張鵬,姜萬君,2
(1中南大學生殖與干細胞工程研究所,長沙410078;2南昌大學醫(yī)學院)
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韓華
抑制miR-29a表達對體外蛻膜化人子宮內膜基質細胞蛻膜催乳素分泌的影響
張鵬1,姜萬君1,2
(1中南大學生殖與干細胞工程研究所,長沙410078;2南昌大學醫(yī)學院)
摘要:目的觀察抑制miR-29a表達對體外蛻膜化人子宮內膜基質細胞蛻膜催乳素(dPRL)分泌水平的影響。方法 收集子宮良性病變患者全子宮切除術中留取的子宮內膜組織,分離子宮內膜基質細胞。取部分細胞分為實驗組與對照組,實驗組用雌二醇(E2)和孕酮(P4)對細胞進行蛻膜化處理,對照組不進行蛻膜化處理;取部分細胞按2×105/mL接種到6孔板中,分為1、2、3組,1組轉染anti-miR-29a,2組轉染control miRNA,3組為空白對照;分別于培養(yǎng)0、24、48、72 h后收集各組細胞。采用real-time PCR法檢測各組細胞中的miR-29a,采用化學發(fā)光法檢測各組細胞培養(yǎng)液上清中的dPRL。結果實驗組培養(yǎng)0、24、48、72 h細胞中miR-29a相對表達量及細胞培養(yǎng)液上清中dPRL水平高于對照組(P均<0.01);實驗組隨培養(yǎng)時間延長,miR-29a表達逐漸上調,培養(yǎng)液上清中dPRL水平增高,各培養(yǎng)時點兩兩相比,P均<0.05。培養(yǎng)24、48、72 h時1組細胞中miR-29a相對表達量低于2、3組,細胞培養(yǎng)液上清中dPRL水平低于2、3組(P均<0.01)。結論 抑制miR-29a表達后,蛻膜化人子宮內膜基質細胞分泌dPRL減少;miR-29a可能參與了子宮內膜基質細胞的蛻膜化過程,并調控子宮內膜基質細胞分泌dPRL。
關鍵詞:微小RNA-29a;蛻膜催乳素;人子宮內膜基質細胞
子宮內膜蛻膜化是指子宮內膜基質細胞受到蛻膜化誘導因子的調節(jié)發(fā)生增生、分化,對胚胎著床及妊娠建立有重要作用[1~4]。近年來發(fā)現(xiàn)miR-29a在子宮內膜癌患者的子宮內膜組織中高表達,并受雌、孕激素調節(jié)[4~9],但miR-29a對子宮內膜基質細胞分化及蛻膜化過程中催乳素(PRL)表達的調控作用尚不明確。2015年2~7月,我們觀察了抑制miR-29a表達對體外蛻膜化人子宮內膜基質細胞分泌蛻膜催乳素(dPRL)水平的影響,為研究miR-29a在子宮內膜基質細胞蛻膜化過程中的作用奠定理論基礎。
1材料與方法
1.1標本與試劑收集子宮良性疾病患者手術切除的正常子宮內膜組織?;颊吣挲g24~43歲,月經周期規(guī)律,無全身系統(tǒng)疾病,術前3個月內未服用激素類藥物。胰蛋白酶、TRIzol試劑盒購自Invitrogen公司,Ⅱ型膠原酶、17-β雌二醇(E2)、孕酮(P4)購自Sigma公司,無酚紅DMEM/F12培養(yǎng)基和FBS購自北京Solarbio公司,real-time PCR試劑盒及逆轉錄試劑盒購自日本TaKaRa公司,抗miR-29a、抗miR對照購自Santa Cruz公司,dPRL化學發(fā)光檢測試劑購自德國拜耳公司。
1.2子宮內膜基質細胞分離培養(yǎng)及鑒定參照文獻[3,8]方法無菌刮取子宮內膜組織,置于含青霉素和鏈霉素的PBS緩沖液中;吹打均勻后離心,用眼科剪將組織剪碎至1 mm3,再次離心后加入0.25%胰酶,分別于4 ℃和37 ℃條件下各消化1 h;加入FBS,終止消化后用PBS溶液沖洗2~3次后離心,去上清;在下層沉淀液中加入5 g/LⅡ型膠原酶,放37 ℃培養(yǎng)箱消化30 min。將細胞懸液經過二次濾網分離基質細胞,用細胞培養(yǎng)液重懸浮細胞,以2×105/mL接種于細胞培養(yǎng)皿中,置入37 ℃、5% CO2培養(yǎng)箱中培養(yǎng),隔天更換1次培養(yǎng)液,原代培養(yǎng)4~5 d,用0.25%胰酶消化后傳代。選擇傳至第4代的子宮內膜基質細胞,進行細胞爬片培養(yǎng),待細胞占玻片80%左右時取出,分別加入一抗和二抗,37 ℃孵育1 h,以0.01 mg/mL 碘化丙啶(PI)37 ℃復染細胞核5~10 min,甘油封片,激光共聚焦顯微鏡下觀察,基質細胞呈多邊形或梭形。
1.3子宮內膜基質細胞的蛻膜化處理取傳代至第4代、純度達到90%的子宮內膜基質細胞,分為實驗組和對照組。實驗組繼續(xù)培養(yǎng),待細胞生長融合度達80%,換含10 nmol/L的E2、1 μmol/L的P4及10%FBS無酚紅DMEM/F12培養(yǎng)基培養(yǎng)。對照組待細胞生長融合度達80%,換含10%FBS無酚紅DMEM/F12培養(yǎng)基培養(yǎng)。分別于培養(yǎng)0、24、48、72 h取兩組細胞。
1.4anti-miR-29a轉染子宮內膜基質細胞轉染前1 d消化收集基質細胞,并按2×105/mL接種到6孔板中,每孔1 mL,待細胞達到80%融合時,將細胞分為1、2、3組。1組轉染anti-miR-29a,2組轉染control miRNA,3組為空白對照。將細胞于37 ℃、5% CO2培養(yǎng)箱中孵育24 h后換正常培養(yǎng)液培養(yǎng)。分別于培養(yǎng)0、24、48、72 h收集各組細胞。
1.5子宮內膜基質細胞中miR-29a檢測采用real-time PCR法。反應條件為95 ℃ 30 s,預變性94 ℃ 5 s,60 ℃ 34 s,共40個循環(huán)。以2-ΔΔCt代表基因相對表達量。
1.6細胞培養(yǎng)液上清中dPRL檢測取實驗組、對照組、1組、2組、3組細胞培養(yǎng)液,800 r/min離心5 min,去除細胞碎片,收集上清。采用化學發(fā)光法檢測dPRL,每組實驗重復3次。
2結果
2.1實驗組與對照組細胞中miR-29a表達及培養(yǎng)液上清中dPRL水平比較實驗組培養(yǎng)0、24、48、72 h細胞中miR-29a表達量及細胞培養(yǎng)液上清中dPRL水平高于對照組(P均<0.01);實驗組隨培養(yǎng)時間延長,miR-29a表達逐漸上調,培養(yǎng)液上清中dPRL水平增高,培養(yǎng)各時點兩兩相比,P均<0.05。見表1。
表1 實驗組與對照組細胞中miR-29a表達及培養(yǎng)液上
注:與對照組同時點相比,*P<0.01;與同組培養(yǎng)0 h時相比,#P<0.05;與同組培養(yǎng)24 h時相比,△P<0.05;與同組培養(yǎng)48 h時相比,☆P<0.05。
2.21、2、3組細胞中miR-29a表達及培養(yǎng)液上清中dPRL水平比較1組培養(yǎng)24、48、72 h細胞中miR-29a相對表達量低于2、3組,細胞培養(yǎng)液上清中dPRL水平低于2、3組(P均<0.01)。見表2。
表2 1、2、3組細胞中miR-29a表達及培養(yǎng)液上清中
注:與1組同時點相比,*P<0.01。
3討論
胚胎著床的重要前提是子宮內膜發(fā)生蛻膜化,即子宮進入容受狀態(tài),此過程涉及到子宮內膜基質細胞的增殖分化[1,8,9]。miRNA是基因表達、修飾、轉錄和翻譯的調節(jié)分子,通過與靶mRNA特異性結合,導致靶mRNA降解或抑制其翻譯[10~13]。miR-29是新近發(fā)現(xiàn)的與纖維化疾病有關的小分子RNA家族。人類miR-29包括miR-29a、miR-29b1、miR-29b2和miR-29c[5,7]。近年來研究[6,13]發(fā)現(xiàn),miR-29a在雌鼠子宮內膜基質細胞蛻膜化的形成及早期妊娠的建立中發(fā)揮重要的調節(jié)作用,而且在E2、P4共同誘導的大鼠子宮內膜基質細胞體外蛻膜化過程中表達上調。
鑒于倫理學方面考慮,無法在體進行蛻膜化及胚胎植入分子調控機理的研究,所以我們分離人子宮內膜基質細胞在體外進行蛻膜化誘導,觀察miR-29a對人子宮內膜基質細胞蛻膜化過程中dPRL分泌水平的影響。本研究結果顯示,實驗組培養(yǎng)0、24、48、72 h細胞中miR-29a相對表達量及細胞培養(yǎng)液上清中dPRL水平高于對照組;實驗組隨培養(yǎng)時間延長,miR-29a表達逐漸上調,培養(yǎng)液上清中dPRL水平增高;1組細胞培養(yǎng)24、48、72 h時miR-29a相對表達量低于2、3組,細胞培養(yǎng)液上清中dPRL水平低于2、3組。上述結果說明,人子宮內膜基質細胞中miR-29a表達量隨著蛻膜化時間的延長逐漸上升,且細胞分泌dPRL水平隨蛻膜化時間的推進而逐漸增高;miR-29a表達下調后,蛻膜化標志分子dPRL的分泌水平也相應下調;miR-29a可能參與了子宮內膜基質細胞體外蛻膜化過程,并調控子宮內膜基質細胞分泌dPRL。對于相關信號通路及具體機制,還有待進一步探索。
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Effect of inhibiting miR-29a expression on prolactin secretion of human endometrial stromal cells during in vitro decidualization
ZHANGPeng1,JIANGWanjun
(1InstituteofReproductive&StemCellEngineering,CentralSouthUniversity,Changsha410078,China)
Abstract:Objective To observe the effect of inhibiting miR-29a expression on prolactin secretion of human endometrial stromal cells (HESCs) during in vitro decidualization. MethodsWe collected the endometrial tissues during panhysterectomy from patients with uterine benign lesions, and used trypsin method to isolate HESCs. The cells were then divided into the experimental group and control group, the experimental group was treated with estradiol (E2) and progesterone (P4), and the control group was not treated. We took part of the cells in 2×105/mL to inoculate into the 6-well plates and and then divided them into groups1, 2 and 3. The group 1 was transfected by anti-miR-29a. Group 2 was transfected by Control-miRNA and group 3 as taken as the blank control. After being cultured for 0 h, 24 h, 48 h, 72 h we collected cells in each group. real-time PCR was used to detect miR-29a in the cells of each group. dPRL in the supernatant of cell culture medium was detected by chemiluminescence method. ResultsThe relative expression of miR-29a in the cells after being cultured for 0 h, 24 h, 48 h, 72 h, and the dPRL level in the cell culture supernatantthe of the experimental group were higher than those of the control group (all P<0.01). In the experimental group, with the prolonged culture time, the miR-29a expression was gradually increased, and the level of dPRL in the culture supernatant was increased. Significant difference was found between every two time points (all P<0.05). The relative expression of miR-29a in the group 1 at 24 h, 48 h and 72 h was lower than that in the groups 2 and 3, and the level of dPRL in the supernatant of group 1 was lower than that in the groups 2 and 3 (all P<0.01). ConclusionsAfter the inhibition of miR-29a expression, human decidual endometrial stromal cells reduce the secretion of dPRL. miR-29a may be involved in the decidual process of HESCs, and regulates the endometrial stromal cells secreting dPRL.
Key words:microRNA-29a; decidual prolactin; human endometrial stromal cells
(收稿日期:2015-09-28)
中圖分類號:R711.51;R321.3
文獻標志碼:A
文章編號:1002-266X(2016)07-0001-03
doi:10.3969/j.issn.1002-266X.2016.07.001
通信作者簡介:姜萬君(1993-),女,學士,主要研究方向為生殖內分泌。E-mail: 18270916871@163.com
作者簡介:第一張鵬(1986-),男,在讀博士,主要研究方向為人類輔助生殖技術。E-mail: zhangpengszyx@csu.edu.cn
基金項目:國家自然科學基金資助項目(31260248)。
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