馬海波，李 恒，唐小三，李曉紅*

(1.山東大學(xué)醫(yī)學(xué)院， 山東 濟(jì)南 250012； 2.山東大學(xué)附屬濟(jì)南市中心醫(yī)院 神經(jīng)內(nèi)科， 山東 濟(jì)南 250013)

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臍血多能干細(xì)胞對阿爾茨海默病患者外周血淋巴細(xì)胞的免疫調(diào)節(jié)作用

馬海波1,2，李 恒2，唐小三2，李曉紅2*

(1.山東大學(xué)醫(yī)學(xué)院， 山東 濟(jì)南 250012； 2.山東大學(xué)附屬濟(jì)南市中心醫(yī)院 神經(jīng)內(nèi)科， 山東 濟(jì)南 250013)

目的研究臍血多能干細(xì)胞(CB-SCs)對阿爾茨海默病(AD)患者外周血淋巴細(xì)胞(LCs)的調(diào)節(jié)作用，初步探討其治療AD的潛能。方法人臍血中分離并培養(yǎng)CB-SCs；AD患者外周血中分離淋巴細(xì)胞；將兩者共培養(yǎng)3 d，其中植物凝集素P(PHA-P)刺激組刺激淋巴細(xì)胞增殖；ELISA法檢測上清液中IL- 1、IL- 4和IL- 10的含量，流式細(xì)胞儀檢測淋巴細(xì)胞亞群中CD4+/CD8+T細(xì)胞的比例、CD4+T細(xì)胞中調(diào)節(jié)性T細(xì)胞的比例及Treg細(xì)胞功能蛋白IL- 10和TGF-β1的陽性率。結(jié)果1)CB-SCs能抑制PHA-P誘導(dǎo)的LCs增殖及LCs聚集；2)CB-SCs共培養(yǎng)組的促炎因子IL- 1明顯下降(P<0.05)，抗炎因子IL- 4和IL- 10明顯上升(P<0.01)；3)CB-SCs可下調(diào)CD4+/CD8+T淋巴細(xì)胞比例(P<0.01)；4)在無PHA-P作用時(shí)CB-SCs可以增加CD4+T淋巴細(xì)胞中Treg細(xì)胞的比例(P<0.01),PHA-P存在時(shí)CB-SCs主要增強(qiáng)Treg細(xì)胞功能蛋白的表達(dá)。結(jié)論CB-SCs在體外對AD患者外周血的淋巴細(xì)胞具有免疫調(diào)節(jié)作用，主要通過增加Treg細(xì)胞的比例及增強(qiáng)其抗炎功能來發(fā)揮作用。

干細(xì)胞；阿爾茨海默??；免疫調(diào)節(jié)；細(xì)胞因子；調(diào)節(jié)性T細(xì)胞

目前認(rèn)為阿爾茨海默病(Alzheimer’s disease, AD)是一種系統(tǒng)炎性反應(yīng)疾病,外周炎性反應(yīng)能導(dǎo)致AD或加劇其進(jìn)展，是AD的一個(gè)重要致病因素[1]。實(shí)驗(yàn)和臨床研究證實(shí)，聯(lián)合外周抗炎或單獨(dú)進(jìn)行外周抗炎能有效治療AD[2- 3]。另有研究發(fā)現(xiàn)臍血多能干細(xì)胞(cord blood-stem cells, CB-SCs)可以通過調(diào)節(jié)外周血的免疫反應(yīng)，有效治療1型糖尿病[4]，但CB-SCs是否同樣可以通過免疫調(diào)節(jié)抑制AD過度的外周炎性反應(yīng)，用于AD的治療，至今未見研究報(bào)道。本實(shí)驗(yàn)通過CB-SCs與重度AD患者的外周血淋巴細(xì)胞共培養(yǎng)，探討CB-SCs對AD的免疫調(diào)節(jié)作用。

1 材料與方法

1.1 材料

臍血樣本取自山東大學(xué)附屬濟(jì)南市中心醫(yī)院產(chǎn)科健康孕產(chǎn)婦，采集產(chǎn)后臍血10例樣本，每例60～120 mL；選擇近3個(gè)月內(nèi)沒有感染性疾病、沒有應(yīng)用任何具有免疫調(diào)節(jié)功能藥物的臨床確診為重度AD的患者10例，每例采集約10 mL空腹靜脈血。本研究經(jīng)醫(yī)院倫理委員會批準(zhǔn)，采集前均告知孕產(chǎn)婦和AD患者及其家屬并簽訂知情協(xié)議。Ficoll淋巴細(xì)胞分離液(Sigma公司)；X-VIVO15無血清培養(yǎng)基(Lonza公司)；細(xì)胞培養(yǎng)皿Petri Dish(Becton Dickinson公司)；RPMI1640培養(yǎng)基、胎牛血清(Invitrogen公司)；紅細(xì)胞裂解液、Foxp3/轉(zhuǎn)錄因子染色液試劑盒和鼠抗人CD34-PE、CD45-PE、CD4-Alexa Fluor 700、CD8-FITC、CD4-FITC、CD25-PerCP-Cy5.5、Foxp3-Alexa Fluor 647、IL- 10-PE-Cy7和TGF-β1-PE(eBioscience公司)；PBS緩沖液、0.25%胰蛋白酶-EDTA、PHA-P、青鏈霉素混合液(×100)(索萊寶公司)；ELISA試劑盒(上海藍(lán)基公司)。

1.2 方法

1.2.1 CB-SCs的分離、培養(yǎng)：依照Zhao等[5]分離培養(yǎng)CB-SCs的實(shí)驗(yàn)方法，采用Ficoll淋巴細(xì)胞分離液分離臍血中的單個(gè)核細(xì)胞，PBS洗1遍，隨后用稀釋9倍的紅細(xì)胞裂解液作用8～12 min去除紅細(xì)胞，PBS洗3遍。加入X-VIVO15無血清培養(yǎng)基，以(0．5～1)×106個(gè)/mL接種于培養(yǎng)皿，置于37 ℃、8% CO2培養(yǎng)箱中孵育10～14 d，待視野下細(xì)胞匯合度達(dá)到80%～90%后,用PBS洗3遍，除去上清液中的懸浮細(xì)胞。貼壁的干細(xì)胞分別進(jìn)行以下鑒定及共培養(yǎng)實(shí)驗(yàn)。

1.2.2 流式細(xì)胞儀鑒定CB-SCs：0.25%胰蛋白酶-EDTA消化貼壁的干細(xì)胞后，10% FBS的X-VIVO15終止消化，PBS洗3遍后，用熒光抗體標(biāo)記CD34和CD45，流式細(xì)胞儀檢測細(xì)胞表面標(biāo)志物CD34和CD45的表達(dá)。

1.2.3 AD患者外周血淋巴細(xì)胞的分離并與干細(xì)胞共培養(yǎng)：用肝素鈉抗凝的一次性負(fù)壓采血容器取約10 mL AD患者空腹靜脈血，同樣方法分離AD患者外周單個(gè)核細(xì)胞。單個(gè)核細(xì)胞接種于含20% FBS的RPMI1640培養(yǎng)基，置于37 ℃、5% CO2全濕條件下培養(yǎng)24 h,收集上清液中懸浮的淋巴細(xì)胞，分為4個(gè)組：1)LCs+PHA-P；2)CB-SCs+LCs+PHA-P；3)LCs；4)CB-SCs+LCs。兩個(gè)共培養(yǎng)組中干細(xì)胞CB-SCs和AD患者外周血淋巴細(xì)胞比例為1∶3，其中作為淋巴細(xì)胞刺激劑的PHA-P濃度為4 mg/L。X-VIVO15無血清培養(yǎng)基培養(yǎng)3 d。

1.2.4 計(jì)數(shù)淋巴細(xì)胞并用ELISA法檢測上清液中的炎性因子：分別收集各培養(yǎng)組細(xì)胞懸液，離心得到的淋巴細(xì)胞計(jì)數(shù)后用于下一步檢測；上清液用于檢測IL- 1、IL- 4和IL- 10的濃度，實(shí)驗(yàn)步驟按ELISA試劑盒說明書操作。

1.2.5 流式細(xì)胞儀檢測細(xì)胞懸液中的CD4+/CD8+T淋巴細(xì)胞比例、調(diào)節(jié)性T細(xì)胞(regulatory T cells, Tregs)比例及其功能蛋白IL- 10及TGF-β1陽性率：各組懸液中的淋巴細(xì)胞做流式細(xì)胞檢測。熒光抗體標(biāo)記CD4和CD8分子，測各組CD4+/CD8+T淋巴細(xì)胞比例；用熒光抗體標(biāo)記細(xì)胞表面分子CD4和CD25以及細(xì)胞內(nèi)因子Foxp3、IL- 10和TGF-β1，測Treg細(xì)胞的比例及細(xì)胞內(nèi)功能蛋白IL- 10和TGF-β1。用BD FACSAriaTMⅢ收集10 000個(gè)細(xì)胞分析。實(shí)驗(yàn)重復(fù)3次。

1.3 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 CB-SCs表面抗原的鑒定及細(xì)胞形態(tài)

原代培養(yǎng)的CB-SCs貼壁生長，呈圓形或橢圓形(圖1A)。CB-SCs標(biāo)志物CD45表達(dá)率達(dá)100%，陰性標(biāo)志物CD34表達(dá)率為2.5%(圖1B)。

2.2 CB-SCs抑制PHA-P誘導(dǎo)的淋巴細(xì)胞增殖

3 d后CB-SCs+LCs+PHA-P組與LCs+PHA-P組相比細(xì)胞數(shù)量明顯下降(P<0.01)。另外，LCs+PHA-P組與LCs組相比細(xì)胞數(shù)顯著上升(P<0.01)(圖2)。

2.3CB-SCs抑制PHA-P誘導(dǎo)的淋巴細(xì)胞聚集反應(yīng)

培養(yǎng)3 d后，LCs組淋巴細(xì)胞無明顯聚集反應(yīng)(圖3A)；PHA-P刺激下的淋巴細(xì)胞出現(xiàn)大量團(tuán)塊狀的聚集(圖3B)；CB-SCs+LCs組無明顯聚集反應(yīng)(圖3C)；CB-SCs+LCs+PHA-P組淋巴細(xì)胞偶爾出現(xiàn)聚集，但細(xì)胞凝集團(tuán)小(圖3D)。

2.4 CB-SCs對上清液中細(xì)胞因子的影響

經(jīng)過3d的培養(yǎng)，CB-SCs+LCs+PHA-P組與LCs+PHA-P組相比，促炎因子IL- 1含量明顯下降(P<0.05)，抗炎因子IL- 4和IL- 10含量明顯上升(P<0.01)(圖4)。

A.morphology of CB-SCs(×100); B.expression of surface marker CD45 and CD34 圖1 CB-SCs表面標(biāo)志物表達(dá)及CB-SCs細(xì)胞形態(tài)Fig 1 Surface marker expression and morphology of CB-SCs

*P<0.01 compared with LCs+PHA-P; #P<0.01 compared with LCs圖2 培養(yǎng)3 d后各組淋巴細(xì)胞數(shù)Fig 2 The number of lymphocytes in each group after cultured for 3 days (±s, n=3)

2.5CB-SCs對淋巴細(xì)胞中T淋巴細(xì)胞亞群的調(diào)節(jié)作用

LCs+PHA-P組CD4+/CD8+T淋巴細(xì)胞的比例為1.47±0.21，顯著高于CB-SCs+LCs+PHA-P組的0.80±0.07(P<0.01,n=3)；LCs組為1.31±0.15，顯著高于CB-SCs+LCs組的0.65±0.10(P<0.01,n=3)，與CB-SCs共培養(yǎng)能降低CD4+/CD8+T淋巴細(xì)胞比例。

CB-SCs+LCs組與LCs組相比，CB-SCs可以提高CD4+T細(xì)胞中CD4+CD25+Foxp3+細(xì)胞的比例(P<0.01)(圖5A～D)和CD4+CD25+T細(xì)胞中Foxp3+的陽性率 (P<0.01)(圖6A)。CB-SCs+LCs+PHA-P組與LCs+PHA-P組相比，CB-SCs上調(diào)CD4+CD25+T細(xì)胞中Foxp3、IL- 10(P<0.01)和TGF-β1的陽性率(P<0.05)(圖6B)，增強(qiáng)Treg細(xì)胞的抑制功能。另外，CB-SCs可使CD4+CD25+Foxp3-細(xì)胞的比例下降(圖5E～H)，降低激活態(tài)CD4+T細(xì)胞的比例。

A.lymphocytes in the LCs group; B.4 mg/L PHA-P stimulated lymphocytes; C.lymphocytes co-cultured with CB-SCs; D.4 mg/L PHA-P stimulated lymphocytes co-cultured with CB-SCs; black arrow.CB-SCs; green arrow.lymphocytes; red arrow.small cell clump

A.cytokines level in the groups of LCs+PHA-P and CB-SCs+LCs+PHA-P; B.cytokines level in the groups of LCs and CB-SCs+LCs; *P<0.05,**P<0.01 compared with the group of LCs+PHA-P

3 討論

AD目前被認(rèn)為是一種系統(tǒng)炎性反應(yīng)疾病[1]，患者外周血中促炎因子IL- 1濃度上升、抗炎因子IL- 4和IL- 10濃度下降[6- 7]。血液中升高的促炎因子能促進(jìn)外周淋巴細(xì)胞及細(xì)胞因子透過血-腦脊液屏障，加速AD的發(fā)生和發(fā)展，并且血液中升高的IL- 1β是罹患AD的危險(xiǎn)因素[6]；研究表明IL- 4能夠抑制Th1細(xì)胞分泌促炎因子，IL- 10能誘導(dǎo)Th0向Th2轉(zhuǎn)化[8],抑制Th17細(xì)胞活性[9]；另有文獻(xiàn)報(bào)道外周血中存在的Th2細(xì)胞能明顯改善AD病理變化及癥狀[10]，而IL- 4和IL- 10是Th2細(xì)胞發(fā)揮免疫抑制功能的主要抗炎因子。本實(shí)驗(yàn)發(fā)現(xiàn)與CB-SCs共培養(yǎng)組上清液中IL- 1濃度下降，而IL- 4和IL- 10濃度升高。因此，CB-SCs對AD患者外周血淋巴細(xì)胞的炎性反應(yīng)具有調(diào)節(jié)作用。

患者外周血中存在淋巴細(xì)胞過度激活的免疫反應(yīng)[11]。本研究結(jié)果顯示，CB-SCs明顯抑制PHA-P刺激引起的淋巴細(xì)胞團(tuán)塊狀凝集，共培養(yǎng)組的淋巴細(xì)胞數(shù)量下降；另外，CB-SCs下調(diào)CD4+/CD8+T淋巴細(xì)胞的比例，降低過度激活淋巴細(xì)胞中CD4+CD25+Foxp3-亞群的比例。CD4+/CD8+T淋巴細(xì)胞比值的下降是免疫抑制狀態(tài)的敏感指標(biāo)[12]，而CD4+CD25+Foxp3-亞群是CD4+T淋巴細(xì)胞激活狀態(tài)的標(biāo)志[11]。據(jù)此，CB-SCs具有明顯的免疫負(fù)向調(diào)節(jié)作用。

A,B.the LCs group; C,D.the CB-SCs+LCs group; E,F.the LCs+PHA-P group; G,H.the CB-SCs+LCs+PHA-P group; UL.the upper-left quadrant; UR.the upper-right quadrant; LL.the lower-left quadrant; LR.the lower-right quadrant

A.groups of LCs and CB-SCs+LCs; B.groups of LCs+PHA-P and CB-SCs+LCs+PHA-P; *P<0.05, **P<0.01 compared with the control group

外周血中發(fā)揮主要免疫抑制作用的細(xì)胞是Treg細(xì)胞。研究發(fā)現(xiàn)重度AD外周血中Treg細(xì)胞的數(shù)目和功能有一定程度的損傷[3]。目前向外周血中注射Treg細(xì)胞的治療作用已經(jīng)在AD[3]及腦卒中引起的神經(jīng)炎性反應(yīng)[13]中得到了驗(yàn)證。本實(shí)驗(yàn)發(fā)現(xiàn)，經(jīng)過與CB-SCs共培養(yǎng)，AD外周血中CD4+CD25+Foxp3+細(xì)胞比例上升，而經(jīng)過PHA-P處理的共培養(yǎng)組中雖未出現(xiàn)Treg細(xì)胞比例的變化，但是CD4+CD25+細(xì)胞中的TGF-β1+和IL- 10+細(xì)胞比例明顯上升，提示Treg細(xì)胞的免疫抑制功能增強(qiáng)。文獻(xiàn)顯示，心內(nèi)注射與干細(xì)胞共培養(yǎng)后分選的CD4+CD25+淋巴細(xì)胞可以改善中樞神經(jīng)炎性反應(yīng)[3]，本結(jié)果與之相吻合。

本實(shí)驗(yàn)通過CB-SCs與淋巴細(xì)胞共培養(yǎng)，發(fā)現(xiàn)CB-SCs在體外環(huán)境下對AD患者外周血淋巴細(xì)胞有明顯的免疫負(fù)向調(diào)節(jié)作用，可以調(diào)節(jié)AD患者外周血淋巴細(xì)胞的免疫和過度炎性反應(yīng)，為臨床應(yīng)用提供了理論依據(jù)。CB-SCs的免疫調(diào)節(jié)作用對AD的治療具有潛在的應(yīng)用價(jià)值，臨床應(yīng)用還需更多系統(tǒng)性的比較研究和安全性評價(jià)。

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Immune-modulation of cord blood-derived multipotent stem cells on lymphocytes of patients with Alzheimer’s disease

MA Hai-bo1,2, LI Heng2, TANG Xiao-san2, LI Xiao-hong2*

(1.Medical College, Shandong University, Ji’nan 250012; 2.Dept. of Neurology, Ji’nan Centre Hospital Affiliated to Shandong University, Ji’nan 250013, China)

ObjectiveTo investigate the regulatory effect of cord blood derived-multipotent stem cells (CB-SCs) on peripheral blood lymphocytes (LCs) of patients with Alzheimer’s disease (AD) and to explore the therapeutic potential of CB-SCs for AD.MethodsCB-SCs were isolated from human cord blood. Lymphocytes were isolated from the peripheral blood of patients with AD. Then, after the two cell populations co-cultured with or without phytohaemagglutinin-P (PHA-P) for 3 days, the levels of cytokines in the supernatant were detected with the ELISA test, the proportions of T-cell subsets were determined by the flow cytometric analysis.Results1)CB-SCs showed an inhibitory effect on lymphocyte proliferation and gathers induced by the PHA-P; 2)Compared with the control group, the level of the pro-inflammatory factor IL- 1 was dramatically decreased (P<0.05), while the release of the anti-inflammatory factors IL- 4 and IL- 10 were significantly increased (P<0.01) in the CB-SCs co-cultured

stem cells; Alzheimer’s disease (AD) ; immune modulation; cytokines; regulatory T cells

2014- 12- 08

：2015- 03- 13

國家自然科學(xué)基金(81373635)；濟(jì)南市科技發(fā)展國際合作項(xiàng)目(201212012)

*通信作者(correspondingauthor)：lxh5231@126.com

1001-6325(2015)05-0654-07

研究論文

R741.05

：A

group. 3) The ratio of CD4+/CD8+T cells in the CB-SCs co-cultured group was significantly lower than that in the control group (P<0.01). 4) Compared with the control group, the proportion of regulatory T cells (Tregs) in the CD4+T cells was higher in the CB-SCs co-cultured group without the stimulation of PHA-P (P<0.01), while the anti-inflammatory proteins expressed in the Tregs was at a higher level with the stimulation of PHA-P.ConclusionsCB-SCs can regulate lymphocytes of patients with Alzheimer’s diseaseinvitro, which is mainly displayed with the increased proportion of Tregs subset and enhanced anti-inflammatoty function of Tregs.