组明显高于ATRA组(t=2.06、2.96、3.03,P<0.05)。以染色指数法计算免疫组化结果,建模各组细胞核增殖抗原(Ki-67,2周3.07±0.64,4周3.67±0.81,8周1.93±0.41)表达明显高于对照组(t=6.93、9.37、4.16,P<0.01),也高于同期ATRA组(2周2.87±0.52,4周3.60±1.21,8周2.10±0.24,t=4.91、6.66、2.14,P<0.05)。实验组Klf5表达(2周4.43±0.70,4周5.67±1.18,8周3.03±0.98)明显高于对照组(t=7.27、9.09、3.83,P<0.01),建模组与ATRA组间KLF5(2周4.43±0.70,4周5.67±1.18,8周3.03±0.98)表达差异无统计学意义(t=0.49、0.17、0.42,P>0.05)。CCK-8实验:72 h Klf5组吸光度(A)值(1.54±0.20)高于其余3组(t=5.62、5.84、8.31,P<0.01),Klf5+ATRA组(1.02±0.14)高于ARTA组(0.80±0.07,t=2.47,P<0.05),对照组(1.04±0.13)高于ATRA组(t=2.70,P<0.05)。划痕实验:以迁移前后划痕距离的比值,klf5组0.098±0.006,对照组0.404±0.009,ATRA组0.597±0.014,Klf5+ATRA 0.265±0.012,组间比较ATRA组高于其余3组(t=4.81、6.12、8.41,P<0.01),Klf5组低于其余3组(t=-8.37、-10.16、-12.25,P值均<0.01)。免疫共沉淀发现Klf5-RARa可以结合形成复合物,以不同浓度ATRA电泳A值进行统计分析,50μmol/ml组A值(1.38±0.15)及70μmol/ml组A值(1.19±0.12)明显低于其余各组(对照组1.88±0.16、10μmol/ml组1.81±0.10、30μmol/ml组1.76±0.13,t=-5.60、-5.10、-4.80、-7.10、-6.80、-6.40,P<0.01)。结论ATRA可以通过对KLF5-RARa结合的阻断作用来抑制平滑肌细胞增殖及迁移,进而预防静脉移植术后血管狭窄。

Objective To study the role and mechanism of all trans retinoic acid(ATRA)regulating Kruppel factor 5(KLF5)in the prevention of stenosis after vein transplantation.Methods New Zealand white rabbits(provided by the Animal Experimental Center of Naval Military Medical University)were randomly divided into two groups:model group(groups A,B,C for 2,4 and 8 weeks,respectively),ATRA group(groups D,E,F for 2,4 and 8 weeks respectively,nasal ATRA 10 mg/day)and blank control group(group G).The transplanted vein samples were obtained and tested by hematoxylin eosin staining and immunohistochemistry.The KLF5 overexpression cell model of human umbilical vein smooth muscle cells(SMCs)was established.The proliferation and migration of SMCs were determined by ATRA intervention,scratch test and cell proliferation test(CCK-8).Immunocoprecipitation confirmed the blocking effect of ATRA on KLF5 rara binding.The measurement data between the two groups were analyzed by two independent sample t-test,the measurement data between multiple groups were processed by one-way ANOVA,and the comparison between the two groups was done by LSD-t test.Results The diameter in groups A,B,C,D,E,F and G was(107.58±18.11),(144.65±26.1),(160.28±28.06),(78.42±9.00),(102.75±16.47),(117.47±38.06)and(35.73±6.04)μm respectively.The diameter in the modeling groups was significantly greater than in the blank control group(t=5.81,8.81,10.08,P<0.01),and that in the modeling groups was significantly greater than in the ATRA groups(t=2.06,2.96,3.03,P<0.05).The expression of Ki67(3.07±0.64 at 2nd week,3.67±0.81 at 4th week and 1.93±0.41 at 8th week)in the modeling groups was significantly higher than that in the blank control group(reference index 1)(t=6.93,9.37 and 4.16,P<0.01),and also higher than that in the ATRA group(2.87±0.52 at 2nd week,3.60±1.21 at 4th week and 2.1±0.24 at 8th week,t=4.91,6.66 and 2.14,P<0.05).The expression of KLF5 in the experimental group(4.43±0.70 at 2nd week,5.67±1.18 at 4th week and 3.03±0.98 at 8th week)was significantly higher than that in the control group(reference index 1)(t=7.27,9.09 and 3.83,P<0.01).There was no significant difference in the expression of KLF5 between the modeling group and ATRA group(4.43±0.70 at 2nd week,5.67±1.18 at 4th week and 3.03±0.98 at 8th week,t=0.49,0.17 and 0.42,P>0.05).For the CCK-8 assay,the absorbance(A)value in KLF5 group(1.54±0.20)was higher than that in the rest three groups(t=5.62,5.84,8.31,P<0.01),that in KLF5+ATRA group(1.02±0.14)was higher than that in ARTA group(0.80±0.07,t=2.47,P<0.05),and that in control group(1.04±0.13)was higher than that in ATRA group(t=2.70,P<0.05).For the scratch test,the ratio of scratch distance before and after migration in KLF5 group was 0.098±0.006,0.404±0.009 in control group,0.597±0.014 in ATRA group,and 0.265±0.012 in KLF5+ATRA group.The ratio of scratch distance before and after migration in ATRA group was higher than the rest three groups(t=4.81,6.12,8.41,P<0.01)and that in KLF5 group was lower than in the rest three groups(t=-8.37,-10.16,-12.25,P<0.01).Immunocoprecipitation showed that KLF5 rara could combine to form a complex.The A values of ATRA electrophoresis at different concentrations were statistically analyzed.The A values in ATRA 50μmol/ml group(1.38±0.15)and 70μmol/ml ARTA group(1.19±0.12)were significantly lower than those in other groups[control group(1.88±0.16),10μmol/ml ARTA group(1.81±0.10),30μmol/ml ARTA group(1.76±0.13),t=-5.60,-5.10,-4.80,-7.10,-6.80,-6.40,P<0.01].It is suggested that a certain concentration of ATRA can block KLF5 rara binding.Conclusion ATRA can inhibit the proliferation and migration of SMCs by blocking the binding of klf5-rara,so as to prevent vascular stenosis after vein transplantation.