程序性细胞死亡因子5在卵巢子宫内膜异位症组织中的表达及意义

doi:10.3877/cma.j.issn.1674-0785.2019.07.001

目的

探讨程序性细胞死亡因子5（PDCD5）在卵巢子宫内膜异位症组织中的表达及PDCD5对异位内膜间质细胞生物学行为的影响。

方法

应用免疫组织化学法、实时定量逆转录酶链式反应法检测PDCD5在正常内膜、卵巢子宫内膜异位症患者异位内膜及在位内膜中的蛋白和mRNA表达水平。进一步分离培养异位内膜间质细胞，采用小干扰RNA敲降PDCD5，分别通过CCK-8法和TUNEL法检测在不同浓度亮丙瑞林作用下异位内膜细胞的增殖和凋亡情况。采用χ²检验比较异位、在位和正常内膜的PDCD5蛋白阳性表达率的差异，异位、在位及正常内膜组织中PDCD5 mRNA相对表达量的两两比较及不同浓度下PDCD5敲降组和对照组的不同时间的活细胞计数、不同浓度亮丙瑞林作用下细胞增殖抑制率以及凋亡率的比较采用t检验。

结果

异位内膜、在位内膜及正常内膜组织中PDCD5蛋白的阳性表达率分别为30%、29%及75%，异位内膜和在位内膜的PDCD5蛋白表达均低于正常内膜，差异均具有统计学意义（χ²=8.120、7.201；P=0.004、0.007）），异位内膜和在位内膜的PDCD5蛋白表达比较，差异无统计学意义（P=0.928）。异位内膜、在位内膜及正常内膜组织中PDCD5 mRNA相对表达量分别为0.24±0.02，0.27±0.03及1.00±0.04，异位内膜和在位内膜的PDCD5 mRNA表达均明显低于正常内膜，差异具有统计学意义（t=-32.098，P<0.001；t=-28.659，P<0.001），异位内膜和在位内膜的PDCD5 mRNA表达比较，差异无统计学意义（t=-1.617，P=0.181）。PDCD5 mRNA表达与蛋白表达水平一致。体外单纯敲降PDCD5对异位内膜间质细胞形态及增殖无明显影响。CCK-8法结果表明，随着亮丙瑞林浓度增加，PDCD5敲降组和对照组的细胞增殖抑制率均逐渐增加，不同浓度下PDCD5敲降组的细胞增殖抑制率明显低于对照组，差异均具有统计学意义（P<0.05）。TUNEL法结果表明，在0.1 μg/ml亮丙瑞林作用下，PDCD5敲降组的细胞凋亡率（0.06±0.01）低于对照组（0.13±0.02），差异具有统计学意义（t=-5.972，P<0.001）。

结论

PDCD5敲降能够减弱异位内膜间质细胞对亮丙瑞林的敏感性，使细胞凋亡减少，增殖增加。PDCD5在卵巢子宫内膜异位症组织中表达下调可能促进了子宫内膜异位症的发生发展。

关键词: 子宫内膜异位症, 程序性细胞死亡因子5, 免疫组织化学, 亮丙瑞林, 间质细胞

Objective

To investigate the expression of programmed cell death 5 (PDCD5) in ovarian endometriosis and the effect of PDCD5 on the biological behavior of ectopic endometrial stromal cells.

Methods

Immunohistochemistry and RT-qPCR were used to detect the expression of PDCD5 protein and mRNA, respectively, in normal endometrium, ectopic endometrium, and eutopic endometrium. Ectopic endometrial stromal cells were isolated and cultured, and then PDCD5 was knocked down by RNA interference. The proliferation and apoptosis of ectopic endometrial stromal cells treated with leuprorelin of different concentrations were detected by CCK-8 assay and TUNEL assay, respectively. The difference in the expression rate of PDCD5 protein in the ectopic, eutopic, and normal endometrium was compared by the χ² test. Pairwise comparisons of the relative expression of PDCD5 mRNA in ectopic, eutopic, and normal endometrium, the number of viable cells between the PDCD5 knockdown group and control group, cell proliferation inhibition rate and apoptosis rate in cells treated with different concentrations of leprorelin, and cell proliferation inhibition rate and apoptosis rate in ectopic, eutopic, and normal endometrium were performed by t-tests.

Results

The positive rates of PDCD5 protein in ectopic endometrium, eutopic endometrium, and normal endometrium were 30%, 29%, and 75%, respectively. The expression of PDCD5 protein in ectopic endopmetrium and eutopic endometrium was both significantly lower than that in normal endometrium (P=0.004 and 0.007, respectively). There was no significant difference in the expression of PDCD5 protein between ectopic endometrium and eutopic endometrium (P=0.928). The relative expression of PDCD5 mRNA in ectopic endometrium, eutopic endometrium, and normal endometrium was 0.24±0.02, 0.27±0.03, and 1.00±0.04, respectively. The expression of PDCD5 mRNA in ectopic endometrium and eutopic endometrium was both significantly lower than that in normal endometrium (t=-32.098, P<0.001; t=-28.659, P<0.001). There was no significant difference in the expression of PDCD5 mRNA between eutopic endometrium and ectopic endometrium (t=-1.617, P=0.181). The trend of mRNA expression is consistent with that of protein expression. Knockdown of PDCD5 had no significant effect on the morphology and proliferation of ectopic endometrial stromal cells in vitro. CCK-8 assay showed that with the increase of leuprorelin concentration, the proliferation inhibition rate of cells in the PDCD5 knock-down group and control group increased gradually, and at the same concentration of leuprorelin, the cell proliferation inhibition rate was significantly lower in the PDCD5 knockdown group than in the control group (P<0.05). TUNEL assay showed that the apoptosis rate of cells treated with leuprorelin at 0.1 μg/ml was significantly in the PDCD5 knockdown group (0.06±0.01) than in the control group (0.13±0.02) (t=-5.972, P<0.001).

Conclusion

PDCD5 knockdown can reduce the sensitivity of ectopic endometrial stromal cells to leuprorelin, resulting in decreased apoptosis and increased cell proliferation. The down-regulation of PDCD5 expression in ovarian endometriosis may promote the occurrence and development of endometriosis.

Key words: Endometriosis, Programmed cell death 5, Immunohistochemistry, Leuprorelin, Stromal cells

表1 3组内膜组织中的PDCD5蛋白表达

组别	例数	（-）	（＋）	（＋＋）	（＋＋＋）	阳性率（%）
正常内膜组	20	5	4	8	3	75
在位内膜组	14	10	3	1	0	29
异位内膜组	20	14	5	1	0	30

表1 3组内膜组织中的PDCD5蛋白表达

组别	例数	（-）	（＋）	（＋＋）	（＋＋＋）	阳性率（%）
正常内膜组	20	5	4	8	3	75
在位内膜组	14	10	3	1	0	29
异位内膜组	20	14	5	1	0	30

图1~3 3组内膜组织中的PDCD5蛋白表达（二氨基联苯胺染色，×400）　图1为正常内膜组织，图2为在位内膜组织，图3为异位内膜组织

图4、5 体外分离培养的异位内膜间质细胞（免疫细胞化学法，×400）　图4为波形蛋白阳性，图5为角蛋白阴性

表2 PDCD5敲降组和对照组在0、24、48、72、96 h活细胞计数比较（×10⁴/ml，±s）

组别	例数	0	24 h	48 h	72 h	96 h
PDCD5敲降组	6	6.84±0.90	10.93±1.47	15.83±2.11	23.83±0.86	35.37±2.06
对照组	6	6.06±0.34	10.76±0.88	16.63±2.37	22.87±2.11	36.77±1.19
t值	?	1.414	0.172	-0.437	0.735	-1.020
P值	?	0.266	0.872	0.685	0.522	0.365

表2 PDCD5敲降组和对照组在0、24、48、72、96 h活细胞计数比较（×10⁴/ml，±s）

组别	例数	0	24 h	48 h	72 h	96 h
PDCD5敲降组	6	6.84±0.90	10.93±1.47	15.83±2.11	23.83±0.86	35.37±2.06
对照组	6	6.06±0.34	10.76±0.88	16.63±2.37	22.87±2.11	36.77±1.19
t值	?	1.414	0.172	-0.437	0.735	-1.020
P值	?	0.266	0.872	0.685	0.522	0.365

图6 PDCD5敲降组和对照组在0、48、96 h的细胞形态（台盼蓝染色，×200）

表3 PDCD5敲降组和对照组在不同浓度亮丙瑞林作用下的细胞增殖抑制率（±s）

组别	0.001 μg/ml	0.01 μg/ml	0.1 μg/ml	1 μg/ml	10 μg/ml
PDCD5敲降组	0.14±0.00	0.19±0.00	0.22±0.02	0.27±0.01	0.31±0.01
对照组	0.22±0.02	0.26±0.01	0.31±0.01	0.37±0.02	0.49±0.00
t值	-9.700	-12.787	-11.282	-11.026	-36.892
P值	<0.001	<0.001	<0.001	<0.001	<0.001

表3 PDCD5敲降组和对照组在不同浓度亮丙瑞林作用下的细胞增殖抑制率（±s）

组别	0.001 μg/ml	0.01 μg/ml	0.1 μg/ml	1 μg/ml	10 μg/ml
PDCD5敲降组	0.14±0.00	0.19±0.00	0.22±0.02	0.27±0.01	0.31±0.01
对照组	0.22±0.02	0.26±0.01	0.31±0.01	0.37±0.02	0.49±0.00
t值	-9.700	-12.787	-11.282	-11.026	-36.892
P值	<0.001	<0.001	<0.001	<0.001	<0.001

图7 PDCD5敲降组和对照组细胞TUNEL阳性表达（TUNEL法染色，×200）

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Significance of expression of programmed cell death 5 in ovarian endometriosis

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Significance of expression of programmed cell death 5 in ovarian endometriosis