腦脈通對(duì)骨髓干細(xì)胞動(dòng)員保護(hù)大鼠腦缺血損傷的影響

1、腦脈通對(duì)骨髓干細(xì)胞動(dòng)員保護(hù)大鼠腦缺血損傷的影響 10-09-04 16:57:00 編輯：studa20作者：李建生 劉敬霞 張新峰 任偉宏 田玉收 王丁超【摘要】 目的 研究腦脈通對(duì)腦缺血大鼠骨髓干細(xì)胞動(dòng)員 (BMSCs) 在血液和腦組織的變化及腦保護(hù)作用的影響。方法 大鼠隨機(jī)分為假手術(shù)組、模型組、腦脈通組、動(dòng)員組、腦脈通+動(dòng)員組，線栓法制備MCAO動(dòng)物模型。皮下注射人重組粒細(xì)胞集落刺激因子(rGCSF)；腦脈通灌胃用藥。檢測(cè)大鼠外周血WBC及CD34+、腦組織CD34+變化；觀察神經(jīng)功能和腦組織病理改變；測(cè)定腦組織含水量和腦梗死面積。結(jié)果 模型組大鼠外周血WBC(8.071.27)109/

2、L和CD34+細(xì)胞(3.170.75)個(gè)/l增加，3 d達(dá)到峰值，各治療組增加更為明顯；聯(lián)合組2和3 d外周血WBC、各時(shí)間點(diǎn)CD34+細(xì)胞均較動(dòng)員組增加。各模型組大鼠腦組織CD34+表達(dá)增強(qiáng)，7 d達(dá)到峰值(33.042.62)個(gè)/l；各聯(lián)合組較動(dòng)員組增強(qiáng)明顯。各模型組大鼠神經(jīng)評(píng)分降低、腦含水量增加、腦梗死面積增大、腦組織病理損傷明顯，以7 d顯著；動(dòng)員組大鼠7和14 d的上述指標(biāo)改善；各聯(lián)合組較動(dòng)員組的改善明顯。結(jié)論 血液WBC數(shù)CD34+計(jì)數(shù)，腦組織、CD34+表達(dá)均顯著顯示，腦缺血可引起B(yǎng)MSCs進(jìn)入外周血并向腦組織歸巢，峰值時(shí)間存在差異；GCSF可使BMSCs分布增加；腦脈通使動(dòng)員后

3、血液和腦組織BMSCs增多、腦組織峰值時(shí)間延長，且使其腦保護(hù)作用增強(qiáng)。 【關(guān)鍵詞】 腦缺血；腦脈通；粒細(xì)胞集落刺激因子；骨髓干細(xì)胞 【Abstract】 Objective To explore the effect of Naomaitong on mobilization of bone marrow stem cells (BMSCs) in blood and brain tissue and the role in protecting brain in rats with cerebral ischemia. Methods Rats were randomly divided i

4、nto different groups. Middle cerebral artery occlusion (MCAO) model was duplicated with nylon thread. Rats in groups of mobilization and model were administrated with rGCSF（10 g-1d-1）through subcutaneous injection before 3 d and after 2 d of operation respectively, once a day. Naomaitong was used th

5、rough intragastric administration. On 2, 3, 7 and 14 d after operation, rats blood were taken through abdominal aorta, then white blood cells (WBCs) and CD34+ cells in peripheral blood were determined. Expression of CD34+ cells in rats brain tissue were detected. Rats state, ratio of weight change a

6、nd general neural function score (GNFS) and brain pathologic change were observed, and then rats brain water ratio (BWR) and cerebral infarction size (CIS) were measured. Results Rats mortality increased after operation and the decreases of weight on 7 d were more obvious. Compared to model groups,

7、decreases of weight in treat groups abated. WBCs（8.071.27） and CD34+ cells（3.170.75） in peripheral blood in model group increased obviously and showed the highest level on 3 d after operation. Increases of WBCs and CD34+ cells in rats of treat groups were more obvious. In comparison with that of mob

8、ilization groups, rats WBCs on 2 and 3 d combination groups increased more significantly and CD34+ cells in each combination group were more higher. Expression of CD34+ cells in brain of rats in model groups increased and showed the highest level on 7 d （33.042.62）and the changes showed more obvious

9、 in each mobilization and combination group, especially in each combination group. Rats GNFS were lower in each model groups and BWR, CIS and brain pathologic increased obviously, especially on 7 d model group. The changes aboved improved significantly on 7 and 14 d mobilization groups. Compared to

10、the changes in mobilization groups, the improvement in each combination group showed more obvious.Conclusions BMSCs could enter peripheral blood and move towards brain tissue after cerebral ischemia and the peak is different. rGCSF could make BMSCs increase both in peripheral blood and brain. Meanwh

11、ile, Naomaitong could increase BMSCs which are mobilized and make the peak of BMSCs in brain prolong, as well as enhance the protection against brain injury after cerebral ischemia. 【Key words】 Cerebral ischemia; Rats; Naomaitong; GCSF; BMSCs骨髓干細(xì)胞(BMSCs)是具有自我更新和多向分化潛能的原始骨髓細(xì)胞，可分化為主要的幾類神經(jīng)細(xì)胞，被用于腦組織損傷的保

12、護(hù)和修復(fù)1。因符合機(jī)體自身的反應(yīng)性修復(fù)機(jī)制，方法簡便、安全且可避免異基因移植的免疫排斥反應(yīng)，BMSCs動(dòng)員在腦缺血損傷的應(yīng)用方面顯示良好前景2，3。粒細(xì)胞集落刺激因子(GCSF)是BMSCs有力的動(dòng)員劑，可使腦缺血損傷后外周血和腦組織的BMSCs增加，并使其保護(hù)腦組織的作用增強(qiáng)。中藥在骨髓干細(xì)胞動(dòng)員保護(hù)腦組織受損方面研究有待進(jìn)一步探索。我們前期研究發(fā)現(xiàn)，中藥腦脈通(由大黃、人參、川芎、葛根組成)可增強(qiáng)BMSCs移植后的腦保護(hù)作用4，本研究擬就其對(duì)BMSCs動(dòng)員后的分布變化及抗腦缺血損傷作用的影響進(jìn)行探討，為中藥在BMSCs動(dòng)員治療腦缺血方面的應(yīng)用提供依據(jù)。1 材料與方法1.1 材料SD大鼠，S

13、PF級(jí)，雌雄各半，34月齡，體重(30050)g，202只，由河南省實(shí)驗(yàn)動(dòng)物中心提供合格證號(hào)：scxk(豫)20050001。人重組粒細(xì)胞集落刺激因子注射液(rGCSF，商品名瑞白，山東齊魯制藥廠，規(guī)格：150 g/支)；熒光標(biāo)記單克隆抗體CD34+ (Santa Cruz產(chǎn)品)；羊抗大鼠IgG生物素(BA1005)、檸檬酸鹽緩沖液(AR0024)、正常山羊血清封閉液(AR0009)、DAB 顯色試劑盒 (AR1022)均由武漢博士德生物工程有限公司提供；腦脈通顆粒 (出膏率15%，由大黃、人參、川芎、葛根組成；河南中醫(yī)學(xué)院藥物分析學(xué)科提供，5 g/袋)。流式細(xì)胞儀(Beckman Coult

14、er Epics，XL)；數(shù)碼相機(jī) (Sony Corpatation，Japan，型號(hào)：DSCF717 2002)；光學(xué)顯微鏡(Olympus optical Co.LTD，japan，型號(hào)：PM10AD)；透射電子顯微鏡(Hitachi，Japan，型號(hào) H7500)；圖像分析系統(tǒng)ImageProplus 5.1(Media Cybernetics Inc，USA，型號(hào)41N510044800)。1.2 方法大鼠按隨機(jī)數(shù)字表法分為假手術(shù)組、模型組、腦脈通組、動(dòng)員組、腦脈通+動(dòng)員組 (聯(lián)合組)。假手術(shù)組10只大鼠，其余4組均為48只；后4組根據(jù)取材時(shí)間又分為2、3、7、14 d組，每組大鼠1

15、2只。分別于術(shù)前3 d和術(shù)后2 d給動(dòng)員組、聯(lián)合組大鼠皮下注射rGCSF(10 gkg1d-1)；假手術(shù)組、模型組和腦脈通組大鼠皮下注射等容積的生理鹽水，每天1次。假手術(shù)組、模型組、動(dòng)員組分別于造模前4 d用生理鹽水灌胃，腦脈通組和聯(lián)合組以生理鹽水制備的腦脈通懸浮液(40.5 mg/ml)灌胃(40.5 mg100 g-1 d-1)，造模前加灌胃1次，術(shù)后每日灌胃1次，直至取材 (灌胃容積為1 ml100 g-1d-1)，每周根據(jù)大鼠體重調(diào)整灌胃藥物的用量和灌胃體積5。各組大鼠均于術(shù)前禁食12 h，不禁水。參照改良的Longa法用線栓阻塞大鼠大腦中動(dòng)脈制備局灶性腦缺血?jiǎng)游锬Ｐ?(MCAO)6。

16、10水合氯醛腹腔注射麻醉大鼠，待完全麻醉后，仰臥位固定大鼠，行頸前正中切口，左側(cè)鈍性分離頸總動(dòng)脈(CCA)；分離頸內(nèi)外動(dòng)脈，穿線備用，結(jié)扎翼顎動(dòng)脈，于頸外動(dòng)脈近動(dòng)脈分叉處剪口，栓線穿入頸內(nèi)動(dòng)脈，緩慢推進(jìn)，直至感覺有阻力為止，穿線成功后縫合皮膚。假手術(shù)組除不穿入栓線外，其余操作相同。手術(shù)過程中保持大鼠肛溫(37.00.5)，保持室溫(261)。術(shù)后注射青霉素鈉(1萬U100 g-1d-1)，以防感染。假手術(shù)組大鼠術(shù)后14 d取材，其余各組分別于術(shù)后2、3、7、14 d觀察大鼠一般狀況，大鼠稱重，進(jìn)行神經(jīng)功能評(píng)測(cè)；麻醉大鼠，腹主動(dòng)脈取血5 ml，注入EDTA3K試管，流式細(xì)胞儀測(cè)定血白細(xì)胞計(jì)數(shù)及C

17、D34+細(xì)胞數(shù)量；4%多聚甲醛溶液經(jīng)升主動(dòng)脈進(jìn)行灌注固定，取出全腦，4生理鹽水沖洗3遍，除去積血，濾紙吸去表面水分，去除嗅球、小腦和腦干。冰盤上迅速分離大腦半球，棄去右側(cè)，取左側(cè)半球，從額極向后冠狀切取腦組織3 mm，待測(cè)腦組織含水量；依次向后冠狀切取腦組織1 mm，迅速投入備好的內(nèi)盛2%的TTC溶液，37避光育孵30 min，然后用10%的甲醛固定15 min，觀察染色效果，待測(cè)腦梗死面積；向后冠狀切取2 mm厚的腦組織投入備好的內(nèi)盛多聚甲醛的小瓶內(nèi)固定，待做腦組織病理和免疫組化檢測(cè)；其余標(biāo)本液氮冷存。1.3 測(cè)定指標(biāo)對(duì)術(shù)后蘇醒至取材時(shí)死亡和存活大鼠進(jìn)行計(jì)數(shù)，計(jì)算各組(包括2、3、7、14

18、d 4個(gè)時(shí)間點(diǎn))死亡大鼠只數(shù)與總體(造模后死亡與存活大鼠總只數(shù))的比率。大鼠死亡率=(死亡只數(shù)/死亡與存活只數(shù)總和)100%。分別于術(shù)后2 d至取材時(shí)間在相應(yīng)時(shí)間點(diǎn)大鼠稱重，采集數(shù)據(jù)的時(shí)間點(diǎn)數(shù)分別為2 d組(1個(gè))、3 d(2個(gè))、7 d(3個(gè))、14 d(4個(gè))，計(jì)算公式為：體重下降率=(術(shù)前體重-術(shù)后體重)/術(shù)前體重100%，體重增長率=(術(shù)后體重-術(shù)前體重)/術(shù)前體重100%。抗凝血液20 l用WBC稀釋液稀釋20倍，取20 l入血細(xì)胞計(jì)數(shù)盤的計(jì)數(shù)室，靜置3 min，待WBC下沉，在低倍鏡下計(jì)四角四個(gè)大方格的有核細(xì)胞，4格總和乘以50則為每立方毫米的有核細(xì)胞計(jì)數(shù)。抽EDTAK3抗凝腹主動(dòng)脈血1 ml輕搖混勻，然后按試管編號(hào)，取樣本全血50 l，加CD34熒光素5 l，輕混，搖勻后避光置室溫20 min，加溶血素250 l，混勻，避光置室溫10 min，加PBS液500 l，混勻，避光置室溫10 min，離心1 500 r/min 5 min，棄上清，每管加鹽水1 ml混勻，應(yīng)用流式細(xì)胞儀進(jìn)行檢測(cè)，計(jì)數(shù)CD34+細(xì)胞平均值(個(gè)/l)。神經(jīng)功能評(píng)測(cè)方法按文獻(xiàn)7從自發(fā)運(yùn)動(dòng)、輕癱實(shí)驗(yàn)、前肢運(yùn)動(dòng)功能檢測(cè)、加強(qiáng)運(yùn)動(dòng)功能檢測(cè)、痛覺、位置覺6個(gè)方面進(jìn)行，總分為18分，癥狀越重，得