粒細(xì)胞集落刺激因子對CD34 骨髓干細(xì)胞在心肌內(nèi)歸巢的影響

1、粒細(xì)胞集落刺激因子對CD34+骨髓干細(xì)胞在心肌內(nèi)歸巢的影響            粒細(xì)胞集落刺激因子對CD34+骨髓干細(xì)胞在心肌內(nèi)歸巢的影響    2008-5-9 12:14:20                     

2、60;                                       作者：馮金華，李玉光，石剛剛，侯玉清，曹世平    【關(guān)鍵詞】  干細(xì)胞；心肌梗死；歸巢；粒細(xì)胞

3、集落刺激因子       【Abstract】 AIM: To explore the possible effects of granulocyte colonystimulating factor (GCSF) on the homing of CD34+ bone marrow stem cell (BMSC) to myocardium. METHODS: Seventeen adult mongrel dogs were randomly assigned to 4 groups. Group 1 (GCSF and MI,

4、 n=5) were treated with 10 g/(kgd)×5 d of GCSF injection sc and then myocardial infract operation; group 2 (GCSF and sham operation, n=4), treated with 10 g/(kgd)×5 d of GCSF injection sc and sham operation; group 3 (nonGCSF and MI, n=4) were injected with 10 g/(kgd)×5 d sc normal sal

5、ine and underwent myocardial infarct operation; group 4 (nonGCSF and sham operation, n=4) were injected with 10 g/(kgd)×5 d sc normal saline and get sham operation. Thoracotomy was performed on the left thorax of each animal and left anterior descending artery was exposed and separated about 1

6、cm. In group 1 and 3 the separated arteries were occluded for 3 h and then reopened, while the other 2 groups were not treated in this way. Every dogs received catheterizing in right femoral artery into the root of aorta arch and a topmodified pigtail catheter cannulating through left jugular superf

7、icial vein into coronary venous sinus. 5 mL blood from each animal aorta and coronary sinus were harvested for two times, in which erythrocytes were lysed. Mononuclear cells were separated with lymphocyte separating solution and CD34+ BMSC were clarified and counted by flow cytometry. Data were calc

8、ulated and managed with SPSS 10.0 statistical package. RESULTS: The amount of the CD34+ stem cells homing to myocardium in each group were (183.0±9.2)×104/L, (71.1±9.2)×104/L, (82.3±9.5)×103/L and (78.5±9.8)×103/L, respectively. We found after comparing the da

9、ta of each group that the difference among every group was very significant (P=0.000, respectively) except between group 3 and 4 (P=0.912). CONCLUSION: GCSF could not only vastly mobilize the BMSC into periphery blood, but also could increase the CD34+ stem cells homing to myocardium independent of

10、myocardial infarct.       【Keywords】 stem cell; myocardial infarction; homing; granulocyte colonystimulating factor       【摘要】 目的：觀察粒細(xì)胞集落刺激因子（GCSF）對CD34+骨髓干細(xì)胞（BMSC）在心肌內(nèi)歸巢的影響. 方法：成齡雜種犬17只，隨機(jī)分4組. A組5只：接受10 g/（kgd），5 d皮下注射和實驗性心梗；B組4只：接受10 g/（k

11、gd），5 d皮下注射和假手術(shù)；C組4只：接受10 g/（kgd），5 d生理鹽水皮下注射和實驗性心梗；D組4只：接受10 g/（kgd），5 d生理鹽水皮下注射和假手術(shù)，對每只動物股動脈插管至主動脈根部，左頸淺靜脈插管至冠狀靜脈竇，分別于造型前后抽血5 mL（抽復(fù)管），裂解紅細(xì)胞，分離單個核細(xì)胞，流式細(xì)胞儀測定CD34+細(xì)胞數(shù)，數(shù)據(jù)分析用SPSS 10.0統(tǒng)計軟件包. 結(jié)果：4個實驗組CD34+ BMSC心肌內(nèi)歸巢數(shù)分別為：（183.0±9.2）×104個/L,（71.1±9.2）×104個/L,（82.3±9.5）×103個/L和

12、（78.5±9.8）×103/L，除C，D組之間差別無統(tǒng)計學(xué)意義（P=0.912）外，其余各組間均有統(tǒng)計學(xué)意義（均為P=0.000). 結(jié)論：GCSF不僅能動員BMSC大量釋放入外周血，同時還能增加CD34+ BMSC在心肌內(nèi)的歸巢.       【關(guān)鍵詞】 干細(xì)胞；心肌梗死；歸巢；粒細(xì)胞集落刺激因子        0引言       心肌梗死（myocardial infarction

13、, MI）已成為第一位致死性疾病，以往認(rèn)為壞死心肌無法治愈，現(xiàn)在研究發(fā)現(xiàn)干細(xì)胞能在體內(nèi)外分化成心肌細(xì)胞1-2，自體骨髓干細(xì)胞（bone marrow stem cell, BMSC）能在心梗時得到動員3-4，并歸巢（homing）到梗死部位分化心肌細(xì)胞，然而正常情況下外周血干細(xì)胞含量很低5，遠(yuǎn)達(dá)不到修復(fù)壞死心肌所需要的濃度，使用粒細(xì)胞集落刺激因子（granulocyte colonystimulating factor, GCSF）動員骨髓可以使大量BMSC進(jìn)入外周血，促進(jìn)其分化成心肌和血管內(nèi)皮細(xì)胞，達(dá)到再生和修復(fù)作用，很顯然只有歸巢到靶部位的干細(xì)胞才有可能分化成心肌和血管內(nèi)皮細(xì)胞，因此干細(xì)胞

14、的歸巢決定了心肌的再生. 我們對GCSF能否影響干細(xì)胞的歸巢進(jìn)行了探討.       1材料和方法       11材料成齡雜種犬17只，雌雄不限，體質(zhì)量17.524.7（平均21.2±2.2）kg，隨機(jī)分為4組，A組（GCSF+MI）；B組（GCSF+sham）；C組（NS+MI）;D組（NS+sham）.       12方法A組（5只）接受GCSF（商品名惠爾血，麒麟昆鵬公司生產(chǎn)）10 g/(kgd)

15、, 5 d皮下注射和MI手術(shù)；B組（4只）接受GCSF 10 g/（kgd），5 d皮下注射和假手術(shù)；C組（4只）接受10 g/(kgd), 5 d生理鹽水皮下注射和MI手術(shù)；D組（4只）接受10 g/(kgd), 5 d生理鹽水皮下注射和假手術(shù).       MI手術(shù)： 每只動物左側(cè)開胸，暴露并分離左冠脈前降支近端約1 cm，阻斷3 h后放開，制作MI模型，MI的判斷用心電監(jiān)護(hù)和心肌聲學(xué)造影（圖14），對每只動物股動脈插管至主動脈根部（冠狀動脈竇）、左頸淺靜脈插管至冠狀竇，分別于造型前后抽冠狀動脈和冠狀靜脈竇血5 mL（均抽復(fù)管），

16、裂解紅細(xì)胞，淋巴細(xì)胞分離液分離單個核細(xì)胞，流式細(xì)胞儀（美國BD公司）測CD34+骨髓單個核細(xì)胞數(shù).       統(tǒng)計學(xué)處理： 所有數(shù)據(jù)采用SPSS 10.0統(tǒng)計軟件包統(tǒng)計處理，計量資料以x±s表示，多組間比較采用方差分析，以P<0.05為統(tǒng)計學(xué)差異判斷標(biāo)準(zhǔn).        2結(jié)果       不同實驗組歸巢前（冠狀動脈竇血）CD34+ BMSC含量和數(shù)量比較見表1.  &

17、#160;    4個組比較后發(fā)現(xiàn)在GCSF動員的基礎(chǔ)上發(fā)生MI，CD34+ BMSC在心肌內(nèi)歸巢的數(shù)量大大增加，只用GCSF動員而不發(fā)生MI的動物（B組）CD34+ BMSC的歸巢數(shù)有所降低（P=0.000），而未經(jīng)GCSF動員組（C,D組）即使發(fā)生MI，CD34+ BMSC在心肌內(nèi)的歸巢數(shù)也微乎其微.表1歸巢前冠狀動脈血CD34+ BMAC含量及數(shù)量比較   3討論       自體BMSC因不引起免疫排斥反應(yīng)、取材方便、無倫理學(xué)糾紛等特有的優(yōu)勢在臨床應(yīng)用中倍受青睞，研究發(fā)現(xiàn)急性心

18、肌梗死本身即可引發(fā)自體BMSC動員3,5，使外周血含量增加，促進(jìn)壞死組織的自發(fā)修復(fù)，但這種應(yīng)急性反應(yīng)所致骨髓動員意義到底多大尚無定論. 本研究顯示單純MI組外周血CD34+ BMSC含量（222±12）×104/L與無心肌梗死組（224±10）×104/L比無明顯差別（P=0.958），兩個組發(fā)生歸巢的CD34+ BMSC數(shù)也無顯著性差異（82±10）×103/L比（78±10）×103/L（P=0.912），提示單純MI本身對干細(xì)胞動員和歸巢的影響不起決定作用.       干細(xì)胞向心肌或血管內(nèi)皮細(xì)胞方向的分化首先需要歸巢到心肌內(nèi)，至于BM