1、粒细胞集落刺激因子能增强骨髓源单个核细胞治疗下肢缺血的作用谷涌泉谷涌泉, 郭连瑞郭连瑞,吴英锋吴英锋, 张建张建,汪忠镐汪忠镐 首都医科大学宣武医院血管外科首都医科大学血管外科研究所 骨髓单个核细胞治疗下肢缺血的临床试验 有利报道越来越多 但是 骨髓采集量过大：达 0.5 to 1 L 需要全麻 对高龄患者仍很危险 单个核细胞的采集量只有109Tateishi-Yuyama, 2002 ; Gu, 2003 ;Huang, 2004 ;Miyamoto, 2004 ;Higashi, 2004粒细胞集落刺激因子 (G-CSF) 是一种细胞因子 能诱导内皮细胞增生和迁移 全身给药能促进心梗区域和
2、缺血肢体生成新生血管Amgen Yamamoto, 1996; Powell, 2005; Huang, 2005； Masuda, 2003 ；Kocher, 2001 目 的 G-CSF的预处理能否增加骨髓中单个核细胞的收获量, 进而减少采髓量，但却增强其肢体血管新生效应? 回顾性研究* 历史对照Group 1Group 2 *G-CSF预处理预处理 + MNC移植MNC移植入选标准 重度间歇跛行 静息痛 溃疡 坏疽入选标准不适合行介入治疗及外科搭桥手术 因为 缺乏流出道或缺乏移植物 介入或外科治疗失败者 一般状况差排除标准 控制不佳的糖尿病(HbA1C6.5g/dl) 五年之内的肿瘤患者
3、, 或术前肿瘤标记物明显升高者(CEA,PSA,CA-153) 心绞痛 器官功能衰竭(心、肝、肾) 脓毒血症 肢体大范围坏疽难以保肢者Group 1: +G-CSFGroup 2 Control患者数(肢体数)35 (43)103 (111)平均年龄71.369.5性别 男 女2312 5935 缺血状态 间歇跛行 静息痛 溃疡 坏疽5 (5)15(19)9 (12)6 (7) 12(13)40(43)26(28)25(27) 病因 糖尿病 非糖尿病 血栓闭塞性脉管炎30 (38)2 (2)3(3) 88 (94)8 (9)7 (8) G-CSF 预处理预处理 300g/d， IH, Qd,
4、for 2 days 白细胞升至23 (15 38) 109 /L 预防心梗及脑血管意外 LMWH ，40-60 mg ,IH, Bid Aspirin ，100 mg ,Qd 采集骨髓单个核细胞采集骨髓单个核细胞 局麻 髂后上棘抽取骨髓 取出骨渣及红细胞 密度梯度离心法（Percol 1.077, 400g, 20min） BM-MNC 移植移植Gp1Gp2IM13(19)67(71)IA16(16)28(30)IM+IA 6 (8) 8 (10)I MI A移植途径 我们既往的研究发现肌注法和动脉腔内移植法两种移植途径的治疗效果没有明显差异。Gu,et al. Chin J Clin Re
5、hab,2004, 8 (35)：7970-2.; Guo,et al. Chin J Clin Rehab, 2005, 9(10):57-9 结果结果BM-MNC 收获量收获量每毫升骨髓的单个核细胞收获量提高10.8倍Vol of BMNo. of BM- MNCsPGp1196 17 ml5.6 2.1 X1090.05TCPO2增加例数 得到随访病人数%Gp1363992%Gp2809089%踝肱指数增加踝肱指数增加0.1 术后术后2月月(P 0.05).45%38%结果（结果（1）- 并发症并发症与使用 G-CSF 相关: 轻度发热：1 例 疲乏无力：2 例 均未经处理而自行康复 B
6、M-MNC 移植后: 心梗：1 例在动员组(术后1周) 死于心衰: 2 例均在未动员组: (分别于术后第2周和第7周) 保肢情况-2月后 -2.3%12.6 %截肢数 随访例数截肢率Gp11432.3%Gp21411112.6%P0.05结果（7） 间歇跛行距离-术后2月术前术前术后术后500mGp I (n=5) 5 (5) 1 4Gp 2(n=10) 10(13) 3(4) 7(9)跛行距离测定: - 常速 -在病房的楼道结果（8） -血管造影（术后4-8周）两组间有显著性差异.侧枝血管增多轻度较多丰富比率Gp1(34)6121391%Gp2(49)6101455%91%55%P0.1 4
7、8%60%TcPO2 增加73%76%截肢率10%24%血管造影(术后6 月)91% ( 19 /21) 74% (20 /27)1年随访结论结论 G-CSF 能明显增加的收获量. G-CSF预处理能减少骨髓采集量，并增强BM-MNC的促血管新生作用. 需要进行多中心前瞻性随机对照研究证实. 谢谢！Granulocyte Colony-stimulating factor can enhance the angiogenic effect of bone marrow mononuclear cells treating limb ischemiaGuo LR, Gu YQ, Wu YF, Z
8、hang J, Wang ZG * Vascular Surgery Department of Xuanwu Hospital; Capital Medical University，Beijing， China Clinical Trials of BM-MNCTo Treat Lower limb Ischemia Favorable reports But: Typically the harvest requires 0.5 to 1 liter Need general anesthesia Risk to elderly patients The MNC yield is onl
9、y about a billion cellsTateishi-Yuyama, 2002 ; Gu, 2003 ;Huang, 2004 ;Miyamoto, 2004 ;Higashi, 2004Granulocyte Colony Stimulating Factor (G-CSF) Cytokine that stimulates the bone marrow Induces endothelial cells to proliferate and migrate Reported to enhance neo-vasculogenesis in infracted hearts an
10、d ischemic limbs. Amgen Yamamoto, 1996; Powell, 2005; Huang, 2005； Masuda, 2003 ；Kocher, 2001 Objective Can pre-treatment with G-CSF enhance the harvest of BM-MNC，then decrease the BM harvest, but enhance the angiogenic efficacy to treat lower limb ischemia? Retrospective Study* historical controlGr
11、oup 1Group 2 *G-CSF + MNCMNCInclusion criteria Severe claudication Rest pain Non-healing ulcer Gangrene Patients were not candidates for endovascular or vascular surgery duo to lack of either suitable conduit lack of suitable anastomotic targets Poor medical condition Exclusion criteria Poorly-contr
12、olled diabetes (HbA1C6.5g/dl) Confirmed malignancy during the past 5 years, or elevated serum tumor markers preoperatively (CEA,PSA,CA-153) Angina End stage organ failure (heart, liver& kidney) Sepsis Limb necrosis requiring amputation independent of blood flowGroup 1: +G-CSFGroup 2 ControlNo. of pa
13、tients (limbs)35 (43)103 (111)Mean Age71.369.5Gender Male Female2312 5935 Ischemic Status of Limbs Claudication Rest Pain Ischemic Ulcer Gangrene 5 (5)15(19)9 (12)6 (7) 12(13)40(43)26(28)25(27) Co-morbidity Diabetes Non-Diabetic Thromboangiitis Obliterans 30 (38)2 (2)3(3) 88 (94)8 (9)7 (8) G-CSF pre
14、-treatment 300g/d IH, Qd, for 2 days WBC increased to 23 (15 38) 109 /L Precaution against MI, stroke LMWH 40-60 mg ,IH, Bid Aspirin 100 mg ,Qd Harvest of BM- MNCs local anesthesia (1% lidocaine ) Aspiration of bone-marrow from the posterior superior iliac crest remove bone spicules and erythrocytes
15、 density gradient centrifugation（Percol 1.077, 400g, 20min） BM-MNC ImplantationGp1Gp2IM13(19)67(71)IA16(16)28(30)IM+IA 6 (8) 8 (10)I MI ADelivery routesNo significant difference in therapeutic effect has been reported between the delivery routes. Gu,et al. Chin J Clin Rehab,2004, 8 (35)：7970-2.; Guo
16、,et al. Chin J Clin Rehab, 2005, 9(10):57-9 ResultsBM-MNC Harvested10.8 fold increase in MNC when corrected for each ml of BM harvested Vol of BMNo. of BM- MNCsPGp1196 17 ml5.6 2.1 X1090.05No. of increase No. of follow-upIncreaserateGp1363992%Gp2809089%Ankle Brachial Index Increase 0.1 2 Months Post
17、-op (P 0.05).45%38%Results（1） - Complications of G-CSF administration: mild fever ：1 patient transient lassitude ：2 patients All of them recovered without intervention. following BM-MNC implantation: myocardial infarction：1 in Gp1 (1 week post-op) died of heart failure: 2 pts In Gp 2: (2 and 7 weeks
18、 post-op) Limb Salvage 2 months post-op -2.3%12.6 %No. of Amputation No. of follow-upAmputation rateGp11432.3%Gp21411112.6%P0.05Results（7） at 2 mon post-op Improvement of pain-free walking distance of claudicantsPre-opPost-op500mGp I (n=5) 5 (5) 1 4Gp 2(n=10) 10(13) 3(4) 7(9)Assessment of pain-free
19、walking distance: - a constant speed -on the same corridor in our wardResults（8） at 4-8 wks post-op -AngiographyThere was significant difference between the two groups.Collateral increaseslightmoderaterichrateGp1(34)6121391%Gp2(49)6101455%91%55%P0.1 48%60%TcPO2 increased73%76%Amputation rate 10%24%A
20、ngiography(6 mon post-op)91% ( 19 /21) 74% (20 /27)One-year follow-upConclusion G-CSF increased the yield of BM-MNC 10.8 fold per ml of bone marrow harvested. The angiogenic benefit could be enhanced despite the smaller volume of BM harvested after G-CSF pre-treatment. A prospective, randomized, controlled study should be strongly recommended. Thank you for your attention！
