干细胞生长因子和粒细胞集落刺激因子与心肌梗死大鼠体内骨髓间充质干细胞的迁移*

背景：外周静脉移植间充质干细胞只有1%~5%的移植细胞能归巢到心肌梗死区域。 目的：观察干细胞生长因子、粒细胞集落刺激因子对骨髓间充质干细胞归巢的影响。 方法：采用贴壁培养法分离培养SD大鼠骨髓间充质干细胞,取传至3~5代细胞。建立SD大鼠急性心肌梗死模型,干细胞生长因子组、粒细胞集落刺激因子组、干细胞生长因子+粒细胞集落刺激因子组在骨髓间充质干细胞移植前3 d和移植后3 d单独或混合皮下注射干细胞生长因子、粒细胞集落刺激因子,骨髓间充质干细胞组不注射细胞因子。 结果与结论：荧光显微镜下观察,骨髓间充质干细胞迁移至心肌梗死组织,骨髓间充质干细胞组、干细胞生长因子组、粒细胞集落刺激因子组迁移至心肌梗死区的骨髓间充质干细胞数量没有明显的区别(P > 0.05),干细胞生长因子+粒细胞集落刺激因子组的骨髓间充质干细胞数量明显高于其他3组(P < 0.05)。免疫荧光组织化学显示,植入的部分骨髓间充质干细胞表达心肌特异蛋白cTnI。结果说明干细胞生长因子和粒细胞集落刺激因子两种细胞因子联合应用可以促进骨髓间充质干细胞归巢至心肌梗死区域,在体内微环境的诱导下,骨髓间充质干细胞能够转化为心肌样细胞。     

细胞因子介导的人脐血单个核细胞体外扩增并重建NOD/SCID小鼠造血及免疫功能

背景：细胞因子介导的脐血造血细胞体外扩增有望能解决脐血移植数量不足的问题。 目的：实验拟确立在无基质培养条件下体外扩增脐血单个核细胞最合适的细胞因子组合及干细胞因子、FLT3配基协同促聚核细胞生成因子对造血及免疫重建功能的影响。 方法：将新鲜脐血标本分离的单个核细胞接种于含有不同细胞因子组合的无血清无基质培养体系中培养7 d,根据不同细胞因子组合分组,将3因子干细胞因子+FLT3配基+促聚核细胞生成因子组合在无血清无基质条件下扩增培养7 d前后的脐血单个核细胞移植给经亚致死量照射的NOD/SCID小鼠。在扩增培养0,7 d检测脐血CD34+细胞数及CD34+CXCR4+,CD34+CD62L+,CD34+CD49d+的细胞数。脐血移植6周后通过流式细胞仪,PCR法检测存活小鼠体内的人源性细胞。 结果与结论：移植6周后,存活小鼠体内均可检测到人源性CD45+细胞,扩增脐血移植组的NOD/SCID小鼠存活率和人特异性基因捡出率均高于新鲜脐血移植组和高于生理盐水移植组 (P < 0.05)。扩增脐血组存活NOD/SCID小鼠骨髓中可检测到人髓系细胞(CD33+),T淋巴细胞(CD4+),B淋巴细胞(CD19+)和人造血干细胞成分(CD34+)细胞的表达。结果提示干细胞因子+FLT3配基+促聚核细胞生成因子3因子组合脐血造血细胞体外扩增是最合适的细胞因子组合,其扩增的脐血单个核细胞能够植入并重建NOD/SCID小鼠的造血及免疫功能。     

基质细胞衍生因子1及其受体CXCR4对人骨髓间充质干细胞向

背景：近年研究发现移植的骨髓间充质干细胞能向颅内创伤、脑卒中、炎症和变性疾病等病灶部位迁移,进而发挥治疗作用,但对于其向病灶定向迁移的具体机制还不十分清楚。 目的：探讨基质细胞衍生因子1及其受体 CXCR4在移植的骨髓间充质干细胞趋向缺血脑组织迁移中的作用。 设计、时间及地点：细胞学体内实验,于2008-02/2009-02在解放军第三军医大学新桥医院中心实验室进行。 材料：骨髓标本取自解放军第三军医大学附属新桥医院血液科收治的15~40岁正常或原发病未累及骨髓患者,三四月龄健康雄性SD大鼠72只由解放军第三军医大学野战外科研究所实验动物中心提供。 方法：密度梯度离心贴壁筛选法分离纯化、体外培养人骨髓间充质干细胞。54只大鼠参照Nagasawa线栓法制备局灶性脑缺血再灌注模型,剩余18只作为假手术组,仅插入线栓10 mm。模型组及假手术组大鼠各取9只,分别于造模后第2,4,8天,采用Real-time PCR和免疫组织化学法定量分析缺血脑组织基质细胞衍生因子1表达变化。剩余36只脑缺血再灌注模型鼠随机分为细胞移植组、溶液对照组,18只/组,于再灌注后24 h分别从尾静脉缓慢注入1 mL人骨髓间充质干细胞悬液(含2×109 L-1个细胞)或1 mL PBS。 主要观察指标：人骨髓间充质干细胞CXCR4 mRNA和蛋白的表达,缺血再灌注后脑组织基质细胞衍生因子1 mRNA和蛋白表达变化,免疫组织化学检测人骨髓间充质干细胞向缺血脑组织的迁移和分布。 结果：RT-PCR结果发现人骨髓间充质干细胞表达CXCR4 mRNA,免疫细胞化学染色发现CXCR4主要表达于人骨髓间充质干细胞的胞膜和胞浆。脑缺血再灌注损伤后2,4,8 d,趋化因子基质细胞衍生因子1 mRNA水平呈上升趋势,与假手术组比较差异有显著性意义(P < 0.05)。经静脉移植的人骨髓间充质干细胞定向迁移到脑损伤区域,并大量分布于基质细胞衍生因子1高表达的缺血半暗区,损伤侧大脑半球人骨髓间充质干细胞数量显著高于对侧半球(P < 0.01)。 结论：基质细胞衍生因子1及其受体CXCR4参与并促进人骨髓间充质干细胞向脑缺血再灌注损伤区的迁移。     

缺氧诱导因子1α小干扰RNA对骨髓间充质干细胞缺氧诱导因子1α、基质细胞衍生因子1α和血管内皮生长因子基因表达的影响*

背景：缺氧可以影响骨髓间充质干细胞分泌细胞因子,其机制可能是通过缺氧诱导因子1α起作用的。 目的：观察缺氧诱导因子1α RNA干扰对骨髓间充质干细胞缺氧诱导因子1α、基质细胞衍生因子1α和血管内皮生长因子基因表达的影响。 方法：贴壁法培养骨髓间充质干细胞,取第3~5代细胞用于实验,倒置显微镜下观察细胞形态,并用流式细胞仪检测其表面标志物CD34、CD44、CD90表达。将骨髓间充质干细胞分四组：常氧对照组(无干预因素)、缺氧组(缺氧24 h)、脂质体对照组(转染空载脂质体后缺氧24 h)、RNA干扰组(转染脂质体介导的RNA干扰序列后缺氧24 h)。RT-PCR法检测骨髓间充质干细胞的缺氧诱导因子1α、基质细胞衍生因子1α和血管内皮生长因子 mRNA表达水平, ELISA检测骨髓间充质干细胞培养上清缺氧诱导因子1α、基质细胞衍生因子1α和血管内皮生长因子蛋白表达水平。 结果与结论：同常氧对照组比较,缺氧组缺氧诱导因子1α、基质细胞衍生因子1 α和血管内皮生长因子基因及蛋白表达增高(P < 0.05);同脂质体对照组比较,RNA干扰组缺氧诱导因子1α、基质细胞衍生因子1α和血管内皮生长因子基因及蛋白表达降低(P < 0.05)。证实了缺氧条件可以使骨髓间充质干细胞血管内皮生长因子和基质细胞衍生因子1α的表达增加,抑制缺氧诱导因子1α的表达可以使血管内皮生长因子和基质细胞衍生因子1α的表达减少,缺氧诱导因子1α很可能是干细胞移植细胞因子分泌的调控因素。     

心肌梗死大鼠体外培养心肌组织对间充质干细胞迁移的作用

背景：间充质干细胞具有改善心脏功能的潜力,间充质干细胞移植后向心脏归巢的机制尚不完全清楚。 目的：观察心肌梗死大鼠体外培养的心肌组织对间充质干细胞迁移的作用及可能机制。 方法：建立大鼠心肌梗死模型,培养大鼠心脏组织块,密度梯度离心及贴壁筛选法分离培养骨髓间充质干细胞,24只SD大鼠随机抽签法分为心肌梗死组、假手术组及正常对照组。假手术组只穿线不结扎,正常对照组不做任何处理。荧光染料DAPI标记间充质干细胞,利用transwell模型进行共培养,共培养48 h。荧光镜下计数迁移细胞数,CD34/CD44免疫细胞化学染色进行迁移细胞定性,免疫荧光检测迁移细胞CXCR4的表达情况,心脏组织切片行免疫组织化学染色检测基质细胞衍生因子1的表达情况并进行平均吸光度分析。 结果与结论：心肌梗死组单位视野迁移细胞数显著高于假手术组(P < 0.05),正常对照组未见迁移细胞。免疫细胞化学染色显示迁移细胞CD44阳性而CD34阴性,符合间充质干细胞特点,其膜受体CXCR4阳性表达。心肌梗死组及假手术组心脏切片基质细胞衍生因子1阳性表达,正常对照组心肌组织不表达基质细胞衍生因子1。心肌梗死组心脏组织基质细胞衍生因子1平均吸光度显著高于其他组别(P < 0.05)。提示心肌梗死后大鼠心脏组织能促进间充质干细胞迁移,这一效应的实现可能与基质细胞衍生因子1-CXCR4轴的作用有关。     

静脉移植骨髓间充质干细胞联合重组人生长激素对充血性心肌病大鼠心肌和血管组织的修复**☆

背景：骨髓间充质干细胞静脉移植后,能否归巢至心脏受损部位和分化为心肌样细胞尚无统一定论。 目的：探讨重组人生长激素联合骨髓间充质干细胞静脉移植对充血性心肌病大鼠心肌和血管新生的影响。 方法：密度梯度离心和贴壁筛选法获得大鼠骨髓间充质干细胞。模型组、细胞移植组、生长激素组、联合组大鼠均在阿霉素诱导下建立心脏衰竭模型。造模后,细胞移植组经静脉注入BrdU标记的骨髓间充质干细胞8×1013 L-1;生长激素组皮下注射重组人生长激素2 U/(kg•d),连续14 d;联合组行骨髓间充质干细胞移植与重组人生长激素注射。4周后取材,BrdU+MHC及BrdU+Actin免疫组化染色确定骨髓间充质干细胞的归巢情况,评价移植细胞向心肌样细胞和血管内皮细胞的分化,苏木精-伊红染色检测血管密度。 结果与结论：与细胞移植组比较,联合组BrdU免疫组化阳性率显著升高(P < 0.001);BrdU+MHC双染和BrdU+Actin双染后心肌样细胞、血管内皮细胞均显著增多(P < 0.001)。与模型组比较,生长激素组、细胞移植组、联合组的总血管密度、微血管密度、毛细血管密度均显著升高(P < 0.001),后3组间比较无明显差异(P > 0.05)。结果证实骨髓间充质干细胞静脉移植后可归巢到心脏,对充血性心肌病大鼠心肌和血管有明显修复作用,能在损伤处区存活、生长,并向心肌样细胞、血管内皮细胞方向分化,增加损伤处血管密度;生长激素可以改善微环境,加强骨髓间充质干细胞向心肌样细胞、血管内皮细胞的转化率。     

骨髓间充质干细胞分泌基质细胞衍生因子1对异体T淋巴细胞和白血病细胞HL-60增殖的影响

背景：骨髓间充质干细胞持续分泌的基质细胞衍生因子1与其受体CXCR4的相互作用，不仅在细胞的炎症反应、造血干细胞的迁移与归巢等生物学过程中发挥作用，还在肿瘤的发生、转移、复发、免疫耐受及血管新生过程中扮演重要角色。 目的：探讨骨髓间充质干细胞分泌的基质细胞衍生因子1对T淋巴细胞及白血病细胞HL-60增殖的影响。 设计：细胞学体外对照观察。 材料：骨髓标本来自本院血液科异基因骨髓移植供者及骨髓象正常的非白血病患者，T淋巴细胞来源于健康志愿者，急性髓性白血病细胞株HL-60为本实验室液氮冷冻保存，CXCR4单克隆抗体12G5为eBioscience公司产品。 方法：应用Ficoll密度梯度离心法分离骨髓间充质干细胞，传至第3代后收集培养5 d的细胞上清，离心去沉淀后备用。应用尼龙棉柱法分离异体外周血T淋巴细胞，调整细胞密度为2×109 L-1备用。T淋巴细胞与骨髓间充质干细胞的共培养实验设立3组：T淋巴细胞组、T淋巴细胞+细胞上清组、T淋巴细胞+细胞上清+单抗组。HL-60细胞与骨髓间充质干细胞的共培养实验设立3组：HL-60细胞组、HL-60细胞+细胞上清组、HL-60细胞+细胞上清+单抗组。 主要观察指标：MTT法检测T淋巴细胞及HL-60细胞在应用单克隆抗体12G5前后的增殖情况。 结果：与T淋巴细胞组比较，T淋巴细胞+细胞上清组、T淋巴细胞+细胞上清+单抗组的吸光度值均明显降低（P < 0.05），后两组间比较差异无显著性意义（P > 0.05）。与HL-60细胞组比较，HL-60细胞+细胞上清组吸光度值明显升高（P < 0.05）；与HL-60细胞+细胞上清组比较，HL-60细胞+细胞上清+单抗组的吸光度值明显降低（P < 0.05）。 结论：加入单克隆抗体12G5阻断基质细胞衍生因子1的生物学效应后，不影响骨髓间充质干细胞上清液抑制T淋巴细胞增殖的效果，但可以抑制白血病细胞HL-60的增殖。     

CXCL12/CXCR4生物学轴对骨髓间充质干细胞修复脊髓损伤的影响☆

背景：研究发现不同途径移植骨髓间充质干细胞均能向脊髓损伤部位迁移,进而发挥治疗作用。 目的：探讨CXCL12/CXCR4生物学轴对骨髓间充质干细胞趋向脊髓损伤部位迁移的作用。 方法：采用改进的脊椎骨破坏法制备脊髓损伤模型。假手术组只打开皮肤,不损伤脊髓且不作任何干预;模型组于造模后第2天采用腰骶鞘内注射5 μL生理盐水;细胞移植组于造模后第2天采用腰骶鞘内移植5 μL骨髓间充质干细胞。 结果与结论：①荧光显微镜下可见,横切的脊髓损伤局部有大量的标记细胞聚集,而在损伤部位远端1 cm处,仅见少量的标记骨髓间充质干细胞。②骨髓间充质干细胞表达中等水平的趋化因子CXCL12,其特异性结合受体CXCR4也有低水平表达。③脊髓损伤7 d后,局部CXCL12表达增强,主要集中在脊髓损伤部位的皮质区域,而在损伤部位1 cm以外的脊髓组织未见大量表达的CXCL12。CXCR4蛋白表达没有明显的时间效应。④检测CXCL12 mRNA的转录水平发现细胞移植组的CXCR4转录水平明显高于假手术组和模型组,损伤后14 d脊髓损伤局部CXCL12的转录水平最强,21 d时降低,CXCL12的局部转录水平明显高于远端。⑤脊髓损伤部位也表达趋化因子CXCR4,但其表达水平没有时程差异。损伤局部的CXCR4转录水平略高于远端,但差异无显著性意义。说明CXCL12/CXCR4生物学轴参与骨髓间充质干细胞向脊髓损伤区域迁移。     

Flk-1+骨髓间充质干细胞移植上调白细胞介素6水平：是否同时加重了胶原诱导性关节炎小鼠的症状？**☆

背景：间充质干细胞的免疫调节作用是被大家普遍认可的,在以往实验中也对Flk-1+骨髓间充质干细胞体外抑制T/B淋巴细胞增殖的能力进行了确认。 目的：验证Flk-1+骨髓间充质干细胞对胶原诱导性关节炎小鼠的治疗作用。 方法：健康10周龄雄性DBA-1(H-2Kq)小鼠18只,随机分为3组：初次免疫后细胞移植组、加强免疫后细胞移植组、模型对照组,3组小鼠均通过尾皮下注射牛Ⅱ型胶原进行初次免疫,21 d后同法进行加强免疫,建立胶原诱导性关节炎模型。密度梯度离心法和贴壁筛选法体外分离DBA-1(H-2Kq)小鼠Flk-1+骨髓间充质干细胞,初次免疫后细胞移植组小鼠在初次免疫后立即经尾静脉输注Flk-1+骨髓间充质干细胞(1~2)×106个/只,加强免疫后细胞移植组小鼠在加强免疫时同法输注等量Flk-1+骨髓间充质干细胞,模型对照组小鼠于初次免疫后0或21 d尾静脉输注等量生理盐水。观察初次免疫后和加强免疫后各组小鼠的爪垫增厚情况、临床评分,检测小鼠关节病理学变化及血清因子质量浓度的动态变化。 结果与结论：与模型对照组比较,初次免疫后细胞移植组爪垫增厚程度及平均临床疾病得分均无明显差异(P > 0.05),均可见明显的滑膜组织损伤和炎症细胞浸润,各血清细胞因子质量浓度基本相似;而加强免疫后细胞移植组爪垫明显增厚(P < 0.01),平均临床疾病得分高达3.35分,滑膜损伤严重,毛细血管增生,在初次免疫后28 d白细胞介素6血清浓度急剧上升(P < 0.1),初次免疫后35 d白细胞介素6血清浓度又明显下降(P < 0.1)。提示在胶原诱导性关节炎小鼠模型中,Flk-1+骨髓间充质干细胞移植不但未取得预期的治疗效果,还在加强免疫后细胞移植组观察到显著地关节炎症状恶化现象,其可能通过上调白细胞介素6血清浓度加重类风湿关节炎小鼠的行为症状。     

可注射型纤维蛋白凝胶转化生长因子β1复合骨髓间充质干细胞移植治疗椎间盘退变☆

摘要 背景：纤维蛋白凝胶主体胶与催化剂未混合前为液态,具有可注射的优点,注射混合后凝固成凝胶状,与髓核相似,并且凝固时间可控性强,作为间充质干细胞的载体植入到椎间盘内有诸多优点。 目的：观察可注射型纤维蛋白凝胶转化生长因子β1复合骨髓间充质干细胞移植抑制椎间盘退变的可行性。 方法：将新西兰大白兔随机分为退变模型组、纤维蛋白凝胶组,骨髓干细胞+纤维蛋白凝胶组。3组采用针刺法诱导建立退变模型后,纤维蛋白凝胶组及干细胞+纤维蛋白凝胶组分别移植入纤维蛋白凝胶转化生长因子β1复合物及骨髓间充质干细胞纤维蛋白凝胶转化生长因子β1复合物,于植入后2,6,10周行CR、MRI及病理检查。 结果与结论：退变模型组与纤维蛋白凝胶组椎间隙高度下降明显,并与时间呈正相关,干细胞+纤维蛋白凝胶组下降较缓慢(P < 0.01)。免疫组织化学及组织学检查显示,退变模型组髓核细胞的数量及Ⅱ型胶原含量进行性减少,细胞凋亡率明显增加,纤维蛋白凝胶组与退变模型组相似,干细胞+纤维蛋白凝胶组髓核细胞数量及Ⅱ型胶原含量较退变模型组及纤维蛋白凝胶组明显增多,细胞凋亡率下降。说明骨髓间充质干细胞联合纤维蛋白凝胶转化生长因子β1能很好抑制椎间盘退变,而单纯纤维蛋白凝胶转化生长因子β1移植不能抑制椎间盘退变。 关键词：纤维蛋白凝胶;骨髓间充质干细胞;转化生长因子β1;椎间盘退变;兔 doi:10.3969/j.issn.1673-8225.2011.03.024     

Effect of stromal cell-derived factor-1/CXCR4 axis in neural stem cell transplantation for Parkinson’s disease

Previous studies have shown that neural stem cell transplantation has the potential to treat Parkinson’s disease,but its specific mechanism of action is still unclear.Stromal cell-derived factor-1 and its receptor,chemokine receptor 4(CXCR4),are important regulators of cell migration.We speculated that the CXCR4/stromal cell-derived factor 1 axis may be involved in the therapeutic effect of neural stem cell transplantation in the treatment of Parkinson’s disease.A Parkinson’s disease rat model was injected with 6-hydroxydopamine via the right ascending nigrostriatal dopaminergic pathway,and then treated with 5μL of neural stem cell suspension(1.5×104/L)in the right substantia nigra.Rats were intraperitoneally injected once daily for 3 days with 1.25 mL/kg of the CXCR4 antagonist AMD3100 to observe changes after neural stem cell transplantation.Parkinson-like behavior in rats was detected using apomorphine-induced rotation.Immunofluorescence staining was used to determine the immunoreactivity of tyrosine hydroxylase,CXCR4,and stromal cell-derived factor-1 in the brain.Using quantitative real-time polymerase chain reaction,the mRNA expression of stromal cell-derived factor-1 and CXCR4 in the right substantia nigra were measured.In addition,western blot assays were performed to analyze the protein expression of stromal cell-derived factor-1 and CXCR4.Our results demonstrated that neural stem cell transplantation noticeably reduced apomorphine-induced rotation,increased the mRNA and protein expression of stromal cell-derived factor-1 and CXCR4 in the right substantia nigra,and enhanced the immunoreactivity of tyrosine hydroxylase,CXCR4,and stromal cell-derived factor-1 in the brain.Injection of AMD3100 inhibited the aforementioned effects.These findings suggest that the stromal cell-derived factor-1/CXCR4 axis may play a significant role in the therapeutic effect of neural stem cell transplantation in a rat model of Parkinson’s disease.This study was approved by the Animal Care and Use Committee of Kunming Medical University,China(approval No.SYXKK2015-0002)on April 1,2014.     

人骨髓及脐血单个核细胞移植构建人源化非肥胖糖尿病/重症联合免疫缺陷

背景：人源化非肥胖糖尿病/重症联合免疫缺陷(nonobese diabetic/severe combined immunodeficient,NOD/SCID)小鼠模型在生物及医学科学研究中被广泛应用,而提高人源化NOD/SCID小鼠模型中的人源化水平是研究者的迫切需要。 目的：探讨提高人源化NOD/SCID小鼠模型人源化水平的措施。 设计、时间及地点：随机对照动物实验,于2008-11/2009-01在广西医科大学实验动物中心及广西医科大学第一附属医院完成。 材料：雄性NOD/SCID小鼠14只,体质量(25.0±1.5)g;1例骨髓标本约6 mL(多部位分层次采集)取自健康志愿者;1例足月脐带血标本大约90 mL,取自广西医科大学第一附属医院产科,为健康产妇。 方法：无菌抗凝的正常骨髓及足月脐带血样本均在采集后6 h内处理。密度梯度离心法分离单个核细胞。将14只小鼠标号电脑随机分为5组：全身照射/环磷酰胺骨髓移植组(n=3),全身照射/环磷酰胺脐血移植组(n=3),全身照射骨髓移植组(n=3),全身照射脐血移植组(n=3),空白对照组(n=2)。将人骨髓或脐血单个核细胞分别移植给经不同方案和剂量预处理的各组小鼠,移植后给小鼠腹腔注射重组人粒-巨噬细胞集落刺激因子及重组人促红细胞生成素。 主要观察指标：各组小鼠输注的细胞数,小鼠存活情况并计算死亡率,移植6周后流式细胞仪检测小鼠骨髓及外周血人CD45+细胞比例。 结果：全身照射/环磷酰胺骨髓移植组、全身照射骨髓移植组和全身照射/环磷酰胺脐血移植组、全身照射脐血移植组小鼠输注人单个核细胞数分别为(0.656 0±0.008 9) ×106和(4.077 5±0.845 0)×106。全身照射/环磷酰胺组死亡率均为100%,全身照射组死亡率均为33.3%。全身照射后再加环磷酰胺,小鼠不能耐受;而全身照射剂量越大,死亡率越高,当达400 cGy时,死亡率100%。存活小鼠移植6周后,流式细胞仪检测小鼠骨髓中人CD45+细胞的比例,全身照射脐血移植组13号和6号小鼠分别为59.61%和21.46%,而全身照射骨髓移植组2号和8号小鼠其比例均为0,空白对照组小鼠比例均为0。而在13号小鼠骨髓细胞中,CD33+细胞比例为13.13%,GlyA+CD45-细胞比例为20.21%。 结论：要建立人源化NOD/SCID小鼠模型,所输入的造血干细胞数量一定要达到阈剂量,而在可耐受的范围内,尽可能提高预处理的强度,并使用人细胞因子可提高人源化NOD/SCID小鼠模型的人源化比例。     

Localization of the alpha-chemokine SDF-1 and its receptor CXCR4 in idiopathic inflammatory myopathies

We studied the distribution of stromal cell-derived factor 1 isoforms alpha and beta, and their receptor CXCR4, in polymyositis, sporadic inclusion body myositis and dermatomyositis using in situ hybridization, immunohistochemistry, immunofluorescence and Western blotting. In control muscle, polymyositis and sporadic inclusion body myositis, stromal cell-derived factor-1alpha expression was noted in muscle fibers, while stromal cell-derived factor-1beta and CXCR4 were predominantly localized to capillaries and arterioles. In dermatomyositis, stromal cell-derived factor-1beta immunoreactivity of blood vessels was focally increased. The vast majority of inflammatory cells in idiopathic inflammatory myopathies were CXCR4 positive. A subset of helper T-cells and macrophages expressed stromal cell-derived factor-1alpha, while only rare inflammatory cells expressed stromal cell-derived factor-1beta. A significant increase of stromal cell-derived factor-1alpha and CXCR4 was observed in protein extracts of idiopathic inflammatory myopathies in comparison with normal controls. The abundance of both CXCR4 and its ligand stromal cell-derived factor-1 implicates their interaction in the pathogenesis of idiopathic inflammatory myopathies and identifies these proteins as possible targets for selective immune therapy.     

SDF-1 (CXCL12) is upregulated in the ischemic penumbra following stroke: association with bone marrow cell homing to injury

The chemokine stromal-derived factor-1 (SDF-1, also known as CXCL12) and its receptor CXCR4 have been implicated in homing of stem cells to the bone marrow and the homing of bone marrow-derived cells to sites of injury. Bone marrow cells infiltrate brain and give rise to long-term resident cells following injury. Therefore, SDF-1 and CXCR4 expression patterns in 40 mice were examined relative to the homing of bone marrow-derived cells to sites of ischemic injury using a stroke model. Mice received bone marrow transplants from green fluorescent protein (GFP) transgenic donors and later underwent a temporary middle cerebral artery suture occlusion (MCAo). SDF-1 was associated with blood vessels and cellular profiles by 24 hours through at least 30 days post-MCAo. SDF-1 expression was principally localized to the ischemic penumbra. The majority of SDF-1 expression was associated with reactive astrocytes; much of this was perivascular. GFP+ cells were associated with SDF-1-positive vessels and were also found in the neuropil of regions with increased SDF-1 immunoreactivity. Most vessel-associated GFP+ cells resemble pericytes or perivascular microglia and the majority of the GFP+ cells in the parenchyma displayed characteristics of activated microglial cells. These findings suggest SDF-1 is important in the homing of bone marrow-derived cells, especially monocytes, to areas of ischemic injury.     

基质细胞衍生因子-1对间质干细胞迁移的影响

目的:观察基质细胞衍生因子-1(SDF-1)在体内外对大鼠骨髓间质干细胞(rMSCs)的趋化诱导作用,探讨SDF-1对rMSCs迁移影响的可能机制。方法:应用体外细胞迁移实验及大鼠脑梗死模型体内移植,观察SDF-1对rMSCs的迁移影响。流式细胞术与RT-PCR检测rMSCs的CXC趋化因子受体4(CXCchemokinereceptor4,CXCR4)表达。结果:在SDF-1存在时,rMSCs迁移活跃,应用抗体封闭CXCR4后,这种迁移显著减弱。体内移植的rMSCs主要聚集在脑梗死灶周围,但在封闭CXCR4后,这种聚集现象大大减弱。流式细胞术示仅小部分rMSCs表面表达CXCR4,但经TritonX-100处理后,表达CXCR4的rMSCs增加。结论:SDF-1可通过CXCR4对rMSCs起趋化作用,针对这种作用可望调控干细胞向靶组织的趋化聚集量,达到治疗目的。     

2', 3'-Cyclic nucleotide 3'-phosphodiesterase cells derived from transplanted marrow stromal cells and host tissue contribute to perineurial compartment formation in injured rat spinal cord

Transdifferentiation of transplanted marrow stromal cells (MSCs) and reactive changes of glial cells in a completely transected rat spinal cord were examined. Marrow stromal cells exhibited 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) at the plasma membrane and this has allowed their identification after transplantation by immunoelectron microscopy. In the control rats, the lesion site showed activated microglia/neural macrophages and some elongated cells, whose cytoplasm was immunoreactive for CNP. Cells designated as CNP1 and apparently host-derived expressed CXCR4. In experimental rats receiving MSCs transplantation, CNP1 cells were increased noticeably. This was coupled with the occurrence of a different subset of CNP cells whose plasma membrane was CNP-immunoreactive and expressed CXCR4. These cells, designated as CNP2, enclosed both myelinated and unmyelinated neurites thus assuming a spatial configuration resembling that of Schwann cells. A remarkable feature was the extensive ramifications of CNP1 cells with long filopodia processes delineating the CNP2 cells and their associated neurites, forming many perineurial-like compartments. Present results have shown that CNP2 cells considered to be MSCs-derived can transform into cells resembling Schwann cells based on their spatial relation with the regenerating nerve fibers, whereas the CNP1 glial cells participate in formation of perineurial compartments, probably serving as conduits to guide the nerve fiber growth. The chemotactic migration of CNP cells either derived from host tissue or MSCs bearing CXCR4 may be attracted by stromal derived factor-1alpha (SDF-1alpha) produced locally. The coordinated cellular interaction between transplanted MSCs and local glial cells may promote the growth of nerve fibers through the lesion site.     

Matrix metalloproteinase-9 and stromal cell-derived factor-1 act synergistically to support migration of blood-borne monocytes into the injured spinal cord

The infiltration of monocytes into the lesioned site is a key event in the inflammatory response after spinal cord injury (SCI). We hypothesized that the molecular events governing the infiltration of monocytes into the injured cord involve cooperativity between the upregulation of the chemoattractant stromal cell-derived factor-1 (SDF-1)/CXCL12 in the injured cord and matrix metalloproteinase-9 (MMP-9/gelatinase B), expressed by infiltrating monocytes. SDF-1 and its receptor CXCR4 mRNAs were upregulated in the injured cord, while macrophages immunoexpressed CXCR4. When mice, transplanted with bone marrow cells from green fluorescent protein (GFP) transgenic mice, were subjected to SCI, GFP+ monocytes infiltrated the cord and displayed gelatinolytic activity. In vitro studies confirmed that SDF-1α, acting through CXCR4, expressed on bone marrow-derived macrophages, upregulated MMP-9 and stimulated MMP-9-dependent transmigration across endothelial cell monolayers by 2.6-fold. There was a reduction in F4/80+ macrophages in spinal cord-injured MMP-9 knock-out mice (by 36%) or wild-type mice, treated with the broad-spectrum MMP inhibitor GM6001 (by 30%). Mice were adoptively transferred with myeloid cells and treated with the MMP-9/-2 inhibitor SB-3CT, the CXCR4 antagonist AMD3100, or a combination of both drugs. While either drug resulted in a 28-30% reduction of infiltrated myeloid cells, the combined treatment resulted in a 45% reduction, suggesting that SDF-1 and MMP-9 function independently to promote the trafficking of myeloid cells into the injured cord. Collectively, these observations suggest a synergistic partnership between MMP-9 and SDF-1 in facilitating transmigration of monocytes into the injured spinal cord.