Effect of ligustrazine on hematopoiesis in bone marrow transplantation mice

Hematopoietic reconstitution after bone marrowtransplantation is a complicated process,whichconcerns with the quality and quantity ofhematopoietic stem cell (HSC) ,and also with therecovery of hematopoietic inducing microenvironment(HIM) .The contact of HSC and HIM is critical forhematopoietic reconstitution.1　MATERIALSAND METHODS1 .1　Materials1 .1 .1　 Animals　　 BALB/c mice of both sexes(clean class,close copulatory series (H- 2 d,MLSb) )were 8— 1 0 weeks old,and 1 7— 2 …     

川芎嗪对骨髓移植小鼠造血细胞SDF-1表达水平的影响

为探讨川芎嗪对骨髓移植(BMT)后造血重建过程中基质细胞来源因子(SDF-1)的作用,建立了典型同基因BMT小鼠模型,胃饲川芎嗪注射液每次2 mg,2次/d,分别于BMT后第7、14 d计数外周血细胞骨髓单核细胞(BMNC)并测定骨髓组织切片中SDF-1的表达.结果表明川芎嗪组BMT后第7、14 d外周血细胞计数、BMNC计数及骨髓组织切片中SDF-1表达水平均高于BMT组.提示川芎嗪能在骨髓移植造血重建早期促进骨髓基质细胞表达SDF-1,可能是促进BMT后造血干细胞(HSC)回髓、加速造血重建的机制之一.     

肝细胞生长因子对骨髓移植小鼠造血重建的促进作用

目的 观察肝细胞生长因子（HGF）对骨髓移植小鼠造血重建的促进作用。方法 用BALB／c小鼠建立同基因骨髓移植模型，随机将BALB／c小鼠分A、B两组，B组移植后叶7d皮下注射HGF，A组皮下注射PBS，移植后1、7、14、21和28d检测小鼠血红蛋白、白细胞、血小板、骨髓单个核细胞及骨髓造血细胞面积。结果 B组血红蛋白在移植后7、14d与同期A组无差异（P〉0．05），B组血红蛋白在移植后21d高于同期A组（P〈0．05）；B组血小板在移植后7、14及21d高于同期A组（P值分别小于0．01、0．01和0．05）：B组白细胞在移植后7、14、21及28d高于同期A组（P值分别小于0．01、0．01、0．05及0．05）；B组骨髓单个核细胞及骨髓造血细胞面积在移植后7、14、21和28d高于同期A组（P值分别小于0．05、0．01、0．01及0．05）。结论 HGF对骨髓移植小鼠造血重建有一定的促进作用。     

Effect of Ligustrazine on the Expression of LFA-1, ICAM-1 Following Bone Marrow Transplantation in Mice

Asablood activatingChinesedrug ,ligustrazinecanimprovethemicroenvironmentofbonemarrow ,increasetheadherentfunctionofstromalcellsandpromotethegrowthofhematopoieticcells[1] .Toin terprettherolesthatligustrazineplaysinbonemar rowhematopoieticreconstitutionan…     

Effects of Ligustrazine on Expression of Bone Marrow Heparan Sulfates in Syngeneic Bone Marrow Transplantation Mice

Hematopoietic reconstitution after bone marrowtransplantation (BMT ) is a complicated process,which concerns notonly with the quality and quanti-ty of hematopoietic stem cells(HSC) ,but also withthe recovery of hematopoietic inducing microenviron-ment (HIM) .The contactof HSC and HIM,whichregulates the proliferation,differentiation and self-maintenance of HSC,is critical for hematopoietic re-constitution.Heparan sulfates (HS) are a kind of gly-cosaminoglycans macromolecules composed of…     

Effects of Ligustrazine on Bone Marrow Microvessel Systemin the Early Period of Acute Radiation Injury in Mice

Ithasbeenprovedbymanystudlesthatthedifferent1ationanddevelopmentofthehematopoieticcellsdependonthesup-portandregulationofbonemarr0wmi-croenvironment,inwhichthevascularsys-temisanimp0rtantpart.Kn0speetalre-portedthatafterhigh-doseirradiationde-stroyedthehematopoieticcellsandthemi-crovesselsysteminbonemarrow,there-coveryofvascularsystemwasessentialforthehematopoiesisrehabilitatioin[l.2].There-fore,itisofgreatsignificancetostudytheeffects0fLigustrazine,akind0fs0called"blood-activatingandstasis-el…     

Effects of Platelet Factor 4 on Expression of Bone Marrow Heparan Sulfate in Syngenic Bone Marrow Transplantation Mice

Platelet factor 4 ( PF4) is a negativehematopoietic factor.It can inhibit the prolifera-tion of endothelial cells and hematopoietic stem/progenitor cells,particularly megakaryoryocyticcells,reversibly[1] ,inhibit DNA synthesis,blockcell cycle progression during S phase and reducethe sensibility of normal hematopoietic stem/pro-genitor cells,but not some cancer or leukemia celllines,to cytotoxic drugs and ionizing radia-tion[2 - 3] ,and it also can cause a population in-crease of the stem cel…     

联合应用IL-2和IL-3的基因疗法增强骨髓移植后骨髓细胞毒活性及抗肿瘤效果的实验研究

目的：探讨联合应用成纤维细胞介导ＩＬ２和ＩＬ３的基因疗法对骨髓移植（ＢＭＴ）后荷瘤小鼠抗肿瘤作用的效果及机理。方法：将分别转染ＩＬ２基因及ＩＬ３基因的ＮＩＨ３Ｔ３细胞以单独或联合方式移植至ＢＭＴ的荷瘤小鼠腹腔内８ｄ后，取出小鼠骨髓细胞检测杀伤活性的变化以及ＩＬ２受体的表达，并观察荷瘤小鼠的存活期。结果：ＩＬ３基因治疗虽然可加速ＢＭＴ后造血重建过程，但对骨髓细胞的ＮＫ、ＬＡＫ活性具有一定的抑制作用；ＩＬ２基因治疗可明显提高骨髓细胞的杀伤活性；联合应用基因治疗后荷瘤小鼠骨髓ＮＫ、ＬＡＫ活性及骨髓细胞ＣＤ２５表达升高，明显延长大剂量化疗后接受ＢＭＴ的荷瘤小鼠的存活期。结论：联合应用ＩＬ２和ＩＬ３的基因疗法能协同提高骨髓细胞ＩＬ２受体表达水平，提高骨髓细胞毒活性，增强ＢＭＴ后的抗肿瘤效果。     

移植前输注供体细胞的小鼠单倍体相合骨髓移植实验

目的建立小鼠非清髓性单倍体相合骨髓移植模型,探讨移植前输注供体细胞的造血干细胞植入效果。方法以CB6F1雌性小鼠为受鼠,C57BL/6雄性小鼠为供鼠,移植前3d经尾静脉输注供鼠脾细胞或骨髓细胞3×107,输注细胞后24h和48h分别通过腹腔注射阿糖胞苷0.015g/d,移植前1d予450cGy全身照射（TBI）,移植当天输注供鼠骨髓有核细胞5×107/只。监测受鼠白细胞恢复、Y染色体性别决定基因（SRY）以及外周血供鼠CD3＋细胞嵌合状态。结果单纯给予TBI小鼠均存活,且移植后30d白细胞恢复正常,TBI＋骨髓细胞移植（TBI＋BMT）组移植后14、30、60d时外周血SRY基因PCR检测结果均阳性,60d供体CD3^＋细胞嵌合率达54.4%。移植前输注供体脾细胞组嵌合率最高,移植后60d达93.5%±4.8%,优于移植前输注供体骨髓细胞组（P〈0.05）。结论 450cGy TBI的非清髓性预处理可以成功建立非清髓性单倍体相合骨髓移植模型,移植前输注供体脾细胞可促进造血干细胞植入。     

小鼠不同来源造血细胞移植的实验研究

本文在小鼠不同来源造血干细胞性能研究的基础上,比较了其不同来源造血细胞的移植特性。依据对受体小鼠经致死量照射后的30天存活率观察,表明小鼠同系胎肝移植和骨髓移植的疗效相似;而异系间移植,则胎肝移植疗效优于骨髓移植;在异系间外周血细胞移植,则移植后死亡率明显高于照射对照组。文章同时对小鼠不同来源造血细胞移植疗效差异的机理进行了分析探讨,并认为对今后临床开展造血细胞移植提供了有价值的实验依据。     

Enhancing Effects of Ligustrazine on Expression of CD31 and Hematopoietic Reconstitution in Syngenic Bone Marrow Transplantation of Mice

Summary： The effect of ligustrazine on the expression of CD31 in syngenic bone marrow transplantation （BMT） mice was studied. Fifty-six Balb/c mice were divided into 3 groups： normal control group. BMT control group, and ligustrazine treated group. Syngenic BMT mouse models were established according to the literatures. In BMT control group and the ligustrazine treated group, the mice were given respecxively orally 0.2 mL saline and 2 mg ligustrazine twice a day. On the 7th, 14th, and 21st day after BMT, the mice were killed. The expression of CD31 on the surface of bone marrow nuclear cells （BMNC） was detected by flow cytometry. Peripheral blood leukocytes, platelets and BMNC were counted. Histological observation of bone marrow was made. The results showed thai in ligustrazine treated group the peripheral blood leukocylcs, platelets and BMNC counts, and the expression of CD31 on the day 7, 14, 21 after BMT were higher than in BMTcontrol group （P〈0.01 or P〈0.05）. In conclusion, ligustrazine could obviously enhance the CD31 expression on the surface of BMNC after syngcnic BMT in mice, which may be one of the mecha- nisms underlying the ligustrazine accelerating hematopoietic reconstitution in syngenic BMT.     

NK细胞受体与MHC I类分子匹配程度对骨髓造血重建影响的初步研究

目的：探讨供鼠NK细胞受体与受鼠MHCⅠ类分子匹配程度对骨髓移植小鼠造血重建的影响。方法分离30只C57BL/c小鼠的骨髓细胞和NK细胞,输给经过预处理的BALB/c和CB6F1小鼠,受鼠分为单纯辐照组、输入骨髓细胞组、输入骨髓细胞和NK细胞组,6只/组,观察不同时间点血常规、生存时间、病理组织学等指标变化,进行组间比较。结果输入骨髓细胞、输入骨髓细胞和NK细胞组小鼠均长期存活,未观察到GVHD病理学改变。移植后第14天时,输入骨髓细胞组和输入骨髓细胞和NK细胞组的BALB/c小鼠（MHCⅠ类分子全不相合）外周血WBC分别为（1.35＆#177;0.33）＆#215;109/L和（1.80＆#177;0.18）＆#215;109/L,差异有统计学意义,血小板计数分别为（79＆#177;19）＆#215;109/L和（117＆#177;13）＆#215;109/L,差异有统计学意义;输入骨髓细胞组和输入骨髓细胞和NK细胞组的CB6F1小鼠（MHCⅠ类分子半相合）外周血 WBC 分别为（1.52＆#177;0.26）＆#215;109/L 和（1.85＆#177;0.34）＆#215;109/L,差异无统计学意义,血小板计数分别为（90＆#177;12）＆#215;109/L 和（113＆#177;15）＆#215;109/L,差异具有统计学意义;BALB/c小鼠白细胞的改善略优于CB6F1小鼠。结论异源反应性NK细胞可以促进骨髓移植小鼠造血功能的恢复,并且这种作用似与NK细胞受体与受鼠MHC I类分子的匹配程度相关。     

Study on the effect of ligustrazine on hematopoietic reconstitution in bone marrow transplantation mice

Hematopoietic reconstitution after bone marrowtransplantation is a complicated process CXCchemokines and their receptors play important rolesin this process. One of these receptors, CXCR4 expresses on hematopoietic stem cells, and is now takenas a "homing factor" in hematopoietic reconstitu--'tionll' 2]. Ligustrazine can protect the bone marrowmicroenvironment after irradiation or immune damage[', 4]. We established allogenic bone marrow transplantation mice models and explored the effects …     

川芎嗪对辐射致血虚证小鼠骨髓细胞蛋白质表达的影响

目的:观察川芎嗪对辐射致血虚证小鼠骨髓细胞蛋白质表达的影响,探讨川芎嗪补血作用的分子机制。方法:采用3.5 Gy60Coγ射线全身1次性照射,制备小鼠血虚证模型;骨髓集落细胞培养,观察并计数粒系-巨噬细胞集落生成单位(CFU-GM)、爆增型红细胞集落生成单位(BFU-E)、红细胞集落生成单位(CFU-E)、混合集落生成单位(CFU-mix)集落数;利用蛋白质组学寻找差异表达蛋白质。结果:辐射后小鼠骨髓CFU-GM、BFU-E、CFU-E、CFU-mix集落数明显减少,川芎嗪能使其显著回升并部分逆转辐射后蛋白质表达的变化,使小鼠骨髓细胞5种蛋白质表达回升,5种回落。结论:川芎嗪可促进辐射致血虚证小鼠骨髓造血祖细胞增殖,调节多种骨髓细胞蛋白质的表达,后者可能是川芎嗪促进造血细胞生长和增殖的重要机制之一。     

参芪扶正注射液对化疗后小鼠造血功能影响的实验研究

目的 探讨参芪扶正注射液对化疗后小鼠造血功能的影响.方法 用5-氟尿嘧啶（5-FU）225 mg/kg腹腔注射复制骨髓抑制动物模型,治疗组给予参芪扶正注射液5 ml/kg,对照组给予同剂量生理盐水.治疗1周后,做血细胞计数、各系造血祖细胞集落（CFU-GM、CFU-E、CFU-MK）培养和骨髓病理检查.结果 参芪扶正注射液能升高外周血细胞计数：治疗组小鼠外周血的红细胞、白细胞和血小板计数均高于对照组（P〈0.05）.促进造血祖细胞增殖：治疗组小鼠的CFU-GM、CFU-E、CFU-MK集落数均大于对照组（P〈0.05）.改善骨髓抑制：骨髓病理检查对照组出现大片空白区,造血细胞稀少,而治疗组造血组织结构较完整,造血细胞量丰富.结论 化疗后小鼠在骨髓抑制、造血功能低下时,参芪扶正注射液能通过促进骨髓各系造血祖细胞的增殖,改善骨髓造血组织增生,从而促进血细胞的生成.     

外源性mFasL-cDNA基因干预后骨髓造血细胞功能的实验研究

目的:探讨FasL基因转移后造血细胞活性是否到影响,骨髓移植(BMT)后受者骨髓造血功能是否受改变。方法:FasL-cDNA经鉴定后,常规收集BalB/c小鼠和BAC小鼠股骨和胫骨骨髓单个核细胞(BMMNC),采用脂质体转移法将FasL基因转入BalB/c小鼠BMMNC,然后按1∶0.625体外与BAC鼠BMMNC混合培养,6d后实验组(第3组)尾静脉输注给经致死量照射的BalB/c小鼠,同时设立:第1组(空白对照组即未移植细胞);第2组(未转染基因BalB/c鼠BMMNC+BAC鼠BMMNC);第4组(同基因BMT组)。观察移植后受者鼠造血功能及细胞来源,移植物抗宿主病(GVHD)及生存率。结果:BMT后10,20d,第4组白细胞及血小板计数明显高于第2、3组(P<0.01);第2、3组间差异不显著(P>0.05);30d,各组间均无显著性差异(P>0.05);第1组小鼠相继于1周内死亡,死亡后取脾观察未见脾结节形成。第2组、第3组存活的11只雌性BalB/c小鼠BMMNC中,Y染色体出现率为(79.35±6.77)%。第2组、第3组、第4组2月生存率分别为30%、80%、100%。第3组生存率明显高于第2组(P<0.01),与第4组无显著差异(P>0.05)。第3组少数及第2组中大部分鼠移植30d出现GVHD表现,两组死亡鼠及第2组存活鼠组织因子改变符合Ⅱ-Ⅲ度GVHD病理变化,第3组存活7只鼠中有Ⅰ度GVHD病理变化。结论:转基因自体造血细胞与异基因供体BMMNC混合培养后移植,能使受者获给供体来源的造血重建,并明显降低GVHD发生。     

Effects of adenovirus mediated vascular endothelial growth factor gene transfer on reconstitution of hematopoiesis in post-bone marrow transplantation mice

Background Bone marrow transplantation (BMT) conditioning procedure is considered as the cause of damage to bone marrow microvasculature and the delay of hematopoiesis recovery. However, hematopoiesis regulation post BMT by vascular endothelial growth factor (VEGF) has not yet been studied. In this study, adenovirus were used to investigate the effects of VEGF gene transfer on preventing damages to bone marrow microenvironment and its promotion of hematopoiesis in post-BMT mice.Methods Recombinant adenovirus (Ad)-enhanced green fluorescent protein (EGFP)/hVEGF165 was injected via tail vein into BALB/c mice undergoing syngeneic BMT. During the different phases post BMT, the distribution of adenovirus and the plasma levels of hVEGF were measured as well as the numbers of white blood cells (WBC), platelet (PLT) and red blood cells (RBC) in peripheral blood. At the same time, the mice were injected with Chinese ink via tail vein, following which the tibias were separated and were used for analysis of bone marrow microvasculature surface area and cellularity.Results Significant expression of EGFP and hVEGF was observed in multiple organs at different phases post BMT, and the plasma level of hVEGF was up to (866.67±97.13) pg/ml. The recovery of WBC, PLT and RBC of the group treated with recombinant adenovirus Ad-EGFP/hVEGF165 were significantly more rapid than those of other BMT groups （P&lt;0.05, respectively）. At the 20th day post BMT, the percentage of bone marrow microvasculature surface area in group treated with VEGF ［(61.2±4.0)%］ returned to normal level ［(62.0±5.0)%, P&gt;0.05］. The restoration of hematopoiesis was retarded more than that of microvasculature. The cellularity of bone marrow in each group was still lower than that of normal control ［(62.3±4.0)%, P&lt;0.05］ at the 30th day post BMT, but the percentage in group treated with VEGF at the 20th and 30th days post BMT ［(46.5±5.0)% and (55.1±4.5)%］ exceeded those of other BMT groups (P&lt;0.05, respectively).Conclusion VEGF gene transfer mediated by adenovirus may protect the hematopoietic microenvironment to promote the restoration of hematopoiesis in post-BMT mice.     

多抗甲素与IL-2协同促进骨髓移植小鼠免疫功能重建的研究

本文报道了给予致死量照射的BARBL/C小鼠移植同系小鼠骨筋细胞后,采取PAA及IL-2联合用药对BMT小鼠免疫功能重建的影响。BMT小鼠分别给予PAA、IL-2或PAA IL-2,然后检查三组BMT小鼠免疫功能重建情况。结果证明,BMT后30天,自然恢复小鼠免疫功能十分低下,但PAA及IL-2用药鼠脾细胞以丝裂原反应明显增强,对SRBC特异的PFC数明显增多,DTH及MLR也明显增强,同时IL-2的产生能力明显增强。提示PAA及IL-2均能单独从多方面促进BMT后免疫功能重建,而PAA IL-2组作用又明显强于PAA及IL-2单独用药组,表现出协同效应。     

放射损伤小鼠骨髓VEGF、CFU-GM、CFU-F的变化及川芎嗪对其影响的研究

目的:探讨放射损伤小鼠骨髓血管内皮细胞生长因子(VEGF)、骨髓粒巨噬细胞集落形成单位(GFU-GM)数和骨髓成纤维细胞集落成单位(CFU-F)的变化意义及川芎嗪对其的影响。方法:健康昆明小鼠经6.0Gy60Coγ照射后,立即喂饲川芎嗪并设对照组和正常组,分别于第3、7、14、21天检测其骨髓VEGF、CFU-GM和CFU-F的变化。结果:照射后第3、7、14 d骨髓VEGF表达水平显著降低,但川芎嗪组高于对照组;随着时间推移骨髓VEGF表达水平逐渐恢复,照射后第21 d川芎嗪组已恢复正常(P>0.05),而照射组仍低于正常组。照射后骨髓CFU-GM和CFU-F明显受到抑制,但川芎嗪组显示了良好的保护作用。结论:放射损伤后小鼠骨髓VEGF的表达变化在造血功能的恢复过程中起重要作用;川芎嗪具有改善骨髓造血功能的作用。     

Study on blocking the leukemia immune escape after BMT by Fas-Fas ligand pathway

Background To investigate if bone marrow transplantation (BMT) with bone marrow mononuclear cells (BMMCs) transducted with murine soluble Fas gene (sFas) using adenovirus vector could block the immune escape of leukemia cells eliminate the residual leukemia cells and reduce their relapse.Methods The recombinant adenovirus vector with murine sFas, adsFas, and the control vector adEGFP were constructed using homologous recombination between two plasmids in Escherichia coli. BMT was carried out after the BMMCs were infected with Adenoviruses. The mice models of leukemia/lymphoma were constructed by inoculating female C57BL/6 mice (H-2b) with 10（5） EL4 cells/mouse through caudal vein. Donors of bone marrow grafts were syngeneic male mice. BMMCs were infected with AdsFas or AdEGFP 24 hours before (Group D or E). The following three groups were simultaneously used: Group A, no BMMCs transplanted; Group B, transplanted with BMMCs not infected with adenoviruses; Group C, only transfusing EL4 cells, neither irradiation nor BMT. The hematopoietic reconstitution, generation of leukemia/lymphoma and the survival rate were observed in all groups after BMT. Results The adenovirus vectors were successfully constructed. The titre of virus after purification was up to 2.5×10（11）pfu/ml. Spleen indices examined 11 days after BMT were not obviously different among Group B, D and E (P&gt;0.05), but indices in Group A were significantly lower than those in the latter three groups (P&lt;0.01). Counts of leukocytes and platelets on +30 day showed mice were reconstituted satisfactorily in Group B and D, but very low in Group C and E. The Y-chromosomes existed 2 months after BMT and examination of bone marrow cytology showed that Group B and D were almost normal, but Group C and E had plenty of lymphoblast-like tumor cells. Tumors were obviously observed in the mice of Group C and E by histopathological examination, but the mice in Group B and D were normal. The survival rates were 0 (0/4) in Group A, 100% in Group B (6/6) and D (16/16), 12.5% (2/16) in Group C and 6.25% (1/16) in Group E respectively. It is demonstrated that, in contrast with the control (Group EGFP), survival rate was significantly increased in the sFas Group (P&lt;0.01). Conclusions The transfer of sFas gene by adenovirus changed the prognosis state of leukemia/lymphoma mice after auto-BMT. The transduction of sFas might block the effect of the immune escape of EL4 cells through FasL. These results could thus provide a new direction to find a way to treat the leukemia and its recurrence after BMT.