【作者】 王超锋；

【导师】 阮狄克；

【作者基本信息】 第四军医大学 ， 外科学， 2010， 博士

【摘要】 椎间盘退变是引起成人腰腿痛、颈肩痛的主要原因。随着人均寿命的延长和社会工作压力增大,椎间盘退变性疾病的发病率明显升高,而目前的保守治疗和外科手术治疗均不能从根本上,解决患者的病情。通过动物实验及临床初步应用证明,椎间盘移植后可以异体存活,并能完全缓解患者的临床症状,就其生物力学方面亦可满足生理活动需要,但植入的异体椎间盘在生化代谢及组织学上有明显的退行性改变,这将导致临床症状的再次出现,并可能出现椎间盘活动度消失等并发症。最近的研究结果显示椎间盘内细胞活性的提高能延缓甚至阻止椎间盘退变性改变。因此本实验拟通过以异体椎间盘为支架材料,以表达外源性人BMP7的髓核细胞为种子细胞,体外构建组织工程椎间盘,并观察组织工程椎间盘的生物活性及功能情况。1.深低温储存温度及时间对异体椎间盘生物活性的影响目的:探讨不同的深低温储存温度及储存时间对异体椎间盘生物活性影响,获得最适合临床应用的保存条件。方法:取犬椎间盘52只,分为对照组、液氮保存组和-80℃保存组。在冷冻保存液中分别储存于液氮及-80℃低温冰箱中,在储存的2w、1m、2m、4m、6m及12m时,通过EB/FAD法检测椎间盘组织中不同部位的细胞存活率,以DMMB法检测髓核组织蛋白多糖(PG)含量,以羟脯氨酸法检测髓核组织中的总胶原含量。结果:各检测时间点两组间外层纤维环细胞活性明显高于内层纤维环及髓核组织(p<0.05),内层纤维环及髓核组织间细胞活性未见明显不同;在内层纤维环及髓核组织中,细胞存活率在储存2w时,可见液氮组明显高于-80℃组(p<0.05),自储存1月起至12个月,两组间均无明显差别(p>0.05)。在储存期间两储存组蛋白多糖含量均逐渐降低,在1m至12m储存期间,液氮保存组蛋白多糖含量均高于-80℃保存组。而两组间总胶原蛋白含量在储存期间随时间逐渐降低,但两组间未见明显差异。结论:利用液氮保存椎间盘组织,无论在细胞总的存活率及PG含量上均优于-80℃,1年的液氮保存期间,细胞存活率及功能蛋白PG含量均在一个恒定的水平,能满足异体椎间盘移植的基本要求,可作为组织工程椎间盘的支架材料。2.重组腺相关病毒人BMP7(rAAV2-hBMP7)病毒载体的构建及鉴定目的:探讨能否利用pSNAV2.0质粒构建系统构建pSNAV2.0-hBMP7穿梭质粒,并包装纯化成可供实验用rAAV-hBMP7病毒载体。方法:利用AgeI和NheI酶切pDC316-hBMP7-IRES-EGFP质粒,回收纯化hBMP7基因片段,以AgeI和NheI酶切载体质粒pSNAV2.0-LacZa,回收纯化pSNAV2.0载体质粒片段,利用T4 DNA连接酶将纯化回收的hBMP7基因与pSNAV2.0载体质粒片段连接构建,并转化入感受态大肠杆菌DH-5α进行扩增纯化,测序鉴定;用Lipofectamine 2000将序列正确的pSNAV2.0- hBMP7质粒转染至BHK-21细胞,G418选择培养,并大量扩增至所需并用HSV1-rc/ΔUL2辅助病毒感染,以PEG8000/NaCl沉淀,以氯仿纯化浓缩、检测滴度及纯度。结果:成功构建了pSNAV2.0- hBMP7质粒,经PCR、酶切和测序鉴定构建正确。利用Lipofectamine 2000将序列正确的pSNAV2.0- hBMP7质粒转染至BHK-21细胞,并通过含有G418的培养基,选择性扩增,并浓缩纯化出了滴度达5.1×1011 v.g、纯度>97%的rAAV-hBMP7病毒载体。结论:利用pSNAV2.0质粒包转系统能成功将外源性基因hBMP7包装成滴度及纯度满足实验要求的rAAV-hBMP7病毒载体。3.介导人骨形成蛋白-7的2型腺相关病毒转染髓核细胞及其对髓核细胞表型的影响目的:以介导外源性人骨形成蛋白-7(hBMP7)基因的2型腺相关病毒(recombinant adeno-associated virus type-2,rAAV2)载体转染犬髓核细胞(canine nucleus pulposus cell, cNP cell),观察转染后髓核细胞hBMP7蛋白表达,并分析髓核细胞生物功能变化。方法:实验组以最佳感染复数(MOI)1×105 vg/cell转染犬髓核细胞,对照组以相同MOI的不含hBMP7基因的rAAV2-EGFP病毒感染。在转染后4d、7d和14d分别以RT-PCR、Wenstenbloting方法分别对两组髓核细胞中hBMP7的mRNA转录及蛋白表达进行检测。在转染后的第7天,检测两组细胞增殖能力。在转染后的4、7和14d,利用real-time PCR、DMMB及Elisa法分别检测蛋白多糖、Ⅰ和Ⅱ胶原的mRNA含量、蛋白多糖含量及Ⅰ和Ⅱ型胶原蛋白含量。结果:在以1×105 vg/cell转染犬髓核细胞后,在4d、7d和14d时均可检测到实验组髓核细胞中高表达hBMP7 mRNA,而对照组,无论在何时间点均不能检测到hBMP7 mRNA的转录,半定量分析显示,7d时mRNA含量较高。Westenbloting蛋白检测结果显示:7d、14d时,实验组均可检测到分子量为55kd特异性hBMP7蛋白的表达,而对照组均未观察到阳性蛋白条带。半定量分析显示:转染后7d时hBMP7蛋白含量相对较高。在转染7天后,转染组与未转染组细胞增殖能力未见明显区别。对实验组髓核细胞的Real-time PCR定量分析发现:蛋白多糖、Ⅱ型胶原mRNA在转染后第4天无明显增加,但在第7天和14天均明显增高。而Ⅰ型胶原mRNA在4、7和14天均未见明显变化。实验组蛋白多糖含量第4天未见明显变化,而第7、14天较对照组分别增高42%及77%。而Ⅰ胶原含量两组间未见明显增加,实验组Ⅱ型胶原含量在第4天未见明显增加,而第7、14天分别较对照组增高63%及94%。结论: rAAV2-hBMP7病毒载体能有效地转染犬髓核细胞并长期稳定表达人骨形成蛋白-7,并能提高犬髓核细胞蛋白多糖及Ⅱ型胶原含量,这将能为组织工程椎间盘提供可选择的基因修饰的种子细胞。4.组织工程椎间盘的体外构建目的:探讨利用液氮储存异体椎间盘及表达外源性人BMP7的髓核细胞体外构建组织工程椎间盘的方法方法:取储存于液氮中2个月的犬椎间盘24只,分为EGFP对照组、1×104、1×105和1×106四组,每组6只。对照组用16G注射器自椎间盘后正中注入1×105的表达EGFP的髓核细胞20ul, 1×104组注入PKH-26标记的表达hBMP7的髓核细胞20ul,其总细胞数为1×104,1×105组为总细胞数1×105的PKH-26标记的表达hBMP7的髓核细胞20ul,1×106组为细胞总数1×106的PKH-26标记的表达hBMP7的髓核细胞20ul。注射后立即置入50ml离心管中,加入30ml完全培养基,分别于培养4、7、14天,从椎间盘形态、细胞存活、及髓核细胞荧光强度、蛋白多糖及胶原含量等方面进行评价。结果:在储存的第4天及7天,椎间盘外形基本未见明显改变,而在储存至14天时,可见椎间盘上下终板有钙质脱落。在各时间点均可见表达绿色荧光蛋白髓核细胞。对不同组、不同时间点的组织工程椎间盘中荧光强度定量后发现:培养第7和14天时,1×105组荧光强度明显高于1×106组和1×104组。而且1×105组培养第7和14天时蛋白多糖及总胶原含量均较另外三组明显增高。结论:利用液氮储存异体椎间盘同1×105转染髓核细胞复合,体外培养7天能构建出较理想的组织工程椎间盘。小结:1.利用液氮保存椎间盘组织,无论在细胞总的存活率及PG含量上均优于-80℃,1年的液氮保存期间,细胞存活率及功能蛋白PG含量均在一个恒定的水平,能满足异体椎间盘移植的基本要求,可作为组织工程椎间盘的支架材料。2.利用pSNAV2.0质粒包转系统能成功将外源性基因hBMP7包装成滴度及纯度满足实验要求的rAAV-hBMP7病毒载体。3. rAAV2-hBMP7病毒载体能有效地转染犬髓核细胞并长期稳定表达人骨形成蛋白-7,并能提高犬髓核细胞蛋白多糖及Ⅱ型胶原含量,这将能为组织工程椎间盘提供可选择的基因修饰的种子细胞。4.利用液氮储存异体椎间盘同1×105转染髓核细胞复合,体外培养7天能构建出生物活性较高组织工程椎间盘。这将能被用于进一步动物实验,以研究组织工程椎间盘的生物活性。更多还原

【Abstract】 Human intervertebral disc degeneration is mainly reason for low back pain and neck-should pain. The ratio of degenerative disc disease step up obviously with lengthening average longevity of human and increasing social work stress. Moreover, the expectant treatment and surgical treatment couldn’t thoroughly solve clinical problem at present. It has been proved that the frozen allogenic intervertebral discs could survive in animal study and clinical trail. Furthermore, the allogenic intervertebral disc could completely relieve clinical symptoms in clinical trail. The motion and stability of the spinal unit is preserved after transplantation of frozen allogenic intervertebral discs. But signs of mild disc degeneration were observed in allogenic intervertebral disc transplantation. This mild disc degeneration will cause clinical symptoms emergence once more. The present study results showed that the change of intervertebral disc degeneration could be prevent, even repair, by increasing the active of nucleus pulposus cell. So in this study, tissue engineering intervertebral disc were constructed by using allograft intervertebral disc with the cannie nucleus pulposus cell expressing bone morphogenetic protein-7. Furthermore, the bioactive and function were also investigate in this study.1. Effects of different storage temperatures and times on cell viability of cryopreserved intervertebral discObjectives. To investigate effects of different storage temperatures and times on cell viability of cryopreserved intervertebral disc and obtain the optimal storage condition.Methods. The 52 canine intervertebral discs were divided into the control group, liquid nitrogen group and -80℃group. The intervertebral discs in liquid nitrogen group and -80℃group were immerged into the freezing preservation medium and storage in liquid nitrogen and -80℃low temperature refrigerator. At the time-points of 2w、1m、2m、4m、6m and 12m, the ratio of different position cell survival were detected by EB/FDA dying method, the PG content of NP tissue were detected by DMMB method, and the collagen content were detected by hydroxyproline method.Results. The ratio of cell survival in outer layer annulus fibrosus is higher than that in inner layer annulus fibrosus and nucleus pulposus in both groups (p<0.05). The ratio of cell survival is not significantly different in inner layer annulus fibrosus and nucleus pulposus. The ratio of cell survival in inner layer annulus fibrosus in liquid nitrogen group is higher that in -80℃group in the 2nd storage week (p<0.05). The ratio of cell survival in the both groups was not different from 1st to 12th storage month (p>0.05). The PG content reduced gradually with storage duration. From 1st to 12th storage month, the PG content in liquid nitrogen is higher than that in -80℃group. The collagen content in both groups reduced gradually in storage duration, and was not different(p>0.05). Conclusion:Both the ratio of cell survival and PG content of intervertebral disc storaged in liquid nitrogen is higher than that of in -80℃. In 1 year storage duration, intervertebral disc storaged in liquid nitrogen maintains the stable ratio of cell survival and PG content, is able to suitable for the basic requirement to allogenic transplantation, is able to be the scaffold of tissue engineering.2. Construction and identification of recombination adeno-associated virus type-2 vector carrying human bone morphogenetic protein-7Objectives: To construct and identification of the recombinant adeno-associated virus type-2 vector carrying human bone morphogenetic protein ( rAAV2-hBMP7).Methods: The recombinant AAV2 packaging plasmid pSNAV2.0 and full-length hBMP7 cDNA was respectively obtained by the simultaneous digestion of plasmid pDC316-BMP7-IRES-EGFP with AgeIase and NheIase. The full-length hBMP7 cDNA and recombinant AAV2 packaging plasmid pSNAV2.0 were then integrated using T4 DNA ligase. The integrated product was used to transfect the competence Bacillus coli DH-5α. The transfected bacillus coli DH-5αwas cultured in agarose culture medium containing ampicil. Monoclonal colonies were selected and inoculated into Luria Broth (LB) culture medium containing ampicil. After 12 hours in culture, plasmid pSNAV2.0-hBMP7 was extracted and the base sequence was analyzed. After constructed and identified plasmid pSNAV2.0-hBMP7, rAAV2-hBMP7 vector were constructed, amplifying and purifying by AAVMaxTM package and purification system. The optimal multiplicity of infection (MOI) for hNP cell were detected by rAAV vector carrying enhanced green fluorescent protein r(AAV2-EGFP).Results: Plasmid pSNAV2.0-hBMP7 was successfully constructed by integrating the full-length hBMP-7 cDNA and recombinant AAV2 packaging plasmid pSNAV2.0 using T4 DNA ligase. Plasmid pSNAV2.0-hBMP7 base sequence was correct by PCR detection and assay of base seguence. The rAAV2-hBMP7 vector was packaged by pSNAV2.0-hBMP7 plasmid using the standard calcium phosphate precipitation method. The titer was determined using quantitative DNA dot blots and purity was examined using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Titers averaged approximately 5.1×1011 vg/ml and purity was >97%.Conclusion:The rAAV2-hBMP7 vector can be successfully constructed by plasmid pSNAV2.0 package system. The titers and purity of the rAAV2-hBMP7 vector is suitable for next experiments.3.Effects of adeno-associated virus-2 mediated human BMP-7 gene transfection on the chondrocytic phenotype of nucleus pulposus cellsObjectives: To investigate the effects of adeno-associated virus-2 expressing human bone morphogenetic protein-7 (rAAV2-hBMP7) on canine nucleus pulposus cells.Methods: Following infection with rAAV-BMP7 vector at multiplicities of infection of 1×105 genomes per cell and subsequent culture, nucleus pulposus cells transferred with hBMP7 gene were assessed semi-qualitatively for BMP7 expression with real-time PCR. At 7 days post-transfection, proliferative ability of nucleus pulposus cells were comparative in the transfection and no-transfection cells. Aggrecan, typeⅠand typeⅡcollagen secreted by nucleus pulposus cells were qualitatively accessed at 4, 7 and 14 days post-transfection in the transfection and control groups.Results: The adeno-associated virus can successfully transfer human BMP7 into the canine NP cell. The NP cell transfected by rAAV-hBMP7 vector express human BMP7 at least 14 days. The expressed human BMP7 promote remarkably accumulation of proteoglycans 42% and 77% (p<0.05) higher than no-transfection cells at 7 and 14 days post-transfection, and typeⅡcollagen 63% and 94% (p<0.05).Conclusion: The rAAV-based gene delivery approach is capable of promoting the expression of proteoglycans and typeⅡcollagen of nucleus pulposus. This approach may be applied for the treatment of degenerative disc disease in the future.4. Construction of Tissue Engineering Intervertebral Disc Using allograft Intervertebral Disc with the Cannie Nucleus Pulposus Cell Expressing Bone morphogenetic protein-7Objective: To construct the tissue engineering intervertebral disc using allograft intervertebral disc with the cannie nucleus pulposus cell expressing hBMP7Methods: The 24 intervertebral discs storage in liquid nitrogen for 2 months were divided into EGFP, 1×104、1×105 and 1×106 groups. EGFP group: 20ul cell suspension including 1×105 nucleus pulposus cells expressing EGFP were injected into intervertebral disc. 1×104 group: 20ul cell suspension including 1×104 nucleus pulposus cells expressing hBMP7 by PKH-26 dying were injected into intervertebral disc. 1×105 group: 20ul cell suspension including 1×105 nucleus pulposus cells expressing hBMP7 by PKH-26 dying were injected into intervertebral disc. 1×106 group: 20ul cell suspension including 1×106 nucleus pulposus cells expressing hBMP7 by PKH-26 dying were injected into intervertebral disc. Intervertebral discs injected were immerged into 30 ml culture medium. The morphous, cell survival, cell fluorescence intensity, PG and collagen content of intervertebral discs in every group were assessed at 4, 7, 14 culture days.Results: The morphous of intervertebral discs in every group didn’t change at 4 and 7 culture days. However, the calcium content in the lamina terminalis of intervertebral disc lost at 14 culture days. In EGFP group, nucleus pulposus cells expressing hBMP7 could be observed at any time points. Results showed that cell fluorescence intensity in 1×105 group was higher than that of 1×106 and 1×104 groups. Moreover, the PG and collagen contents in 1×105group were higher than that of 1×106 and 1×104 groups.Conclusion: The ideal tissue engineering intervertebral disc could be constructed using allograft intervertebral disc storaged in liquid combined with the cannie nucleus pulposus cell expressing hBMP7, and then be cultured for 7days in vitro.Summary:1. Both the ratio of cell survival and PG content of intervertebral disc storaged in liquid nitrogen is higher than that of in -80℃. In 1 year storage duration, intervertebral disc storaged in liquid nitrogen maintains the stable ratio of cell survival and PG content, is able to suitable for the basic requirement to allogenic transplantation, is able to be the scaffold of tissue engineering.2. The rAAV2-hBMP7 vector can be successfully constructed by Plasmid pSNAV2.0 package system. The titers and purity of the rAAV2-hBMP7 vector is suitable for next experiments.3. The rAAV-based gene delivery approach is capable of promoting the expression of proteoglycans and typeⅡcollagen of nucleus pulposus. This approach may be applied for the treatment of degenerative disc disease in the future. 4. The ideal tissue engineering intervertebral disc could be constructed using allograft intervertebral disc storaged in liquid combined with the cannie nucleus pulposus cell expressing hBMP7, and then be cultured for 7days in vitro.更多还原