外周血内皮祖细胞改善肢体缺血的研究

【关键词】  内皮；干细胞；移植；外周血

    【Abstract】  AIM: To estimate the effect of human peripheral blood derivedendothelial progenitor cell(EPC) transplantation on the repairment of limb ischemia.  METHODS: The mononuclear cells from human peripheral blood  were cultured in M199 medium supplemented with 10% fetal bovine serum, 20 ng/mL VEGF and 4ng/mL bFGF in vitro. The specific markers of EPC were analyzed by FCS at day 6 and 14  during culture respectively. The bilateral femoral artery, great saphenous artery, iliac circumflex artery, and muscular branch of 15 Balb/c nude mice were ligated to cause limb ischemia. The 6day  adherence cells marked by CMDiI were multipoint injected into the left local muscles of ischemic limbs(EPC group) and M199 was multipoint injected into the right local muscles of ischemic limbs (M199 group) in order to evaluate the therapeutic effect. Seven days after operation, 60 μg FITCUEA was injected via tail vein.One hour later, the mice were killed.The heart,liver,lung,spleen,kidney and limb gastrocnemius were taken and examined with fluorescence microscopy. The number of capillaries of ischemic limbs was measured by factor Ⅷ related antigen immunohistochemical staining.  RESULTS: The cultured cells at day 6  expressed KDR, CD133, CD34 and vWF,and the positive rates of KDR, CD133,  CD34 and vWF were (46.4±9.3)%, (33.8±11.7)%,(73.5±6.3)%and (38.9±8.2)%, respectively. At the 14th day  during culture, the cells expressed KDR, CD34 and vWF and the positive rates of KDR,CD34 and vWF were (81.5±7.6)%,(88.9±5.4)%  and  (78.3±13.6)%, respectively; the positive rate of CD133 dropped to (3.8±8.7)%.  The necrosis and density of capillary of the ischemic limbs of nude mice were improved more obviously in the EPC group than that in the M199 group (P<0.05). Red and green fluorescence were observed in the paraffin section of ischemic limbs of nude mice in the EPC group. CONCLUSION: We can culture and obtain EPC from mononuclear cells of human peripheral blood in vitro under a certain condition. The transplanted EPC can incorporate into the local capillary networks and repair the ischemia of posterior limbs of nude mice.

    【Keywords】  endothelium; stem cells; transplantation；peripheral blood

    【摘要】 目的： 研究人外周血来源内皮祖细胞(EPC)移植对改善肢体缺血的作用. 方法： 人外周血单个核细胞在体外诱导扩增6 d和14 d后，分别检测其EPC特异性标志的表达，并将荧光染料标记后的培养第6日的贴壁细胞通过缺血局部多点注射移植到后肢缺血的裸鼠动物模型体内，以评价其效果. 结果： 人外周血单个核细胞经体外诱导分化，培养第6日的贴壁细胞表达KDR, CD133, CD34和vWF，流式细胞仪检测其阳性率分别为（46.4±9.3）%, （33.8±11.7）%, （73.5±6.3）%和（38.9±8.2）%；培养第14日的贴壁细胞KDR, CD34和vWF的表达阳性率均增高，而CD133表达阳性率明显降低，流式细胞仪检测其阳性率分别为（81.5±7.6）%, （88.9±5.4）%, （78.3±13.6）%和（3.8±8.7）%；移植EPC后裸鼠缺血后肢的坏死情况和毛细血管密度均较对照组明显改善(P<0.05)；移植EPC后缺血后肢肌肉石蜡切片中可见分散不均的红色和黄绿色双色荧光的细胞掺入. 结论： 从人外周血单个核细胞中可诱导出EPC，而且移植的EPC可以定向整合到后肢缺血局部，明显改善裸鼠后肢缺血.

    【关键词】　内皮；干细胞；移植；外周血

    1997年Asahara等［1］在 Science杂志发表报道内皮祖细胞(endothelial progenitor cells, EPC)分离成功，并在体内证明其血管生成的能力. 从此EPC受到了研究者的普遍关注，国内外关于EPC的研究方兴未艾［2-3］. 骨髓、外周血和脐血来源的EPC能在体外扩增并分化成功能性内皮细胞，在修复心肌梗死患者的心脏，治疗临床肢体缺血、冠状动脉疾病、脑中风，改善糖尿病患者的血管形成能力，抑制肿瘤血管生成，以及作为基因治疗导向载体和靶细胞等方面，具有广阔的应用前景［4-7］. 我们拟将人外周血单个核细胞在体外特定生长因子的调控下诱导分化为EPC，并且通过注射荧光染料标记后的EPC于动物体内，观察其对改善肢体缺血的作用，以期指导临床实践.

    1材料和方法

    1.1材料100 mL/L胎牛血清（四季青，杭州），20 μg/L的VEGF和4 μg/L的bFGF（PEPROTECH,英国）；Balb/c无胸腺裸鼠(7～8 wk, 18.5～22 g，第四军医大学实验动物研究中心)；碳花青荧光染料(CMDiI，Molecular Probes，美国)；荆豆凝集素(UEA，VECTOR LABORATORIES，美国)；vWF（博士德，武汉）.

    1.2方法

    1.2.1细胞培养和鉴定采用Ficoll密度梯度离心法获取人外周血单个核细胞，洗涤后以M199培养液重悬细胞，培养液含100 mL/L胎牛血清，20 μg/L的VEGF和4 μg/L的bFGF，以5×108/L的密度接种于铺有10 g/L明胶的培养瓶中，细胞培养箱培养48 h后弃去贴壁细胞，悬浮细胞离心后用M199培养液重悬，以2×108/L的密度接种于一次性培养瓶中. 每3 d换液，用Hanks液洗掉未贴壁的细胞后加入M199培养液继续培养. 在培养的第6日和第14日分别对细胞计数，并制作流式细胞仪检测标本，共12份，分别做KDR, CD133, CD34和vWF的流式细胞仪检测，无荧光抗体的培养细胞做为阴性对照.

    1.2.2双后肢缺血模型的建立Balb/c无胸腺裸鼠饲养于SPF级层流架中. 超净台中用20 mg/L的戊巴比妥钠按照50 mg/kg腹腔注射麻醉,沿腹股沟中点至膝内侧作约2 cm长的切口,分离、显露股神经血管鞘及其分支,显微镜下分离出双侧的股动脉及大隐动脉、旋髂外动静脉及股动静脉肌支共四处,分别结扎后缝合切口.

    1.2.3细胞的示踪和移植在培养第6日的准备移植的贴壁细胞培养瓶中加入碳花青荧光染料，使CMDiI浓度为2 mg/L，在37℃孵育5 min，4℃ 15 min, PBS洗2次后，2.5 g/L胰酶/EDTA消化，离心后重悬于M199培养液中. 后肢缺血裸鼠共15只，左后肢局部多点注射扩增后的贴壁细胞，共5 mL，细胞总量5×105，为EPC治疗组；右后肢局部多点注射M199培养液，共5 mL，为M199对照组. 移植标记细胞后的第8日,脱臼处死裸鼠. 处死前1 h，从尾静脉注入20 mg/L FITC标记的的荆豆凝集素3 mL. 取心、肝、肺、脾、肾及双后肢小腿肌肉40 g/L甲醛固定后作石蜡切片，脱蜡后荧光显微镜下观察.

    1.2.4裸鼠后肢情况的检测手术后每日观察裸鼠后肢色泽、温度的变化和活动情况. 双后肢小腿肌肉石蜡切片做vWF免疫组化染色，在光学显微镜下,每张切片随机计数5个×200倍视野内的棕黄色染色管状数取其平均数，表示新生的毛细血管数.

    统计学处理:  计量资料以x±s表示，组间统计处理采用配对t检验，计数资料采用Fisher确切概率检验分析(n=30). 所有统计分析采用 SPSS11.0软件完成.