【作者】 王志涛；

【导师】 张建宁；

【作者基本信息】 天津医科大学 ， 外科学， 2010， 博士

【摘要】 颅脑创伤(traumatic brain injury, TBI)是一种致死率和致残率都很高的疾病,给人类个体、家庭乃至整个社会都带来了许多的负担。对于颅脑创伤的治疗,人们一直致力于对损伤脑组织保护的研究,以减轻原发性和继发性脑损伤。利用干细胞治疗中枢神经系统损伤,是目前学术界广泛关注的神经损伤治疗新策略,它为颅脑创伤后的神经修复治疗带来了新的曙光。目前研究的重点为调控干细胞治疗的安全性和有效性。近年来,研究人员发现骨髓间充质干细胞(bone marrow stromal cell, BMSC)是一种优秀的载体细胞,它取自自体骨髓,取材方便,且体外易培养,增殖快。并且由于来源于自体,BMSCs免疫原性较弱,能够抑制混合淋巴细胞反应,由它诱导而来的组织在进行移植时不存在组织配型及免疫排斥等问题,亦不涉及伦理道德问题,因此通过体外培养扩增后的BMSCs是组织工程中理想的种子细胞。它在体内或体外诱导条件下可以向神经元方向转化,也可以分泌神经生长因子,如神经生长因子(neural growth factor, NGF)、脑源性神经生长因子(brain-derived neurotrophic factor,BDNF)等,从而发挥对损伤神经组织的修复作用。但BMSC也会因其生存周期短且不稳定而影响其发挥神经保护功能。本研究通过比较,筛选出脑创伤修复相关基因脑源性神经生长因子(BDNF)；进一步通过转染阳离子脂质体介导的外源性端粒酶表达,使BMSC永生化,获得了hTERT调控的的永生化大鼠BMSC,避免细胞过早衰老,稳定其在体内外的生长。我们进一步构建BDNF高效表达载体,并转染Ad5-BDNF至hTERT-BMSC,证明了转染BDNF的永生化BMSC在体外通过高表达BDNF具有一定的损伤细胞修复作用。在此基础上,还借助大鼠TBI模型和立体定向技术研究TBI组织内该细胞系统的生物学特征与转归,证明了其在大鼠体内的安全性和对TBI治疗的有效性,为BMSC治疗TBI步入临床奠定基础。本研究分为三个部分。第一部分研究是通过原代贴壁培养法获得性状稳定的大鼠BMSCs,利用流式细胞术检测其表面标记抗原,我们进一步通过体外诱导的方法检测rBMSCs的向成骨细胞、成脂肪细胞和神经样细胞方向的分化潜能。研究结果显示,原代rBMSCs易于提取和体外纯化、扩增,鉴定其标记物符合BMSCs的特征,其在体外能可通过诱导向脂肪样细胞、成骨样细胞及神经元样细胞分化,提示其具有多向分化潜能,证实rBMSCs培养成功。第二部分研究是通过转染pLXSN质粒介导的人端粒酶逆转录酶催化亚单位(human telomerase reverse transcriptase hTERT)来增强BMSCs端粒酶的活性,获得了Ad5-hTERT调控的永生化大鼠BMSC,利用ELISA方法检测转染细胞的端粒酶活性。同时,通过构建腺病毒介导的BDNF高效表达载体Ad5-BDNF,将Ad5-BDNF复合体转染至hTERT-BMSC,证明了转染BDNF的永生化BMSC在体外具有一定程度的损伤细胞修复作用,为Ad5-BDNF-BMSC的体内实验奠定基础。第三部分研究中我们选取国际上较为普遍采用的大鼠液压颅脑损伤模型,在1.5-2.0atm条件下制备大鼠中型颅脑创伤模型,同时借助于立体定向细胞移植技术,将共转染BDNF和hTERT基因的BMSCs移植到损伤脑组织区,观察细胞移植治疗后大鼠生存期变化、神经损伤评分(neurologic severity score, NSS)的变化以及损伤区脑组织水肿在核磁共振成像(MRI)的变化,评价基因修饰的BMSCs在大鼠液压颅脑创伤模型体内的安全性和对TBI治疗的有效性,为BDNF和BMSCs联合治疗TBI步入临床奠定基础。上述研究结果表明：大鼠BMSCs易于分离、培养和扩增,长期体外培养BMSCs可出现过早老化和过度增殖等生物学性状不稳定现象；hTERT正以表达载体pLXSN-S-hTERT转染至大鼠BMSC,可以使BMSC的端粒酶活性增强,促进BMSCs增殖,并保持在长期传代培养过程中形状稳定性,是建立BMSCs永生化细胞系的安全可靠而有效的方法；Ad-BDNF-hTERT-BMSCs在体内外可以高表达BDNF,能够有效减少受损神经元的的凋亡,提高脑损伤大鼠的生存率,并能促进其神经功能的康复,为BDNF和BMSCs治疗TBI步入临床奠定基础。更多还原

【Abstract】 Traumatic brain injury (TBI) is a disease of high fatality and mutilation rate, which has brought a lot of burden to the human individual, family and society. For the treatment of traumatic brain injury, people have been committed to the protection of brain tissue damage in order to reduce primary and secondary brain injury. Using stem cells to treat central nervous system damage, is the academic attention of a new strategy for treatment of nerve injury, which has brought a new dawn to the nerve recover after brain trauma. The current focus of the study is the regulation of stem cell therapy for safety and effectiveness. In recent years, researchers found that bone-marrow mesenchymal stem cells (BMSCs) are excellent carrier cells, which are derived from autologous bone marrow, easy to culture in vitro, and the proliferation of fast. And as derived from autologous, the immunogenicity of BMSCs is weak and they could inhibit the mixed lymphocyte reaction. Thus, the problem of tissue typing and immune exclusion does not exist when transplantation, nor is there any ethical issues. Therefore, BMSCs after amplification in vitro are ideal seed cells in tissue engineering area. BMSCs could transform into neurons in vivo or in vitro, and also secrete nerve growth factor which play on the repair of nerve tissue injury, for example, neural growth factor (NGF), brain-derived neurotrophic factor (BDNF) and so on. However, because of their short life cycle BMSCs are unstable to play a stable neuroprotective function.We selected BDNF as the treatment gene related to the repair of brain injury by comparing before scientific studies. Then we establish the rat immortalized BMSCs line by transfection of expression plasmids of exogenous human telomerase reverse transcriptase (hTERT) mediated with the cationic liposome. We further build high BDNF expression vector—Ad5-BDNF, transfect Ad5-BDNF to the hTERT-BMSCs and Proved that BDNF has a role in cell repair of damage in vitro. On this basis, we also use rat fluid percussion brain injury model and the stereotactic equipment to study the biological characteristics and outcome of the transfected cells. The results prove its safety in vivo and the efficacy of treatment for TBI, which establish a foundation for BMSC into the clinical treatment of TBI. This study is divided into three parts. The first part of the study is to get stable rat BMSCs by adherent primary culture method. We Use flow cytometry to detect the surface antigen markers of BMSCs and further detect the differentiation potential of BMSCs into osteoblasts, adipocytes and neural-like cells in vitro. The results show that primary rBMSCs are easy to extract, purification, and amplification in vitro. The Identified markers are consistent with the characteristics of BMSCs. And BMSCs could be induced to fat-like cells, osteoblast-like cells and neuron-like cells in vitro, which implying that they have more potential to differentiate and confirming rBMSCs successful culture.The second part of the study is by transfection of pLXSN plasmid-mediated human telomerase reverse transcriptase (human telomerase reverse transcriptase hTERT) to enhance the activity of BMSCs telomerase. We Use ELISA method to assay telomerase activity and get the immortalized rat BMSCs controlled by hTERT. Meanwhile, by constructing efficient adenovirus-mediated vector—Ad5-BDNF and transfecting Ad5-BDNF complex into hTERT-BMSCs, we demonstrate that BDNF transfected immortalized BMSC in vitro has a certain degree repair of injured neuron cells.In the third part of the study, we selecte the more widely used internationally fluid percussion brain injury model in rats to establish the rat medium traumatic brain injury model with the 1.5-2.0atm condition. At the same time by means of stereotactic cell transplantation technology, we migrate BMSCs co-transfected by hTERT and BDNF gene to damaged rat brain areas, and observe the changes in rat survival, neurologic severity score (NSS), and the brain tissue edema in the magnetic resonance imaging (MRI). Thus, we could evaluate the safety and effectiveness of gene-modified BMSCs treatment on TBI, and establish a good foundation of TBI clinical treatment with BMSCs and BDNF.The total study results show that rat BMSCs are easily isolated, cultured and expanded. Long-term cultured BMSCs can be premature aging and excessive proliferation and other biological characteristics of instability. Transfecting hTERT expression vector—pLXSN-S-hTERT to rat BMSCs could enhance the telomerase activity of BMSCs and maintain long-term subculture in the process. This is a safe and effective method of establishment of immortalized BMSCs line. Ad-BDNF-hTERT-BMSCs can highly expresse BDNF in vivo, which could reduce apoptosis of damaged neurons, improve the survival rate in brain damage rats, and promote the rehabilitation of the neurological function. The results of this study provide an experimental basis for the clinical application of BMSCs and BDNF更多还原