内皮化小口径人工血管的研究

目的 探讨成人大隐静脉内皮细胞(HSVECs)种植到人工血管内表面的可行性.方法 酶消化法获取成人大隐静脉内皮细胞,在体外扩增培养13～15 d,将扩增培养的内皮细胞种植于纤维蛋白胶预衬的聚四氟乙烯(ePTFE)人工血管内表面,继续体外培养9～12 d.在不同的时间点,分别剪取部分内皮化的人工血管,行荧光显微镜和扫描电镜检查.结果 内皮细胞种植于人工血管后,扫描电镜下可见细胞在血管表面黏附、生长、增殖.平均孵育12 d后,人工血管腔面见一层均匀的基质,其表面有内皮细胞单层,内皮细胞排列紧密,呈梭形.结论 成人大隐静脉内皮细胞可以种植到人工血管,在体外增殖形成内皮细胞单层,达到内皮化的效果.     

纯化自体外周血CD34+细胞移植治疗下肢重度缺血

目的 探讨纯化自体外周血CD34+细胞移植治疗下肢重度缺血的安全性、可行性和有效性.方法 2009年5月至2010年3月采用纯化自体外周血CD34+细胞移植治疗下肢重度缺血7例,其中血栓闭塞性脉管炎6例,结节性红斑伴血栓形成1例,年龄23～54岁,平均(39±11)岁;均不具备血管重建条件.经G-CSF动员后第5天采集外周血单个核细胞,分选获得纯化CD34+细胞,下肢肌肉局部注射,观察不良反应和缺血缓解情况.结果 7例患者均获技术和保肢成功,移植细胞数(7.1±2.3)×105/kg[(4.6×105～1×106)/kg].7例患者均获随访,随访时间6～14个月,平均(8±3)个月.术后1个月静息痛均明显缓解,Wong-Baker FACES疼痛评分由术前平均7.1±2.0(4～10)降至1.1±1.1(0～2),P=0.0000.无痛步行时间术前平均(4±4)min(1～10 min),术后3个月延长至(12±7)min(5～21 min),P=0.04,术后6个月(20±12)min(6～40 min),P=0.02.术前踝肱指数0.54±0.18(0.41～0.87),术后3个月提高至0.66±0.13(0.52～0.86),P=0.17,术后6个月0.72±0.13(0.56～0.91),P=0.07.6例溃疡中,3例直径＜2 cm者完全愈合,另3例直径＞2 cm者明显缩小.经皮氧分压术前(29±14)mmHg(10～52 mm Hg),术后3个月(46±14)mm Hg(27～63 mm Hg),P=0.04,术后6个月(57±10)mm Hg(41～66 mm Hg),P=0.001.无严重不良反应.结论 初步结果显示纯化自体外周血CD34+细胞移植治疗下肢重度缺血安全,可行,有效.

Abstract：

Objective To evaluate the safety, feasibility and efficacy of transplantation of purified peripheral blood CD34+ cells in treatment of critical ischemia of the lower extremities. Methods From May 2009 to March 2010, seven cases of critical ischemia of the lower extremities received purified peripheral blood CD34+ cells transplantation, among those 6 were caused by thromboangiitis obliterans and 1 by thrombosis coexistent with nodular erythema. Mean age was ( 39 ± 11 ) years ( range 23 - 54 ), and all patients were not suitable for surgical or endovascular revascularization. G-CSF was subcutaneously injected for 5 days before apheresis for peripheral blood mononuclear cells. Then CliniMACS system was used to isolate the CD34+ cells. If the number of CD34+ cells was between 105/kg and 106/kg , they were all intramuscular injected into patients' calf and foot. Results Technical success and limb salvage were achieved in all cases. The mean number of transplanted cells was (7. 1 ±2.3) × 105/kg [ range(4.6 ×105 -1 × 106 )/kg]. All cases were followed-up, ranging from 6 - 14 months (mean 8 ± 3 months). One month after transplantation, the rest pain was obviously relieved in all cases, and the Wong-Baker FACES pain rating scale score significantly decreased from 7. 1 ±2. 0(4 - 10)to 1. 1 ± 1.1 (0 -2) ,P =0. 0000. The pain-free walking distance was significantly improved from (4 ± 4) min (range 1 -10 min)to (12 ± 7 ) min (range 5 - 21min , P =0.04) at 3 months and(20.4 ± 12.5) min(range 6 -40 min, P = 0.02) at 6 months, respectively. The ankle-brachial index increased from 0. 54 ± 0. 18 ( range 0. 41 - 0. 87 ) to 0.66 ±0. 13(range 0. 52-0. 86 , P=0. 17)at 3 months and 0.72 ±0. 13(range 0.56 -0.91, P=0. 07)at 6 months, respectively. Of 6 cases with the toe ulcer, the ulcer was healed in 3 and apparently shrank in 3. Transcutaneous partial oxygen pressure rose from (29 ± 14) mm Hg(range 10 -52 mm Hg)to 46 ±14 mm Hg ( range 27 - 63 mm Hg, P = 0. 04) at 3 months and (57 ± 10) mm Hg( range 41 - 66 mm Hg, P =0.001) at 6 months, respectively. No serious complications were found either perioperatively or postoperatively. Conclusions Transplantation of purified peripheral blood CD34+ cells is safe, feasible and effective in the treatment of critical ischemia of the lower extremities.     

聚己内酯-碳酸亚乙酯［Poly(CL-EC)］和血管内皮生长因子(VEGF)静电混纺支架的构建及其生物学性能

 目的  以聚己内酯-碳酸亚乙酯［Poly(CL-EC)］共聚物混合血管内皮生长因子(vascular endothelial growth factor,VEGF),采用静电纺的方法构建纳米支架并检测其生物学性能。方法  按照EC/CL共聚物比例为1∶9、1∶6、1∶4,Poly(CL-EC)浓度分别为5%、10%、15%电纺纤维膜,分析电纺纤维膜的表征和力学性能。然后将VEGF按照0 ng/g、10 ng/g、100 ng/g、1 μg/g的质量比与Poly (CL-EC)溶液混合,电纺制备纳米支架。对混纺膜进行细胞增殖和黏附试验、乳酸脱氢酶(lactate dehydrogenase,LDH)释放试验、间接溶血试验和皮下植入试验等检测。结果  根据Poly (CL-EC)电纺纤维膜的表征和力学性能,我们选用EC/CL比例为1∶6的10% Poly(CL-EC)与VEGF构建混合电纺膜。细胞增殖和黏附试验证实Poly(CL-EC)/VEGF电纺膜具有良好的细胞相容性,尤其是血管内皮细胞;LDH释放试验、接触溶血试验和体内植入试验显示该材料无细胞毒性、有较好的血液相容性和很低的异物反应。结论  静电纺构建的Poly(CL-EC)/VEGF具有良好的生物学性能,能够作为组织工程支架材料。     

同步辐射成像技术对肢体微血管相位衬度成像的实验研究

目的 应用同步辐射光源类同轴相位成像技术检测大鼠后肢肌肉的微血管.方法 取SD大鼠8只分别用生理盐水和硫酸钡对后肢动脉进行灌注,对不同厚度的标本进行普通X线和同步辐射相位衬度成像.另取1只大鼠,在不灌注对比剂的情况下检测肝脏、肺叶和肢体的血管.结果 未灌注对比剂的肝脏和灌注生理盐水或硫酸钡的后肢可见血管显影.和普通X线成像比较,灌注硫酸钡的后肢标本可显示更清晰的血管树,可显示的最小血管直径为9 μm.结论 同步辐射成像技术有助于检测肢体微血管或新生血管.     

内皮祖细胞促动脉内皮损伤修复的研究

目的评价骨髓源性内皮祖细胞(progenitor endothelial cells,EPCs)移植促动脉损伤内皮修复的效果。方法将菲立磁(superparam agnetic iron oxide,SPIO)和绿色荧光蛋白(green fluorescent protein,GFP)双标记的EPC局部注入损伤颈动脉段腔内。术后1d、7d和28d行磁共振(magnetic resonance imaging,MRI)扫描、组织学检查及内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)检测。结果MRI扫描发现移植部位出现低信号区。移植组HE、普鲁士蓝、vWF免疫组化染色、GFP荧光、偶氮蓝(Evans blue)染色均提示EPC可粘附于内膜损伤处,并随时间分化为内皮细胞;同时eNOS表达随时间变化而增多。结论菲立磁标记内皮祖细胞可促进动脉损伤内皮的修复,并可为MRI示踪。     

实验犬浅静脉内皮细胞原位获取及体外培养

背景:自体血管内皮细胞数量有限且体外长期培养传代后存在功能老化,是目前制约组织工程血管发展的问题.目的:实验拟验证通过体外分离和大规模培养方式为组织工程动脉支架体外内皮化提供血管内皮细胞的可行性.设计、时间及地点:重复测量设计,实验于2007-03/12在复旦大学附属中山医院中心实验室完成.材料:健康杂种犬6只,雌雄不拘,体质量35～40kg,用于获取血管内皮细胞.方法:将犬麻醉后前肢隐静脉原位插管,采用Ⅰ型胶原酶消化法获取血管内皮细胞,体外培养、传代并大规模扩增.采用微格法每日计数培养的细胞总数.主要观察指标:倒置显微镜观察细胞生长情况,透射电镜和细胞免疫化学进行细胞鉴定,检测原代和传代细胞的条件培养液中6-酮-前列腺素F1°和Ⅷ因子相关抗原的含量.结果:倒置显微镜下观察静脉内皮细胞呈典型的"纺锤样"梭形细胞,单层细胞贴壁生长至融合时呈铺路石样排列.透射电镜下见内皮细胞胞浆中特征性Weibel-Palade小体.细胞免疫化学显示血管内皮细胞Ⅷ因子相关抗原抗体染色阳性.原代及传代细胞培养上清液中6-酮-前列腺素F1°和Ⅷ因子相关抗原的含量差异无显著性意义(P＞0.05).结论:实验成功培养、传代并大规模扩增静脉血管内皮细胞.所培养的静脉内皮细胞可以为组织工程动脉支架体外内皮化提供足够数量和功能良好的细胞.