【作者】 王锋；

【导师】 刘文革；

【作者基本信息】 福建医科大学 ， 外科学， 2007， 硕士

【摘要】 碱性成纤维细胞生长因子bFGF (FGF-2) (basic fibroblast growth factor)是FGF家族的成员之一,具有促血管形成、诱导胚胎发育、促进神经生长等功能,bFGF在体内分布广泛,尤其是在神经组织中含量丰富。目前已经有大量的研究证实碱性成纤维细胞生长因子对脊髓损伤(spinal cord injury SCI)的再生和修复具有重要作用,但由于急性脊髓损伤后bFGFR表达时程早于bFGF转录合成增加,因此限制了机体内源性bFGF发挥保护缺血损伤神经元的作用,这提示治疗脊髓损伤时需在bFGFR高表达时程提供足够bFGF。而通过局部或全身给予外源性bFGF均难以达到理想的治疗效果,本研究正是为解决这一问题而作的基础研究。将bFGF基因以质粒pcDNA3.1为载体,通过脂质体转染法导入雪旺细胞(schwann cell,SC),使外源bFGF基因能持续稳定高表达,在体内合成分泌bFGF,这将可能为损伤脊髓及时提供足量bFGF、为改善脊髓损伤局部微环境奠定基础。本文的研究内容主要包括以下三个部分:1、用混合酶消化法获取高纯度的雪旺细胞;2、PCR扩增bFGF基因片段,构建重组质粒bFGF- pcDNA3.1;3、将pcDNA3.1-bFGF质粒通过脂质体转染法导入雪旺细胞,用ELISA方法检测实验组、空载体组及空白对照组雪旺细胞培养上清中bFGF的表达水平;应用RT -PCR方法检测外源基因bFGFmRNA的表达情况。本研究所取得的结果及结论:1、通过大鼠坐骨神经预变性、混合酶消化法及G-418筛选,可以培养获取高纯度的雪旺细胞。2、选择安全性较高的高拷贝真核表达载体pcDNA3.1,可以将外源性bFGF基因片段与pcDNA3.1进行重组,获取含目的基因片段的真核表达体系。3、将重组bFGF- pcDNA3.1质粒通过脂质体转染法导入雪旺细胞后外源性bFGF基因能即时、持续、稳定表达,极大提高bFGF的表达水平。结论:(1)将bFGF基因以pcDNA3.1为载体通过脂质体转染导入雪旺细胞,能使雪旺细胞在体外培养过程中表达分泌bFGF;(2)经过bFGF基因修饰的雪旺细胞能持续稳定高水平的表达分泌bFGF;但在体外培养过程中因细胞生长条件限制而可能出现细胞bFGF分泌水平降低现象;(3)将bFGF基因与雪旺细胞移植载体相结合,具有独特的优势,不但能为损伤脊髓及时提供稳定高水平的bFGF,还可为神经轴突再生提供支持和引导,这将为损伤脊髓神经元的修复、再生提供极为有利的条件,为临床脊髓损伤的治疗开辟新的前景。更多还原

【Abstract】 bFGF (FGF-2) (basic fibroblast growth factor) is one member of FGF family which related to the angiogeneration , fetus growing and the neuro-generation. bFGF generally exists throughout the body especially in nerves. Nowadays, lots of the investigations have shown the significance of bFGF at the spinal regeneration and rehabilitation after injury. However ,after acute spinal injury, bFGFR expresses earlier than bFGF which diminishes its protection for the ischemic neuron. Therefore, during spinal injury treatment, the abundant bFGF supply before bFGFR expression reaches its climax is of the significant importance. But, unfortunately, neither local nor general application of the external bFGF can reach the ideal result. Based on the fact above mentioned ,our research is trying to make some improvements. We reconstruct a new plasmid of bFGF- pcDNA3.1 and then transfected it into the schwann cell (SC) by liposome so that to achieve the high and stable expression of bFGF .That both supply abundant bFGF and improve the local minimal environment.Our research contains 3 parts1. Obtain the highly purified schwann cell.2. reconstruct the plasmid bFGF- pcDNA3.13. Transfected the pcDNA3.1-bFGF into the schwann cell (SC) by liposome, then test the bFGF expression level in the schwann cell culture supernatant of experiment group, empty carrier group and control group respectively by ELISA . Exam the bFGF mRNA expression by RT -PCRResults1.Highly purified schwann cell can be obtained from mouse sciatic nerve by pre-degeneration, coenzyme digestion and G-418 bolting. 2.the eukaryotic expression system of destination gene and activity is reconstructed by bFGF and pcDNA3.1 that is of high security and replica.3. After bFGF- pcDNA3.1 has been transfected into schwann cell by liposome, the prompt, persistant, stable and high expression of bFGF can be obtainedConclusion :1. bFGF can be expressed and excreted into the supernatant after bFGF- pcDNA3.1 transfected into schwann cell by liposome.2. schwann cell modified by bFGF- pcDNA3.1 can express and excrete bFGF highly and stably ,but we also observe the reduction of bFGF expression when schwann cell is developed in vitro due to the limitation of culture environment.3. It is of unique advantages to connect schwann cell and bFGF- pcDNA3, which not only expresses bFGF enough and stably in trauma spine but supports and leads to the neuro-axon regeneration. Therefore, it offers the significant conditions for the damaged spinal neuron’s rehabilitation and regeneration and shows the bright future for the clinical treatment of spinal injury.更多还原