负压伤口疗法对糖尿病足轻、中度缺血创面肉芽组织转化生长因子-β1表达的影响

doi:10.3877/cma.j.issn.1673-9450.2019.01.005

目的

探讨负压伤口疗法(NPWT)对糖尿病足轻、中度缺血创面肉芽组织转化生长因子-β1(TGF-β1)表达的影响。

方法

选取2014年1月至2018年2月在解放军白求恩国际和平医院接受NPWT治疗的43例糖尿病足患者，据踝肱指数(ABI)分为中度缺血组23例(0.5≤ABI<0.7)和轻度缺血组20例(0.7≤ABI<0.9)。于治疗前及治疗14 d后分别取创面肉芽组织，标本分成3份，1份于4%多聚甲醛溶液中固定，4 ℃保存，标本收齐后行免疫组织化学染色，分析TGF-β1平均光密度；另外2份于液氮中转送至-80 ℃冰箱中保存，其中1份行Western blotting法，分析TGF-β1蛋白表达情况，另1份行Real-time PCR，分析TGF-β1 mRNA表达情况。2组间数据比较采用独立样本t检验。

结果

经免疫组织化学染色分析TGF-β1表达，中度缺血组和轻度缺血组经14 d治疗后TGF-β1平均光密度均高于治疗前，差异有统计学意义(P值均小于0.05)，且相较于中度缺血组变化幅度[(38.71±12.78)%]，轻度缺血组[(63.09±10.96)%]升高幅度显著，差异均有统计学意义(t＝－6.66，P<0.05)；经Western blotting法分析TGF-β1蛋白表达水平，中度缺血组和轻度缺血组经14 d治疗后TGF-β1蛋白表达均高于治疗前，差异均有统计学意义(P值均小于0.05)，且相较于中度缺血组变化幅度[(50.31±25.64)%]，轻度缺血组变化幅度[(85.44±27.78)%]升高幅度显著，差异有统计学意义(t＝－4.31，P<0.05)；经Real-time PCR法分析TGF-β1 mRNA表达水平，中度缺血组和轻度缺血组经14 d治疗后TGF-β1 mRNA表达均高于治疗前，差异均有统计学意义(P值均小于0.05)，且相较于中度缺血组变化幅度[(73.85±41.22)%]，轻度缺血组[(129.78±36.71)%]升高幅度显著，差异有统计学意义(t＝－4.67，P<0.05)。

结论

NPWT能促进糖尿病足轻、中度缺血创面肉芽组织TGF-β1的表达，且对糖尿病足轻度缺血创面肉芽组织TGF-β1表达的促进作用优于糖尿病足中度缺血创面。

关键词: 负压伤口疗法, 糖尿病足, 转化生长因子β1, 踝肱指数

Objective

To explore the expression of the TGF-β1 of diabetic foot wound granulation tissue treated by negative pressure wound therapy(NPWT), which with mild or moderate peripheral artery disease.

Methods

Forty three patients at Bethune International Peace Hospital of People′s Liberation Army with diabetic foot ulcers from January 2014 to February 2018 were enrolled in this study and divided into moderate ischemia group [23 cases, 0.5≤ankle-brachial index (ABI)<0.7] and mild ischemia group (20 cases, 0.7≤ABI<0.9) according to the ABI. Granulation tissue biopsies were taken before and 14 days after treatment, which were all divided into three parts: one part were preserved in 4% paraformaldehyde at 4 ℃ for immunohistochemical staining when all specimens collected; the other two parts were transferred in liquid nitrogen to the laboratory immediately and preserved in refrigerator at -80 ℃ for Western blotting and Real-time PCR respectively. Date were compared with independent sample t test.

Result

Immunohistochemical staining showed that optical density of TGF-β1 after 14 days treatment in both moderate and mild ischemia group were higher than those before treatment, the differences were statistically significant (with P values below 0.05), with the increase in mild ischemia group (63.09±10.96)% more significant than moderate ischemia group[(38.71±12.78)%], the difference was statistically significant (t=-6.66, P<0.05). Western blotting showed those the expression of TGF-β1 protein after 14 days treatment were higher than that before treatment in both moderate and mild ischemia group, the differences were statistically significant (with P values below 0.05), compared with the increase in mild ischemia group (50.31±25.64)%, the degree of change in the mild ischemic group [(85.44±27.78)%] was significant, the difference was statistically significant (t=-4.31, P<0.05). Real-time PCR showed that the expression of TGF-β1 gene after 14 days treatment were higher than those before treatment in both moderate and mild ischemia group, the differences were statistically significant (with P values below 0.05). Compared with the change amplitude of the moderate ischemic groups [(73.85±41.22)%], the increase the mild ischemic group [(129.78±36.71)%] was significant, the difference was statistically significant (t=-4.67, P<0.05).

Conclusion

NPWT can promote the expression of TGF-β1 in granulation tissue of diabetic patients with mild and moderate ischemic wounds, moreover, the promotion of TGF-β1 expression in granulation tissue of diabetic foot with mild ischemic wound is better than that of moderate ischemic wound.

Key words: Negative-pressure wound therapy, Diabetic foot, Transforming growth factor-beta1, Ankle brachial index

表1 2组糖尿病足患者一般资料比较

组别	例数	性别(例)		年龄(岁，±s)	空腹血糖(mmol/L, ±s)	糖化血红蛋白(%, ±s)
组别	例数	男	女	年龄(岁，±s)	空腹血糖(mmol/L, ±s)	糖化血红蛋白(%, ±s)
中度缺血组	23	13	10	58.39±6.56	9.97±3.38	10.00±2.07
轻度缺血组	20	8	12	59.00±6.67	8.29±3.92	10.06±1.91
t/χ²值	?	1.17^b		0.30^a	1.51^a	0.10^a
P值	?	0.28		0.77	0.14	0.92
组别	例数	溃疡时间(d, ±s)	谷丙转氨酶(U/L, ±s)	肌酐(mmol/L, ±s )
中度缺血组	23	56.30±19.51	17.75±7.75	64.13±19.90
轻度缺血组	20	54.65±18.60	16.21±7.86	61.85±20.48
t/χ²值	?	0.36^a	0.87^a	0.37^a
P值	?	0.72	0.39	0.71

表1 2组糖尿病足患者一般资料比较

组别	例数	性别(例)		年龄(岁，±s)	空腹血糖(mmol/L, ±s)	糖化血红蛋白(%, ±s)
组别	例数	男	女	年龄(岁，±s)	空腹血糖(mmol/L, ±s)	糖化血红蛋白(%, ±s)
中度缺血组	23	13	10	58.39±6.56	9.97±3.38	10.00±2.07
轻度缺血组	20	8	12	59.00±6.67	8.29±3.92	10.06±1.91
t/χ²值	?	1.17^b		0.30^a	1.51^a	0.10^a
P值	?	0.28		0.77	0.14	0.92
组别	例数	溃疡时间(d, ±s)	谷丙转氨酶(U/L, ±s)	肌酐(mmol/L, ±s )
中度缺血组	23	56.30±19.51	17.75±7.75	64.13±19.90
轻度缺血组	20	54.65±18.60	16.21±7.86	61.85±20.48
t/χ²值	?	0.36^a	0.87^a	0.37^a
P值	?	0.72	0.39	0.71

图1 2组糖尿病足患者NPWT治疗前后创面肉芽组织TGF-β1免疫组织化学染色情况(×400)。A示中度缺血组治疗前TGF-β1表达情况；B示治疗14 d后，中度缺血组TGF-β1较治疗前表达明显增多；C示轻度缺血组治疗前TGF-β1表达：D示治疗14 d后，轻度缺血组TGF-β1表达明显增多；箭头示TGF-β1免疫组织化学染色阳性区域；NPWT为负压伤口疗法；TGF-β1为转化生长因子-β1

表2 2组糖尿病足患者NPWT治疗前后TGF-β1平均光密度值统计结果(±s)

组别	例数	治疗前	治疗14 d后	治疗前后变化值	变化幅度(%)	t₁值	P₁值
中度缺血组	23	0.24±0.03	0.34±0.03	0.12±0.03	38.71±12.78	－20.26	<0.05
轻度缺血组	20	0.28±0.03	0.45±0.03	0.17±0.02	63.09±10.96	－10.26	<0.05
t₂值	?	－4.25	－11.92	-8.12	-6.66	?	?
P₂值	?	<0.05	<0.05	<0.05	<0.05	?	?

表2 2组糖尿病足患者NPWT治疗前后TGF-β1平均光密度值统计结果(±s)

组别	例数	治疗前	治疗14 d后	治疗前后变化值	变化幅度(%)	t₁值	P₁值
中度缺血组	23	0.24±0.03	0.34±0.03	0.12±0.03	38.71±12.78	－20.26	<0.05
轻度缺血组	20	0.28±0.03	0.45±0.03	0.17±0.02	63.09±10.96	－10.26	<0.05
t₂值	?	－4.25	－11.92	-8.12	-6.66	?	?
P₂值	?	<0.05	<0.05	<0.05	<0.05	?	?

图2 Western blotting法分析2组糖尿病足患者经NPWT治疗前后创面肉芽组织TGF-β1蛋白表达情况

表3 2组糖尿病足患者NPWT治疗前后TGF-β1蛋白表达变化(±s)

组别	例数	治疗前	治疗14 d后	治疗前后变化值	变化幅度(%)	t₁值	P₁值
中度缺血组	23	0.07±0.02	0.11±0.02	0.03±0.02	50.31±25.64	－5.56	<0.05
轻度缺血组	20	0.10±0.03	0.19±0.04	0.09±0.03	85.44±27.78	－7.86	<0.05
t₂值	?	－4.70	－7.89	－7.44	－4.31	?	?
P₂值	?	<0.05	<0.05	<0.05	<0.05	?	?

表3 2组糖尿病足患者NPWT治疗前后TGF-β1蛋白表达变化(±s)

组别	例数	治疗前	治疗14 d后	治疗前后变化值	变化幅度(%)	t₁值	P₁值
中度缺血组	23	0.07±0.02	0.11±0.02	0.03±0.02	50.31±25.64	－5.56	<0.05
轻度缺血组	20	0.10±0.03	0.19±0.04	0.09±0.03	85.44±27.78	－7.86	<0.05
t₂值	?	－4.70	－7.89	－7.44	－4.31	?	?
P₂值	?	<0.05	<0.05	<0.05	<0.05	?	?

表4 2组糖尿病足患者NPWT治疗前后TGF-β1 mRNA表达情况(±s)

组别	例数	治疗前	治疗14 d后	治疗前后变化值	变化幅度(%)	t₁值	P₁值
中度缺血组	23	1.04±0.19	1.77±0.38	0.73±0.37	73.85±41.22	－8.32	<0.05
轻度缺血组	20	1.26±0.16	2.85±0.38	1.59±0.38	129.78±36.71	－17.28	<0.05
t₂值	?	－4.03	－9.32	－7.55	－4.67	?	?
P₂值	?	<0.05	<0.05	<0.05	<0.05	?	?

表4 2组糖尿病足患者NPWT治疗前后TGF-β1 mRNA表达情况(±s)

组别	例数	治疗前	治疗14 d后	治疗前后变化值	变化幅度(%)	t₁值	P₁值
中度缺血组	23	1.04±0.19	1.77±0.38	0.73±0.37	73.85±41.22	－8.32	<0.05
轻度缺血组	20	1.26±0.16	2.85±0.38	1.59±0.38	129.78±36.71	－17.28	<0.05
t₂值	?	－4.03	－9.32	－7.55	－4.67	?	?
P₂值	?	<0.05	<0.05	<0.05	<0.05	?	?

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Effect of negative pressure wound therapy on the expression of transforming growth factor-beta1 in granulation tissue with mild and moderate ischemia wound of diabetic foot

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Effect of negative pressure wound therapy on the expression of transforming growth factor-beta1 in granulation tissue with mild and moderate ischemia wound of diabetic foot