加壓運(yùn)動(dòng)對(duì)羊骨髓間充質(zhì)干細(xì)胞增殖、分化的影響

楊玉輝，孫紹騫，佐藤義昭，劉吉平，張　軍，王宗強(qiáng)*

(1.吉林大學(xué)中日聯(lián)誼醫(yī)院,吉林 長(zhǎng)春130033；2.日本加壓運(yùn)動(dòng)學(xué)會(huì)，日本加壓醫(yī)療學(xué)會(huì)；3.東方朝日運(yùn)動(dòng)發(fā)展(北京)有限公司)

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加壓運(yùn)動(dòng)對(duì)羊骨髓間充質(zhì)干細(xì)胞增殖、分化的影響

楊玉輝1△，孫紹騫1，佐藤義昭2△，劉吉平3，張軍3，王宗強(qiáng)1*

(1.吉林大學(xué)中日聯(lián)誼醫(yī)院,吉林 長(zhǎng)春130033；2.日本加壓運(yùn)動(dòng)學(xué)會(huì)，日本加壓醫(yī)療學(xué)會(huì)；3.東方朝日運(yùn)動(dòng)發(fā)展(北京)有限公司)

目的探索加壓運(yùn)動(dòng)對(duì)羊骨髓間充質(zhì)干細(xì)胞增值、分化的影響。方法將60只波爾山羊隨機(jī)分成實(shí)驗(yàn)組和對(duì)照組，實(shí)驗(yàn)組每周進(jìn)行2次加壓運(yùn)動(dòng)，對(duì)照組非加壓下運(yùn)動(dòng)，6個(gè)月后獲取羊骨髓，然后采用percoll密度梯度離心法分離并吸取上中層界面間以單個(gè)核細(xì)胞為主的白色云霧層進(jìn)行培養(yǎng),觀察細(xì)胞形態(tài)和增殖速率；兩組細(xì)胞均用5-氮雜胞苷誘導(dǎo)其定向分化為心肌樣細(xì)胞,對(duì)誘導(dǎo)后的細(xì)胞進(jìn)行免疫熒光檢測(cè)細(xì)胞上心肌特異性抗原a-actin表達(dá)情況。結(jié)果從骨髓中分離出的白色云霧層中的細(xì)胞能貼壁、能傳代且生長(zhǎng)狀態(tài)良好。此外，經(jīng)過5-氮雜胞苷誘導(dǎo)后細(xì)胞均表達(dá)心肌細(xì)胞特異性抗原。實(shí)驗(yàn)組骨髓間充質(zhì)干細(xì)胞在培養(yǎng)過程中細(xì)胞多呈小而圓的形態(tài)，且增殖速率比對(duì)照組骨髓間充質(zhì)干細(xì)胞快，而對(duì)照組細(xì)胞多呈多角形或梭形且增殖速率相對(duì)較慢。結(jié)論成功的分離并培養(yǎng)了骨髓間充質(zhì)干細(xì)胞,并在體外成功誘導(dǎo)了骨髓間充質(zhì)干細(xì)胞向心肌樣細(xì)胞的分化。長(zhǎng)期處于加壓運(yùn)動(dòng)條件下的骨髓間充質(zhì)干細(xì)胞比非加壓運(yùn)動(dòng)條件下更傾向于增殖，而分化能力相對(duì)弱一些。

加壓運(yùn)動(dòng)；骨髓間充質(zhì)干細(xì)胞；增殖；分化

(ChinJLabDiagn,2016,20:1240)

骨髓間充質(zhì)干細(xì)胞 (BMSCs)是中胚層來源的具有高度增殖、自我更新的能力及多向分化潛能的多能干細(xì)胞。進(jìn)一步研究證明骨髓間充質(zhì)干細(xì)胞體外還可以向心肌、神經(jīng)元、神經(jīng)膠質(zhì)細(xì)胞分化[1]，體內(nèi)移植后可以遷移至損傷部位(多數(shù)為缺血、缺氧的環(huán)境)修復(fù)相應(yīng)的組織。細(xì)胞移植治療為不可逆性心臟疾病提供了全新的治療策略[2]，骨髓間充質(zhì)干細(xì)胞目前被認(rèn)為是一類最理想的進(jìn)行細(xì)胞移植治療的種子細(xì)胞[3]，常被用作基因治療的載體細(xì)胞[4,5]。異體骨髓間充質(zhì)干細(xì)胞移植會(huì)產(chǎn)生免疫排斥反應(yīng)。自體血干細(xì)胞數(shù)量有限，如何促進(jìn)自體骨髓間充質(zhì)干細(xì)胞的增殖并釋放入血就變得至關(guān)重要，尤其是大型哺乳動(dòng)物，近幾年關(guān)于鼠、兔等小型動(dòng)物骨髓間充質(zhì)干細(xì)胞的研究較多[6,7]，對(duì)大型動(dòng)物羊的骨髓間充質(zhì)干細(xì)胞的研究報(bào)道還較少[8]，因此對(duì)大型動(dòng)物羊骨髓間充質(zhì)干細(xì)胞體外如何更好誘導(dǎo)其定向分化的研究具有重要意義。

1　材料與方法

1.1實(shí)驗(yàn)材料

實(shí)驗(yàn)動(dòng)物：健康波爾山羊；5-氮雜胞苷 (sigma，美國)，一抗:鼠源抗α骨骼肌肌動(dòng)蛋白抗體(anti alpha skeletal muscle actin antibody) (abcam，英國)，二抗:驢抗小鼠免疫球蛋白g (Dylight 488 affinipure donkey anti-mouse IgG(H+L))(EarthOx,llc，美國)，0.25%胰酶(GIBCO，美國),IMDM (GIBCO，美國),胎牛血清(Hyclon，美國)

1.2方法

1.2.1加壓運(yùn)動(dòng)選取24-30月齡健康波爾山羊60只，體重在30-35 kg，隨機(jī)分成實(shí)驗(yàn)組和對(duì)照組，每組30只。采用日本佐藤義昭先生發(fā)明的KAATSU加壓運(yùn)動(dòng)設(shè)備將實(shí)驗(yàn)組山羊左側(cè)后肢根部綁縛加壓帶，壓力范圍60-100 mmHg，壓力值上限以不引起山羊跛行為準(zhǔn)。兩組山羊每周進(jìn)行兩次行走訓(xùn)練，每次持續(xù)15 min，其余時(shí)間統(tǒng)一圈養(yǎng)，飲食無差別。

1.2.2取羊骨髓，分離培養(yǎng)骨髓間充質(zhì)干細(xì)胞 6個(gè)月后無菌條件下取羊左后肢股骨及脛骨，剖開骨干，用IMDM培養(yǎng)液沖出骨髓。分離出的骨髓用1.082 g/ml比重的 Percoll 細(xì)胞分離液，500×g/min 速度下密度梯度離心 25 min，取中間單核細(xì)胞層，用0.01M PBS充分洗滌，加入培養(yǎng)基制成單細(xì)胞懸液；計(jì)數(shù)，按 1.0×106/ml 濃度接種于完全培養(yǎng)基(含體積分?jǐn)?shù)為10%FBS的IMDM和青、鏈霉素各 100 U/ml)中。置于37℃細(xì)胞培養(yǎng)箱中培養(yǎng)，24 h后去除非貼壁細(xì)胞，每3天換液 1 次。

1.3羊骨髓間充質(zhì)干細(xì)胞傳代培養(yǎng)

待細(xì)胞達(dá)80%-90%融合后，加入PBS洗一遍后,用濃度為0.25% 胰蛋白酶在37℃條件下消化細(xì)胞，待細(xì)胞形態(tài)開始皺縮，用含10%胎牛血清的IMDM培養(yǎng)基終止消化,移入離心管，1 200 r/min離心5 min后，傳至兩皿中繼續(xù)培養(yǎng),約每3天換液一次，直至貼壁細(xì)胞接近80%-90%融合，再重復(fù)上述操作，反復(fù)傳代擴(kuò)增。

1．4生長(zhǎng)曲線

骨髓間充質(zhì)干細(xì)胞用含10% 胎牛血清的IMDM培養(yǎng)基培養(yǎng)，每隔24 h 在倒置顯微鏡下觀察并進(jìn)行計(jì)數(shù)，每個(gè)樣品測(cè)3次,取平均值，分別計(jì)算實(shí)驗(yàn)組和對(duì)照組所有樣品平均值的均數(shù)。連續(xù)觀察12 d以存活細(xì)胞數(shù)(萬/ml) 對(duì)培養(yǎng)時(shí)間(d) 作圖，即得生長(zhǎng)曲線。

1.5誘導(dǎo)羊骨髓間充質(zhì)干細(xì)胞向肌源細(xì)胞定向分化

取培養(yǎng)至第二代的羊骨髓間充質(zhì)干細(xì)胞，加入10 μ mol/L 5-氮雜胞苷誘導(dǎo)24 h后,更換新的含10% FBS的IMDM培養(yǎng)基，培養(yǎng)14 d后,免疫熒光法檢測(cè)心肌細(xì)胞特異性抗原a- actin表達(dá)情況.

1.6免疫熒光實(shí)驗(yàn)

取來誘導(dǎo)后的細(xì)胞棄去培養(yǎng)液，用4%甲醛室溫固定30 min后用磷酸鹽緩沖液洗3次，每次15 min；然后用1% (vol/vol) Triton配成含5% (wt/vol) BSA抗體稀釋液，用抗體稀釋液1∶100比例稀釋鼠源抗a- actin抗體,將稀釋好的抗體加到細(xì)胞中，4度孵育過夜；第二天用磷酸鹽緩沖液洗3次,每次15 min。再用抗體稀釋液1∶200比例稀釋二抗(驢抗小鼠免疫球蛋白g)，將二抗加到細(xì)胞中室溫避光孵育1 h，再用磷酸鹽緩沖液洗3次，每次15 min，然后用5%甘油封片，置于熒光顯微鏡下觀察。

2　結(jié)果

2.1實(shí)驗(yàn)過程中實(shí)驗(yàn)組和對(duì)照組分別有9只、10只羊因?yàn)榧膊∷劳龆撀洌瑢?shí)際嚴(yán)格完成實(shí)驗(yàn)的羊分別是實(shí)驗(yàn)組21只、對(duì)照組20只。

2.2羊骨髓形態(tài)學(xué)特征和加壓運(yùn)動(dòng)對(duì)骨髓間充質(zhì)干細(xì)胞分化的影響

骨髓間充質(zhì)干細(xì)胞在培養(yǎng)48 h內(nèi)貼壁，細(xì)胞形態(tài)不一、呈圓形和梭形，隨著培養(yǎng)時(shí)間延長(zhǎng)，細(xì)胞形態(tài)以梭形居多，細(xì)胞增長(zhǎng)速度較快，3-5 d傳代一次。實(shí)驗(yàn)組和對(duì)照組的骨髓間充質(zhì)干細(xì)胞同樣條件進(jìn)行培養(yǎng)，隨著培養(yǎng)時(shí)間延長(zhǎng)，細(xì)胞間形態(tài)差異逐漸增大，培養(yǎng)第12 d時(shí)顯微鏡下觀察，發(fā)現(xiàn)實(shí)驗(yàn)組更多比例的細(xì)胞呈圓形，而對(duì)照組圓形細(xì)胞比例較少，大部分細(xì)胞呈梭形,見圖1。

2.3生長(zhǎng)曲線

計(jì)數(shù)結(jié)果顯示，培養(yǎng)第1-2 d時(shí)細(xì)胞數(shù)目沒有變化為增殖潛伏期，第3-9 d細(xì)胞數(shù)目快速增加，進(jìn)入指數(shù)生長(zhǎng)期，在9-12 d細(xì)胞數(shù)目增長(zhǎng)緩慢,見圖2。

2.4免疫熒光檢測(cè)心肌特異性抗原表達(dá)情況

經(jīng)5-氮雜胞苷誘導(dǎo)24 h后培養(yǎng)14 d時(shí)免疫熒光染色發(fā)現(xiàn)，細(xì)胞呈纖維性，且均呈綠色，表明經(jīng)誘導(dǎo)后的細(xì)胞均強(qiáng)表達(dá)心肌細(xì)胞特異性抗原α-actin，提示羊骨髓間充質(zhì)干細(xì)胞正處于或已完成向心肌樣細(xì)胞分化過程,見圖3。

圖1　實(shí)驗(yàn)組和對(duì)照組的骨髓間充質(zhì)干細(xì)胞培養(yǎng)第12天的形態(tài)圖(X 4倍)

圖2　實(shí)驗(yàn)組和對(duì)照組羊骨髓間充質(zhì)干細(xì)胞的生長(zhǎng)曲線圖

圖3　骨髓間充質(zhì)干細(xì)胞經(jīng)5-氮雜胞苷誘導(dǎo)14天后免疫熒光檢測(cè)圖 (X 40倍)

3　討論

從羊骨髓中分離提取的細(xì)胞，能貼壁，且能傳代，并且在適宜的條件下誘導(dǎo)能定向分化，提示已經(jīng)成功培養(yǎng)羊骨髓間充質(zhì)干細(xì)胞。羊骨髓間充質(zhì)干細(xì)胞在5-氮雜胞苷誘導(dǎo)下表達(dá)出心肌特異性抗原α-actin，提示骨髓間充質(zhì)干細(xì)胞正處于或已完成向心肌樣細(xì)胞分化，對(duì)于骨髓間充質(zhì)干細(xì)胞經(jīng)5-氮雜胞苷誘導(dǎo)后向心肌樣細(xì)胞分化的機(jī)制還不明確，目前研究認(rèn)為：5-氮雜胞苷是一種去甲基化藥物，能夠引起DNA 中某些胞嘧啶去甲基化，從而誘導(dǎo) BMSCs 向心肌細(xì)胞分化[9,10]。上述實(shí)驗(yàn)結(jié)果表明羊骨髓間充質(zhì)干細(xì)胞在體外培養(yǎng)時(shí)表現(xiàn)出較高的增殖速率和定向分化的潛能，為大型動(dòng)物的骨髓間充質(zhì)干細(xì)胞在體外在適宜的條件下誘導(dǎo)其定向分化提供了一定的可行性，為其在動(dòng)物醫(yī)學(xué)和人類醫(yī)學(xué)的臨床應(yīng)用提供參考。此外，另有研究表明羊骨髓間充質(zhì)干細(xì)胞免疫原性弱，且易于外源基因的導(dǎo)入和表達(dá)，因而在組織工程、細(xì)胞和基因治療等領(lǐng)域得到廣泛的應(yīng)用，羊與其它哺乳動(dòng)物如人、牛、兔、大鼠等類似，對(duì)于以后利用羊骨髓間充質(zhì)干細(xì)胞作轉(zhuǎn)基因克隆供體細(xì)胞的選擇及胚胎發(fā)育的研究奠定了基礎(chǔ)，將成為具有廣闊應(yīng)用前景的一類成體干細(xì)胞。同時(shí)發(fā)現(xiàn)實(shí)驗(yàn)組和對(duì)照組的骨髓間充質(zhì)干細(xì)胞在培養(yǎng)過程中出現(xiàn)了生長(zhǎng)形態(tài)上的差異，實(shí)驗(yàn)組的骨髓間充質(zhì)干細(xì)胞經(jīng)多次傳代培養(yǎng)后形態(tài)上多呈小而圓形，與剛從骨髓中分離的原代細(xì)胞形態(tài)更接近，更傾向于增殖。骨髓間充質(zhì)干細(xì)胞主要來源于骨髓，骨髓是一典型的低氧環(huán)境，同時(shí)實(shí)驗(yàn)組動(dòng)物經(jīng)過加壓運(yùn)動(dòng)造成了一種低氧代謝的狀態(tài)。缺氧條件下可能通過反饋刺激使骨髓基質(zhì)干細(xì)胞增殖能力增強(qiáng)，但是分化能力受到抑制，而對(duì)照組動(dòng)物未經(jīng)加壓運(yùn)動(dòng)，常氧條件下的骨髓間充質(zhì)干細(xì)胞多呈梭形，經(jīng)誘導(dǎo)后向心肌樣細(xì)胞分化，提示我們常氧條件下的骨髓間充質(zhì)干細(xì)胞比其在缺氧條件下更傾向于分化。有關(guān)加壓運(yùn)動(dòng)對(duì)骨髓間充質(zhì)干細(xì)胞影響的研究還需深入探討，如加壓運(yùn)動(dòng)的壓力值、運(yùn)動(dòng)時(shí)間和頻次為多少更利于其增殖或分化，這樣就可以根據(jù)臨床需要設(shè)計(jì)加壓運(yùn)動(dòng)的各種條件，以調(diào)控骨髓間充質(zhì)干細(xì)胞的增殖和分化。

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Effects of KAATSU Training on proliferation and differentiation of goat bone marrow mesenchymal stem cells

YANG Yu-hui,SUN Shao-qian,Sato Voshiaki,et al.

(China-JapanUnionHospitalofJilinUniversity,Changchun130033,China)

ObjectiveTo explore the effects of KAATSU Training on proliferation and differentiation of goat bone marrow mesenchymal stem cells.Methods60 Boer goats were randomly divided into experimental group and control group,the experimental group were given KAATSU Training twice a week,non-KAATSU Training twice a week for the control group,6 months later,we got the goat bone marrow,and then separated and absorbed the white cloud layer which mainly contained the mononuclear cell in the upper-middle interface with the method of percoll-density gradient centrifugation,cultured and observed the cell morphology and the proliferation rate;the cells of the two groups were induced into cardiomyocyte like cells by the 5-azacytidine,The cells which had been differentiated were detected the expression of the cardiac specific antigen a-actin by immunofluorescence assay.ResultsThe cells isolated from the bone marrow in the white cloud layer were adherent,generated and grew well,in addition,the cells which induced by 5-azacytidine could express the cardiac specific antigen.The bone marrow mesenchymal stem cells of the experimental group were small and round,and their proliferation rate was faster than the control group’s,though the shape of the cells in the control group were polygonal or triangular,and the proliferation rate were slow.ConclusionIt has been succeeded both in separation and cultivation of the bone marrow mesenchymal stem cells,and also induced the proliferation of turning into cardiomyocyte like cells in vitro.The bone marrow mesenchymal stem cells in pressurization motion for a long period of time were more easy to proliferate than the cells in non-pressurization motion,but the differentiative capability were low.

KAATSU Training;Bone marrow mesenchymal stem cells;Proliferation,Differentiation

1007-4287(2016)08-1240-04

Q813

A

2015-12-20)

△并列為第一作者