CXCR4-FAK信号通路在低氧预处理骨髓间充质干细胞向大鼠脊髓缺血再灌注损伤组织迁移和粘附中的作用

目的 探讨CXC趋化因子受体4-粘着斑激酶(CXCR4-FAK)信号通路在低氧预处理骨髓间充质于细胞(BMSC)向大鼠脊髓缺血再灌注损伤组织迁移和粘附中的作用.方法 第一部分重组腺病毒介导绿色荧光蛋白基因转染成功的大鼠原代BMSC,以1×106个/ml密度接种于24孔培养板(1 ml/孔),采用随机数字表法,将其分为5组(n=18):对照组(C组)、常氧培养组(N组)、低氧预处理组(H组)、低氧预处理+ CXCR4拮抗剂AMD3100组(HA组)和低氧预处理+FAK抑制剂粘着斑相关非激酶组(HF组).N组BMSC在常氧环境中培养36 h;H组BMSC在低氧环境中培养24h后在常氧环境中继续培养12 h;HA组和HF组于低氧预处理前分别加入5 μg/ml AMD3100和10 μg/ml粘着斑相关非激酶.测定BMSC中基质细胞衍生因子-1α(SDF-1α)、CXCR4和磷酸化FAK(p-FAK)表达、BM-SC向SDF-1α迁移能力和BMSC与纤维粘连蛋白(FN)粘附能力.第二部分 雄性SD大鼠216只,体重300 ～ 350 g,其中210只采用阻断胸主动脉合并体循环低血压的方法制备脊髓缺血再灌注损伤模型.取脊髓缺血再灌注大鼠36只,分别于模型制备前、再灌注12h、1、3、5、7 d(T0-5)时处死6只大鼠,取腰段脊髓组织,检测SDF-1α含量.其余脊髓缺血再灌注大鼠180只,采用随机数字表法,将其分为5组(n=36),于再灌注即刻分别鞘内注射C组DMEM培养液300μl和N组、H组、HA组、HF组1×106个/ml BMSC悬液300μl.分别于T0-5时取6只大鼠,进行神经行为学评分,然后取腰段脊髓组织,测定BMSC聚集度.结果 与C组比较,N组BMSC的SDF-1α、CXCR4、p-FAK表达及其向SDF-1α迁移能力、与FN粘附能力、神经行为学评分和脊髓组织BMSC聚集度差异无统计学意义(P＞0.05);与N组比较,H组BMSC的SDF-1α、CXCR4和p-FAK表达上调,向SDF-1α迁移能力、与FN粘附能力、神经行为学评分和脊髓组织BMSC聚集度升高(P＜0.05),HA组和HF组BMSC的p-FAK表达及其向SDF-1α迁移能力、与FN粘附能力、神经行为学评分和脊髓组织BMSC聚集度差异无统计学意义(P＞0.05);与H组比较,HA组和HF组BMSC的p-FAK表达下调,向SDF-1x迁移能力、与FN粘附能力、神经行为学评分和脊髓组织BMSC聚集度降低(P＜0.05).与T0时比较,T2.3时脊髓组织SDF-1α含量升高(P＜0.05).结论 低氧预处理通过CXCR4-FAK信号通路促进BMSC向大鼠脊髓缺血再灌注损伤组织迁移,并与之粘附,从而发挥脊髓保护作用.     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

大鼠骨髓间充质细胞经SDF-1预处理后移植对急性心肌梗死的治疗效果

Objective To investigate the effects of preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) on the levels of apoptosis of bone marrow stromal cells (BMSC) treated with hypoxia plus serum deprivation, and observe the therapeutic efficacy of cellular transplant with BMSC preconditioned with SDF-1 in rats with acute myocardial infarction. Methods BMSC were cultured with the whole marrow-adherence way. RT-PCR and immunohistochemistry were used to determine the expression of CXCR4. BMSC were incubated in medium for 24 h with 10 and 100 μg/L SDF-1 respectively, then treated with hypoxia plus serum deprivation for 6 h. The levels of apoptosis were detected by flow cytometry and TUNEL method. Acute myocardial infarction (AMI) model was established in SD rats, and BMSC preconditioned or non-preconditioned with SDF-1 were transplanted into border zone around infarct area, then heart function was measured after two weeks by ultrasonography. Results BMSC exhibited the CXCR4 expression. The number of apoptotic cells was significantly reduced in SDF-1 PC group than in control group (P<0.05), and 100μg/L SDF-1 PC group had the lowest level of apoptosis. AMI model was established successfully. Two weeks after BMSC transplant, significant improvement in cardiac function was observed in 100 μg/L SDF-1 PC group as compared with the non-PC group (P<0.05). Conclusions PC with the chemokine SDF-1 suppresses the apoptosis of BMSC treated with hypoxia plus serum deprivation. SDF-1 PC is a novel approach for enhancing therapeutic efficacy of cellular transplant in rats with AML     

基质细胞衍生因子-1对神经干细胞的趋化作用

目的 观察基质细胞衍生因子-1(SDF-1)对神经干细胞(NSCs)迁移的影响.方法 由GFP转基因SD大鼠胚胎脑组织获取NSCs并进行传代培养,免疫细胞化学染色法检测SDF-1特异性受体CXCR4的表达,利用Blind-Well小室体外迁移体系观察不同浓度的SDF-1(0、1、10、50、100、500、1000μg/L)对NSCs定向迁移数量的影响,随后分别使用CXCR4激动剂和阻断剂处理NSCs,再次利用上述方法观察最适浓度SDF-1时NSCs的迁移.结果 成功分离培养得到能够稳定表达GFP的NSCs,且CXCR4在该种NSCs上有表达.体外趋化实验结果表明,SDF-1对NSCs有较强的趋化作用,随着SDF-1浓度的升高,发生迁移的细胞数量也随之增加,并于SDF-1浓度为500μg/L时达到最高峰;CXCR4特异性激动剂和阻断剂分别能够增强和减弱SDF-1对NSCs定向迁移的趋化作用.结论 SDF-1与其特异性受体CXCR4相互作用,能够对NSCs的定向迁移产生靶向性作用.     

黄芪与当归对体外培养糖尿病骨髓干细胞增殖和血管内皮生长因子表达的影响

目的：观察黄芪、当归及二者合用对体外培养大鼠骨髓干细胞增殖的影响,并探讨其可能机制。方法：自2009年7月至2010年2月,选择5只雄性200～220gSD大鼠高糖高脂饲料喂养4周后,给予链脲佐菌素（STZ）按照30mg/kg腹腔内注射2次诱发2型糖尿病,1周后检测血糖≥16.7mmol/L为2型糖尿病造模成功。密度梯度法分离5只大鼠骨髓干细胞,分为空白对照组（A组）、黄芪组（B组）、当归组（C组）、黄芪当归组（D组）。空白对照组加入无血清DMEM100μl,黄芪组、当归组、黄芪当归组分别加入等量的用无血清DMEM配制的黄芪煎液、当归煎液、黄芪当归合煎液,终浓度分别为黄芪1100mg/L,当归1100mg/L,黄芪当归组含黄芪1100mg/L、当归220mg/L。各组细胞培养14d后,采用MTT法测定细胞增殖,ELISA法检测细胞培养上清液VEGF蛋白浓度,Western-Blot检测骨髓干细胞VEGF蛋白表达。结果：与空白对照组比较,黄芪组和黄芪当归组的骨髓干细胞增殖水平、上清液VEGF蛋白浓度、骨髓干细胞VEGF蛋白表达水平均明显增高（P〈0.05或P〈0.01）,且黄芪当归组的以上作用均较黄芪组明显增强（P〈0.05或P〈0.01）。结论：单用黄芪或其与当归合用均具有刺激体外培养骨髓干细胞增殖的作用,可能与其促进VEGF蛋白表达增加有关,但单用当归无上述作用。     

Effect of erythropoietin on the oriented chemotaxis of bone-marrow mesenchymal stem cells under acute kidney injury microenvironment

Objective To investigate the migration of bone-marrow mesenchymal stem cells (BMSCs) under acute kidney injury (AKI) microenvironment in vitro and the effect of erythropoietin (EPO) intervention, and to explore its underlying mechanism. Methods Renal tubular epithelial cells (RTECs) were cultured in hypoxia/ re-oxygenation (HR) condition for 12 h, respectively, in order to establish HR-RTEC. BMSCs and RTECs were co-cultured by Transwell system and were divided into 7 groups: control group (group①, only BMSC cultured), BMSC-RTEC co-culturing group (group②), BMSC-HR-RTEC co-culturing ＋EPO intervention groups (group③to group⑦, EPO concentration: 0, 1, 5, 10, 50 IU/ml). All the groups were cultured for 48 h and the number of migrating BMSCs was detected. Western blotting was applied for the detection of SDF-1 expression in RTECs and p-MAPK and MAPK levels in BMSCs. SDF-1 concentration in the RTECs culture supernatant was tested by ELISA. Results The number of BMSCs migrating to the low chamber where HR-RTECs were cultured was increased, and EPO intervention further enhanced this migration which reached the peak at the concentration of 10 IU/ml [Compared with group③, (46.67±7.37) cells vs (19.00±2.37) cells, P＜0.05]. Intracellular expression level and the secreated level of SDF-1 in HR-RTECs in group③ were higher than those in RTECs of group② [0.37±0.01 vs 0.19±0.01, P＜0.05; (61.64±4.88) μg/L vs (35.26±8.78) μg/L, P＜0.05]. EPO intervention increased above SDF-1 levels and reached the peak at the concentration of 10 IU/ml [group⑥ vs group③：(173.53±14.66) μg/L vs (61.64±4.88) μg/L, P＜0.05], accompanied with enhanced phosphorylation of MAPK in BMSCs. Conclusions AKI microenvironment has obvious chemotaxis effect on BMSCs, and EPO intervention can strengthen this effect. The increased SDF-1 level and enhanced phosphorylation of MAPK, the downstream signal protein of SDF-1/CXCR4 axis, are the possible mechanism for EPO performance.     

SDF-1/CXCR4轴在骨髓间充质干细胞移植促进胰岛再生中的作用

目的 观察同种异体骨髓间充质干细胞(MSCs)移植入受体后,基质细胞衍生因子(SDF)-1/CXCR4轴在促进残存胰岛及其周围新生血管增殖中的作用.方法 对大鼠MSCs进行体外培养、鉴定.链脲佐菌素(STZ)诱导的糖尿病大鼠随机分为A组(MSCs移植组)、B组(MSCs移植+SDF-I/CXCR4轴阻断剂AMD组)和C组(糖尿病对照组),另设D组(正常大鼠对照组).移植MSCs后第30天取出各组大鼠胰腺和血清,胰腺组织采用苏木素-伊红(HE)染色和免疫组织化学法观察CD31、增殖细胞核抗原(PCNA)、胰腺干细胞标志物(PDX)-1在胰腺组织的表达水平.血糖仪检测血糖水平、放免法检测胰岛素水平、酶联免疫吸附试验(ELISA)检测SDF-1水平.结果 (1)A组残存胰岛周围可见新生血管,CD31、PCNA、PDX-1染色阳性率分别为(71.2±5.3)%、(76.5±4.5)%、(69.8±6.7)%;B组残存胰岛周围基本未见新生血管,CD31、PCNA、PDX-1染色阳性率分别为(7.4±2.1)%、(5.5±3.7)%、(8.8±2.9)%,两组比较差异有统计学意义(P＜0.05).(2)移植后第25天,A组血糖浓度基本正常,低于B组和C组,而胰岛素水平明显高于B组和C组(P＜0.05).(3)A组与B组血清SDF-1水平差异无统计学意义(P＞0.05),但都明显高于C组(P＜0.05).结论 MSCs促进胰岛再生和新生血管形成,AMD3100能抑制MSCs的作用,进而提示SDF-1/CXCR4轴在胰岛再生和血管形成中具有重要作用.

Abstract：

Objective To investigate the role of stromal cell derived factor-1 (SDF-1)/CXCR4axis in recipients' remnant islets regeneration and neovascularization after the transplantation of allogeneic bone marrow mesenchymal stem cells (MSCs). Methods MSCs were isolated from SD rats, cultured in vitro and identified by testing the phenotypes with flow cytometry ( FCM ). The diabetic rats induced by streptozotozin were randomly divided into group A ( MSCs transplant group), group B ( MSCs transplant +AMD group) and group C ( DM control group). Group D serve as the normal control. The pancreata were removed and blood serum was retrieved from each group simultaneously at the 13th day after MSCs transplant. The expression of CD31, proliferating cell nuclear antigen (PCNA) and PDX-1 in each group of pancreas tissue was detected by using immunohistochemistry, and the morphological changes in the isletswere observed by Hematoxylin and Eosin (HE) staining. Serum glucose and insulin levels were determined by blood glucose monitor, radioimmunoscintigraphy, and SDF-1 in serum was by enzyme linked immunosorbent assay (ELISA). Results Neovascularization was observed in the remnant islets of the recipient pancreatic tissue and CD31 -positive cells (71.2 ± 5.3 ) %, PCNA-positive cells ( 76. 5 ± 4. 5 ) %, PDX-1-positive cells (69. 8 ±6. 7)% were highly expressed in group A. As compared with group A, seldom-positive cells[CD31 (7.4±2. 1)%, PCNA (5.5 ±3.7)% and PDX-1 (8.8 ±2.9)%]and rarely neovascularization were observed in group B (P ＜0. 05 ). Serum glucose level in group A was lower than that in group B and group C, but serum insulin level in group A was significantly higher than that in group B and group C (P ＜ 0. 05 ). There was no significant difference between group A and group B in serum SDF-1level ( P ＞ 0. 05 ), but that was higher in groups A and B than in group C ( P ＜ 0. 05 ). Conclusion Obviously, MSCs promote recipient neovascularization surrounding the islets, which enhances the proliferation and regeneration of remnant islets. AMD 3100 has the function of intervening SDF-1/CXCR4 axis,which inhibits the effect of MSCs on promoting islets regeneration. It is suggested that SDF-1/CXCR4 axis may play an important role in vascularization and islets regeneration.     

前列腺素E1抗大鼠骨髓间充质干细胞凋亡的最佳浓度探讨

目的探讨在体外条件下前列腺素E1抗大鼠骨髓间充质干细胞(BMSCs)凋亡的最佳浓度。方法提取SD大鼠骨髓间充质干细胞,取P3-P5代的大鼠骨髓间充质干细胞在体外缺血清缺氧条件下进行培养,将前列腺素E1分别以0μg/L(对照组)、1μg/L、10μg/L、20μg/L、30μg/L、40μg/L、50μg/L、100μg/L的浓度作用于大鼠骨髓间充质干细胞,并设置48 h,72 h二个时间点,用流式细胞检测的方法测定每个时间点及每个浓度组的大鼠骨髓间充质干细胞凋亡率,最后对数据进行统计分析。结果在各个时间点中,质量浓度为1μg/L、10μg/L、20μg/L、30μg/L、40μg/L、50μg/L、100μg/L的前列腺素E1均可减少大鼠骨髓间充质干细胞的凋亡,其中20μg/L浓度组的大鼠骨髓间充质干细胞凋亡率最低;前列腺素E1在作用48 h后,大鼠骨髓间充质干细胞凋亡率最低。结论体外缺血清缺氧条件下,前列腺素E1抗大鼠骨髓间充质干细胞凋亡的最佳浓度是20μg/L,且其抗凋亡作用在48 h时达到高峰。     

AMD3100体外阻断基质细胞衍生因子1/趋化因子受体4信号通路对人关节软骨细胞分泌基质金属蛋白酶3、9、13水平的影响

目的探讨趋化因子受体4(chemokine receptor 4,CXCR4)特异性拮抗剂AMD3100体外阻断基质细胞衍生因子1(stromal cell derived factor 1,SDF-1)/CXCR4信号通路对人关节软骨细胞分泌基质金属蛋白酶(matrixmetalloproteinase,MMP)3、9、13水平的影响,明确AMD3100的作用机制。方法取12例骨关节炎(osteoarthritis,OA)患者144块软骨组织(OA软骨组)和12例创伤性截肢患者144块正常软骨组织(正常软骨组)(Mankin评分均为0或1),根据添加培养液不同,每组再分为A、B、C 3个亚组。A亚组含1 000 nmol/L AMD3100及100 ng/mL SDF-1的DMEM液,B亚组含1 000 nmol/L MAB310及100 ng/mL SDF-1的DMEM液,C亚组含100 ng/mL SDF-1的DMEM液。体外培养2、4 d后,ELISA法测定培养液内MMP-3、9、13含量,RT-PCR检测软骨组织中MMP-3、9、13 mRNA表达。结果 ELISA法及RT-PCR检测示:同组相同时间点A亚组MMP-3、9、13含量及mRNA表达均显著低于B、C亚组(P<0.05)。同一时间点相同亚组OA软骨组MMP-3、9、13含量及mRNA表达均显著高于正常软骨组(P<0.05)。结论 SDF-1通过SDF-1/CXCR4信号通路可诱导人关节软骨中MMP-3、9、13的表达和释放;AMD3100可阻断SDF-1/CXCR4信号通路,使软骨细胞MMP-3、9、13 mRNA表达水平及分泌量降低,但AMD3100不能使退变的OA软骨分泌MMP-3、9、13恢复至正常软骨水平。     

基质细胞衍生因子-1及其受体CXCR4对结肠癌肝转移潜能的影响

目的 探讨趋化因子基质细胞衍生因子-1(SDF-1)及其受体CXCR4对结肠癌肝转移潜能的影响.方法 采用Western-blot法检测不同结肠癌细胞株中CXCR4蛋白及不同组织中SDF-1蛋白的表达,MTT法检测SDF-1及抗CXCR4单抗对结肠癌细胞HT-29增殖能力的影响,体外趋化实验检测HT-29细胞定向迁移能力的变化.建立裸鼠结肠癌肝转移瘤模型,观察CXCR4特异性拮抗剂AMD3100对裸鼠肝转移率和转移瘤数目的 影响.结果 HT-29细胞表达较高强度的CXCR4蛋白,而肝组织表达高强度的SDF-1蛋白.与对照组相比,SDF-1可以诱导HT-29细胞增殖(0.76±0.11 vs0.38±0.06,P<0.05),抗CXCR4单抗对SDF-1的诱导增殖具有显著的抑制作用(0.42±0.08 vs0.76±0.11,P<0.05);SDF-1可促进HT-29细胞的趋化迁移,抗CXCR4单抗可显著抑制SDF-1诱导下HT-29细胞的迁移能力(104.6±18.3 vs 148.8±26.2,P<0.05).AMD3100治疗组裸鼠结肠癌肝转移率显著低于对照组(40% vs 100%,P<0.05),平均瘤结节数目显著低于对照组(7.8±2.6 vs 22.4±8.6,P<0.05).结论 SDF-1/CXCR4生物轴参与了结肠癌肝转移过程,拮抗CXCR4功能可抑制裸鼠结肠癌肝转移,其机制与抑制CXCR4能够有效阻断结肠癌细胞在SDF-1诱导下的细胞增殖和定向迁移有关.     

Effect of CXCR4-overexpressing bone marrow-derived mesenchymal stem cells on the repair of the co-cultured hypoxia/re-oxygenation renal tubular epithelial cells and its possible mechanism

Objective CXCR4-overexpressing bone marrow-derived mesenchymal stem cells (CXCR4-BMSC) were constructed and co-cultured with hypoxia/re-oxygenation pretreated renal tubular epithelial cells (HR-RTEC). Repair of HR-RTEC was detected and the possible mechanism was also discussed. Methods CXCR4-BMSC (CXCR4-BMSC/eGFP, eGFP as the tracer gene) and null-BMSC (BMSC/eGFP) were obtained by gene transfection technique, and the level of CXCR4 in the transfected cells was detected. RTEC was cultured under hypoxia/re-oxygenation condition for 12 h, respectively, to obtain HR-RTEC, which was used to simulate AKI in vitro. BMSC and HR-RTEC were co-cultured for 12 h, and the proportion of apoptotic cells among the HR-RTEC was assayed by immunofluorescence technique. Western blot was used to test the protein levels of cleaved Caspase-3 and Bcl-2. The number of migrating BMSC was also assayed. After culturing with the HR-RTEC culture supernatant, the expression of cytokeratin 18 (CK18) in BMSC was tested by immunofluorescence staining. Cytokines including bone morphogenetic protein-7 (BMP-7), hepatic growth factor (HGF) and interleukin-10 (IL-10) in the BMSC culture supernatant were detected by ELISA method. Results Expression of CXCR4 was enhanced in CXCR4-BMSC. Proportions of the apoptotic cells among HR-RTEC after being co-cultured with BMSC, CXCR4-BMSC and null-BMSC were all decreased, especially in the C/H group. The decreased cleaved Caspase-3 and enhanced Bcl-2 were also observed in HR-RTEC. The number of migrating CXCR4-BMSC was the highest. Proportions of CK18+ cells in BMSC, CXCR4-BMSC and null-BMSC were all low and showed no difference. However, CXCR4 overexpression in BMSC stimulated secretions of BMP-7, HGF and IL-10. Conclusions CXCR4-overexpressing BMSC has more repair effect on the co-cultured HR-RTEC, the enhanced migration ability and secretion ability of CXCR4-BMSC are the possible mechanisms.     

Stromal cell-derived factor-1 binding to its chemokine receptor CXCR4 on precursor cells promotes the chemotactic recruitment, development and survival of human osteoclasts

Osteoclasts (Oc) derive from hematopoietic precursors present in the circulation and bone marrow, and they differentiate into multinucleated bone-resorbing cells in response to the dual essential signals receptor activator of NF-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) primarily provided by bone marrow stromal cells (BMSC) and osteoblasts (Ob). However, little is known about signals that direct Oc precursors from the circulation into bone or control their migration within the marrow. Stromal cell-derived factor-1 (SDF-1 or CXCL12) is a chemokine highly expressed by bone endothelium, BMSC, and immature Ob that is essential for the normal homing, early development, and survival of various hematopoietic progenitor cells. We investigated whether SDF-1 and its unique chemokine receptor CXCR4 were involved in regulating human Oc precursor chemotaxis, development, function, or survival. CXCR4 was highly expressed by freshly isolated human monocyte (MN) populations, in vitro generated Oc and Oc-like cells, and mature Oc isolated from human femoral bones. SDF-1 markedly stimulated the chemotactic recruitment of circulating human MN capable of generating bone-resorptive Oc, leading to a 4-fold increase in Oc formation and greater bone pit resorption after their M-CSF + RANKL induced differentiation compared to spontaneously migrating cells. SDF-1 also directly promoted early (but not later) stages of Oc development via stimulating precursor cell numbers, multinucleated cell fusion, increased cell size, and tartrate-resistant acid phosphatase (TRAP) activity in a similar, but non-additive, fashion to M-CSF + RANKL. While SDF-1 did not cause full development of bone-resorbing Oc or stimulate the resorptive function of mature Oc directly, it also did not interfere with any actions promoted by M-CSF + RANKL. In mature human Oc, SDF-1 proved equally as effective as M-CSF + RANKL for preventing Oc apoptosis induced by cytokine withdrawal. In both cases, Oc survival was accompanied by analogous rises in the mRNA ratios for anti-apoptotic Bcl-xL and Bfl-1 relative to pro-apoptotic Bax, and by marked protein suppression of the critical pro-apoptotic signal Bim. These findings demonstrate for the first time that SDF-1 chemoattracts circulating human Oc precursors capable of developing into bone-resorptive Oc, and that it can stimulate MN cell fusion and TRAP activity, mimic M-CSF + RANKL in early osteoclastogenic effects, substitute for M-CSF + RANKL in maintaining the survival of mature human Oc, and suppress Oc expression of Bim protein. Thus, high levels of SDF-1 produced by bone endothelium, BMSC, and Ob may selectively target circulating Oc precursors into bone and stimulate their marrow migration into suitable perivascular stromal sites for their early development, RANKL differentiation, and survival. Consequently, SDF-1 may be a key factor linking bone vascular cells, BMSC, Ob, and Oc in the normal homeostatic regulation of bone development and remodeling.     

Transfection of antisense p38 alpha gene ameliorates myocardial cell injury mediated by hypoxia and burn serum

BACKGROUND: Myocardial damage occurs immediately following severe burns even before significant reduction in blood volume. This phenomenon is called postburn "shock heart" ("cardiac shock"), the pathogenesis of which is unclear. This study was designed to investigate the role of antisense p38 alpha gene transfection in ameliorating hypoxia and burn serum-mediated myocardial cell injury. METHODS: A model of myocardial cells cultured under hypoxia and with burn serum was established. The cells were divided into control group (group C), the group cultured under hypoxia plus burn serum (group HS), and the group treated with antisense p38 alpha gene transfection (group A-p38 alpha) and cultured under hypoxia plus burn serum. Burn serum was collected from Wistar rats with 40% TBSA III degree burns. Hypoxia was produced using a mixed gas with 1% O(2). Antisense p38 alpha gene recombinants were constructed and expression of p38 alpha kinase, and NF-kappaB subunits p50, p65 and I kappa B alpha in myocardial cells were detected by Western blot. Myocardial viability was determined by tetrazolium colorimetry (MTT). Apoptosis was detected by flow cytometry. Lactate dehydrogenase (LDH) activity in cell culture supernatants was determined. Changes in TNFalpha and IL-1 beta mRNA expression were detected by RT-PCR. RESULTS: Activation of p38 alpha kinase, expression of NF-kappaB p50, NF-kappaB p65 and I kappa B protein, and TNFalpha and IL-1 beta were downregulated significantly following antisense p38 alpha gene transfection into myocardial cells treated with hypoxia plus burn serum. Myocardial apoptosis and LDH activity in cell culture supernatants decreased markedly and myocardial viability increased significantly in the antisense p38 alpha gene treated group. CONCLUSIONS: Results demonstrated that transfection of antisense p38 alpha gene diminishes myocardial cell injury mediated by hypoxia and burn serum, suggesting a new target for the prevention and treatment of myocardial damage after burn.