郭婷婷

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·論著·

肝硬化脾切除患者外周血T淋巴細(xì)胞亞群的變遷及其免疫功能變化

郭婷婷

目的了解肝硬化患者行脾切除術(shù)后外周T淋巴細(xì)胞亞群的變遷，分析其免疫功能情況。方法采用流細(xì)胞術(shù)檢測(cè)75例肝硬化患者脾臟切除前后外周血總T細(xì)胞及其各亞群細(xì)胞的絕對(duì)含量。結(jié)果與脾切除前相比較，脾切除后患者T淋巴細(xì)胞絕對(duì)含量顯著降低(t=4.57，P<0.01)；CD3陽(yáng)性T細(xì)胞、CD4陽(yáng)性T細(xì)胞及CD8陽(yáng)性T細(xì)胞百分含量顯著降低，分別由切除前的(63.47±6.45)%、(33.08±3.05)%、(22.45±7.85)%降至(48.62±5.85)%，(26.28±3.87)%及(15.89±4.84)%，差異有統(tǒng)計(jì)學(xué)意義(t值分別為8.67、5.32、5.87，P均<0.01)；而CD4/CD8比值由(1.47±0.43)升至(1.69±0.53)，差異有統(tǒng)計(jì)學(xué)意義(t=2.58，P<0.05)。脾切除前白細(xì)胞分化抗原CD3<50% 6例；CD4<27% 15例，CD8<15% 11例；CD4/CD8<0.71 4例；脾切除后CD3<50% 28例，CD4<27% 38例，CD8<15% 24例，CD4/CD8<0.71占6.7%(5例)；與脾切除前相比較，脾切除后CD3陽(yáng)性、CD4陽(yáng)性及CD8陽(yáng)性淋巴細(xì)胞亞群異常率顯著升高(t值分別為3.59、6.44及4.89，P<0.01)。此外，與脾切除前相比，脾切除后其T淋巴細(xì)胞分泌的一系列細(xì)胞因子如IL-2、IL-4、IFN-γ及TNF-α均顯著低于脾切除前。結(jié)論肝硬化脾切除患者存在明顯的T細(xì)胞免疫功能紊亂，各亞群胞數(shù)量顯著降低，CD3陽(yáng)性T細(xì)胞明顯下降，CD4陽(yáng)性T細(xì)胞相對(duì)于CD8陽(yáng)性細(xì)胞的功能亢進(jìn)，隨著病情加重CD4陽(yáng)性T細(xì)胞的免疫應(yīng)答能力進(jìn)一步減退。

肝硬化；脾切除；T細(xì)胞亞群；免疫功能

肝硬化是目前臨床肝膽科最常見的疾病之一，是在酒精等損害因素作用下引起的肝細(xì)胞彌漫性變性或壞死，繼而出現(xiàn)肝纖維組織增生和肝細(xì)胞結(jié)節(jié)形成[1-2]。損傷因素的長(zhǎng)期存在而未經(jīng)正規(guī)有效治療最終導(dǎo)致肝小葉結(jié)構(gòu)和正常血液循環(huán)途徑被改變，導(dǎo)致患者后期出現(xiàn)一系列不同程度的門靜脈高壓、肝功能障礙及肝衰竭、脾功能亢進(jìn)及脾衰竭[3-4]。脾臟屬于人體的次級(jí)淋巴器官，當(dāng)機(jī)體受到抗原刺激后，淋巴細(xì)胞會(huì)以脾臟作為場(chǎng)所進(jìn)行免疫應(yīng)答并產(chǎn)生免疫效應(yīng)分子[5-6]。然而，肝硬化患者由于門靜脈高壓的形成，脾臟血液循環(huán)發(fā)生障礙，造成脾臟腫大和功能亢進(jìn)，最終導(dǎo)致細(xì)胞免疫功能的紊亂[7-8]。目前臨床上針對(duì)此類疾病已廣泛開展脾切除術(shù)，并取得了較好的療效，但其對(duì)免疫功能的影響研究尚未深入。本研究采用流式細(xì)胞術(shù)及ELISA對(duì)75例肝硬化脾切除患者外周血T淋巴細(xì)胞亞群及其細(xì)胞因子的分泌水平進(jìn)行檢測(cè)和分析，探討其對(duì)機(jī)體免疫功能的影響，為患者病情評(píng)估及療效監(jiān)測(cè)提供一定理論依據(jù)。

資料和方法

一、研究對(duì)象

選取珠海市人民醫(yī)院住院肝硬化并行脾切除術(shù)的75例患者，所有患者均符合《慢性乙型肝炎防治指南(2010年)》中肝硬化的診斷標(biāo)準(zhǔn)，并排除HIV抗體或梅毒抗體檢測(cè)陽(yáng)性或曾行潑尼松等免疫抑制治療的患者。75例患者中男42例，女33例，年齡36～86歲，平均年齡48歲。所有患者入院后均完善肝硬化相關(guān)檢查，積極預(yù)防并治療相關(guān)病因及誘因，完善術(shù)前準(zhǔn)備，擇期行賁門周圍血管離斷術(shù)。術(shù)后行護(hù)肝、抗感染治療，并予以補(bǔ)液、抑酸等支持治療?；颊叱鲈汉箅S訪5年，每半年到院復(fù)查1次，檢測(cè)術(shù)后患者免疫功能變化并觀察其臨床癥狀改善狀況。該研究獲得醫(yī)院醫(yī)學(xué)倫理部門批準(zhǔn)，所有患者及其家屬均被告知研究方案并簽署紙質(zhì)版知情同意書。

二、方法

治療前采集所有患者靜脈血2～3 mL，先予以EDTA-K2混合抗凝，然后采用BD十一通道流式細(xì)胞儀及配套試劑行T細(xì)胞亞群的檢測(cè)，分別測(cè)定T細(xì)胞亞群CD3+、CD4+及CD8+的含量并計(jì)算其所占百分比，具體檢測(cè)及實(shí)驗(yàn)方案按文獻(xiàn)[9-10]進(jìn)行。

三、T淋巴細(xì)胞的培養(yǎng)

取上述患者外周血4 mL加入含2 mL Ficoll的離心管中，緩緩加入Ficoll上層，400×g離心20 min。取PBMC層，PBS洗滌后，用1640培養(yǎng)液重懸單核細(xì)胞，取樣計(jì)數(shù)并調(diào)至至106個(gè)/mL加入10%胎牛血清，然后置于體積分?jǐn)?shù)為0.05的CO2培養(yǎng)箱中進(jìn)行培養(yǎng)。

四、ELISA檢測(cè)(雙抗夾心法)

上述細(xì)胞培養(yǎng)48 h后，離心取上清液50 μL加入取包被好的96孔板中，其中陰性對(duì)照組加入50 μL PBS，陽(yáng)性對(duì)照組加入50 μL標(biāo)準(zhǔn)品，將其置于室溫下振蕩反應(yīng)2 h，PBS洗三遍后加入50 μL一抗稀釋液(1∶500)；PBS洗三次加入50 μL辣根過(guò)氧化物酶二抗，將其置于室溫下振蕩反應(yīng)1 h；PBS洗三遍后在所有孔內(nèi)加入顯色劑A、顯色劑B各50 μL，在微孔振蕩器上震蕩10 s，混勻，封蓋后將反應(yīng)板置37 ℃孵育30 min；在所有反應(yīng)孔里加入50 μL終止液，振蕩反應(yīng)5 s，使之充分混勻，用酶標(biāo)儀(波長(zhǎng)450 nm)讀數(shù)。

五、統(tǒng)計(jì)學(xué)方法

結(jié)　　果

一、脾切除前后外周血T淋巴細(xì)胞亞群變遷

與脾切除前相比較，脾切除后患者T淋巴細(xì)胞絕對(duì)含量顯著降低(t=4.57，P<0.01)，其絕對(duì)含量由切除前的(1.46±0.38)×109/L降至(0.43±0.09)×109/L；CD3陽(yáng)性T細(xì)胞百分含量亦明顯降低，其百分含量由切除前的(63.47±6.45)%降至(48.62±5.85)%，差異有統(tǒng)計(jì)學(xué)意義(t=8.67，P<0.01)；CD4陽(yáng)性T細(xì)胞百分含量由(33.08±3.05)%降至(26.28±3.87)%，與脾切除前相比，差異有統(tǒng)計(jì)學(xué)意義(t=5.32，P<0.01)；CD8陽(yáng)性T細(xì)胞百分含量由(22.45±7.85)%降至(15.89±4.84)%，與脾切除前相比，差異有統(tǒng)計(jì)學(xué)意義(t=5.87，P<0.01)；而CD4/CD8比值由(1.47±0.43)升至(1.69±0.53)，差異有統(tǒng)計(jì)學(xué)意義(t=2.58，P<0.05)。

脾切除前白細(xì)胞分化抗原CD3陽(yáng)性T細(xì)胞百分?jǐn)?shù)<50% 6例；CD4陽(yáng)性T細(xì)胞百分?jǐn)?shù)<27% 15例，CD8陽(yáng)性T細(xì)胞百分?jǐn)?shù)<15% 11例；CD4/CD8<0.71 4例；脾切除后CD3陽(yáng)性T細(xì)胞百分?jǐn)?shù)<50% 28例，CD4陽(yáng)性T細(xì)胞百分?jǐn)?shù)<27% 38例，CD8陽(yáng)性T細(xì)胞百分?jǐn)?shù)<15% 24例，CD4/CD8<0.71占6.7%(5例)；與脾切除前相比較，脾切除后CD3陽(yáng)性、CD4陽(yáng)性及CD8陽(yáng)性T淋巴細(xì)胞亞群異常率均顯著升高，差異有統(tǒng)計(jì)學(xué)意義(t值分別為3.59、6.44和4.89，P<0.01)。

二、細(xì)胞因子的分泌水平

脾切除前和脾切除后T淋巴細(xì)胞分泌的一系列細(xì)胞因子IL-2、IL-4、IFN-γ及TNF-α分別為1.00±0.13、1.00±0.16、1.00±0.21、1.00±0.15和0.56±0.14、0.63±0.09、0.71±0.08、0.48±0.14，脾切除后顯著低于脾切除前的細(xì)胞因子分泌水平，差異有統(tǒng)計(jì)學(xué)意義(t值分別為4.32、3.85、5.47及4.77，P<0.01)。

三、脾切除后相關(guān)并發(fā)癥

2例脾切除4年左右表現(xiàn)為CD3明顯低下(18%～35%)，多次復(fù)查淋巴細(xì)胞亞群各指標(biāo)無(wú)明顯變化。脾切除后1年腹腔嚴(yán)重感染致死1例；脾切除5年內(nèi)伴肺部重度感染致死3例；外傷后脾切除4年合并肺結(jié)核2例；切脾除后第3年、5年、6年發(fā)生肝癌各1例；脾切除后第4年前列腺癌和宮頸癌各1例。

討　　論

抗原入侵體內(nèi)或體內(nèi)原有抗原經(jīng)血液循環(huán)刺激機(jī)體T淋巴細(xì)胞產(chǎn)生細(xì)胞免疫，在這過(guò)程中，T細(xì)胞的活化即效應(yīng)T細(xì)胞的產(chǎn)生均在脾臟進(jìn)行[11-12]。脾臟作為機(jī)體最大的外周免疫器官，其對(duì)機(jī)體正常免疫功能的維持是十分重要的。行脾切除術(shù)后的慢性肝硬化門靜脈高壓患者會(huì)發(fā)生機(jī)體免疫系統(tǒng)紊亂，惡性腫瘤及嚴(yán)重感染。研究發(fā)現(xiàn)，CD3+、CD4+、CD8+T細(xì)胞亞群在維持血液正常免疫細(xì)胞循環(huán)功能中起到關(guān)鍵作用，肝硬化患者CD3陽(yáng)性和CD4陽(yáng)性細(xì)胞的表達(dá)水平與其總生存期呈正相關(guān)[13-14]。本研究表明，肝硬化脾切除患者外周血總T細(xì)胞及各亞群T細(xì)胞的百分含量和絕對(duì)含量均顯著降低，淋巴細(xì)胞亞群異常率顯著上升。慢性肝硬化患者行脾切除術(shù)后免疫功能的紊亂原因多重，第一，可能與術(shù)后T細(xì)胞生成受到抑制有關(guān)；第二，原有T細(xì)胞總量及其各亞群數(shù)量減少，T細(xì)胞免疫功能整體下降；第三，CD4陽(yáng)性T細(xì)胞所占百分比升高，CD4/CD8比值顯著升高，從而導(dǎo)致CD4陽(yáng)性T細(xì)胞功能亢進(jìn)。

T細(xì)胞亞群中CD4陽(yáng)性T起中心調(diào)節(jié)作用，而CD8陽(yáng)性T細(xì)胞則發(fā)揮抑制性T細(xì)胞和效應(yīng)T細(xì)胞作用，兩者的數(shù)量減少會(huì)大大削弱機(jī)體保護(hù)自我排除異己的免疫應(yīng)答反應(yīng)[15]，因此為減少脾切除術(shù)后CD3、CD4、NK細(xì)胞數(shù)量下降造成的免疫功能低下，對(duì)于肝硬化脾切除患者在給予相應(yīng)治療的同時(shí)應(yīng)進(jìn)行相應(yīng)的免疫功能的干預(yù)，也能減少各種感染與腫瘤的發(fā)生。T細(xì)胞分泌的一系列細(xì)胞因子如IL-2、IL-4、IFN-γ及TNF-α，不僅可正向調(diào)節(jié)免疫效應(yīng)細(xì)胞殺傷活性，促進(jìn)T細(xì)胞的增殖，通過(guò)正反饋調(diào)節(jié)進(jìn)一步促進(jìn)相關(guān)細(xì)胞因子的分泌，提高機(jī)體免疫力[11,13]；此外，細(xì)胞因子還可直接參與腫瘤細(xì)胞殺傷及抗感染機(jī)體免疫過(guò)程，在一定程度上反映了T細(xì)胞的免疫功能狀態(tài)[16]。本研究發(fā)現(xiàn)，肝硬化脾切除患者不僅伴有外周血總T細(xì)胞及各亞群T細(xì)胞的百分含量和絕對(duì)含量的異常，其T淋巴細(xì)胞分泌的一系列細(xì)胞因子IL-2、IL-4、IFN-γ及TNF-α均顯著低于脾切除前。因此，定期檢測(cè)外周血總T細(xì)胞及各亞群T細(xì)胞的百分含量和絕對(duì)含量及其免疫功能狀態(tài)，有助于及時(shí)掌握脾切除患者T細(xì)胞的免疫功能，對(duì)于病情判斷、療效監(jiān)測(cè)及預(yù)后評(píng)估等均具有積極意義。

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(本文編輯：茹素娟)

Alteration of T-cell subsets in peripheral blood and immunologic function in liver cirrhosis patients with splenectomy

GuoTing-ting.

DepartmentofGastroenterology,ZhuhaiPeople'sHospital,Guangdong519000,China

ObjectiveTo investigate the alteration of peripheral blood T lymphocyte subsets and varied immune function in liver cirrhosis patients after splenectomy. MethodsThe absolute quantity of peripheral blood T lymphocyte subsets was detected using flow cytometry in 75 patients with liver cirrhosis before and after splenectomy. ResultsCompared with the prior to splenectomy, obviously decrease in absolute value of T cell was received in patients after splenectomy. The percentages of CD3+, CD4+ and CD8+ T cells were decreased from (63.47±6.45)%, (33.08±3.05)%, (22.45±7.85)% to (48.62±5.85)%, (26.28±3.87)%, (15.89±4.84)% respectively. However, CD4+/CD8+ ratio rose dramatically from (1.47±0.43) to (1.69±0.53) (t=P<0.05). The percentage of patients, which the frequencies of CD3+, CD4+ and CD8+ T cells were less than 50%, 27%, and 15% respectively, was increased from 8%, 20% and 14.7% to 37.3%, 50.7% and 32%, respectively. Additionaly, the percentage of patients with CD4/CD8<0.71 increased from 5.3% to 6.7%. Compared to before splenectomy, abnormal percentage of lymphocyte subsets after splenectomy, including CD3+、CD4+ and CD8+, was increased significantly (t=3.59, 6.44 and 4.89 respectively, allP<0.01), meanwhile the levels of cytokines secreted by T cells after splenectomy, including IL-2, IL-4, IFN-γ and TNF-a, were decreased significantly. ConclusionThere might be immune disorders in liver cirrhosis patients after splenectomy, such as apparent reduction in quantity of T lymphocyte subsets and hyperfunction of CD4+ T cell compared to CD8+ T cell. As the disease condition aggravates, the immune response ability of CD4+ T cell could be further weaken. Patients after splenectomy showing significant abnormality of lymphocyte subsets should be alert to the occurrence of tumor or infection.

Liver cirrhosis; Splenectomy; T-cell subsets; Immune function

519000廣東珠海市人民醫(yī)院消化科

2016-04-22)