【作者】 林健清；

【导师】 张志坚；

【作者基本信息】 福建医科大学 ， 神经病学， 2009， 博士

【摘要】 第一部分构建hIL10和EGFP基因的双顺反子重组腺病毒载体及其在骨髓间质干细胞中的表达目的构建带有hIL-10基因的腺病毒载体,并感染大鼠骨髓间质干细胞(rat mesenchymal stem cells,rMSCs)后,进一步检测hIL-10表达从而获得有生物活性hIL-10修饰的rMSCs(hIL10-rMSCs)。方法以pSNAV2.0-hIL10重组质粒为模板,PCR扩增获得hIL10cDNA片段,经酶切连接到带有EGFP标记的pDC316-IRES-EGFP-lacZalpha载体质粒上,PmeI线性化重组质粒pDC316-hIL10-IRES-EGFP ,与腺病毒包装系统:AdMax转染293包装细胞,包装产生复制缺陷型重组腺病毒,经反复感染293细胞扩增病毒后,进行离子交换法纯化病毒,并测定病毒颗粒数、滴度。所获重组腺病毒感染rMSCs后,通过荧光显微镜和流式细胞仪观察测定重组腺病毒感染rMSCs的感染率,Western- blotting法测定rMSCs hIL-10蛋白表达水平。结果经PCR鉴定、限制性酶切分析及序列测定,证明已正确构建重组穿梭质粒pDC316-hIL10-IRES-EGFP和重组腺病毒质粒Ad-hIL10-IRES-EGFP。扩增纯化后,测得重组腺病毒颗粒数为3.2×1011VP/mL ,和OD260/OD280值约为2.0,滴度为1.1×1010 CCID50/mL;重组腺病毒感染rMSCs后,在荧光显微镜下和流式细胞仪上观察结果,发现明亮绿色荧光,表达呈感染复数和时间依赖性,感染复数为100时,感染率高达92.9%;通过蛋白免疫印迹法测得的hIL10蛋白亦呈感染复数和时间依赖性表达。结论已成功构建含hIL10和EGFP基因的双顺反子重组腺病毒载体,其对rMSCs具较高感染率并能很好地表达目的基因hIL10,这为下一步hIL-10免疫抑制治疗研究奠定了实验基础。第二部分脊髓胶质细胞及前炎性细胞因子在长春新碱致神经病理性疼痛中的作用及机制?目的探讨脊髓星形胶质细胞和小胶质细胞以及前炎性细胞因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和肿瘤坏死因子α(TNFα)在长春新碱致神经病理性疼痛发生发展中的作用及机制。方法雄性SD大鼠随机分为模型组和对照组。模型组通过大鼠腹腔内重复注射长春新碱建立神经病理性疼痛模型;以第1次注射长春新碱为第1天,模型组第1～5天和第8～12天腹腔注射0.1mg·(kg·d)-1长春新碱注射液(以生理盐水稀释至1mL)。对照组注射1mL生理盐水,余处理同模型组。通过检测机械性痛阈(PWT)和热痛敏潜伏期(PWL)来评价动物痛行为学改变。采用免疫组织化学、逆转录多聚酶链反应(RT-PCR)等方法,检测脊髓星形胶质细胞GFAP和小胶质细胞OX-42的表达以及脊髓前炎性细胞因子IL-1β、IL-6及TNFαmRNA表达的变化,评价脊髓小胶质细胞和星形胶质细胞活化情况及脊髓前炎性细胞因子表达与疼痛行为改变的关系。结果与对照组比较,模型组大鼠腹腔内注射长春新碱第11天出现PWT明显降低和第9天出PWL明显降低(P<0.05),并呈进行性下降趋势;随着时间进程,免疫组化结果显示模型组动物脊髓小胶质细胞和星形胶质细胞依次活化,小胶质细胞活化开始于注射长春新碱第7天,星形胶质细胞活化开始于注射长春新碱第14天;模型组动物脊髓IL-1β、IL-6和TNFαmRNA表达较对照组明显增加(P<0.05)。结论大鼠腹腔内重复注射长春新碱成功地建立了化学药物致神经病理性疼痛模型;长春新碱诱发的脊髓胶质细胞活化及前炎性细胞因子IL-1β、IL-6和TNFα的表达增加,可能在神经病理性疼痛的产生和维持中起着重要的作用。第三部分鞘内注射人IL-10基因修饰的间质干细胞对长春新碱致神经病理性疼痛大鼠的镇痛作用目的人IL-10基因修饰的间质干细胞(hIL10-MSCs)经皮鞘内注射入长春新碱致神经病理性疼痛大鼠蛛网膜下腔,观察其在蛛网膜下腔存活情况及其对大鼠的镇痛作用及可能机制。方法长春新碱反复腹腔注射制备神经病理性疼痛大鼠模型。模型大鼠60只随机分为hIL10-MSCs组、空载体-MSCs组及对照组,每组20只。hIL10-MSCs组和空载体-MSCs组分别经皮鞘内注射hIL10-MSCs细胞悬液(1×106cells/mL)以及空载体-MSCs细胞悬液(1×106cells/mL)各30uL;对照组经皮鞘内注射生理盐水30uL。各组大鼠分别于鞘内注射前,鞘内注射后3、7、14天测定机械性痛阈(PWT)和热痛敏潜伏期(PWL),同时应用荧光激发、ELISA法、免疫组化及WB技术检测hIL10-MSCs在蛛网膜下腔存活情况,腰段脊髓hIL10蛋白及脊髓胶质细胞GFAP、OX-42、IL-1β、IL-6和TNFα表达情况。结果与对照组和空载体-rMSCs组相比,hIL10-MSCs组鞘内注射后3、7、14天PWT及PWL均明显升高,差异有统计学意义(p<0.05),而空载体-MSCs组与对照组比较差异无统计学意义(p>0.05);hIL10-MSCs组和空载体-MSCs组鞘内注射细胞7、14天后在荧光激发下可见绿色荧光细胞,而对照组则未见绿色荧光细胞;对照组、空载体-MSCs组各时间点均未检测到人白介素10蛋白表达,hIL10-MSCs组腰段脊髓组织人白介素10含量在鞘内注射细胞后3、7、14天分别为1.24±0.25ng/mg、2.24±0.30 ng/mg、1.82±0.29 ng/mg。与对照组及空载体-MSCs组相比,hIL10-MSCs组腰段脊髓胶质细胞GFAP、OX-42表达明显降低,IL-1β、IL-6和TNFα表达也明显降低(p<0.05),而这些指标在空载体-MSCs组与对照组比较则无明显差异(p>0.05)。结论鞘内注射hIL10-MSCs基因工程干细胞可在蛛网膜下腔存活并能通过表达人白介素10,抑制脊髓胶质细胞活化,减少脊髓前致炎细胞因子(IL-1β、IL-6和TNFα)的释放在一定程度上减轻长春新碱大鼠神经病理性疼痛。更多还原

【Abstract】 Part One Construction of hIL10 Gene Recombinant Adenovirs Vector and Its Expression on Mesenchymal Stem CellsObjectives To obtain intact and errorless human interleukin-10(hIL10) gene. And construct adenovir(Ad) vector carrying the hIL10 gene, and then infect the rat mesenchymal stem cells (rMSCs) to get hIL10 gene-modified rMSCs.Methods The hIL-10 gene was constructed by PCR with pSNAV2.0-hIL10 recombinant plasmid as template, enzyme digestion and ligation, and inserted the obtained hIL10cDNA fragment into shuttle vector pDC316-IRES-EGFP-lacZalpha linearization with PmeI and supercoil adenovirus skeleton plasmid pAdMax for homologous recombination.293 cells was transfected with the obtained recombinant adenovirus vector pAd/ hIL10 in mediation of liposme. The replication defective recombinant adenovirus were propagated by repeat infection of 293 cells and purified by ion exchange method, then the number of viral particles was counted and the purity and titer were determined. After the the rat mesenchymal stem cells(rMSCs) were infected by the acquired Ad, the expression of hIL10 was observed with fluorine microscopy, flow cytometry and Western-blotting method.Results Both recombinant shuttle plasmid pDC316-hIL10-IRES-EGFPand recombinant adenovirus vectorAd-hIL10-IRES-EGFP were correctly constructed, and proved by PCR ,restriction analysis and sequencing. After propagation and purification ,the virus particle count , OD260/OD280 and titer of recombinant adenovirus were3.2×1011VP/mL,2.0 and1.1×1010CCID50/mL respectively. The light green fluorescence in infected rMSCs was observed under fluorine microscopy, and the expression of EGFP and hIL10 protein were in a multiplicity of infection(MOI)and time-dependent manner.Conclusions Ad vector carrying the hIL10 and EGFP gene were constructed successfully.The hIL10 gene-modified rMSCs could express hIL10 to a higher degree,which laid a foundation for further study on adenovirus vector mediated immunosuppressive therapy withhIL10 gene. Part Two Roles of spinal glia and proinflammatory cytokines in rats with vincristine induced neuropathic painObjective To investigate the roles and mechanism of spinal glia and proinflammatory cytokinesIL-1β,IL-6 and TNFαin the rat model of neuropathic pain induced by vincristine.Methods SD rats were randomly divided into two groups(model group and control group).The rat neuropathic pain model was established by repeated peritoneal injection of vincristine. The day of first injection of vincristine was defined as the first day. The rats of model group were peritoneally injected vincristine in the dose of 0.1mg·(kg·d)-1 on the days of the first day to the fifth day and the eighthday to the tweleveth day. The rats of control group were peritoneally injected with normal saline(NS) in the same volume . The thermal hyperalgesia (PWL) and mechanical hyperalgesia (PWT) of the rats were measured, and the expression levels of IL-1β, IL-6 and TNFαin the spinal cord of neuropathic pain models and controls were measured with histochemical staining and reverse transcription polymerase chain reaction (RT-PCR).Results The peritoneal injection of vincristine led to progressive thermal and mechanical hyperalgesia along time course. Histochemical staining showed that the GFAP and OX-42 optical density ratios of the spinal dorsal horn were increased with time course after vincristine injection.The levels of IL-1β,IL-6 and TNFαwere up-regulated in the spinal cord with time course.Conclusion These results demonstrated that the rat neuropathic pain model could be successfully established by repeated peritoneal injection of vincristine. In this model, vincristine induced spinal microglia and astrocyte activations, which promoted the release of IL-1β,IL-6 and TNFα, thus contributing to the maintenance of neuropathic pain. Part Three Intrathecal MSCs modified human interleukin-10 gene therapy for vincristine induced neuropathic pain in ratsObjectives To transplant hIL-10 gene-modified MSCs to the rats with vincristine induced neuropathic pain by intrathecal injection, then observe the viability of MSCs in the subarachnoid space and the analgesic effect, and explore the mechanism of vincristine induced neuropathic pain in rats.Methods Sixty model rats were randomly divided into three groups(n=20, each): hIL10-MSCs group, no-load-MSCs group and control group, and received intrathecal (IT) injection of 30uL hIL10-MSCs suspension(1×106cells/mL), 30uL no- load-MSCs suspension(1×106cells/mL)and 30uL normal saline(NS) respectively in corresponding groups. Paw withdrawl threshold(PWT)and paw withdrawl latency(PWL) were measured on the 3, 7 and 14 days after IT injection.The viability of hIL10-MSCs and no-load-MSCs , the activation of glial cell, and the expression of hIL10, TNFa ,IL-1βand IL-6 in the lumbar segment of spinal cord were identified with immunohistochemical staining , fluorescence excitation , ELISA and Western- blotting respectively.Results PWT and PWL were significantly increasd in rats treated with hIL10-MSCs compared with that of control group and no-load-MSCs group (p<0.05), whereas there was not difference found between no-load-MSCs group and control group(p>0.05). The green fluorescence was seen in the hIL10-MSCs group and the no-load-MSCs group on the 7 and 14 days after IT injection, but no green fluorescence was found in the control group. The expression of hIL10 was found in the hIL10-MSCs group, but not in the no-load-MSCs and control group. Compared with control group and no-load-MSCs group, the activity of gliaI cells and the expression of TNFa,IL-1βand IL-6 were significantly decreased in hIL10-MSCs group(p<0.05).There was not difference found between no-load-MSCs group and control group(p>0.05).Conclusion Intrathecal MSCs modified human interleukin-10 gene therapy can ease pain induced by vincristine, this effect may depend on the inhibition of the activity of spinal gliaI cells and reduction of the release of TNFa ,IL-1βand IL-6 in the lumbar segment of spinal cord by expressing hIL-10 protein of MSCs modified human interleukin-10 gene living in the subarachnoid space for long time.更多还原