定向激活HIF-1协同脂肪干细胞对糖尿病小鼠皮肤创伤修复的促进作用

doi:10.3877/cma.j.issn.2095-1221.2018.02.005

目的

探讨定向激活HIF-1协同脂肪干细胞（ASCs）对糖尿病小鼠损伤修复的影响及机制。

方法

8周龄雄性SPF级C57BJ/L小鼠68只，行腹腔注射1%链脲佐菌素（STZ，60?mg/?kg）的柠檬酸-柠檬酸钠缓冲液，3 d后空腹血糖大于16.7 mmol/L的小鼠用于实验。在小鼠背部做直径为1 cm的圆形皮肤全层创口，将动物随机分为空载质粒（EV）组，CA5-HIF-1α转染（CA5）组，EV+saline组，CA5+saline组，EV+ASCs组，CA5+ASCs组。于给药后第0、3、7、10、14、17和21天，观察并记录创口面积大小，利用激光多普勒血流成像（LDPI）检测血流灌注情况，荧光定量PCR法检测HIF-1α及VEGF mRNA表达水平，HE染色检测血管密度，Western blot法检测VEGE蛋白表达水平。用Bonferroni或Tukey post hoc方差分析法进行统计学分析。

结果

CA5-HIF-1α基因注射剂量为125 μg时，局部HIF-1α mRNA表达水平较高为10.40±0.22（F = 19.48，P = 0.025），故选用125 μg用于后续实验。于给药后第3 ~ 14天，CA5组小鼠的伤口面积分别为7?828.92±294.28、7?285.97±118.24、4?050.41±301.97、1?292.35±101.14小于EV组9?062.00±225.75、8?534.42±189.35、5?634.59±198.06、2?308.15±245.36（F?=?41.37、32.16、27.29、25.16，P?=?0.028、0.034、0.038、0.042）。CA5组小鼠皮肤血流于第10、17天（253.06±8.34、250.59±10.13）均大于EV组（158.31±9.98、169.73±7.28）（F?=?21.53、26.08，P?=?0.038、0.032）。在治疗后3 ~ 14天，CA5+saline组（7?656.92±177.03、7?163.83±128.24、4?238.23±228.36、1?316.52±90.75）、EV+ASCs组(7?593.64±192.12、7?233.08±146.86、4?097.58±227.91)及CA5+ASCs组的伤口面积小于对照组（6?745.25±203.16、6?159.35±168.72、3?682.06±257.30）（F?=?39.58、44.09、34.67，P?=?0.031、0.028、0.037），且CA5+ASCs组最小。本研究还发现CA5+saline组（262.05±9.34、248.45±11.13）、EV+ASCs组（215.33±10.75、185.82±10.47）及CA5+ASCs组（322.54±12.27、292.49±9.57）的小鼠皮肤血流于第10天和第17天均大于对照组（161.30±5.64、134.57±8.67）（F?=?29.15、17.38，P?=?0.026、0.034），CA5+ASCs组血流增加幅度最大。此外，CA5+saline组、EV+ASCs组及CA5+ASCs组血管密度大于对照组，且CA5+ASCs组最高。CA5+saline组、EV+ASCs组及CA5+ASCs组VEGF mRNA及蛋白表达水平分别为2.03±0.14、2.16±0.13、3.41±0.18和1.75±0.12、1.82±0.06、2.96±0.14，高于对照组1.05±0.02和1.03±0.05（F?=?34.08、29.53，P?=?0.019、0.021），且CA5+ASCs组最高。

结论

定向激活HIF-1基因协调脂肪干细胞可促进糖尿病小鼠创伤修复，可能与其调控VEGF表达有关。

关键词: HIF-1α, 脂肪干细胞, 糖尿病, 小鼠, 创面愈合, VEGF

Objective

To explore the effect of combination of HIF-1α gene transfection and Adipose stem cells (ASCs) on diabetic wound healing in mice.

Methods

Sixty-eight SPF C57BJ/L mice were intravenously treated with 1% streptozotocin (STZ) at a dose of 60 mg/?kg versus body weight for 3 days. Animals with fasting blood glucose concentration higher than 16.7?mmol/L were employed in the following experiment. Subsequently, on the back of mice, round-thickness skin wounds with 1 cm were made. The mice were randomly divided into empty vector group (EV), a plasmid DNA construct expressing a stabilized mutant form of HIF-1α (CA5-HIF-1α) vector transfected group (CA5), EV+ saline group, CA5+saline group, EV+ASCs group, CA5+ASCs group. On the 3rd, 10rd, 17rd, 21st days after treatment, the area of the wounds was measured. The dermal blood flow was measured by Laser Doppler perfusion imaging (LDPI). The levels of HIF-1α and VEGF mRNA were measured by quantitative Real-time PCR (qRT-PCR). The blood vessel density was detected by H&E staining. The level of VEGF protein was measured by Western blot. The statistical analysis was used with the Bonferroni or Tukey post hoc variance analysis method.

Results

The level of HIF-1α mRNA expression treated with 125 μg CA5-HIF-1α gene was the highest among all doses (10.40±0.22) (F = 19.48, P = 0.025) so we selected 125 μg HIF-1α gene for the following experiments. By days 3 ~ 14, the wound area in CA5 group (7?828.92±294.28, 7?285.97±118.24, 4?050.41±301.97, 1?292.35±101.14) was smaller than the EV group (9?062.00±225.75, 8?534.42±189.35, 5?634.59±198.06, 2?308.15±245.36) (F?=?41.37, 32.16, 27.29, 25.16, P?=?0.028, 0.034, 0.038, 0.042). Besides, in the 10th and 17th days, the blood flow of mice in CA5 group (253.06±8.34, 250.59±10.13) was significantly greater than that of the EV group (158.31±9.98, 169.73±7.28) (F?=?21.53, 26.08, P?=?0.038, 0.032). ASCs were shown to have promotive effects on the wound healing. Therefore, we further explored the effects of combination of HIF-1α gene and ASCs therapy on skin wound healing in diabetic mice. We found that the wound area of CA5+saline group (7?656.92±177.03, 7?163.83±128.24, 4?238.23±228.36, 1?316.52±90.75), EV+ASCs group (7?593.64±192.12, 7?233.08±146.86, 4?097.58±227.91), CA5+ASCs group (7?593.64±192.12, 7?233.08±146.86, 4?097.58±227.91) were significantly smaller than the control group (6?745.25±203.16, 6?159.35±168.72, 3?682.06±257.30) (F?=?39.58, 44.09, 34.67, P?=?0.031, 0.028, 0.037) on 3 ~ 14 days after treatment. Besides, CA5+ASCs group showed the smallest wound area. In addition, we also found that the blood flow of CA5+saline group (262.05±9.34, 248.45±11.13), EV+ASCs group (215.33±10.75, 185.82±10.47), CA5+ASCs group (322.54±12.27, 292.49±9.57) significantly increased on day 10 compared to the control group (161.30±5.64, 134.57±8.67) (F = 29.15, 17.38, P = 0.026, 0.034). Moreover, the blood flow in combination group increased sharpest. CA5+saline group, EV+ASCs group, CA5+ASCs group showed an obvious increase in vessel density and the combined group had the highest increase in vessel density. We also found that the expression levels of VEGF mRNA and protein in CA5+saline group, EV+ASCs group, CA5+ASCs group (2.03±0.14, 2.16±0.13, 3.41±0.18) and (1.75±0.12, 1.82±0.06, 2.96±0.14) were significantly higher than those in the control group (1.05±0.02) and (1.03±0.05) (F = 34.08, 29.53, P = 0.019, 0.021). The combined group showed the highest levels of VEGF expression.

Conclusion

Targeted activation of HIF-1 gene coordinated ASCs can promote the wound healing of diabetic mice, and its mechanism may be related to regulation of VEGF expression.

Key words: HIF-1α, Adipose stem cells, Diabetic, Mice, Wound healing, VEGF

图1 CA5-HIF-1α示意图

表1 HIF-1α、VEGF、GAPDH基因的引物

基因	上游引物（5'-3'）	下游引物（5'-3'）
HIF-1α	CCACAGGACAGTACAGGATG	TCAAGTCGTGCTGAATAATACC
VEGF	CTCGCAGTCCGAGCCGGAGA	GCAGCCTGGGACCACTTGGC
GAPDH	ATCATCCCTGCATCCACT	ATCCACGACGGACACATT

表1 HIF-1α、VEGF、GAPDH基因的引物

基因	上游引物（5'-3'）	下游引物（5'-3'）
HIF-1α	CCACAGGACAGTACAGGATG	TCAAGTCGTGCTGAATAATACC
VEGF	CTCGCAGTCCGAGCCGGAGA	GCAGCCTGGGACCACTTGGC
GAPDH	ATCATCCCTGCATCCACT	ATCCACGACGGACACATT

图2 不同处理组小鼠HIF-1α mRNA表达水平的比较

表2 不同处理组经定向激活HIF-1基因治疗后糖尿病小鼠伤口面积的比较（像素，±s）

分组	治疗后时间
分组	第0天	第3天	第7天	第10天	第14天	第17天	第21天
EV组	9 125.48±117.53	9 062.00±225.75	8 534.42±189.35	5 634.59±198.06	2 308.15±245.36	462.42±22.61	12.65±0.03
KGF组	8 962.06±184.37^*	8 351.02±164.56^*	7 724.10±205.61^*	4 396.48±175.69^*	1 785.17±231.58^*	198.13±14.02^*	13.26±0.02^*
CA5组	9 067.26±247.03^*	7 828.92±294.28^*	7 285.97±118.24^*	4 050.41±301.97^*	1 292.35±101.14^*	69.61±18.57^*	9.25±0.25^*
F值	39.25	41.37	32.16	27.29	25.16	45.49	28.17
P值	0.031	0.028	0.034	0.038	0.042	0.024	0.037

表2 不同处理组经定向激活HIF-1基因治疗后糖尿病小鼠伤口面积的比较（像素，±s）

分组	治疗后时间
分组	第0天	第3天	第7天	第10天	第14天	第17天	第21天
EV组	9 125.48±117.53	9 062.00±225.75	8 534.42±189.35	5 634.59±198.06	2 308.15±245.36	462.42±22.61	12.65±0.03
KGF组	8 962.06±184.37^*	8 351.02±164.56^*	7 724.10±205.61^*	4 396.48±175.69^*	1 785.17±231.58^*	198.13±14.02^*	13.26±0.02^*
CA5组	9 067.26±247.03^*	7 828.92±294.28^*	7 285.97±118.24^*	4 050.41±301.97^*	1 292.35±101.14^*	69.61±18.57^*	9.25±0.25^*
F值	39.25	41.37	32.16	27.29	25.16	45.49	28.17
P值	0.031	0.028	0.034	0.038	0.042	0.024	0.037

图3 CA5-HIF-1α基因治疗组（CA5组）与空白对照组（EV组）小鼠创面血流量比较

表3 不同处理组经HIF-1α基因联合ASCs治疗后糖尿病小鼠伤口面积的比较（像素，±s）

分组	治疗后时间
分组	第0天	第3天	第7天	第10天	第14天	第17天	第21天
EV+Saline	9 080.12±120.41	8 989.25±175.03	8 320.72±176.05	5 590.61±208.28	2 473.08±163.82	405.76±16.78	42.13±0.05
CA5+Saline	9 117.46±241.58^*	7 656.92±177.03^*	7 163.83±128.24^*	4 238.23±228.36^*	1 316.52± 90.75^*	186.36±17.95^*	26.08±0.13^*
EV+ASCs	9 134.50±254.33^*	7 593.64±192.12^*	7 233.08±146.86^*	4 097.58±227.91^*	1 488.53± 86.25^*	160.48±18.72^*	31.45±0.28^*
CA5+ASCs	8 994.27±184.72^*	6 745.25±203.16^*	6 159.35±168.72^*	3 682.06±257.30^*	997.64±183.71^*	108.67±10.58^*	19.25±0.36^*
F值	27.15	39.58	44.09	34.67	29.52	35.05	31.68
P值	0.046	0.031	0.028	0.037	0.043	0.035	0.041

表3 不同处理组经HIF-1α基因联合ASCs治疗后糖尿病小鼠伤口面积的比较（像素，±s）

分组	治疗后时间
分组	第0天	第3天	第7天	第10天	第14天	第17天	第21天
EV+Saline	9 080.12±120.41	8 989.25±175.03	8 320.72±176.05	5 590.61±208.28	2 473.08±163.82	405.76±16.78	42.13±0.05
CA5+Saline	9 117.46±241.58^*	7 656.92±177.03^*	7 163.83±128.24^*	4 238.23±228.36^*	1 316.52± 90.75^*	186.36±17.95^*	26.08±0.13^*
EV+ASCs	9 134.50±254.33^*	7 593.64±192.12^*	7 233.08±146.86^*	4 097.58±227.91^*	1 488.53± 86.25^*	160.48±18.72^*	31.45±0.28^*
CA5+ASCs	8 994.27±184.72^*	6 745.25±203.16^*	6 159.35±168.72^*	3 682.06±257.30^*	997.64±183.71^*	108.67±10.58^*	19.25±0.36^*
F值	27.15	39.58	44.09	34.67	29.52	35.05	31.68
P值	0.046	0.031	0.028	0.037	0.043	0.035	0.041

图4 不同处理组小鼠创面血流量比较

图5 荧光显微镜下观察各组小鼠皮肤创面的血管密度（HE染色　×50）

图6 不同处理组小鼠的VEGF表达水平的比较

图7 不同处理组小鼠VEGF蛋白表达Western blot检测

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Targeted activation of HIF-1 gene coordinated ASCs can promote the wound healing of diabetic mice

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Targeted activation of HIF-1 gene coordinated ASCs can promote the wound healing of diabetic mice