【作者】 陈奇；

【导师】 李锋；

【作者基本信息】 华中科技大学 ， 外科学， 2011， 博士

【摘要】 研究目的SDF-1/CXCR4通过PI3K/Akt途径在BMSCs向损伤脊髓组织迁徙的诱导作用及影响因素。研究方法1.用Percoll密度离心法从大鼠骨髓内分离出间充质干细胞并进行传代,以免疫组化的方法(SABC法)进行鉴定。2.用Allen打击法建立大鼠急性脊髓损伤模型。3.用Real-time PCR检测BMSCs中编码CXCR4和P13K基因的存在。4.用Western Bolt检测BMSCs表面CXCR4和P13K的蛋白表达。5.用Transwell小室迁徙实验为主要的体外实验方法,研究SDF-1/CXCR4对BMSCs的体外迁徙诱导作用并精确测定SDF-1的最佳诱导浓度。实验分为：阴性对照组(上室加入BMSCs细胞悬液,下室加入不含SDF-1的血清培养基)；实验组(上室加入BMSCs细胞悬液,下室加入含不同浓度SDF-1的血清培养基)；CXCR4阻断组(上室加入CXCR4抑制剂AMD3100预处理过的BMSCs细胞悬液,下室加入不同浓度的SDF-1的血清培养基);试剂对照组(上室加入CXCR4抑制剂AMD3100预处理过的BMSCs细胞悬液,下室加入不含SDF-1的血清培养基)。用不同干预措施后急性脊髓损伤模型大鼠的脊髓功能恢复情况(后肢运动BBB评分)研究SDF-1/CXCR4对BMSCs向脊髓损伤组织迁徙的影响。实验分为：空白对照组(模型大鼠不给予任何处理)；阴性对照组(模型大鼠尾静脉注射生理盐水)；试剂对照组(模型大鼠尾静脉注射AMD3100); BMSCs移植组(模型大鼠尾静脉注射BMSCs细胞悬液)、CXCR4抑制组(模型大鼠尾静脉注射CXCR4抑制剂预处理过的BMSCs细胞悬液)。6.用Transwell小室迁徙实验为主要的体外实验方法,研究PI3K/Akt途径在SDF-1/CXCR4趋化BMSCs过程中的影响。实验分为：阴性对照组(上室加入BMSCs细胞悬液,下室加入不含SDF-1的血清培养基),试剂AMD3100对照组(上室加入CXCR4抑制剂AMD3100预处理过的BMSCs细胞悬液,下室加入不含SDF-1的血清培养基),CXCR4抑制组(上室加入CXCR4抑制剂AMD3100预处理过的BMSCs细胞悬液,下室加入含SDF-1的血清培养基),试剂LY294002对照组(上室加入PI3K抑制剂LY294002预处理过的BMSCs细胞悬液,下室加入不含SDF-1的血清培养基)),PI3K抑制组(上室加入PI3K抑制剂LY294002预处理过的BMSCs细胞悬液,下室加入含SDF-1的血清培养基),外源性处理组(上室加入CXCR4抑制剂AMD3100预处理过的BMSCs细胞悬液和外源性PI3K产物PIP3,下室加入含SDF-1的血清培养基)。用不同干预措施后急性脊髓损伤模型大鼠的脊髓功能恢复情况(后肢运动BBB评分)研究SDF-1/CXCR4对BMSCs向脊髓损伤组织迁徙的影响。实验分为：空白对照组(模型大鼠不给予任何处理)；阴性对照组(模型大鼠尾静脉注射生理盐水)；试剂AMD3100对照组(模型大鼠尾静脉注射AMD3100)；试剂LY294002对照组(模型大鼠尾静脉注射LY294002)；BMSCs移植组(模型大鼠尾静脉注射BMSCs细胞悬液)；CXCR4抑制组(模型大鼠尾静脉注射CXCR4抑制剂AMD3100预处理过的BMSCs细胞悬液)；PI3K抑制组(模型大鼠尾静脉注射PI3K抑制剂LY294002预处理过的BMSCs细胞悬液)；外源性处理组(模型大鼠尾静脉注射CXCR4抑制剂AMD3100预处理过的BMSCs细胞悬液和外源性PI3K产物PIP3)。7.本研究中,均值以x±s表示。用方差分析(Analysis of variance, ANOV)分析多组间的差异,组间的两两比较用q检验进行分析。所有数据用统计分析软件包(Statistic Package for Social Science, SPSS)17.0处理,以P<0.05认为差异具有统计学意义。研究结果1. BMSCs的细胞形态及免疫组化鉴定：典型的BMSCs呈长梭形或多角形的成纤维细胞样,有长短、粗细不一的胞浆突起。细胞早期呈集落样生长,集落呈放射状或漩涡状,后期细胞逐渐密集,集落不可分辨。免疫组化显示CD34-、CD44+、CD54+、CD99+。2. Allen打击法建立大鼠急性损伤模型：用70g.cm的能量对大鼠T10-11水平脊髓进行垂直打击,打击瞬间可见大鼠四肢及躯体抽搐,尾部呈痉挛样摆动,表示打击成功。3-5min后可见受损伤脊髓呈暗红色,有出血、水肿表现。建模后饲养一周,后肢运动BBB评分从术前的18.73±0.85降至术后的3.04±0.65,差异具有统计学意义(P<0.05)。3. BMSCs中CXCR4和PI3K的基因存在和蛋白表达：BMSCs存在CXCR4基因和PI3K基因,Real-time PCR的扩增ACT值分别为16.47和13.43。CXCR4和PI3K在BMSCs中均有蛋白表达。4. SDF-1/CXCR4对BMSCs迁徙的诱导作用：在体外实验中,试剂对照组和阴性对照组迁徙到下室的细胞数没有明显差异(P>0.05),试剂AMD3100本身对BMSCs的迁徙没有影响。在此基础上,相比阴性对照组,下室中加入SDF-1无血清培养基的小室,迁徙到下室的细胞数都有增加(P<0.05),且随着下室中SDF-1浓度的增加而增加。SDF-1浓度为144.5±4.4ng/ml时达到最佳迁徙效果,继续增加SDF-1浓度,迁徙到下室的细胞数也不会随之增加。AMD3100阻断CXCR4后,SDF-1对BMSCs不再有诱导迁徙作用(P>0.05)。5. P13K/Akt途径对SDF.1/CXCR4诱导BMSCs迁徙的影响：在体外实验中,验证试剂AMD3100和LY294002对BMSCs的自然迁徙没有影响的基础上(P>0.05),CXCR4抑制组和PI3K抑制组迁徙到下室的细胞数均小于正常的SDF-1趋化组(P<0.05),而在CXCR4抑制组中外源性的加入PI3K产物PIP3,则又可以观察到SDF-1的趋化作用。在体内实验中,验证了试剂AMD3100、LY294002以及注射操作对实验结果没有影响(P>0.05),向模型大鼠尾静脉注射PI3K抑制剂LY294002预处理过的BMSCs悬液与注射CXCR4抑制剂AMD3100预处理过的BMSCs悬液,模型大鼠的脊髓功能恢复程度均相同,均介于移植BMSCs组和阴性对照组之间(P<0.05)。在CXCR4抑制组中外源性的加入PI3K产物PIP3,模型大鼠的脊髓功能恢复与不抑制CXCR4时的效果相同(P>0.05)。研究结论1. Percoll密度离心法可以从大鼠长骨骨髓中分离出纯度较高的BMSCs。2. Allen打击法能够建立符合要求的大鼠急性损伤模型。3. SDF-1/CXCR4可以诱导BMSCs向受损的脊髓组织迁徙。4.SDF-1浓度为144.5na/ml时SDF-1/CXCR4对BMSCs的迁徙诱导效果达到最佳。5. PI3K/Akt途径是SDF-1/CXCR4诱导BMSCs向受损脊髓组织迁徙的下游机制。更多还原

【Abstract】 ObjectsTo investigate the effect of SDF-1/CXCR4 induced migration of bone marrow mensenchymal stem cells (BMSCs) to injured spinal cord via PI3K/Akt pathway in rats.Methods1. Isolated BMSCs from bone marrow of rat by density gradient centrifugation with percoll, and transfered these cells from generation to generation. Identified these cells with immunohistochemistry (Sreptavidin-biotin complex, SABC)2. Built model of rat with acute spinal cord injury by Allen’s method.3. Detected gene of CXCR4 and PI3K in BMSCs with Real-time PCR.4. Detected expression of CXCR4 and PI3K in BMSCs with Western Bolt5. Took Transwell migration experiment as the method of studying migration of BMSCs induced by SDF-1/CXCR4 in vitro, and got the peak concentration of SDF-1. Groups were divided into negative control group (cell suspension of BMSCs in upper room, serum culture medium without SDF-1 in lower room), experimental group (cell suspension of BMSCs in upper room, serum culture medium with SDF-1 of different concentration in lower room), CXCR4 blocked group (cell suspension of BMSCs pre-treated with AMD3100 in upper room, serum culture medium with SDF-1 of different concentration in lower room), and reagent control group (cell suspension of BMSCs pre-treated with AMD3100 in upper room, serum culture medium without SDF-1 in lower room). Studied the migration of BMSCs to injured spinal cord in vivo by compared the recoveries of spinal cord function of model rats with different treatments. Groups were divided into blank control group (no treatment given), negative control group (injected normal saline through caudal vein), AMD3100 control group (injected AMD3100 through caudal vein), BMSCs transplanted group (injected BMSCs through caudal vein), and CXCR4 blocked group (injected BMSCs pre-treated with AMD3100 through caudal vein).6. Took Transwell migration experiment as the method of studying the effect of PI3K/Akt pathway in the migrating of BMSCs induced by SDF-1/CXCR4 in vitro. Groups were divided into negative control group (cell suspension of BMSCs in upper room, serum culture medium without SDF-1 in lower room), AMD3100 control group (cell suspension of BMSCs pre-treated with AMD3100 in upper room, serum culture medium without SDF-1 in lower room), LY294002 control gourp (cell suspension of BMSCs pre-treated with LY294002 in upper room, serum culture medium without SDF-1 in lower room), CXCR4 blocked group (cell suspension of BMSCs pre-treated with AMD3100 in upper room, serum culture medium with SDF-1 in lower room), PI3K blocked group (cell suspension of BMSCs pre-treated with LY294002 in upper room, serum culture medium with SDF-1 in lower room), and exogenous PIP3 added group (cell suspension of BMSCs pre-treated with AMD3100 and exogenous PIP3 in upper room, serum culture medium with SDF-1 in lower room). Studied the migration of BMSCs to injured spinal cord by compared the recoveries of spinal cord function of model rats with different treatments. Groups were divided into blank control group (no treatment given), negative control group (injected normal saline through caudal vein), AMD3100 control group (injected AMD3100 through caudal vein), LY294002 control group (injected LY294002 through caudal vein), BMSCs transplanted group (injected BMSCs through caudal vein), CXCR4 blocked group (injected BMSCs pre-treated with AMD3100 through caudal vein), PI3K blocked group (injected BMSCs pre-treated with LY294002 through caudal vein), and exogenous PIP3 added group (injected BMSCs pre-treated with AMD3100 and exogenous PIP3 through caudal vein).7. Means were showed with x±s in this study. Analysis of variance (ANOV) was used to compare difference among more than two groups, q-test was used to compare difference between two groups. All data were treated with Statistic Package for Social Science (SPSS) 17.0. Results were accepted wiht P<0.05.Results1. Cytomorphology and results of immunohistochemistry of BMSCs:typical cytomorphology of BMSCs was similar with fibrocyte, long spindle or polygon with long or short cytoplasm protuberant. Cells gathered to colonies, which were radial or gyrate, in early growth period. Cell colonies became different to identify with the increase of cell number. CD34-、CD44+、CD54+、CD99+ were the results of immunohistochemistry.2. Rat model of acute spinal cord injury built by Allen’s method:spinal cord of rat was hit with the energy of 70g-cm. When its spinal cord was hit, limbs and body of the rat shocked and its tail wiggled. Spinal cord changed to dark red with bleeding and edema 3 to 5 minutes later. One week later, the BBB scores of model rats declined from 18.73±0.85 pre-operation to 3.04±0.65 post-operation, the difference was significant (P<0.05). 3. The existences and expressions of CXCR4 and PI3K genes in BMSCs:the existence of CXCR4 and PI3K genes were detected with real-time PCR, changes of cycle threshold (ΔCT) were 16.47 and 13.43. Expressions of CXCR4 and PI3K were detected with western blot.4. Migration of BMSCs to injured spinal cord induced by SDF-1/CXCR4:AMD3100 and was proved to have no influence to experiment (P>0.05). In vitro experiment, more cells migrated to lower room in experimental group compared with negative control group (P<0.05), and cell number increased with concentration of SDF-1. Best effect was observed when SDF-1 reached the concentration of 144.5±4.4ng/ml. SDF-1 didn’t induce the migration of BMSCs when CXCR4 was blocked with AMD3100.5. Influence of PI3K/Akt pathway to the migration of BMSCs to injured spinal cord induced by SDF-1/CXCR4:AMD3100 and LY294002 were proved to have no influences to experiment (P>0.05). In vitro experiment, less cells migrated to lower room in CXCR4 blocked group and PI3K blocked group compared with SDF-1 control group (P<0.05). However, there was no difference between exogenous PIP3 added group and SDF-1 group (P>0.05). In vivo experiment, recoveries of spinal cord function model rats in CXCR4 blocked group and PI3K blocked group was similar with each other (P>0.05), both were between that in BMSCs transplanted group and negative group (P<0.05). Recoveries of spinal cord function model rats in exogenous PIP3 added group were the same as that in BMSCs transplanted group (P>0.05).Conclusion1. BMSCs of higher purity can be isolated from bone marrow of rat by density gradient centrifugation with percoll.2. Rat model of acute spinal cord injury can be build with Allen’s method.3. SDF-1/CXCR4 can induce the migration of BMSCs to injured spinal cord.4. Best effect of migration can be obtained when the concentration of SDF-1 reach 144.5ng/ml. 5. PI3K/Akt pathway is the downstream mechanism of migration of BMSCs to injured spinal cord induced by SDF-1/CXCR4.更多还原