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课件

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,郭连瑞,吴英锋,张建,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

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0.5to1L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

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缺血肢体生成新生血管AmgenYamamoto,1996;Powell,2005;Huang,2005；Masuda,2003；Kocher,2001粒细胞集落刺激因子(G-CSF)AmgenYamamo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?

目的G-CSF的预处理能否增加骨髓中单个核细胞的收获量回顾性研究*历史对照Group1Group2*G-CSF预处理

+MNC移植MNC移植回顾性研究*历史对照Group1Group2*G-C入选标准重度间歇跛行静息痛溃疡坏疽入选标准重度间歇跛行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入选标准不适合行介入治疗及外科搭桥手术排除标准控制不佳的糖尿病(HbA1C>6.5g/dl)五年之内的肿瘤患者,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排除标准控制不佳的糖尿病(HbA1C>6.5g/dl)Group1:+G-CSFGroup2Control患者数(肢体数)35(43)103(111)平均年龄71.369.5性别

男

女23125935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)Group1:Group2患者数(肢体数)35(43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课件G-CSF预处理300µg/d，IH,Qd,for2days白细胞升至23(15~38)×109/L预防心梗及脑血管意外LMWH，40-60mg,IH,BidAspirin，100mg,QdG-CSF预处理300µg/d，IH,Qd,for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（Percol1.077,400g,20min）

采集骨髓单个核细胞局麻BM-MNC移植Gp1Gp2IM13(19)67(71)IA16(16)28(30)IM+IA6(8)8(10)IMIABM-MNC移植Gp1Gp2IM13(19)67(71)I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,etal.ChinJClinRehab,2004,8(35)：7970-2.;Guo,etal.ChinJClinRehab,2005,9(10):57-9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结果

BM-MNC收获量每毫升骨髓的单个核细胞收获量提高10.8倍VolofBMNo.ofBM-MNCsPGp1196±17ml5.6±2.1X109<0.001Gp2377±39ml1.0±0.9X109结果

BM-MNC收获量每毫升骨髓的单个核细胞收获量提高1主观症状的改善

移植后2月

-

主观症状--疼痛,冷感和麻木感

(P=0.011)95%84%完全缓解改善总体改善主观症状的改善

移植后2月

-主观症状--疼痛,冷TcPO2

术后2月89%)92%)P>0.05TCPO2增加例数

得到随访病人数%Gp1363992%Gp2809089%TcPO2

术后2月89%)92%)P>0.05TCPO2踝肱指数增加>0.1

术后2月(P>0.05).45%38%踝肱指数增加>0.1

术后2月(P>0.05).45%3结果（1）---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BM-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)结果（1）---并发症与使用G-CSF相关: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%P<0.05保肢情况---2月后

-2.3%12.6%截肢数结果（7）间歇跛行距离---术后2月

术前术后<100m100-500m>500mGpI(n=5)5(5)14Gp2(n=10)10(13)3(4)7(9)跛行距离测定:

---常速---在病房的楼道结果（7）间歇跛行距离---术后2月

术前术后<100m1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%P<0.001结果（8）---血管造影（术后4-8周）两组间有显著性差异术前术后术前术后术前8周后术前8周后（G-CSF动员组）术后（G-CSF动员组）术后Gp1(35)Gp2(103)获随访数32(40肢体)71(72肢体)死亡率012.7%症状消失30%

改善53%

总体:83%.消失31%改善33%

总体:64%ABI增加>0.148%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Gp1(35)Gp2(103)获随访数32(40肢体)7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.结论G-CSF能明显增加的收获量.谢谢！谢谢！GranulocyteColony-stimulatingfactorcanenhancetheangiogenic

effect

ofbonemarrowmononuclearcells

treatinglimbischemia

GuoLR,GuYQ,WuYF,ZhangJ,

Wang

ZG*VascularSurgeryDepartmentofXuanwuHospital;CapitalMedicalUniversity，Beijing，ChinaGranulocyteColony-stimulating

ClinicalTrials

ofBM-MNC

ToTreatLowerlimbIschemiaFavorablereportsBut:Typicallytheharvestrequires0.5to1literNeedgeneralanesthesiaRisktoelderlypatientsTheMNCyieldisonlyaboutabillioncellsTateishi-Yuyama,2002;Gu,2003;Huang,2004;Miyamoto,2004;Higashi,2004

ClinicalTrialsofBM-MNC

ToGranulocyteColonyStimulatingFactor(G-CSF)CytokinethatstimulatesthebonemarrowInducesendothelialcellstoproliferateandmigrateReportedtoenhanceneo-vasculogenesisininfractedheartsandischemiclimbs.AmgenYamamoto,1996;Powell,2005;Huang,2005；Masuda,2003；Kocher,2001GranulocyteColonyStimulatingObjectiveCanpre-treatmentwithG-CSFenhancetheharvest

ofBM-MNC，thendecreasetheBMharvest,butenhancetheangiogenicefficacytotreatlowerlimbischemia?

ObjectiveCanpre-treatmentwitRetrospectiveStudy*historicalcontrolGroup1Group2*G-CSF

+MNCMNCRetrospectiveStudy*historicaInclusioncriteria

SevereclaudicationRestpainNon-healingulcerGangreneInclusioncriteriaSevereclauPatientswerenotcandidatesforendovascularorvascularsurgeryduotolackofeithersuitableconduitlackofsuitableanastomotictargetsPoormedicalcondition

PatientswerenotcandidatesfExclusioncriteriaPoorly-controlleddiabetes(HbA1C>6.5g/dl)Confirmedmalignancyduringthepast5years,orelevatedserumtumormarkerspreoperatively(CEA,PSA,CA-153)AnginaEndstageorganfailure(heart,liver&kidney)SepsisLimbnecrosisrequiringamputationindependentofbloodflowExclusioncriteriaPoorly-contrGroup1:+G-CSFGroup2ControlNo.ofpatients(limbs)35(43)103(111)MeanAge71.369.5GenderMaleFemale23125935IschemicStatusofLimbsClaudicationRestPainIschemicUlcerGangrene5(5)15(19)9(12)6(7)12(13)40(43)26(28)25(27)Co-morbidityDiabetesNon-DiabeticThromboangiitisObliterans30(38)2(2)3(3)88(94)8(9)7(8)Group1:Group2No.ofpatient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课件G-CSFpre-treatment300µg/dIH,Qd,for2daysWBCincreasedto23(15~38)×109/LPrecautionagainstMI,strokeLMWH40-60mg,IH,BidAspirin100mg,QdG-CSFpre-treatment300µg/dIH,HarvestofBM-MNCslocalanesthesia(1%lidocaine)Aspirationofbone-marrowfromtheposteriorsuperioriliaccrestremovebonespiculesanderythrocytesdensitygradientcentrifugation（Percol1.077,400g,20min）

HarvestofBM-MNCslocalanestBM-MNCImplantationGp1Gp2IM13(19)67(71)IA16(16)28(30)IM+IA6(8)8(10)IMIABM-MNCImplantationGp1Gp2IM13(DeliveryroutesNosignificantdifferenceintherapeuticeffecthasbeenreportedbetweenthedeliveryroutes.Gu,etal.ChinJClinRehab,2004,8(35)：7970-2.;Guo,etal.ChinJClinRehab,2005,9(10):57-9DeliveryroutesNosignificantResults

BM-MNCHarvested10.8fold

increase

inMNCwhencorrectedforeachmlofBMharvested

VolofBMNo.ofBM-MNCsPGp1196±17ml5.6±2.1X109<0.001Gp2377±39ml1.0±0.9X109Results

BM-MNCHarvested10.8fImprovementof

SubjectiveSymptoms

2MonthsPost-op

-

Subjectivesymptoms--Pain,coolnessandnumbness(P=0.011)95%84%ImprovementofSubjectiveSympTcPO2

2MonthsPost-op89%)92%)P>0.05No.ofincreaseNo.offollow-upIncreaserateGp1363992%Gp2809089%TcPO2

2MonthsPost-op89%)92%AnkleBrachialIndexIncrease>0.1

2MonthsPost-op

(P>0.05).45%38%AnkleBrachialIndexIncreaseResults（1）

-ComplicationsofG-CSFadministration:mildfever：1patienttransientlassitude：2patientsAllofthemrecoveredwithoutintervention.followingBM-MNCimplantation:

myocardialinfarction：1inGp1(1weekpost-op)diedofheartfailure:2ptsInGp2:(2and7weekspost-op)Results（1）

-ComplicationsofLimbSalvage

2monthspost-op

-2.3%12.6%No.ofAmputationNo.offollow-upAmputationrateGp11432.3%Gp21411112.6%P<0.05LimbSalvage

2monthspost-opResults（7）at2monpost-op

Improvementofpain-freewalkingdistanceofclaudicants

Pre-opPost-op<100m100-500m>500mGpI(n=5)5(5)14Gp2(n=10)10(13)3(4)7(9)Assessmentofpain-freewalkingdistance:

---aconstantspeed---onthesamecorridorinourwardResults（7）at2monpost-opImResults（8）at4-8wkspost-op

--AngiographyTherewassignificantdifferencebetweenthetwogroups.CollateralincreaseslightmoderaterichrateGp1(34)6121391%Gp2(49)6101455%91%55%P<0.001Results（8）at4-8wkspost-op

Pre-opPost-opPre-opPost-opBeforeoperation8weeksafteroperationBeforeoperation8weeksafterApatientinGroup1（withG-CSFpre-treatment）8weekspost-opApatientinGroup18weekspGp1(35)Gp2(103)No.offollow-up32pts(40limbs)71pts(72limbs)Mortalityrate012.7%Symptomsresolved30%improved53%total:83%.resolved31%improved33%total:64%ABIincreased>0.148%60%TcPO2increased73%76%Amputationrate10%24%Angiography(6monpost-op)91%(19/21)74%(20/27)One-yearfollow-upGp1(35)Gp2(103)No.offollow-ConclusionG-CSFincreasedtheyieldofBM-MNC10.8foldpermlofbonemarrowharvested.TheangiogenicbenefitcouldbeenhanceddespitethesmallervolumeofBMharvestedafterG-CSFpre-treatment.Aprospective,randomized,controlledstudyshouldbestronglyrecommended.ConclusionG-CSFincreasedtheThankyouforyourattention！Thankyou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,郭连瑞,吴英锋,张建,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

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0.5to1L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

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缺血肢体生成新生血管AmgenYamamoto,1996;Powell,2005;Huang,2005；Masuda,2003；Kocher,2001粒细胞集落刺激因子(G-CSF)AmgenYamamo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?

目的G-CSF的预处理能否增加骨髓中单个核细胞的收获量回顾性研究*历史对照Group1Group2*G-CSF预处理

+MNC移植MNC移植回顾性研究*历史对照Group1Group2*G-C入选标准重度间歇跛行静息痛溃疡坏疽入选标准重度间歇跛行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入选标准不适合行介入治疗及外科搭桥手术排除标准控制不佳的糖尿病(HbA1C>6.5g/dl)五年之内的肿瘤患者,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排除标准控制不佳的糖尿病(HbA1C>6.5g/dl)Group1:+G-CSFGroup2Control患者数(肢体数)35(43)103(111)平均年龄71.369.5性别

男

女23125935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)Group1:Group2患者数(肢体数)35(43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课件G-CSF预处理300µg/d，IH,Qd,for2days白细胞升至23(15~38)×109/L预防心梗及脑血管意外LMWH，40-60mg,IH,BidAspirin，100mg,QdG-CSF预处理300µg/d，IH,Qd,for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（Percol1.077,400g,20min）

采集骨髓单个核细胞局麻BM-MNC移植Gp1Gp2IM13(19)67(71)IA16(16)28(30)IM+IA6(8)8(10)IMIABM-MNC移植Gp1Gp2IM13(19)67(71)I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,etal.ChinJClinRehab,2004,8(35)：7970-2.;Guo,etal.ChinJClinRehab,2005,9(10):57-9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结果

BM-MNC收获量每毫升骨髓的单个核细胞收获量提高10.8倍VolofBMNo.ofBM-MNCsPGp1196±17ml5.6±2.1X109<0.001Gp2377±39ml1.0±0.9X109结果

BM-MNC收获量每毫升骨髓的单个核细胞收获量提高1主观症状的改善

移植后2月

-

主观症状--疼痛,冷感和麻木感

(P=0.011)95%84%完全缓解改善总体改善主观症状的改善

移植后2月

-主观症状--疼痛,冷TcPO2

术后2月89%)92%)P>0.05TCPO2增加例数

得到随访病人数%Gp1363992%Gp2809089%TcPO2

术后2月89%)92%)P>0.05TCPO2踝肱指数增加>0.1

术后2月(P>0.05).45%38%踝肱指数增加>0.1

术后2月(P>0.05).45%3结果（1）---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BM-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)结果（1）---并发症与使用G-CSF相关: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%P<0.05保肢情况---2月后

-2.3%12.6%截肢数结果（7）间歇跛行距离---术后2月

术前术后<100m100-500m>500mGpI(n=5)5(5)14Gp2(n=10)10(13)3(4)7(9)跛行距离测定:

---常速---在病房的楼道结果（7）间歇跛行距离---术后2月

术前术后<100m1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%P<0.001结果（8）---血管造影（术后4-8周）两组间有显著性差异术前术后术前术后术前8周后术前8周后（G-CSF动员组）术后（G-CSF动员组）术后Gp1(35)Gp2(103)获随访数32(40肢体)71(72肢体)死亡率012.7%症状消失30%

改善53%

总体:83%.消失31%改善33%

总体:64%ABI增加>0.148%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Gp1(35)Gp2(103)获随访数32(40肢体)7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.结论G-CSF能明显增加的收获量.谢谢！谢谢！GranulocyteColony-stimulatingfactorcanenhancetheangiogenic

effect

ofbonemarrowmononuclearcells

treatinglimbischemia

GuoLR,GuYQ,WuYF,ZhangJ,

Wang

ZG*VascularSurgeryDepartmentofXuanwuHospital;CapitalMedicalUniversity，Beijing，ChinaGranulocyteColony-stimulating

ClinicalTrials

ofBM-MNC

ToTreatLowerlimbIschemiaFavorablereportsBut:Typicallytheharvestrequires0.5to1literNeedgeneralanesthesiaRisktoelderlypatientsTheMNCyieldisonlyaboutabillioncellsTateishi-Yuyama,2002;Gu,2003;Huang,2004;Miyamoto,2004;Higashi,2004

ClinicalTrialsofBM-MNC

ToGranulocyteColonyStimulatingFactor(G-CSF)CytokinethatstimulatesthebonemarrowInducesendothelialcellstoproliferateandmigrateReportedtoenhanceneo-vasculogenesisininfractedheartsandischemiclimbs.AmgenYamamoto,1996;Powell,2005;Huang,2005；Masuda,2003；Kocher,2001GranulocyteColonyStimulatingObjectiveCanpre-treatmentwithG-CSFenhancetheharvest

ofBM-MNC，thendecreasetheBMharvest,butenhancetheangiogenicefficacytotreatlowerlimbischemia?

ObjectiveCanpre-treatmentwitRetrospectiveStudy*historicalcontrolGroup1Group2*G-CSF

+MNCMNCRetrospectiveStudy*historicaInclusioncriteria

SevereclaudicationRestpainNon-healingulcerGangreneInclusioncriteriaSevereclauPatientswerenotcandidatesforendovascularorvascularsurgeryduotolackofeithersuitableconduitlackofsuitableanastomotictargetsPoormedicalcondition

PatientswerenotcandidatesfExclusioncriteriaPoorly-controlleddiabetes(HbA1C>6.5g/dl)Confirmedmalignancyduringthepast5years,orelevatedserumtumormarkerspreoperatively(CEA,PSA,CA-153)AnginaEndstageorganfailure(heart,liver&kidney)SepsisLimbnecrosisrequiringamputationindependentofbloodflowExclusioncriteriaPoorly-contrGroup1:+G-CSFGroup2ControlNo.ofpatients(limbs)35(43)103(111)MeanAge71.369.5GenderMaleFemale23125935IschemicStatusofLimbsClaudicationRestPainIschemicUlcerGangrene5(5)15(19)9(12)6(7)12(13)40(43)26(28)25(27)Co-morbidityDiabetesNon-DiabeticThromboangiitisObliterans30(38)2(2)3(3)88(94)8(9)7(8)Group1:Group2No.ofpatient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课件G-CSFpre-treatment300µg/dIH,Qd,for2daysWBCincreasedto23(15~38)×109/LPrecautionagainstMI,strokeLMWH40-60mg,IH,BidAspirin100mg,QdG-CSFpre-treatment300µg/dIH,HarvestofBM-MNCslocalanesthesia(1%lidocaine)Aspirationofbone-marrowfromtheposteriorsuperioriliaccrestremovebonespiculesanderythrocytesdensitygradientcentrifugation（Percol1.077,400g,20min）

HarvestofBM-MNCslocalanestBM-MNCImplantationGp1Gp2IM13(19)67(71)IA16(16)28(30)IM+IA6(8)8(10)IMIABM-MNCImplantationGp1Gp2IM13(DeliveryroutesNosignificantdifferenceintherapeuticeffecthasbeenreportedbetweenthedeliveryroutes.Gu,etal.ChinJClinRehab,2004,8(35)：7970-2.;Guo,etal.ChinJClinRehab,2005,9(10):57-9DeliveryroutesNosignificantResults

BM-MNCHarvested10.8fold

increase

inMNCwhencorrectedforeachmlofBMharvested

VolofBMNo.ofBM-MNCsPGp1196±17ml5.6±2.1X109<0.001Gp2377±39ml1.0±0.9X109Results

BM-MNCHarvested10.8fImprovementof

SubjectiveSymptoms

2MonthsPost-op

-

Subjectivesymptoms--Pain,coolnessandnumbness(P=0.011)95%84%ImprovementofSubjectiveSympTcPO2

2MonthsPost-op89%)92%)P>0.05No.ofincreaseNo.offollow-upIncreaserateGp1363992%Gp2809089%TcPO2

2MonthsPost-op89%)92%AnkleBrachialIndexIncrease>0.1

2MonthsPost-op

(P>0.05).45%38%AnkleBrachialIndexIncreaseResults（1）

-ComplicationsofG-CSFadministration:mildfever：1patienttransientlassitude：2patientsAllofthemrecoveredwithoutintervent