碱性调宁蛋白对糖尿病大鼠烧伤创面成纤维细胞增殖与凋亡的影响

Effect of calponin on fibroblast proliferation and apoptosis in burn wound of diabetic rats

目的探讨碱性调宁蛋白在糖尿病大鼠烧伤创面愈合过程中的表达情况及其对成纤维细胞增殖与凋亡的影响。方法选64只雄性Wistar大鼠,按随机数字表法分为实验组和对照组,每组32只。实验组大鼠,腹腔注射链脲佐菌素60 mg/kg,制作糖尿病大鼠模型;对照组大鼠,腹腔注射0.9%氯化钠溶液1.0 mL。适应性饲养2周后将2组大鼠均制作为深Ⅱ度烫伤模型(以下称为烧伤),伤后立即液体复苏、常规治疗。观察两组大鼠的创面愈合时间及在伤后第7、14、21、28天的创面愈合率,创周切取组织、制作切片,行苏木精-伊红染色观察组织形态学变化,免疫组织化学SP法分析碱性调宁蛋白表达,成纤维细胞增殖细胞核抗原(PCNA)的表达,原位末端转移酶标记(TUNEL)法测定成纤维细胞凋亡情况。对数据行方差分析和t检验。结果实验组大鼠创面平均愈合时间[(25.6±2.8)d]长于对照组大鼠创面平均愈合时间[(19.1±2.1)d],差异有统计学意义(t=10.49,P<0.05);在伤后第7、14、21、28天,实验组大鼠创面愈合率分别为(18.8±4.6)%、(37.6±7.9)%、(66.3±11.4)%、(85.4±4.8)%,明显小于对照组[(40.8±4.5)%、(62.1±6.7)%、(90.2±9.7)%、(95.4±5.5)%,差异均有统计学意义(t=9.69、6.69、4.51、3.87,P值均小于0.05)。伤后第7、14、21天,两组大鼠创面逐渐愈合,创面组织中成纤维细胞、新生微血管均逐渐增多,且对照组大鼠未愈合创面组织中成纤维细胞、新生微血管均较实验组增多。伤后第7、14、21、28天,实验组大鼠碱性调宁蛋白在烧伤创面组织中的表达分别为1.43±0.07、1.17±0.11、0.73±0.06、0.77±0.07,均高于对照组大鼠(0.75±0.09、0.60±0.10、0.63±0.06、0.64±0.10),差异均有统计学意义(t=17.58、10.92、3.44、3.10,P值均小于0.05)。实验组大鼠成纤维细胞PCNA在烧伤创面组织中表达分别为654.19±102.80、820.84±112.86、766.68±156.63、232.75±55.37,较对照组(766.85±73.30、1322.22±121.44、1112.35±142.32、740.79±106.90)低,差异均有统计学意义(t=2.52、8.55、4.62、11.94,P值均小于0.05)。伤后第7、14、21、28天,实验组细胞凋亡数分别为58.51±10.89、41.53±9.95、27.28±6.58、20.13±4.23,较对照组(30.70±6.41、25.31±5.74、17.46±5.36、15.29±4.10)高,差异均有统计学意义(t=6.23、4.00、3.27、2.32,P值均小于0.05)。结论碱性调宁蛋白在糖尿病大鼠烧伤创面愈合过程中表达水平增高,可能调控创面组织中成纤维细胞的增殖和(或)凋亡水平,是糖尿病创面延迟愈合的原因之一。

Objective To discuss the expression of calponin in burn wound healing process of diabetic rats and its effect on the proliferation and apoptosis of fibroblasts.Methods Sixty-four Wistar male rats were selected and divided into the experimental group and the control group according to the random number table method,with 32 rats in each group.Rats in the experimental group were given intraperitoneal injections with streptozotoci by 60 mg/kg to create diabetic animal model.Rats in the control group were given 0.9%sodium chloride solution with 1.0 mL.Deep partial-thickness scald models(hereinafter referred to as burns)were produced in all rats at the time of adaptive feeding after 2 weeks.Liquid recovery and routine treatment were immediately followed after injury.Then the rats were under observation for calculation of wound healing time and the rates on post injury day 7,14,21,28.The tissue samples of wound were collected for observation of the histomorphology changes with hematoxylin-eosin staining,for analysis of the expression of calponin and proliferating cell nuclear antigen(PCNA)of fibroblasts by immunohistochemical SP method,and for measure of the apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL)assay.Data were processed with analysis of variance and t test.Results The average wound healing time of the experimental group[(25.6±2.8)d]was longer than that of the control group[(19.1±2.1)d],the difference was statistically significant(t=10.49,P<0.05).On post injury day 7,14,21,28,the wound healing rates of the rats in the experimental group[(18.8±4.6)%,(37.6±7.9)%,(66.3±11.4)%,(85.4±4.8)%]were obviously lower than those of the rats in the control group[(40.8±4.5)%,(62.1±6.7)%,(90.2±9.7),(95.4±5.5)%],and the differences were statistically significant(t=9.69,6.69,4.51,3.87;with P values below 0.05).On post injury day 7,14,21,the wound lasted and would heal gradually;fibroblasts and nascent capillaries in unhealed wound tissue of rats in the two groups both gradually increased,and more fibroblasts,and nascent capillaries were seen in unhealed wound tissue of rats in the control group.On post injury day 7,14,21,28,the expressions of calponin in the experimental group(1.43±0.07,1.17±0.11,0.73±0.06,0.77±0.07)were higher than those in the control group(0.75±0.09,0.60±0.10,0.63±0.06,0.64±0.10),and the differences were statistically significant(t=17.58,10.92,3.44,3.10;with P values below 0.05).The expressions of PCNA in fibroblasts of the the experimental group(654.19±102.80,820.84±112.86,766.68±156.63,232.75±55.37)were lower than those of the control group(766.85±73.30,1322.22±121.44,1112.35±142.32,740.79±106.90),and the differences were statistically significant(t=2.52,8.55,4.62,11.94;with P values below 0.05).The expressions of fibroblast apoptosis in the experimental group(58.51±10.89,41.53±9.95,27.28±6.58,20.13±4.23)were higher than those in the control group(30.70±6.41,25.31±5.74,17.46±5.36,15.29±4.10),and the differences were statistically significant(t=6.23,4.00,3.27,2.32;with P values below 0.05).Conclusion The increased expression of calponin during burn wound healing in diabetic rats may regulate the proliferation and(or)apoptosis of fibroblasts in the wound tissue,which is one of the reasons for delayed healing of diabetic wounds.