周 娟, 肖小敏
(暨南大學附屬第一醫(yī)院婦產(chǎn)科,廣東 廣州 510632)
妊娠期高血壓疾病患者子宮蛻膜NK細胞表型分析
周 娟, 肖小敏△
(暨南大學附屬第一醫(yī)院婦產(chǎn)科,廣東 廣州 510632)
目的觀察妊娠期高血壓疾病患者子宮蛻膜自然殺傷細胞(dNK 細胞)的表型。方法選取2008年8月至2009年3月在廣州市暨南大學附屬第一醫(yī)院婦產(chǎn)科因妊娠期高血壓疾病行剖宮產(chǎn)的單胎妊娠孕婦20例作為妊娠期高血壓組,隨機選取同期在此院因社會心理因素行選擇性剖宮產(chǎn)的正常單胎妊娠孕婦15例作為正常對照組。收集孕晚期子宮蛻膜組織,機械研磨加梯度離心法提取蛻膜內(nèi)單核細胞,流式細胞技術(FCM)篩選出dNK細胞,并檢測dNK細胞表面CD56及CD16的表達情況。結果(1)妊娠期高血壓組與正常對照組CD56brightCD16-CD3-dNK細胞的數(shù)量均多于CD56dimCD16+CD3-dNK細胞,且差異顯著(均P<0.01)。(2)妊娠期高血壓組與正常對照組CD56brightCD16-CD3-dNK細胞所占比例無顯著差異(P>0.05),CD56dimCD16+CD3-dNK細胞所占比例亦無顯著差異(P>0.05)。(3)妊娠期高血壓組與正常對照組dNK細胞表面CD16分子的表達率并無顯著差異(P>0.05)。結論妊娠期高血壓疾病患者與正常孕婦孕晚期子宮蛻膜內(nèi)dNK細胞表型無明顯改變,均以CD56brightCD16-CD3-亞型為主。
子宮蛻膜; 自然殺傷細胞; 妊娠高血壓; 母-胎界面; 細胞表型
妊娠期高血壓疾病是產(chǎn)科最常見及嚴重的妊娠合并癥之一,研究證實此病的發(fā)生與母體免疫系統(tǒng)功能混亂密切相關[1]。母-胎界面是母體與胎兒成分直接接觸的部位,其局部免疫微環(huán)境在妊娠的建立、維持以及臨產(chǎn)的發(fā)動中起著重要的作用。子宮蛻膜自然殺傷細胞(decidual natural killer cells, dNK cells)是母-胎界面最主要的免疫細胞[2],在局部有重要的免疫調(diào)節(jié)作用。研究表明其主要分為CD56brightCD16-CD3-與CD56dimCD16+CD3-2個亞型:前者屬分泌型細胞,可分泌各種細胞/化學因子、血管生成因子等,有利于妊娠的建立、維持[3];而后者具有細胞毒性殺傷作用,殺傷非“己”物質(zhì)。兩者的平衡對于正常妊娠至關重要。已知正常妊娠者體內(nèi)的dNK細胞以CD56brightCD16-CD3-亞型為主[4]。而在病理性妊娠如反復自發(fā)性流產(chǎn)患者體內(nèi),dNK細胞以CD56dimCD16+CD3-亞型為主,造成子宮局部的免疫殺傷作用增強,從而導致胚胎發(fā)育異常、壞死以至流產(chǎn)[5]。有學者[6]提出:既然妊娠期高血壓和反復自發(fā)性流產(chǎn)在病理生理學上存在某些相似之處,是否其dNK細胞同樣以CD56dimCD16+CD3-亞型為主?Wilczynski等[7]報道本病患者dNK表型存在變化,CD56dimCD16+CD3-亞型增加。而Zhou等[8]則報道dNK細胞表型并未改變??紤]到母-胎界面在妊娠中的重要性等各種因素,本研究著重對妊娠期高血壓疾病患者孕晚期子宮蛻膜內(nèi)dNK細胞的表型進行了研究。
1材料
1.1標本 選取2008年8月至2009年3月在廣州市暨南大學附屬第一醫(yī)院因妊娠期高血壓疾病行剖宮產(chǎn)的單胎妊娠孕婦20例作為妊娠期高血壓組。除妊娠期高血壓疾病外, 無其它妊娠合并癥及并發(fā)癥。既往無高血壓及肝臟疾病史;無腎臟疾病、器官移植、免疫治療及輸血史;月經(jīng)規(guī)則,末次月經(jīng)清楚。隨機選取同期在此院因社會心理因素行選擇性剖宮產(chǎn)的正常單胎妊娠孕婦15例作為正常對照組。2組孕婦的平均年齡、孕齡及孕產(chǎn)次等均無顯著差異。術前取得上述剖宮產(chǎn)者的同意,在其胎盤娩出后,刮取胎盤附著部位宮壁的蛻膜組織。
1.2主要試劑 RPMI-1640培養(yǎng)液(含Hepes、L-谷氨酰胺)購于Gibco,臺盼藍購于Sigma, 小鼠抗人CD56-PE、CD16-FITC、CD3-PE-Cy5及小鼠IgG1-FITC、IgG1-PE、IgG1-PE-Cy5均購于BD PharMingen。胎牛血清(FBS)、人淋巴細胞分離液(Ficoll)和雙抗(青霉素、鏈霉素)均購于天津灝洋生物有限公司。
2方法
2.1細胞懸液的制備 所取蛻膜組織用RPMI-1640培養(yǎng)液(含青霉素105U/L和鏈霉素100 mg/L)洗滌2次(盡量洗去組織中的血液、胎糞、胎脂等物質(zhì)),放入裝有RPMI-1640完全培養(yǎng)基(含10%胎牛血清)的小燒杯中。用眼科剪將組織反復剪碎直至大小約1 mm×1 mm×1 mm,研磨棒輕輕研磨組織, 200目濾網(wǎng)過濾收集濾液。將人淋巴細胞分離液(Ficoll)平鋪于15 mL無菌離心管中,將收集的濾液按照1∶1的比例平鋪于分離液上(形成清楚的分界面)。20 ℃下2 000 r/min離心20 min。離心后吸取淋巴細胞分離液與濾液間的云霧樣絮狀物層并置于另一支無菌離心管中。RPMI-1640完全培養(yǎng)基洗滌2次(20℃下1 500 r/min離心5 min),最后1次離心棄上清液后加入RPMI-1640完全培養(yǎng)基吹打混合均勻,調(diào)整細胞濃度約為109cells/L,制成細胞懸液。取少許細胞懸液,臺盼藍染液染色檢測細胞活力>90%。
2.2dNK細胞表型檢測 每份標本編2支試管,每管分別加入100 μL細胞懸液。每份標本的1號管中加入對照試劑(小鼠IgG1-PE、小鼠IgG1-FITC及小鼠IgG1-PE-Cy5)各20 μL,2號管中加入小鼠抗人CD56-PE、CD16-FITC及CD3-PE-Cy5各20 μL標記dNK(CD56+CD3-)細胞、CD56brightCD16-CD3-dNK細胞及CD56dimCD16+CD3-dNK細胞,室溫下孵育25 min。磷酸鹽緩沖溶液(PBS)洗滌2次(室溫下1 500 r/min離心5 min),離心去上清后每管內(nèi)加300 μL PBS重懸細胞。
上機檢測:激光管預熱30 min后用熒光微球(Beckman-Coulter)調(diào)整儀器,使各放大器接收信號的HCV值<2%,以1號管作為空白定標,計算10 000個細胞,記錄標本的陽性細胞百分率和平均熒光強度。
3統(tǒng)計學處理
采用SPSS 13.0軟件對所有數(shù)據(jù)進行統(tǒng)計學處理分析。妊娠期高血壓組與正常對照組內(nèi)的比較采用配對資料的秩和檢驗;妊娠期高血壓組與正常對照組間的比較采用兩樣本比較的秩和檢驗,結果以中位數(shù)、四分位間距(QR)、最大值、最小值表示。
流式細胞技術分選出熒光抗體標記后CD56表達陽性、CD3表達陰性(CD56+CD3-)的細胞即dNK細胞。根據(jù)dNK細胞表面CD56表達水平的高低及是否表達CD16,將其分為CD56brightCD16-CD3-dNK細胞及CD56dimCD16+CD3-dNK細胞。
1妊娠期高血壓組
孕晚期子宮蛻膜dNK細胞仍以CD56brightCD16-CD3-亞型為主,其數(shù)量(55.0%±20.1%)多于CD56dimCD16+CD3-亞型(9.4%±8.7%),兩者差異顯著差異(P<0.01),見表1。
表1妊娠期高血壓組CD56brightCD16-CD3-dNK細胞與CD56dimCD16+CD3-dNK細胞的比較
Table 1. The comparison between the CD56brightCD16-CD3-dNK cells and CD56dimCD16+CD3-dNK cells in the HDCP group(n=20)
SubgroupMedianMaximumMinimumQRCD56brightCD16-62.5%82.2%7.3%21.7%CD56dimCD16+7.9%**43.5%2.4%4.8%**
**P<0.01vsCD56brightCD16-.
2正常對照組
孕晚期子宮蛻膜dNK細胞亦以CD56brightCD16-CD3-亞型為主,其數(shù)量(59.5%±17.6%)多于CD56dimCD16+CD3-亞型(8.9%±7.8%),兩者差異顯著(P<0.01),見表2。
表2正常對照組CD56brightCD16-CD3-dNK細胞與CD56dimCD16+CD3-dNK細胞的比較
Table 2. The comparison between the CD56brightCD16-CD3-dNK cells and CD56dimCD16+CD3-dNK cells in the normal control group(n=15)
SubgroupMedianMaximumMinimumQRCD56brightCD16-62.6%86.2%26.8%21.6%CD56dimCD16+7.6%**31.5%0.6%4.9%*
**P<0.01vsCD56brightCD16-.
3妊娠期高血壓組與正常對照組的比較
(1)2組dNK細胞均以CD56brightCD16-CD3-亞型為主,分別為(59.5%±17.6%)和(55.0%±20.1%),無顯著差異(P>0.05),見表3、圖1。
(2)2組CD56dimCD16+CD3-dNK細胞的數(shù)量分別為(8.9%±7.8%)和(9.4%±8.7%),亦無顯著差異(P>0.05),見表4、圖1。
(3)2組dNK細胞表面CD16的表達率分別為(13.6%±8.3%)和(12.9%±9.5%),無顯著差異(P>0.05),見表5、圖2。
表3妊娠期高血壓組與正常對照組間CD56brightCD16-CD3-dNK細胞的比較
Table 3. CD56brightCD16-CD3-dNK cells in HDCP group and normal control group
GroupnMedianMaximumMinimumQRHDCP2062.5%82.2%7.3%21.7%Normalcontrol1562.6%86.2%26.8%21.6%
Figure 1. FCM analysis of CD56-positive and CD3-negative (CD56+CD3-) cells in uterine decidual NK(dNK) cells. According to the expression of CD56 on the surface of dNK cells and whether CD16 were expressed, we sorted out the CD56brightCD16-CD3-dNK cells (shown as the R3 domain) and the CD56dimCD16+CD3-dNK cells (shown as the R4 domain).There was no statistical difference between HDCP group and normal control group,in neither CD56brightCD16-CD3-dNK cells nor CD56dimCD16+CD3-dNK cells.
圖1妊娠期高血壓組與正常對照組之間CD56brightCD16-CD3-dNK細胞及CD56dimCD16+CD3-dNK細胞的比較
表4妊娠期高血壓組與正常對照組間CD56dimCD16+CD3-dNK細胞的比較
Table 4. CD56dimCD16+CD3-dNK cells in HDCP group and normal control group
GroupnMedianMaximumMinimumQRHDCP207.9%43.5%2.4%4.8%Normalcontrol157.6%31.5%0.6%4.9%
表5妊娠期高血壓組與正常對照組dNK細胞表面CD16表達情況的比較
Table 5. The expression of CD16 on the surface of dNK cells between HDCP group and normal control group
GroupnMedianMaximumMinimumQRHDCP2011.0%46.3%2.8%6.1%Normalcontrol1511.5%33.6%1.0%9.4%
Figure 2. dNK cells were screened by FCM. The A1 and A2 domains represent the expression of CD16 on the surface of dNK cells from HDCP group and normal control group, respectively. There was no significant difference in the expression of CD16 on dNK cells between the two groups.
圖2妊娠期高血壓組與正常對照組dNK細胞表面CD16表達情況的比較
研究證實子宮蛻膜dNK細胞主要包括2大亞群:CD56brightCD16-CD3-dNK細胞及CD56dimCD16+CD3-dNK細胞。2種細胞在細胞表型、基因表達及功能上均存在很大差別。CD56brightCD16-CD3-dNK細胞屬分泌型細胞,具有低細胞毒性的特點。通過產(chǎn)生各種細胞因子(如白細胞介素8、干擾素-γ)、血管源性分子(如血管內(nèi)皮生長因子、胎盤生長因子及血管生成素2)等生物活性物質(zhì)參與妊娠的免疫耐受[9]、子宮螺旋動脈的構建[10],調(diào)節(jié)絨毛外滋養(yǎng)細胞(EVT)的侵潤及胎盤的發(fā)育形成[11,12]。CD56dimCD16+CD3-dNK細胞內(nèi)含有大量的穿孔素、顆粒酶等殺傷性物質(zhì),主要起細胞毒性殺傷作用。它不僅可以通過直接釋放預先儲存的殺傷性物質(zhì)來殺傷靶細胞,還可通過抗體依賴性細胞毒性作用(ADCC)來實現(xiàn)。其表面表達的CD16可作為低親和力的 FcgRIII,與已結合到靶細胞上的IgG抗體的Fc段相結合,增強NK細胞的殺傷作用[13]。姓娠期高血壓的發(fā)病機制尚不清楚[14]。長期以來有關病理性妊娠的大量研究表明,子宮局部免疫微環(huán)境的異常是導致妊娠失敗的主要原因之一,清楚了解此處的微循環(huán)特點對了解疾病的發(fā)生機制及預防治療有著重要的意義。本實驗在對母-胎界面dNK細胞進行研究時發(fā)現(xiàn)妊娠期高血壓疾病患者孕晚期子宮蛻膜dNK細胞的表型未發(fā)生明顯改變,仍以CD56brightCD16-CD3-dNK細胞為主,妊娠期高血壓疾病患者與正常孕婦dNK細胞表面CD16的表達率并無顯著差異。
然而,即使是同一表型的細胞,在功能上也不一定完全一致。已知dNK細胞表面表達2種類型的受體:殺傷細胞活化性受體(killer activating receptors,KARs)和殺傷細胞抑制性受體(killer inhibiting receptors,KIRs),其與相應配體如絨毛外滋養(yǎng)細胞表面表達的HLA-C、HLA-E及HLA-G等相互作用后,能上調(diào)/下調(diào)dNK細胞的功能,從而產(chǎn)生不同的生物學效應。Hiby等[15]指出抑制dNK細胞會增加患子癇前期的可能性;Hanna等[12]認為活化的dNK細胞可通過分泌NK細胞來源的生長因子及化學因子促進滋養(yǎng)細胞侵潤、蛻膜血管形成從而防止子癇前期的發(fā)生。不僅如此,dNK細胞本身的反應性亦可影響其生物學效應(如對于趨化信號的反應性可影響其在子宮內(nèi)的募集)。因此,我們推測是否因為CD56brightCD16-CD3-dNK細胞功能異常導致了本病的發(fā)生,認為有必要對dNK細胞功能進行進一步深入研究。
綜上所述,妊娠期高血壓疾病患者與正常孕婦孕晚期子宮蛻膜dNK細胞的表型均以CD56brightCD16-CD3-亞型為主。推測可能dNK細胞自身功能異常與本病發(fā)生有關。
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PhenotypeofdecidualNKcellsinwomenwithhypertensivedisordercomplicatingpregnancy
ZHOU Juan, XIAO Xiao-min
(DepartmentofObstetricsandGynecology,TheFirstAffiliatedHospitalofJinanUniversity,Guangzhou510632,China.E-mail:hellen118@163.com)
AIM: To investigate the phenotype of uterine decidual natural killer cells (dNK cells) in women with hypertensive disorder complicating pregnancy (HDCP).METHODSAll the study subjects were collected from Department of Obstetrics and Gynecology, The First Affiliated Hospital of Jinan University, Guangzhou, China. Twenty cases of singleton pregnancy who underwent caesarean section because of HDCP were selected as HDCP group, and 15 cases of singleton pregnancy
selective caesarean section because of social-psychological concerns were also randomly selected as normal control group. The decidual tissues were sampled immediately after caesarean section. The mononuclear cells were extracted from the tissues by means of mechanic grinding and gradient centrifugation. The technique of flow cytometry was used for dNK cell sorting and the expression of CD56 and CD16 on the surface of cells was also examined.RESULTSIn HDCP group and normal control group, the proportions of CD56brightCD16-CD3-dNK cells were significantly higher than those of CD56dimCD16+CD3-dNK cells (P<0.01). Neither the CD56brightCD16-CD3-subset nor the CD56dimCD16+CD3-subset had statistical difference between HDCP group and normal control group. No significant difference of CD16 expression on the surface of dNK cells between HDCP group and normal control group was observed (P>0.05).CONCLUSIONThe phenotypes of dNK cells from the women with HDCP and from healthy pregnant women are both dominated by CD56brightCD16-CD3-subset, without significant difference.
Uterine decidua; Natural killer cells; Hypertension,pregnancy-induced; Maternal-fetal interface; Cell phenotype
R714.2; R363.2
A
10.3969/j.issn.1000-4718.2011.01.036
1000-4718(2011)01-0183-04
2010-05-26
2010-11-10
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