文献类型：学位论文

中文题名：细胞因子修饰的MSCS联合红景天促进脊髓损伤修复的实验研究

英文题名：Experimental Study on the Cell Factors-modified MSCs Combined with Rhodiola Rosea to Improve the Recovery of Spinal Cord Injury

作者：王博[1];

第一作者：王博

机构：[1]甘肃中医学院;

第一机构：甘肃中医药大学

导师：哈小琴;甘肃中医学院

授予学位：硕士

语种：中文

中文关键词：高海拔;脊髓损伤;红景天;间充质干细胞;角质生长因子;低氧诱导因子;细胞移植;腺病毒

外文关键词：high altitude;spinal cord injury;Rhodiola;mesenchymal stem cells;keratinocyte growth factor;hypoxia inducible factor;cell transplantation;adenovirus

年份：2014

摘要：目的：在低氧环境下，对雌性大鼠脊髓撞击损伤模型，用中药红景天联合应用角质细胞生长因子/(Keratinocyte Growth Factor，KGF/)和低氧诱导因子-1（hypoxia induciblefactor-1，HIF）基因修饰的骨髓间充质干细胞（marrow-mesenchymal stem cells， MSCs）进行防治，动态评估脊髓损伤修复愈合的速度、质量及功能重建情况，将为低氧环境下脊髓再生修复及功能重建提供新的治疗策略。 方法： （1）雄性大鼠MSCs的分离、培养及鉴定，选用CD34、CD45、CD90、CD105抗体进行骨髓间充质干细胞的鉴定。（2）MSCs外源基因的修饰，MSCs分别用200MOI的Ad-KGF-HIF、Ad-KGF、Ad-HIF感染MSCs，72h收集细胞备用。（3）大鼠脊髓损伤模型建立，应用Allen打击法致雌性Wistar大鼠脊髓损伤，创建脊髓撞击损伤模型。（4）细胞移植及红景天的应用，模型大鼠分为7组：生理盐水对照组/(a/)、红景天组/(b/)、MSCs移植组/(c/)、Ad-HIF修饰的MSCs移植组/(d/)、Ad-KGF修饰的MSCs移植组/(e/)、Ad-KGF-HIF修饰的MSCs移植组/(f/)、中药红景天联合Ad-KGF-HIF修饰的MSCs组/(g/)。（5）组织学观察及功能评估， HE染色观察脊髓出血、水肿；神经尼氏体亚甲蓝染色观察神经细胞肿胀、变性、坏死变化；免疫组织化学法检测KGF和HIF蛋白的表达；功能评估标准采用改良Gale联合评分法（改良CBS法）进行评分。（6）RT-PCR法检测腺病毒及性别决定基因sry的表达丰度，以评价移植病毒的生物分布及移植细胞参与修复组织的量；原位杂交法检测Sry基因的分布定位，以确定MSCs细胞移植成功。 结果： 1、雄性大鼠MSCs的分离、培养后鉴定：结果CD105和CD90两种表面分子表达呈阳性，CD34和CD45两种表面分子表达呈阴性； 2、Ad-KGF-HIF、Ad-KGF、Ad-HIF感染MSCs修饰后，ELISA法检测到细胞中两种外源基因的表达，且两种细胞因子的表达上清可刺激低氧条件下培养的成骨细胞增殖； 3、用Allenˊs打击法成功建立了雌性Wistar大鼠脊髓损伤模型； 4、细胞移植及红景天应用后，组织学观察结果：HE染色，3~6周a组、b组、c组有较多空腔且无明显变化， d组、e组空腔减少未见新生组织，f组、g组的空腔有较多新生组织，12周a组、b组仍有较多空腔，神经纤维及细胞坏死，在横断处附近形成较多空泡，f组、g组无明显空腔，空泡小而少。神经尼氏体亚甲蓝染色，1-12周，a组、b组神经细胞损伤最严重且修复很少c组d组、e组神经有修复但不明显，f组、g组神经细胞逐渐修复，神经细胞数量增多。术后1周、3周、6周、12周评估功能指标，c组、d组、e组、f组、g组运动功能均优于红景天组与对照组，其中g组较为明显； 5、sry基因：RT-PCR法检测sry基因，移细胞移植组sry基因检测量均较高。原位杂交检测sry基因的移植后脊髓组织中的表达量，细胞移植组均表达，g组表达量高于其它组移植细胞的监测。 结论： 1、密度梯度离心法成功分离MSCs，重组腺病毒感染后可成功表达具有生物活性的HIF和KGF两种外源基因； 2、成功建立了脊髓损伤模型； 3、初步验证了在低氧环境下大鼠脊髓损伤模型中，HIF和KGF双基因修饰的大鼠MSCs联合红景天的防治方案对于促进脊髓损伤的修复具有较好的效果； 4、检测到HIF和KGF基因在局部有表达，在损伤修复组织检测到移植细胞的存在，表明转入的基因及细胞均参与了脊髓损伤的修复；
Objective：At hypoxic environment, female rats spinal cord injury model were treatedwith Rhodiola rosea extract combined with or without keratinocyte growth factor /(KGF/)and//or hypoxia inducible factor /(HIF/) gene-modified bone marrow mesenchymal stem cells/(MSCs/) with followed by dynamic assessment of spinal cord injury healing and functionalrecovery of motor neuron were assessed, which provides insight into the therapeuticprospective of spinal cord regeneration and functional reconstruction under hypoxicenvironment. Method：The bone marrow mesenchmal stem cells from male Winstar rats were isolatedby density gradient centrifugation, and transfected with either control adenovirus or KGF andHIF-carrying adenovirus. The female rat spinal cord injury model was established withmodified Allen method and70Winstar rats randomly divided into7groups: MSCstransplantation group, Ad-HH-MSCs transplantation group, Ad-HH-MSCs transplantationcombined Rhodiola group, saline control group, and the Rhodiola only group. The successfultransplantation of MSCs was determined with in situ hybridization of sex-determining geneSry gene. The spinal cord hemorrhage and edema were observed under light microscopy afterHE staining. The swelling, degeneration, and necrosis of nerve cells were analyzed by nerveNissl methylene blue staining. The distribution and participation of injury repair oftransplanted cells and the effect of Rhodiola were evaluated by detecting the expression ofKGF and HIF protein by immunohistochemistry and examining the levels of adenovirus andsry by fluorescence quantitative real-time PCR. Data were expressed as x±s. Thedifferences between groups were analyzed with student t-test using SPSS19.0statisticalsoftware. A p-value less than0.05was considered statistically significant. Result: 1、HE staining observation: no significant difference was observed among groups oneweek after transplantation; three and six weeks after injury, saline control group had morecavities whereas neuronal regeneration and tissue repair were observed in the spinal cord ofrats received MSCS transplant;12weeks after injury, the spinal cord tissue of saline controlgroup rats were darker with more cavities many microcapsules in the white matter, necrosis of nerve fibers and cells, large vacuoles in the vicinity of transection while that from ratsreceived MSCS transplant and//or Rhodiola was lighter with no significant cavities, vacuolesless and small, and denser nerve fibers and more neurons. The combined treatment ofAd-HH-MSCs transplantation and Rhodiola had the best results. 2、Nerve cell swelling, degeneration, necrosis and Nissl body changes were analyzedunder light microscopy with nerve Nissl methylene blue staining. There was no significantdifference among groups1and3weeks after injury. The number of neurons was slightly morein treatment groups compared to saline control. At week12post-injury, serious neuronaldamage, neuron reduction, and increased vacuoles were observed in saline control whereasgradual neuronal regeneration was seen in rats received MSCs and//or Rhodiola. 3、Immunohistochemical staining was employed to detect the KGF and HIF proteinlevels in the spinal cord of rats at1,2, and3weeks after injury. The KGF and HIF proteinlevels were low in saline control and Rhodiola group while there were significantlyincreased in MSCs, Ad-HH-MSCs, and Ad-HH-MSCs plus Rhodiola groups, the levels ofthose proteins peaked in the first week. The difference among groups was statisticallysignificant. 4、The expression levels of adenoviral gene and sry were analyzed by fluorescencequantitative PCR. There was no significant difference at week1and3after injury, at week6and12, the expression of adenoviral gene and sry was undetectable in saline control andRhodiola groups but strongly expressed in Ad-HH-MSCs and Ad-HH-MSCs combined withRhodiola groups. The difference among groups was statistically significant. 5、Sry gene expression in injured rat spinal cord was visualized by in situ hybridization.Sry mRNA was detected in MSCs, Ad-HH-MSCs, and Ad-HH-MSCs plus Rhodiola groupsand the abundance in those groups was in ascending order. Sry mRNA was not detected insaline and Rhodiola groups. Conclusions： 1、MSCs are successfully isolated through density gradient centrifugation. Once infectedby recombinant adenovirus, they can express HIF and KGF, two foreign genes that canexpress biological activities. 2、pinal cord injury model are successfully established. 3、It is tested and verified preliminarily that in the spinal cord jury model of rats in lowoxygen environment, for rats with HIF and KGF gene modification, the control scheme ofMSCs with rhodiola rosea is effective to the reparation of the injury. 4、It has been found that HIF and KGF have regional expression and transplanted cellsexist in injury repairing mechanism, both of which indicate that genes and cells havefunctioned in spinal cord injury repairing.