周 娟， 肖小敏

(暨南大學(xué)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(xì)胞表型分析

周 娟， 肖小敏△

(暨南大學(xué)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(xì)胞(dNK 細(xì)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(xué)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(jī)選取同期在此院因社會(huì)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(duì)照組。收集孕晚期子宮蛻膜組織，機(jī)械研磨加梯度離心法提取蛻膜內(nèi)單核細(xì)胞，流式細(xì)胞技術(shù)(FCM)篩選出dNK細(xì)胞，并檢測(cè)dNK細(xì)胞表面CD56及CD16的表達(dá)情況。結(jié)果(1)妊娠期高血壓組與正常對(duì)照組CD56brightCD16-CD3-dNK細(xì)胞的數(shù)量均多于CD56dimCD16+CD3-dNK細(xì)胞，且差異顯著(均P<0.01)。(2)妊娠期高血壓組與正常對(duì)照組CD56brightCD16-CD3-dNK細(xì)胞所占比例無(wú)顯著差異(P>0.05)，CD56dimCD16+CD3-dNK細(xì)胞所占比例亦無(wú)顯著差異(P>0.05)。(3)妊娠期高血壓組與正常對(duì)照組dNK細(xì)胞表面CD16分子的表達(dá)率并無(wú)顯著差異(P>0.05)。結(jié)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(xì)胞表型無(wú)明顯改變，均以CD56brightCD16-CD3-亞型為主。

子宮蛻膜; 自然殺傷細(xì)胞； 妊娠高血壓； 母-胎界面； 細(xì)胞表型

妊娠期高血壓疾病是產(chǎn)科最常見(jiàn)及嚴(yán)重的妊娠合并癥之一，研究證實(shí)此病的發(fā)生與母體免疫系統(tǒng)功能混亂密切相關(guān)[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(dòng)中起著重要的作用。子宮蛻膜自然殺傷細(xì)胞(decidual natural killer cells, dNK cells)是母-胎界面最主要的免疫細(xì)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(gè)亞型：前者屬分泌型細(xì)胞，可分泌各種細(xì)胞/化學(xué)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(xì)胞毒性殺傷作用，殺傷非“己”物質(zhì)。兩者的平衡對(duì)于正常妊娠至關(guān)重要。已知正常妊娠者體內(nèi)的dNK細(xì)胞以CD56brightCD16-CD3-亞型為主[4]。而在病理性妊娠如反復(fù)自發(fā)性流產(chǎn)患者體內(nèi)，dNK細(xì)胞以CD56dimCD16+CD3-亞型為主，造成子宮局部的免疫殺傷作用增強(qiáng)，從而導(dǎo)致胚胎發(fā)育異常、壞死以至流產(chǎn)[5]。有學(xué)者[6]提出：既然妊娠期高血壓和反復(fù)自發(fā)性流產(chǎn)在病理生理學(xué)上存在某些相似之處，是否其dNK細(xì)胞同樣以CD56dimCD16+CD3-亞型為主？Wilczynski等[7]報(bào)道本病患者dNK表型存在變化，CD56dimCD16+CD3-亞型增加。而Zhou等[8]則報(bào)道dNK細(xì)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(duì)妊娠期高血壓疾病患者孕晚期子宮蛻膜內(nèi)dNK細(xì)胞的表型進(jìn)行了研究。

材 料 和 方 法

1材料

1.1標(biāo)本 選取2008年8月至2009年3月在廣州市暨南大學(xué)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(wú)其它妊娠合并癥及并發(fā)癥。既往無(wú)高血壓及肝臟疾病史；無(wú)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(jī)選取同期在此院因社會(huì)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(duì)照組。2組孕婦的平均年齡、孕齡及孕產(chǎn)次等均無(wú)顯著差異。術(shù)前取得上述剖宮產(chǎn)者的同意，在其胎盤(pán)娩出后,刮取胎盤(pán)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(gòu)于Gibco，臺(tái)盼藍(lán)購(gòu)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(gòu)于BD PharMingen。胎牛血清(FBS)、人淋巴細(xì)胞分離液(Ficoll)和雙抗(青霉素、鏈霉素)均購(gòu)于天津?yàn)笊镉邢薰尽?/p>

2方法

2.1細(xì)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(fù)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(guò)濾收集濾液。將人淋巴細(xì)胞分離液(Ficoll)平鋪于15 mL無(wú)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(xì)胞分離液與濾液間的云霧樣絮狀物層并置于另一支無(wú)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(xì)胞濃度約為109cells/L，制成細(xì)胞懸液。取少許細(xì)胞懸液，臺(tái)盼藍(lán)染液染色檢測(cè)細(xì)胞活力>90%。

2.2dNK細(xì)胞表型檢測(cè) 每份標(biāo)本編2支試管，每管分別加入100 μL細(xì)胞懸液。每份標(biāo)本的1號(hào)管中加入對(duì)照試劑(小鼠IgG1-PE、小鼠IgG1-FITC及小鼠IgG1-PE-Cy5)各20 μL，2號(hào)管中加入小鼠抗人CD56-PE、CD16-FITC及CD3-PE-Cy5各20 μL標(biāo)記dNK(CD56+CD3-)細(xì)胞、CD56brightCD16-CD3-dNK細(xì)胞及CD56dimCD16+CD3-dNK細(xì)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(xì)胞。

上機(jī)檢測(cè)：激光管預(yù)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(hào)的HCV值<2%，以1號(hào)管作為空白定標(biāo)，計(jì)算10 000個(gè)細(xì)胞，記錄標(biāo)本的陽(yáng)性細(xì)胞百分率和平均熒光強(qiáng)度。

3統(tǒng)計(jì)學(xué)處理

采用SPSS 13.0軟件對(duì)所有數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)處理分析。妊娠期高血壓組與正常對(duì)照組內(nèi)的比較采用配對(duì)資料的秩和檢驗(yàn)；妊娠期高血壓組與正常對(duì)照組間的比較采用兩樣本比較的秩和檢驗(yàn)，結(jié)果以中位數(shù)、四分位間距(QR)、最大值、最小值表示。

結(jié) 果

流式細(xì)胞技術(shù)分選出熒光抗體標(biāo)記后CD56表達(dá)陽(yáng)性、CD3表達(dá)陰性(CD56+CD3-)的細(xì)胞即dNK細(xì)胞。根據(jù)dNK細(xì)胞表面CD56表達(dá)水平的高低及是否表達(dá)CD16，將其分為CD56brightCD16-CD3-dNK細(xì)胞及CD56dimCD16+CD3-dNK細(xì)胞。

1妊娠期高血壓組

孕晚期子宮蛻膜dNK細(xì)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(jiàn)表1。

表1妊娠期高血壓組CD56brightCD16-CD3-dNK細(xì)胞與CD56dimCD16+CD3-dNK細(xì)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(duì)照組

孕晚期子宮蛻膜dNK細(xì)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(jiàn)表2。

表2正常對(duì)照組CD56brightCD16-CD3-dNK細(xì)胞與CD56dimCD16+CD3-dNK細(xì)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(duì)照組的比較

(1)2組dNK細(xì)胞均以CD56brightCD16-CD3-亞型為主，分別為(59.5%±17.6%)和(55.0%±20.1%)，無(wú)顯著差異(P>0.05)，見(jiàn)表3、圖1。

(2)2組CD56dimCD16+CD3-dNK細(xì)胞的數(shù)量分別為(8.9%±7.8%)和(9.4%±8.7%)，亦無(wú)顯著差異(P>0.05)，見(jiàn)表4、圖1。

(3)2組dNK細(xì)胞表面CD16的表達(dá)率分別為(13.6%±8.3%)和(12.9%±9.5%)，無(wú)顯著差異(P>0.05)，見(jiàn)表5、圖2。

表3妊娠期高血壓組與正常對(duì)照組間CD56brightCD16-CD3-dNK細(xì)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(duì)照組之間CD56brightCD16-CD3-dNK細(xì)胞及CD56dimCD16+CD3-dNK細(xì)胞的比較

表4妊娠期高血壓組與正常對(duì)照組間CD56dimCD16+CD3-dNK細(xì)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(duì)照組dNK細(xì)胞表面CD16表達(dá)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(duì)照組dNK細(xì)胞表面CD16表達(dá)情況的比較

討 論

研究證實(shí)子宮蛻膜dNK細(xì)胞主要包括2大亞群：CD56brightCD16-CD3-dNK細(xì)胞及CD56dimCD16+CD3-dNK細(xì)胞。2種細(xì)胞在細(xì)胞表型、基因表達(dá)及功能上均存在很大差別。CD56brightCD16-CD3-dNK細(xì)胞屬分泌型細(xì)胞，具有低細(xì)胞毒性的特點(diǎn)。通過(guò)產(chǎn)生各種細(xì)胞因子(如白細(xì)胞介素8、干擾素-γ)、血管源性分子(如血管內(nèi)皮生長(zhǎng)因子、胎盤(pán)生長(zhǎng)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(dòng)脈的構(gòu)建[10]，調(diào)節(jié)絨毛外滋養(yǎng)細(xì)胞(EVT)的侵潤(rùn)及胎盤(pán)的發(fā)育形成[11,12]。CD56dimCD16+CD3-dNK細(xì)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(xì)胞毒性殺傷作用。它不僅可以通過(guò)直接釋放預(yù)先儲(chǔ)存的殺傷性物質(zhì)來(lái)殺傷靶細(xì)胞，還可通過(guò)抗體依賴性細(xì)胞毒性作用(ADCC)來(lái)實(shí)現(xiàn)。其表面表達(dá)的CD16可作為低親和力的 FcgRIII，與已結(jié)合到靶細(xì)胞上的IgG抗體的Fc段相結(jié)合，增強(qiáng)NK細(xì)胞的殺傷作用[13]。姓娠期高血壓的發(fā)病機(jī)制尚不清楚[14]。長(zhǎng)期以來(lái)有關(guān)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(dǎo)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(diǎn)對(duì)了解疾病的發(fā)生機(jī)制及預(yù)防治療有著重要的意義。本實(shí)驗(yàn)在對(duì)母-胎界面dNK細(xì)胞進(jìn)行研究時(shí)發(fā)現(xiàn)妊娠期高血壓疾病患者孕晚期子宮蛻膜dNK細(xì)胞的表型未發(fā)生明顯改變，仍以CD56brightCD16-CD3-dNK細(xì)胞為主，妊娠期高血壓疾病患者與正常孕婦dNK細(xì)胞表面CD16的表達(dá)率并無(wú)顯著差異。

然而，即使是同一表型的細(xì)胞，在功能上也不一定完全一致。已知dNK細(xì)胞表面表達(dá)2種類型的受體：殺傷細(xì)胞活化性受體(killer activating receptors，KARs)和殺傷細(xì)胞抑制性受體(killer inhibiting receptors，KIRs)，其與相應(yīng)配體如絨毛外滋養(yǎng)細(xì)胞表面表達(dá)的HLA-C、HLA-E及HLA-G等相互作用后，能上調(diào)/下調(diào)dNK細(xì)胞的功能，從而產(chǎn)生不同的生物學(xué)效應(yīng)。Hiby等[15]指出抑制dNK細(xì)胞會(huì)增加患子癇前期的可能性；Hanna等[12]認(rèn)為活化的dNK細(xì)胞可通過(guò)分泌NK細(xì)胞來(lái)源的生長(zhǎng)因子及化學(xué)因子促進(jìn)滋養(yǎng)細(xì)胞侵潤(rùn)、蛻膜血管形成從而防止子癇前期的發(fā)生。不僅如此，dNK細(xì)胞本身的反應(yīng)性亦可影響其生物學(xué)效應(yīng)(如對(duì)于趨化信號(hào)的反應(yīng)性可影響其在子宮內(nèi)的募集)。因此，我們推測(cè)是否因?yàn)镃D56brightCD16-CD3-dNK細(xì)胞功能異常導(dǎo)致了本病的發(fā)生，認(rèn)為有必要對(duì)dNK細(xì)胞功能進(jìn)行進(jìn)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(xì)胞的表型均以CD56brightCD16-CD3-亞型為主。推測(cè)可能dNK細(xì)胞自身功能異常與本病發(fā)生有關(guān)。

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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

△通訊作者 Tel:020-38688660；E-mail：hellen118@163.com