巴曲酶对下肢深静脉血栓患者内皮祖细胞的影响

目的 探讨巴曲酶(DF-521)对下肢深静脉血栓(LEDVT)患者外周血内皮祖细胞(EPC)数量及其体外功能的影响.方法 将LEDVT患者随机分为对照组和治疗组,每组各18例.对照组仅给予低分子肝素(4000 U/12 h)和尿激酶(300 000 U/d)治疗,而治疗组给予相同剂量的尿激酶、低分子肝素和DF-521(5 U/2 d),分别于治疗7 d和3个月后评定各组疗效.于治疗前和治疗7 d后取患者外周血,通过荧光激活细胞分选检测外周血中EPC(CD34+VEGFR2+)的比例.应用密度梯度离心法分离健康人外周血单个核细胞,将其诱导培养成内皮祖细胞(EPC).将EPC分为实验组(4组,1×10-2、2×10-2、4×10-2、8×10-2U/ml DF-521)和对照组(培养基).采用MTT比色法、一氧化氮(NO)试剂盒、体外血管形成试剂盒以及流式细胞仪检测细胞表型,透射电镜观察细胞超微结构.分析不同浓度DF-521对EPC增殖、NO分泌、血管形成以及向内皮细胞分化能力的影响.结果 治疗7 d后,治疗组和对照组的疗效差异无统计学意义(66.7%比66.7%,P>0.05),而3个月后治疗组则优于对照组(94.4%比66.7%,P<0.05).治疗7d后,治疗组患者外周血EPC含量显著高于治疗前(0.64±0.05比0.42±0.02,P<0.05),并且显著高于治疗后对照组的含量(0.64±0.05比0.42±0.03,P<0.05).对照组治疗前后无明显差异(0.41±0.03比0.42±0.03,P>0.05).DF-521各实验组EPC吸光度A值、NO浓度、血管管腔样结构数均高于对照组(P<0.05),而且各实验组的吸光度A值和NO浓度随着DF-521浓度的增加而升高(r=0.978、0.981,均P<0.05),4×10-2 U/ml DF-521组血管管腔样结构数量最多.各实验组CD31和vWF的表达均高于对照组(P<0.05),但各实验组之间差异无统计学意义(P>0.05).透视电镜结果显示,8×10-2 U/mlDF-521组有内皮细胞的特有结构帕怀特小体.结论 DF-521可以改善LEDVT患者的预后,增加患者外周血EPC的数量,在体外环境下改善EPC的活性及功能,并促进EPC向内皮细胞分化.

Effect of batroxobin on endothelial progenitor cells in patients with lower extremity deep venous thrombosis

Objective To investigate the effect of batroxobin (DF-521) on peripheral counts and in vitro capabilities of endothelial progenitor cells (EPCs) in patients with lower extremity deep venous thrombosis (LEDVT). Methods Patients with LEDVT were recruited and randomized into the control group (n=18) and the therapy group (n=18). The control group was given low-molecular-weight heparin (4000 U/12h)and urokinase (300 000 U/d),whereas the therapy group received DF-521 (5 U/2 d) in addition to the same dosages of heparin and urokinase as in the control group. The efficacy of two groups was evaluated at 7days and at 3 months after treatment. Before and on day 7 of treatment,blood samples were collected from patients and determined for the percentage of EPCs (CD34+ VEGFR2 + ) by fluorescence-activated cell sorting. From healthy blood samples,peripheral blood mononuclear (PBMN) cells were separated by density gradient centrifugation and induction-cultured into EPCs. The EPCs in vitro were divided into 4 experimental groups (incubated with various concentrations of DF-521:1 × 10-2,2× 10-2,4× 10-2,8× 10-2 U/ml) and a control group (culture medium only). The effects of DF-521 on proliferation,nitric oxide (NO) release,angiogenesis and differentiation of EPCs were observed by MTT assay,nitric oxide kits,tube formation assay,the phenotypes by flow cytometry,and cell ultrastrueture by transmitting electron microscopy (TEM).Results The treatment efficacy was statistically comparable on day 7 (66.7% vs 66.7%,P>0.05) but appeared better in the therapy group than that in the control group at 3 months (94.4% vs 66.7%,P<0.05).On day 7 of treatment,the therapy group showed an increase in peripheral EPCs percentage counts compared with baseline (0.64±0.05 vs 0.42±0.02,P<0.05) and with the control group (0.64±0.05 vs 0.42±0.03,P<0.05),while the control group did not show any change in EPCs count (0.41±0.03 vs 0.42±0.03,P>0.05).The absorbance (A value),NO level and number of lumen-like structure were greater in all experimental EPCs groups than those in the control group (P<0.05). Moreover,there was an increasing trend for A value and NO level along with higher level of DF-521 (r=0.978 and 0.981,both P<0.05). Incubation with 4×10-2U/ml DF-521 was associated with the greatest number of lumen-like structure identified. Compared with the control group,the expressions of CD31 and vWF were higher in all experimental groups (P<0.05) but appeared comparable between each other (P>0.05). TEM study found endothelium-specific Weibel Palade body in the group treated with 8×10-2 U/ml DF-521. Conclusion Treatment with DF-521 may significantly improve the prognosis of LEDVT,increase the peripheral EPCs count,increase the in vitro activities and capacities of EPCs,and promote differentiation of EPCs to endothelial cells.