脐血间充质干细胞心肌移植对梗死区CD4^+T、CD8^+T淋巴细胞分布的影响

The Effect of Allogeneic Canine Cord Blood Mesenchymal Stem Cells Transplantation on Distribution of CD4^+T and CD8^+T in Infarcted Regions of Hearts

目的研究犬脐血间充质干细胞(cbMSC)心肌移植对梗死区CD4+T、CD8+T淋巴细胞分布的影响。方法取犬脐带血,密度梯度离心法分离出单个核细胞,贴壁培养法扩增间质干细胞;采用随机数字表法将成年雄性杂种犬36只分为实验组和对照组,采用结扎冠状动脉左前降支的方法建立急性心肌梗死动物模型;实验组取第4代细胞用5-氮杂胞苷诱导72h,LacZ报告基因标记后经冠状动脉移植入犬心脏的梗死区域,对照组注射等体积的IMDM培养液;采用免疫组织化学法检测表达β-gal的移植细胞以及梗死区域CD4+T、CD8+T的分布,应用Image-Proplus5.1软件进行图象分析。结果异体犬cbMSC移植心肌梗死区后可长期存活;图象分析显示,实验组移植后第2、4、8周时,CD4+T的累积光密度值(IOD)为44.35±7.03,19.29±4.11和20.27±3.51,CD4+T与CD8+T的IOD比值为0.63±0.12,0.51±0.15和0.66±0.08;对照组CD4+T的IOD值为65.78±10.27,28.02±2.59和29.79±6.83,CD4+T与CD8+T的IOD比值为1.28±0.20,1.34±0.09和1.50±0.16,两组比较实验组明显低于对照组。检测表明,实验组移植后第2、4、8周时CD8+T的IOD为69.88±7.84,37.80±8.83和30.81±7.42,高于对照组的51.28±10.01,20.87±4.50和19.91±2.87。结论初步的实验结果表明,犬cbMSC的同种异体移植能产生免疫耐受,其机制可能与其抑制CD4+T,减少局部浸润的CD4+T数量,降低局部CD4+T/CD8+T比值有关。

Objective To study the effect of allogeneic canine cord blood mesenehymal stem cells （cbMSC） transplantation on the distribution of CD4^＋ T and CD8^＋ T in infracted area of hearts. Methods Mononuclear cells of cord blood were isolated by density gradient centrifugation and amplified by adherent culture. 36 adult male dogs were divided into experimental group and control group. Animal models of acute myocardial infarction were established by ligating anterior descending coronary artery. The fourth generations of mesenchymal stem cells （MSC） were transplanted into infarcted area of hearts by left anterior descending coronary artery after 72h induced by 5-aza and transfected by LacZ. The survival of transplanted cells in hearts can be confirmed by i3-gal expression. CD4^＋ T and CD8^＋ T cells distributed in infarcted area were detected by immunohistochemical staining method. The Image-Plus 5.1 software was used to analyze the images. Results Cells transplanted into infarcted area could survive for a long time. 2, 4, 8 weeks after transplantation, the IOD of CD4^＋Tin experimental group were 44.35±7.03, 19.29±4.11 and 20.27±3.51 respectively, and the CD4＋T/CDS＋T ratios were 0. 63±0. 12, 0. 51±0. 15 and 0. 66±0.08. In control group, the IOD of CD4^＋T at 2, 4, 8 weeks after transplantation were 65.78±10.27, 28. 02±2.59, 29. 79± 6.83, and theCD4^＋T/CD8^＋Tratios were 1.28±0.20, 1.34±0.09 and 1.50±0.16. ThelOD of CD4^＋T and CD4^＋ T/CD8^＋T ratio in experimental group were significantly lower than that in control group. In experimental group the IOD of CD8^＋T at 2, 4, 8 weeks after transplantation were 69.88±7.84 , 37.80±8.83 and 30.81±7.42, higher than that in control group which were 51. 28 ± 10. 01, 20. 87 ± 4. 50 and 19. 91 ± 2. 87. Conclusion The preliminary results indicated that allogeneic cbMSC transplanted in infarcted area can escape from immune rejection, its mechanism may be associated withdecreasing the amount of CD4^＋T cells infiltrated in periphery of infarcted area and maintaining CD4^＋ T/CD8^＋ T ratios at a lower level.