[1]林念慈,孙绍裘,李益亮,等.桃红四物汤对兔自体皮片移植模型Delta-like 4表达的影响[J].中医正骨,2015,27(11):1-7.
　LIN Nianci,SUN Shaoqiu,LI Yiliang,et al.Impact of Taohong Siwu Tang(桃红四物汤)on Delta-like 4 expression in the autologous skin graft model of rabbits[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2015,27(11):1-7.
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桃红四物汤对兔自体皮片移植模型Delta-like 4表达的影响()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第27卷
期数:
2015年11期
页码:
1-7
栏目:
基础研究
出版日期:
2015-11-30

文章信息/Info

Title:
Impact of Taohong Siwu Tang(桃红四物汤)on Delta-like 4 expression in the autologous skin graft model of rabbits
作者:
林念慈1; 孙绍裘2; 李益亮2; 徐无忌2; 陈希龙2; 周昭辉2
1.广东省江门市新会区中医院,广东 江门 529100;
2.湖南中医药大学第二附属医院,湖南 长沙 410005
Author(s):
LIN Nianci1; SUN Shaoqiu2; LI Yiliang2; XU Wuji2; CHEN Xilong2; ZHOU Zhaohui2
1.Traditional Chinese Medicine Hospital of Xinhui district in Jiangmen City,Jiangmen 529100,Guangdong,China
2.The Second Affiliated Hospital of Hunan University of Traditional Chinese Medicine,Changsha 410005,Hunan,China
关键词:
桃红四物汤;  皮肤移植;  Notch信号通路;  兔;  动物实验
Keywords:
Taohong Siwu Tang;  skin transplantation;  Notch Signaling Pathway;  rabbits;  animal experimentation
摘要:
目的:观察桃红四物汤对兔自体皮片移植模型Delta-like 4表达的影响。方法:将75只新西兰大白兔(10～12月龄)随机分为假手术组、模型组、低剂量组、中剂量组和高剂量组,每组15只。模型组、低剂量组、中剂量组和高剂量组动物进行自体皮片移植手术,假手术组仅在对应区域作切口,不进行皮下组织分离。造模术后假手术组和模型组动物以蒸馏水灌胃,低剂量组、中剂量组和高剂量组均以制备好的桃红四物汤汤剂灌胃(每次给药量分别为2.5 g·kg-1、5 g·kg-1、10 g·kg-1),每天2次。分别在药物干预开始后2、5、8 d从每组随机选取5只动物,心脏采血后处死,切取移植皮片吻合区皮片组织。采用酶联免疫吸附实验测定血清Delta-like 4蛋白含量; 所取皮片组织分成2份,一份制成石蜡切片,HE染色后测定血管密度,另一份采用逆转录聚合酶链式反应法测定Delta-like 4 mRNA含量。结果:①血清Delta-like 4蛋白含量。药物干预开始后2 d,5组动物血清Delta-like 4蛋白含量比较,差异无统计学意义[(30.58±3.56)ng·mL-1,(32.31±2.66)ng·mL-1,(33.53±1.75)ng·mL-1,(36.00±1.49)ng·mL-1,(35.63±1.87)ng·mL-1,F=2.669,P=0.062]。药物干预开始后5、8 d,5组动物血清Delta-like 4蛋白含量比较,组间差异均有统计学意义[(30.56±1.35)ng·mL-1,(33.52±2.50)ng·mL-1,(37.96±1.83)ng·mL-1,(48.74±2.34)ng·mL-1,(49.05±2.86)ng·mL-1,F=46.239,P=0.000;(29.26±2.20)ng·mL-1,(32.98±1.43)ng·mL-1,(35.24±1.97)ng·mL-1,(38.02±1.72)ng·mL-1,(36.06±2.50)ng·mL-1,F=8.394,P=0.000]; 模型组分别与假手术组和低剂量组比较,差异均无统计学意义(5 d:P=0.112,P=0.269; 8 d:P=0.340,P=0.182); 中、高剂量组血清Delta-like 4蛋白含量均高于模型组(5 d:P=0.000,P=0.000; 8 d:P=0.020,P=0.006); 中、高剂量组比较,组间差异均无统计学意义(P=0.862,P=0.245)。药物干预后5 d,中、高剂量组血清Delta-like 4蛋白含量均高于低剂量组(P=0.000,P=0.000)。药物干预开始后8 d,中、高剂量组Delta-like 4蛋白含量与低剂量组比较,差异均无统计学意义(P=0.105,P=0.623)。②移植皮片组织中Delta-like 4 mRNA含量。药物干预开始后2、5、8 d,5组动物移植皮片中Delta-like 4 mRNA含量比较,组间差异均有统计学意义(0.26±0.03,0.29±0.04,0.34±0.06,0.42±0.03,0.39±0.04,F=7.072,P=0.001; 0.25±0.03,0.34±0.04,0.39±0.03,0.48±0.06,0.45±0.06,F=12.028,P=0.001; 0.25±0.06,0.30±0.04,0.35±0.03,0.44±0.03,0.43±0.04,F=10.813,P=0.000); 模型组分别与假手术组和低剂量组比较,差异均无统计学意义(2 d:P=0.452,P=0.162; 5 d:P=0.320,P=0.158; 8 d:P=0.171,P=0.219); 模型组Delta-like 4 mRNA含量低于中、高剂量组(2 d:P=0.000,P=0.001; 5 d:P=0.001,P=0.009; 8 d:P=0.001,P=0.002); 中、高剂量组比较,组间差异均无统计学意义(P=0.426,P=0.330,P=0.725)。药物干预开始后2 d,中、高剂量组与低剂量组比较,差异均无统计学意义(P=0.111,P=0.167); 药物干预开始后5、8 d,中、高剂量组Delta-like 4 mRNA含量均高于低剂量组(5 d:P=0.024,P=0.025; 8 d:P=0.013,P=0.028)。③移植皮片组织中血管密度。药物干预开始后2、5 d,5组动物移植皮片中每个视野(×400)中血管数量比较,组间差异均有统计学意义[(5.37±0.36)个,(2.55±0.41)个,(2.83±0.24)个,(3.31±0.26)个,(3.53±0.67)个,F=4.084,P=0.014;(5.62±0.56)个,(4.25±0.68)个,(4.80±0.74)个,(5.88±0.42)个,(5.92±0.48)个,F=4.600,P=0.009]; 模型组均低于假手术组(P=0.001,P=0.011),模型组与低剂量组比较,差异均无统计学意义(P=0.527,P=0.268); 中、高剂量组比较,组间差异均无统计学意义(P=0.620,P=0.939)。药物干预开始后2 d,中、高剂量组与模型组比较,差异均无统计学意义(P=0.102,P=0.131); 中、高剂量组与低剂量组比较,差异均无统计学意义(P=0.297,P=0.352)。药物干预开始后5 d,中、高剂量组高于模型组(P=0.003,P=0.003); 中、高剂量组与低剂量组比较,差异均无统计学意义(P=0.038,P=0.033)。药物干预开始后8 d,5组动物移植皮片中每个视野中血管数量比较,差异无统计学意义[(6.18±0.33)个,(5.86±0.66)个,(6.03±0.40)个,(6.27±0.53)个,(6.27±0.59)个,F=0.334,P=0.852]。结论:桃红四物汤能上调兔自体皮片移植模型Delta-like 4的表达,促进血管新生。但本研究未能明确桃红四物汤的剂量及作用时间与其上调Delta-like 4表达作用的关系,需要在今后的研究中进一步探讨。
Abstract:
Objective:To observe the impact of Taohong Siwu Tang(桃红四物汤,THSWT)on Delta-like 4 expression in the autologous skin graft model of rabbits.Methods:Seventy-five New Zealand rabbits which ranged in age from 10 to 12 months were randomly divided into sham-operated group,model group,low-dose group,middle-dose group and high-dose group,15 cases in each group.The autologous skin transplantation surgery were performed on rabbits in model group,low-dose group,middle-dose group and high-dose group,while sham operation were performed on rabbits in sham-operated group.After the modeling operation,the rabbits in sham-operated group and model group were intragastric administrated with distilled water; while the others in low-dose group,middle-dose group and high-dose group were intragastric administrated with the prepared THSWT in dosages of 2.5,5 and 10 g/kg at a time,twice a day.Five rabbits were randomly selected from each group and the blood samples were obtained from the heart at 2,5 and 8 days after the beginning of the drug intervention respectively.The rabbits were executed and their anastomotic skin graft tissues were fetched out.The serum content of Delta-like 4 protein were measured by using enzyme-linked immunosorbent assay(ELISA).The acquired skin tissues were divided into 2 parts,and one of them were maded into paraffin sections and the blood vessel density were measured after HE staining,while the other portion were applied to measure the Delta-like 4 mRNA level by using reverse transcription-polymerase chain reaction(RT-PCR).Results:There was no statistical difference in serum content of Delta-like 4 protein between the 5 groups at 2 days after the beginning of the drug intervention(30.58+/-3.56,32.31+/-2.66,33.53+/-1.75,36.00+/-1.49,35.63+/-1.87 ng/ml,F=2.669,P=0.062).There was statistical difference in serum content of Delta-like 4 protein between the 5 groups at 5 and 8 days after the beginning of the drug intervention(30.56+/-1.35,33.52+/-2.50,37.96+/-1.83,48.74+/-2.34,49.05+/-2.86 ng/ml,F=46.239,P=0.000; 29.26+/-2.20,32.98+/-1.43,35.24+/-1.97,38.02+/-1.72,36.06+/-2.50 ng/ml,F=8.394,P=0.000).There was no statistical difference in serum content of Delta-like 4 protein between model group and sham-operated group and between model group and low-dose group(5d:P=0.112,P=0.269; 8d:P=0.340,P=0.182).The serum content of Delta-like 4 protein were higher in middle-dose group and high-dose group compared to model group(5d:P=0.000,P=0.000; 8d:P=0.020,P=0.006).There was no statistical difference in serum content of Delta-like 4 protein between middle-dose group and high-dose group(P=0.862,P=0.245).The serum content of Delta-like 4 protein were higher in middle-dose group and high-dose group compared to low-dose group at 5 days after the beginning of the drug intervention(P=0.000,P=0.000).There was no statistical difference in serum content of Delta-like 4 protein between middle-dose group and low-dose group and between high-dose group and low-dose group at 8 days after the beginning of the drug intervention(P=0.105,P=0.623).There was statistical difference in mRNA level of Delta-like 4 in skin graft between the 5 groups at 2,5 and 8 days after the beginning of the drug intervention(0.26+/-0.03,0.29+/-0.04,0.34+/-0.06,0.42+/-0.03,0.39+/-0.04,F=7.072,P=0.001; 0.25+/-0.03,0.34+/-0.04,0.39+/-0.03,0.48+/-0.06,0.45+/-0.06,F=12.028,P=0.001; 0.25+/-0.06,0.30+/-0.04,0.35+/-0.03,0.44+/-0.03,0.43+/-0.04,F=10.813,P=0.000).There was no statistical difference in Delta-like 4 mRNA level between model group and sham-operated group and between model group and low-dose group(2 d:P=0.452,P=0.162; 5 d:P=0.320,P=0.158; 8 d:P=0.171,P=0.219).The Delta-like 4 mRNA level were lower in model group compared to middle-dose group and high-dose group(2 d:P=0.000,P=0.001; 5 d:P=0.001,P=0.009; 8 d:P=0.001,P=0.002).There was no statistical difference in Delta-like 4 mRNA level between middle-dose group and high-dose group(P=0.426,P=0.330,P=0.725).There was no statistical difference in Delta-like 4 mRNA level between middle-dose group and low-dose group and between high-dose group and low-dose group at 2 days after the beginning of the drug intervention(P=0.111,P=0.167).The Delta-like 4 mRNA level was higher in middle-dose group and high-dose group compared to low-dose group at 5 and 8 days after the beginning of the drug intervention(5 d:P=0.024,P=0.025; 8 d:P=0.013,P=0.028).There was statistical difference in the number of blood vessels in skin graft under the optical microscope(×400)between the 5 groups at 2 and 5 days after the beginning of the drug intervention(5.37+/-0.36,2.55+/-0.41,2.83+/-0.24,3.31+/-0.26,3.53+/-0.67,F=4.084,P=0.014; 5.62+/-0.56,4.25+/-0.68,4.80+/-0.74,5.88+/-0.42,5.92+/-0.48,F=4.600,P=0.009).The number of blood vessels was less in model group compared to sham-operated group(P=0.001,P=0.011),and there was no statistical difference in the number of blood vessels between model group and low-dose group(P=0.527,P=0.268)and between middle-dose group and high-dose group(P=0.620,P=0.939).At 2 days after the beginning of the drug intervention,there was no statistical difference in the number of blood vessels between middle-dose group and model group(P=0.102)and between high-dose group and model group(P=0.131)and between middle-dose group and low-dose group(P=0.297)and between high-dose group and low-dose group(P=0.352).At 5 days after the beginning of the drug intervention,the number of blood vessels was more in middle-dose group and high-dose group compared to model group(P=0.003,P=0.003),while no statistical difference was found between middle-dose group and low-dose group and between high-dose group and low-dose group(P=0.038,P=0.033).At 8 days after the beginning of the drug intervention,there was no statistical difference in the number of blood vessels in skin graft under the optical microscope(×400)between the 5 groups(6.18+/-0.33,5.86+/-0.66,6.03+/-0.40,6.27+/-0.53,6.27+/-0.59,F=0.334,P=0.852).Conclusion:THSWT can up-regulate Delta-like 4 expression and promote angiogenesis in autologous skin graft model of rabbits.However,we cann't clarify the relationship between the role of THSWT in up-regulating Delta-like 4 expression and its dose and action time,which need to be further studied.

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备注/Memo

备注/Memo:
2015-07-16收稿 2015-10-07修回
基金项目:国家自然科学基金项目(81373658)
通讯作者:孙绍裘 E-mail:54sunshaoqiu@163.com
更新日期/Last Update: 2015-11-30