脂多糖诱导对内皮细胞释放内皮细胞微粒的影响

doi:10.3877/cma.j.issn.1673-9450.2017.02.006

目的

探讨脂多糖刺激对内皮细胞释放内皮细胞微粒(EMP)的影响。

方法

取人脐静脉内皮细胞培养，分为正常对照组(无脂多糖诱导)和不同浓度脂多糖(浓度分别为0.1、1.0、10.0、20.0 μg/mL)组，脂多糖诱导24 h后收集细胞培养液并消化内皮细胞制成细胞悬液。离心法提取细胞培养液内的EMP，加入特异性荧光抗体(anti-CD31-CFS、anti-CD51-PE、anti-CD54-FITC、anti-CD144-PE、anti-CD62E-APC)。采用流式细胞仪检测脂多糖诱导内皮细胞释放荧光标记EMP的变化以及内皮细胞的凋亡率。对数据行方差分析、t检验、Dunnett-t检验。

结果

对照组和0.1、1.0、10.0、20.0 μg/mL脂多糖组CD31^＋EMP数量水平分别为(1.23±0.43)×10⁶、(1.92±0.64)×10⁶、(2.55±0.78)×10⁶、(3.96±0.78)×10⁶、(6.96±1.80)×10⁶，组间比较差异有统计学意义(F＝5.83，P<0.05)，CD31^＋EMP占总体EMP的比值分别为(4.57±1.33)%、(5.92±1.24)%、(6.93±1.61)%、(7.44±1.09)%、(13.44±3.39)%，组间比较差异有统计学意义(F＝3.43，P<0.05)。与对照组比较，10.0、20.0 μg/mL脂多糖组CD31^＋EMP数量水平明显升高，差异均有统计学意义(t＝－3.06、－3.61，P值均小于0.05)；20.0 μg/mL脂多糖组CD31^＋EMP数量水平高于0.1、1.0 μg/mL脂多糖组，差异均有统计学意义(P值均小于0.05)。CD31^＋EMP占总体EMP的比值20.0 μg/mL脂多糖组高于对照组和0.1 μg/mL脂多糖组，差异均有统计学意义(P值均小于0.05)。对照组和0.1、1.0、10.0、20.0 μg/mL脂多糖组CD54^＋EMP数量水平分别为(4.70±1.01)×10⁶、(9.20±3.34)×10⁶、(8.83±1.70)×10⁶、(17.18±3.78)×10⁶、(17.73±4.98)×10⁶，组间比较差异有统计学意义(F＝3.35，P<0.05)。与对照组比较，10.0、20.0 μg/mL脂多糖组CD54^＋EMP数量水平明显升高，差异均有统计学意义(t＝－3.19、－3.00，P值均小于0.05)。对照组和0.1、1.0、10.0、20.0 μg/mL脂多糖组CD144^＋EMP数量水平分别为(3.93±1.22)×10⁶、(6.80±1.36)×10⁶、(8.03±2.53)×10⁶、(17.22±4.52)×10⁶、(11.05±5.80)×10⁶，组间比较差异有统计学意义(F＝3.17，P<0.05)。10.0 μg/mL脂多糖组CD144^＋EMP数量水平高于对照组及0.1、1.0 μg/mL脂多糖组，差异均有统计学意义(P值均小于0.05)。20.0 μg/mL脂多糖组CD62E^＋EMP数量水平为(2.95±0.26)×10⁶，明显高于对照组(1.26±0.26)×10⁶，差异有统计学意义(t＝－4.45，P<0.05)。各组内皮细胞凋亡率比较差异无统计学意义(F＝0.17，P>0.05)。

结论

脂多糖损伤内皮细胞可表现为表达特异性标记的EMP的大量释放，可能是脓毒症内皮细胞损伤机制之一。

关键词: 脂多糖类, 内皮细胞, 脐静脉, 微粒

Objective

To investigate the significance of changes of endothelial microparticle(EMP) induced by lipopolysaccharide.

Methods

Human umbilical vein endothelial cell (HUVEC) was cultured. The cells were divided into control group (without lipopolysaccharide) and lipopolysaccharide group (0.1, 1.0, 10.0, 20.0 μg/mL). The cell culture supernatant were collected after 24 h incubation with or without lipopolysaccharide. The cells were digested and suspended. EMP was collected by series of centrifugation and incubated with CFS anti-CD31, PE anti-CD51, FITC anti-CD54, PE anti-CD144, APC anti-CD62E. Flow cytometry was performed to analyze by detecting the expression of the fluorescenct labeling EMP and the measurement of apoptosis of endotheliums. Data were processed with analysis of variance, t and Dunnett-t test.

Results

The counts of CD31⁺EMP in control group and lipopolysaccharide group (0.1, 1.0, 10.0, 20.0 μg/mL) were respectively (1.23±0.43)×10^6, (1.92±0.64)×10^6, (2.55±0.78)×10^6, (3.96±0.78)×10^6, (6.96±1.80)×10^6, there were significant differences (F=5.83, P<0.05), and the ratios of CD31⁺EMP were respectively (4.57±1.33)%, (5.92±1.24)%, (6.93±1.61)%, (7.44±1.09)%, (13.44±3.39)%, there were significant differences (F=3.43, P<0.05). Compared with that of control group, the counts of CD31⁺EMP were obviously increased in 10.0, 20.0 μg/mL lipopolysaccharide groups (with t value respectively -3.06, -3.61, P values were less than 0.05). In 20.0 μg/mL lipopolysaccharide group, the counts of CD31⁺EMP were higher than that in 0.1, 1.0 μg/mL lipopolysaccharide groups (P<0.05), and the ratios of CD31⁺EMP were higher than that in control group (t=-2.74, P<0.05) and in 0.1 μg/mL lipopolysaccharide group (P<0.05). The counts of CD54⁺EMP in control group and lipopolysaccharide group (0.1, 1.0, 10.0, 20.0 μg/mL) were respectively (4.70±1.01)×10^6, (9.20±3.34)×10^6, (8.83±1.70)×10^6, (17.18±3.78)×10^6, (17.73±4.98)×10^6, there were significant differences (F=3.35, P<0.05). Compared with that of control group, the counts of CD54⁺EMP were obviously increased in 10.0, 20.0 μg/mL lipopolysaccharide groups (with t value respectively -3.19, -3.00, P values were less than 0.05). The counts of CD144⁺EMP in control group and lipopolysaccharide group (0.1, 1.0, 10.0, 20.0 μg/mL) were respectively (3.93±1.22)×10^6, (6.80±1.36)×10^6, (8.03±2.53)×10^6, (17.22±4.52)×10^6, (11.05±3.80)×10^6, there were significant differences (F=3.17, P<0.05). The counts of CD144⁺EMP were higher in 10.0 μg/mL lipopolysaccharide group than that in control group and that in 0.1, 1.0 μg/mL lipopolysaccharide groups (P values were less than 0.05). In 20.0 μg/mL lipopolysaccharide group, the counts of CD62E⁺EMP were (2.95±0.26)×10^6, which were higher than that in control group whose counts were (1.26±0.26)×10⁶(t=-4.45, P<0.05). Comparing the apoptosis rate of endothelial cell in each group, there were no significant differences (F=0.17, P>0.05).

Conclusion

The increase of EMP levels suggests that the specific bioactivity of each antigen-positive EMP may participate in regulating endothelial function induced by lipopolysaccharide, and they may be one of the mechanisms of endothelial dysfunction in sepsis.

Key words: Lipopolysaccharides, Endothelial cells, Umbilical veins, Microparticles

表1 CD31^＋EMP数量和比值变化(±s)

组别	CD31^＋EMP(×10⁶)	CD31^＋EMP(%)
对照组	1.23±0.43	4.57±1.33
0.1 μg/mL脂多糖组	1.92±0.64^ae	5.92±1.24^ce
1.0 μg/mL脂多糖组	2.55±0.78^ae	6.93±1.61^c
10.0 μg/mL脂多糖组	3.96±0.78^b	7.44±1.09^c
20.0 μg/mL脂多糖组	6.96±1.80^b	13.44±3.39^d
F值	5.83	3.43
P值	<0.05	<0.05

表1 CD31^＋EMP数量和比值变化(±s)

组别	CD31^＋EMP(×10⁶)	CD31^＋EMP(%)
对照组	1.23±0.43	4.57±1.33
0.1 μg/mL脂多糖组	1.92±0.64^ae	5.92±1.24^ce
1.0 μg/mL脂多糖组	2.55±0.78^ae	6.93±1.61^c
10.0 μg/mL脂多糖组	3.96±0.78^b	7.44±1.09^c
20.0 μg/mL脂多糖组	6.96±1.80^b	13.44±3.39^d
F值	5.83	3.43
P值	<0.05	<0.05

表2 CD54^＋EMP数量和比值变化(±s)

组别	CD54^＋EMP(×10⁶)	CD54^＋EMP(%)
对照组	4.70±1.01	17.92±3.29
0.1 μg/mL脂多糖组	9.20±3.34^a	27.63±6.21^c
1.0 μg/mL脂多糖组	8.83±1.70a	24.93±3.04^c
10.0 μg/mL脂多糖组	17.18±3.78^b	32.15±5.53^c
20.0 μg/mL脂多糖组	17.73±4.98^b	33.77±8.65^c
F值	3.35	1.37
P值	<0.05	>0.05

表2 CD54^＋EMP数量和比值变化(±s)

组别	CD54^＋EMP(×10⁶)	CD54^＋EMP(%)
对照组	4.70±1.01	17.92±3.29
0.1 μg/mL脂多糖组	9.20±3.34^a	27.63±6.21^c
1.0 μg/mL脂多糖组	8.83±1.70a	24.93±3.04^c
10.0 μg/mL脂多糖组	17.18±3.78^b	32.15±5.53^c
20.0 μg/mL脂多糖组	17.73±4.98^b	33.77±8.65^c
F值	3.35	1.37
P值	<0.05	>0.05

表3 CD144^＋EMP数量和比值变化(±s)

组别	CD144^＋EMP(×10⁶)	CD144^＋EMP(%)
对照组	3.93±1.22	16.00±6.11
0.1 μg/mL脂多糖组	6.80±1.36^ad	22.77±5.06^c
1.0 μg/mL脂多糖组	8.03±2.53^ad	23.81±8.24^c
10.0 μg/mL脂多糖组	17.22±4.52^b	32.08±7.62^c
20.0 μg/mL脂多糖组	11.05±3.80^a	21.58±7.82^c
F值	3.17	0.71
P值	<0.05	>0.05

表3 CD144^＋EMP数量和比值变化(±s)

组别	CD144^＋EMP(×10⁶)	CD144^＋EMP(%)
对照组	3.93±1.22	16.00±6.11
0.1 μg/mL脂多糖组	6.80±1.36^ad	22.77±5.06^c
1.0 μg/mL脂多糖组	8.03±2.53^ad	23.81±8.24^c
10.0 μg/mL脂多糖组	17.22±4.52^b	32.08±7.62^c
20.0 μg/mL脂多糖组	11.05±3.80^a	21.58±7.82^c
F值	3.17	0.71
P值	<0.05	>0.05

表4 CD62E^＋EMP数量和比值变化(±s)

组别	CD62E^＋EMP(×10⁶)	CD62E^＋EMP(%)
对照组	1.26±0.26	4.80±0.67
0.1 μg/mL脂多糖组	2.10±0.50^a	6.72±0.99^c
1.0 μg/mL脂多糖组	2.87±0.68^a	8.31±1.76^c
10.0 μg/mL脂多糖组	7.60±3.60^a	13.76±5.85^c
20.0 μg/mL脂多糖组	2.95±0.26^b	5.66±0.35^c
F值	2.07	1.48
P值	>0.05	>0.05

表4 CD62E^＋EMP数量和比值变化(±s)

组别	CD62E^＋EMP(×10⁶)	CD62E^＋EMP(%)
对照组	1.26±0.26	4.80±0.67
0.1 μg/mL脂多糖组	2.10±0.50^a	6.72±0.99^c
1.0 μg/mL脂多糖组	2.87±0.68^a	8.31±1.76^c
10.0 μg/mL脂多糖组	7.60±3.60^a	13.76±5.85^c
20.0 μg/mL脂多糖组	2.95±0.26^b	5.66±0.35^c
F值	2.07	1.48
P值	>0.05	>0.05

表5 CD51^＋EMP数量和比值变化(%，±s)

组别	CD51^＋EMP(×10⁶)	CD51^＋EMP(%)
对照组	5.38±1.80	19.80±5.57
0.1 μg/mL脂多糖组	5.49±2.83^a	15.70±5.57^b
1.0 μg/mL脂多糖组	7.70±2.86^a	20.43±5.68^b
10.0 μg/mL脂多糖组	14.71±4.52^a	27.17±7.12^b
20.0 μg/mL脂多糖组	12.58±3.26^a	24.27±6.06^b
F值	1.79	0.53
P值	>0.05	>0.05

表5 CD51^＋EMP数量和比值变化(%，±s)

组别	CD51^＋EMP(×10⁶)	CD51^＋EMP(%)
对照组	5.38±1.80	19.80±5.57
0.1 μg/mL脂多糖组	5.49±2.83^a	15.70±5.57^b
1.0 μg/mL脂多糖组	7.70±2.86^a	20.43±5.68^b
10.0 μg/mL脂多糖组	14.71±4.52^a	27.17±7.12^b
20.0 μg/mL脂多糖组	12.58±3.26^a	24.27±6.06^b
F值	1.79	0.53
P值	>0.05	>0.05

表6 内皮细胞凋亡率变化(±s)

组别	Annexin V＋/PI－
对照组	16.43±8.55
0.1 μg/mL脂多糖组	15.15±6.22
1.0 μg/mL脂多糖组	20.85±9.41
10.0 μg/mL脂多糖组	20.33±11.28
20.0 μg/mL脂多糖组	26.03±15.17
F值	0.17
P值	>0.05

表6 内皮细胞凋亡率变化(±s)

组别	Annexin V＋/PI－
对照组	16.43±8.55
0.1 μg/mL脂多糖组	15.15±6.22
1.0 μg/mL脂多糖组	20.85±9.41
10.0 μg/mL脂多糖组	20.33±11.28
20.0 μg/mL脂多糖组	26.03±15.17
F值	0.17
P值	>0.05

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Changes of endothelial microparticles induced by lipopolysaccharide

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Changes of endothelial microparticles induced by lipopolysaccharide