蔣凌云，覃愛(ài)平，歐奇志，靳玉甫，杭馥，覃金春，謝偉，莫馥華

(廣西醫(yī)科大學(xué)第一附屬醫(yī)院生殖醫(yī)學(xué)研究中心，南寧530021)

?

不同濃度去氫表雄酮對(duì)小鼠蛻膜化子宮內(nèi)膜基質(zhì)細(xì)胞增殖及Dtprp mRNA表達(dá)的影響

蔣凌云，覃愛(ài)平，歐奇志，靳玉甫，杭馥，覃金春，謝偉，莫馥華

(廣西醫(yī)科大學(xué)第一附屬醫(yī)院生殖醫(yī)學(xué)研究中心，南寧530021)

目的觀察不同濃度去氫表雄酮(DHEA)對(duì)小鼠蛻膜化子宮內(nèi)膜基質(zhì)細(xì)胞增殖及蛻膜催乳素相關(guān)蛋白(Dtprp)mRNA表達(dá)的影響。方法體外分離培養(yǎng)妊娠第4天小鼠的子宮內(nèi)膜基質(zhì)細(xì)胞。將細(xì)胞分為實(shí)驗(yàn)1組、實(shí)驗(yàn)2組、實(shí)驗(yàn)3組、實(shí)驗(yàn)4組、模型組、對(duì)照組；各實(shí)驗(yàn)組與模型組以E2、P4誘導(dǎo)蛻膜化，實(shí)驗(yàn)1、2、3、4組分別加入0.1、1、10、50 μmol/L的DHEA，模型組不加DHEA，對(duì)照組培養(yǎng)液不給予E2、P4及DHEA；培養(yǎng)72 h后采用real-time PCR法檢測(cè)細(xì)胞中的Dtprp mRNA，分別于培養(yǎng)24、48 h后倒置顯微鏡下觀察細(xì)胞形態(tài)變化。取各實(shí)驗(yàn)組、模型組的細(xì)胞懸液接種于96孔板，調(diào)零組不接種細(xì)胞；培養(yǎng)72 h后檢測(cè)細(xì)胞增殖能力。結(jié)果實(shí)驗(yàn)1、2、3、4組細(xì)胞中Dtprp mRNA相對(duì)表達(dá)量分別為0.978 0±0.258 0、0.764 0±0.135 0、0.250 0±0.022 0、0.013 0±0.000 0，模型組與對(duì)照組分別為1.000 0±0.000 0、0.000 2±0.000 0；實(shí)驗(yàn)3、4組Dtprp mRNA相對(duì)表達(dá)量與模型組相比，P均<0.05；實(shí)驗(yàn)2組與實(shí)驗(yàn)4組相比，P<0.05。對(duì)照組細(xì)胞呈成纖維細(xì)胞樣；模型組細(xì)胞形態(tài)更飽滿(mǎn)，胞質(zhì)增多；實(shí)驗(yàn)1、2組細(xì)胞形態(tài)與模型組近似，實(shí)驗(yàn)3、4組細(xì)胞呈多角形，胞質(zhì)減少。實(shí)驗(yàn)1、2、3、4組細(xì)胞增殖能力分別為1.222 8±0.038 2、0.790 5±0.207 1、0.592 5±0.229 8、0.265 8±0.037 4，模型組為1.149 7±0.106 2，調(diào)零組校正A值為0.087 1±0.006 3；實(shí)驗(yàn)3、4組與模型組相比，P均<0.05；實(shí)驗(yàn)1組與實(shí)驗(yàn)2、3、4組相比，P均<0.05；實(shí)驗(yàn)2組與實(shí)驗(yàn)4組相比，P<0.05。結(jié)論0.1、1 μmol/L DHEA對(duì)蛻膜化子宮內(nèi)膜基質(zhì)細(xì)胞增殖及Dtprp mRNA表達(dá)無(wú)明顯影響，而10、50 μmol/L DHEA可抑制細(xì)胞增殖，并下調(diào)Dtprp mRNA表達(dá)。

去氫表雄酮；子宮內(nèi)膜基質(zhì)細(xì)胞；蛻膜細(xì)胞反應(yīng)；蛻膜催乳素相關(guān)蛋白

蛻膜化是子宮內(nèi)膜基質(zhì)細(xì)胞分化為蛻膜細(xì)胞的過(guò)程，對(duì)胚胎著床和妊娠有重要作用[1,2]。去氫表雄酮(DHEA)是人體最豐富的甾體激素，具有調(diào)節(jié)血管內(nèi)皮細(xì)胞功能、改善胰島素敏感性、改善中樞神經(jīng)系統(tǒng)功能、調(diào)節(jié)免疫及血脂代謝等多種生物學(xué)效應(yīng)[3]。國(guó)外研究[4]證實(shí)高濃度(50、100 μmol/L)DHEA可抑制子宮內(nèi)膜蛻膜化，不利于胚胎著床和妊娠。2013年9月～2015年7月，我們觀察了不同濃度DHEA對(duì)體外蛻膜化小鼠子宮內(nèi)膜基質(zhì)細(xì)胞增殖及蛻膜化標(biāo)志物[5]蛻膜催乳素相關(guān)蛋白(Dtprp)mRNA表達(dá)的影響，現(xiàn)報(bào)告如下。

1　材料與方法

1.1實(shí)驗(yàn)動(dòng)物清潔級(jí)中國(guó)昆明小白鼠400只(廣西醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心)，6～8周齡，體質(zhì)量30～35 g，于發(fā)情當(dāng)日雌雄2∶1合籠，次日晨8:00～9:00檢查陰栓，發(fā)現(xiàn)陰栓計(jì)為妊娠第1天。

1.2小鼠子宮內(nèi)膜基質(zhì)細(xì)胞的分離與鑒定參考文獻(xiàn)[6]方法并略作改動(dòng)。取妊娠第4天小鼠，斷頭處死后取子宮，剔除多余脂肪及結(jié)締組織；用含100 IU/mL雙抗的PBS沖洗2遍，剪開(kāi)雙側(cè)子宮角暴露宮腔，剪碎移入15 mL離心管，離心去除PBS；加入與組織碎塊等體積的消化液，在37 ℃的CO2溫箱消化2 h，每15 min振蕩30 s；用含10%普通胎牛血清的無(wú)酚紅DMEM/F12培養(yǎng)液終止消化，吸管吹打數(shù)次；靜置離心管3 min，吸取上層細(xì)胞懸液，過(guò)200目濾網(wǎng)，PBS沖洗離心管內(nèi)組織塊2次，沖洗液過(guò)200目濾網(wǎng)；將上述細(xì)胞濾液1 000 r/min離心7 min，棄上清，用加雙抗的含10%普通胎牛血清DMEM/F12培養(yǎng)液重懸，臺(tái)盼藍(lán)染色，細(xì)胞以2×105/孔接種入24孔板、12×105/瓶接種入T25培養(yǎng)瓶，置于37 ℃、5%CO2培養(yǎng)箱培養(yǎng)。1～2 h內(nèi)基質(zhì)細(xì)胞貼壁后吸出原培養(yǎng)液，依次用PBS和新鮮待加入培養(yǎng)液潤(rùn)洗2次，加入新鮮培養(yǎng)液繼續(xù)培養(yǎng)。待24孔板中細(xì)胞爬滿(mǎn)80%板底面積時(shí)，進(jìn)行免疫細(xì)胞熒光染色[7]，鑒定子宮內(nèi)膜基質(zhì)細(xì)胞。

1.3DHEA對(duì)子宮內(nèi)膜基質(zhì)細(xì)胞中Dtprp mRNA表達(dá)及細(xì)胞形態(tài)的影響觀察

1.3.1細(xì)胞分組與DHEA用法將細(xì)胞分為實(shí)驗(yàn)1組、實(shí)驗(yàn)2組、實(shí)驗(yàn)3組、實(shí)驗(yàn)4組、模型組與對(duì)照組。實(shí)驗(yàn)1、2、3、4組與模型組以10 nmol/L E2、1 μmol/L P4誘導(dǎo)蛻膜化，其中實(shí)驗(yàn)1、2、3、4組分別加入0.1、1、10、50 μmol/L的DHEA，模型組不加DHEA；對(duì)照組培養(yǎng)液不給予E2、P4及DHEA處理，加入0.1%乙醇。各組每隔24 h換培養(yǎng)液。培養(yǎng)72 h后檢測(cè)Dtprp mRNA。

1.3.2各組細(xì)胞中Dtprp mRNA檢測(cè)提取細(xì)胞總RNA，逆轉(zhuǎn)錄為cDNA，進(jìn)行real-time PCR反應(yīng)。Dtprp mRNA上游引物序列為5′-AGAGCCAGAAATCACTGCCACTCTA-3′，下游引物序列為5′-AGGAGTGATCCATGCACCCATAA-3′；內(nèi)參GAPDH上游引物序列為5′- GGTGAAGGTCGGTGTGAACG-3′，下游引物序列為5′-CTCGCTCCTGGAAGATGGTG-3′。以2-ΔΔCt表示目的基因相對(duì)表達(dá)量。

1.3.3細(xì)胞形態(tài)觀察分別于培養(yǎng)24、48 h后，在倒置顯微鏡下觀察細(xì)胞形態(tài)變化。

1.4DHEA對(duì)子宮內(nèi)膜基質(zhì)細(xì)胞增殖的影響觀察取“1.3.1”中各實(shí)驗(yàn)組、模型組的細(xì)胞懸液，接種于96孔板，細(xì)胞約8×104個(gè)/孔。調(diào)零組不接種細(xì)胞，只加入含E2、P4的1%碳吸附胎牛血清DMEM/F12培養(yǎng)液。置于37 ℃、5% CO2培養(yǎng)箱中培養(yǎng)。培養(yǎng)72 h后吸出各孔培養(yǎng)液，每孔加入無(wú)酚紅DMEM/F12培養(yǎng)液100 μL及CCK-8試劑10 μL，繼續(xù)培養(yǎng)2 h，ELISA法檢測(cè)吸光度值(A460值)，將模型組及實(shí)驗(yàn)組各孔測(cè)得的A460值減去調(diào)零孔A460值，得到校正的A值，表示細(xì)胞增殖能力。

2　結(jié)果

2.1各組細(xì)胞中Dtprp mRNA表達(dá)比較實(shí)驗(yàn)1、2、3、4組細(xì)胞中Dtprp mRNA相對(duì)表達(dá)量分別為0.978 0±0.258 0、0.764 0±0.135 0、0.250 0±0.022 0、0.013 0±0.000 0，模型組與對(duì)照組分別為1.000 0±0.000 0、0.000 2±0.000 0；實(shí)驗(yàn)3、4組Dtprp mRNA相對(duì)表達(dá)量與模型組相比，P均<0.05；實(shí)驗(yàn)2組與實(shí)驗(yàn)4組相比，P<0.05。

2.2各組細(xì)胞形態(tài)變化對(duì)照組子宮內(nèi)膜基質(zhì)細(xì)胞生長(zhǎng)旺盛，呈長(zhǎng)梭形；模型組細(xì)胞出現(xiàn)蛻膜化，形態(tài)更飽滿(mǎn)，胞質(zhì)增多；實(shí)驗(yàn)1、2組細(xì)胞形態(tài)與模型組近似，實(shí)驗(yàn)3、4組細(xì)胞呈多角形，胞質(zhì)明顯減少。

2.3各組細(xì)胞增殖能力比較實(shí)驗(yàn)1、2、3、4組增殖能力分別為1.222 8±0.038 2、0.790 5±0.207 1、0.592 5±0.229 8、0.265 8±0.037 4，模型組增殖能力為1.149 7±0.106 2，調(diào)零組校正A值為0.087 1±0.006 3；實(shí)驗(yàn)3、4組與模型組相比，P均<0.05；實(shí)驗(yàn)1組與實(shí)驗(yàn)2、3、4組相比，P均<0.05；實(shí)驗(yàn)2組與實(shí)驗(yàn)4組相比，P<0.05。

3　討論

DHEA主要在人腎上腺皮質(zhì)網(wǎng)狀帶合成，大部分以硫酸去氫表雄酮的形式進(jìn)入血循環(huán)。DHEA有廣泛的生物學(xué)效應(yīng)。蛻膜化是子宮內(nèi)膜基質(zhì)細(xì)胞分化為蛻膜細(xì)胞的過(guò)程。研究[2]證實(shí)，蛻膜組織能提供胚胎發(fā)育所需的生長(zhǎng)因子和細(xì)胞因子，調(diào)節(jié)滋養(yǎng)細(xì)胞侵襲，并發(fā)揮免疫調(diào)節(jié)作用。有研究[8]指出，DHEA可用于絕經(jīng)后女性預(yù)防骨質(zhì)疏松及改善陰道萎縮的替代治療。也有學(xué)者[9]將DHEA用于卵巢低反應(yīng)性患者從而改善卵巢反應(yīng)性，但效果和安全性有待進(jìn)一步驗(yàn)證。

正常人血清DHEA水平一般在30 nmol/L以下[10]，多囊卵巢綜合征患者血清DHEA水平可達(dá)正常人的2倍。國(guó)外研究顯示，100 μmol/L DHEA可抑制腎上腺髓質(zhì)祖細(xì)胞增殖，而0.1、1、10 μmol/L DHEA對(duì)細(xì)胞活性無(wú)顯著影響[11]。藥理濃度(1～200 μmol/L)的DHEA可抑制HepG2細(xì)胞系增殖，而生理濃度(1 μmol/L以下)的DHEA可促進(jìn)細(xì)胞增殖[12]。有學(xué)者[4]發(fā)現(xiàn)，10 μmol/L及以上濃度的DHEA可抑制細(xì)胞葡萄糖-6-磷酸脫氫酶的活性。我們推測(cè)不同濃度DHEA對(duì)子宮內(nèi)膜基質(zhì)細(xì)胞葡萄糖-6-磷酸脫氫酶活性的抑制作用存在差別。

本研究利用原代分離培養(yǎng)的小鼠子宮內(nèi)膜基質(zhì)細(xì)胞，在培養(yǎng)液中加入雌、孕激素誘導(dǎo)蛻膜化，發(fā)現(xiàn)不同濃度DHEA對(duì)小鼠子宮內(nèi)膜基質(zhì)細(xì)胞增殖和體外蛻膜化的影響程度不同。實(shí)驗(yàn)3、4組Dtprp mRNA相對(duì)表達(dá)量及細(xì)胞增殖能力低于模型組，細(xì)胞呈多角形、胞質(zhì)減少，提示0.1、1 μmol/L DHEA不抑制子宮內(nèi)膜基質(zhì)細(xì)胞增殖和體外蛻膜化，而10、50 μmol/L DHEA則可明顯抑制細(xì)胞增殖及體外蛻膜化。我們推測(cè)，人在疾病狀態(tài)下補(bǔ)充DHEA可能不會(huì)對(duì)子宮內(nèi)膜蛻膜化產(chǎn)生影響。然而，DHEA的生理功能及作用機(jī)制仍未完全闡明，上述結(jié)論仍需要進(jìn)一步去證實(shí)。

[1] Zhu H,Hou CC,Luo LF,et al.Endometrial stromal cells and decidualized stromal cells: origins,transformation and functions[J].Gene,2014,551(1):1-14.

[2] Ramathal CY,Bagchi IC,Taylor RN,et al.Endometrial decidualization: of mice and men[J].Semin Reprod Med,2010,28(1):17-26.

[3] Traish AM,Kang HP,Saad F,et al.Dehydroepiandrosterone (DHEA)--a precursor steroid or an active hormone in human physiology[J].J Sex Med,2011,8(11):2960-2982.

[4] Frolova AI,O′N(xiāo)eill K,Moley KH.Dehydroepiandrosterone inhibits glucose flux through the pentose phosphate pathway in human and mouse endometrial stromal cells,preventing decidualization and implantation[J].Mol Endocrinol,2011,25(8):1444-1455.

[5] Kimura F,Takakura K,Takebayashi K,et al.Messenger ribonucleic acid for the mouse decidual prolactin is present and induced during in vitro decidualization of endometrial stromal cells[J].Gynecol Endocrinol,2001,15(6):426-432.

[6] Zhao KQ,Lin HY,Zhu C,et al.Maternal Smad3 deficiency compromises decidualization in mice[J].J Cell Biochem,2012,113(10):3266-3275.

[7] Zhang L,Guo W,Chen Q,et al.Adam12 plays a role during uterine decidualization in mice[J].Cell Tissue Res,2009,338(3):413-421.

[8] Lin LT,Tsui KH,Wang PH.Clinical application of dehydroepiandrosterone in reproduction: A review of the evidence[J].J Chin Med Assoc,2015,78(8):446-453.

[9] Rutkowski K,Sowa P,Rutkowska-Talipska J,et al.Dehydroepiandrosterone (DHEA): hypes and hopes[J].Drugs,2014,74(11):1195-1207.

[10] Warner M,Gustafsson JA.DHEA - a precursor of ERbeta ligands[J].J Steroid Biochem Mol Biol,2015,145:245-247.

[11] Chung KF,Qin N,Androutsellis-Theotokis A,et al.Effects of dehydroepiandrosterone on proliferation and differentiation of chromaffin progenitor cells[J].Mol Cell Endocrinol,2011,336(1-2):141-148.

[12] Teng Y,Litchfield LM,Ivanova MM,et al.Dehydroepiandrosterone-induces miR-21 transcription in HepG2 cells through estrogen receptor beta and androgen receptor[J].Mol Cell Endocrinol,2014,392(1-2):23-36.

Effects of different concentrations of dehydroepiandrosterone on proliferation and Dtprp mRNA expression of decidualized endometrial stromal cells in mice

JIANG Lingyun,QIN Aiping,OU Qizhi,JIN Yufu,HANG Fu,QIN Jinchun,XIE Wei,MO Fuhua

(The First Affiliated Hospital of Guangxi Medical University,Nanning 530021,China)

ObjectiveTo observe the effects of different concentrations of dehydroepiandrosterone (DHEA) on proliferation and decidual prolactin-related protein (Dtprp) mRNA expression of decidualized endometrial stromal cells in mice.MethodsPrimary endometrial stromal cells were isolated and cultured in vitro from mice on day 4 of pregnancy.The cells were divided into the experimental group 1,group 2,group 3,group 4,model group and control group.The experimental groups and the model group were treated with estradiol (E2) and progesterone (P4) to induce in vitro decidualization.The experimental groups 1,2,3 and 4 were exposed to 0.1,1,10 and 50 μmol/L DHEA,respectively.The control group was not treated with E2,P4and DHEA.The expression of Dtprp mRNA was detected by real-time PCR after 72-hour culture.Morphological changes were observed under inverted microscope after 24 and 48 h.The cells of the experimental groups and model group were cultured in 96-well plates except the blank wells.The cell proliferation ability was measured using a cell counting kit-8 after 72 h.ResultsThe relative expression of Dtprp mRNA in the experimental groups 1,2,3 and 4 was 0.978 0±0.258 0,0.764 0±0.135 0,0.250 0± 0.022 0 and 0.013 0±0.000 0,respectively; while that in the model group and control group was 1.000 0±0.000 0 and 0.000 2±0.000 0.The expression of Dtprp mRNA in both group 3 and group 4 was lower than that of the model group (all P<0.05).Significant difference was found between the experimental group 2 and group 4 (P<0.05).The cells in the control group showed a fibroblast-like appearance.Cells in the model group enlarged and had an increased amount of cytoplasm.The appearance of cells in the experimental group 1 and group 2 was similar to that of the model group.Cells in the experimental group 3 and 4 became polygonal with less cytoplasm.The corrected absorbance of the experimental groups 1,2,3,4,model group and backgroud absorbance were 1.222 8± 0.038 2,0.790 5±0.207 1,0.592 5±0.229 8,0.265 8±0.037 4,1.149 7±0.106 2 and 0.087 1±0.006 3,respectively.Significant difference was found between experimental groups 3,4 and the model group,between experimental group 1 and groups 2,3 and 4,between experimental group 2 and group 4 (all P<0.05).ConclusionThe proliferation and Dtprp mRNA expression of mouse endometrial stromal cells are not significantly affected by 0.1 and 1 μmol/L DHEA,while 10 and 50 μmol/L DHEA inhibits cell proliferation and down-regulates Dtprp mRNA expression.

dehydroepiandrosterone; endometrial stromal cells; decidual reaction; decidual prolactin-related protein

國(guó)家自然科學(xué)基金資助項(xiàng)目(81260096)。

蔣凌云(1989-)，女，在讀碩士研究生，主要研究方向?yàn)樯硟?nèi)分泌。E-mail：jianglingyun2013@sina.com

簡(jiǎn)介：覃愛(ài)平(1963-)，女，教授，主要研究方向?yàn)樯硟?nèi)分泌、輔助生殖技術(shù)。E-mail: aini02456@126.com

10.3969/j.issn.1002-266X.2016.19.002

R339.2

A

1002-266X(2016)19-0005-03

2015-11-24)