bFGF基因修饰雪旺细胞bFGF表达的实验研究
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。
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 parts
1. Obtain the highly purified schwann cell.
2. reconstruct the plasmid bFGF- pcDNA3.1
3. 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 -PCR
Results
1.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 obtained
Conclusion :
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.
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。
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 parts
1. Obtain the highly purified schwann cell.
2. reconstruct the plasmid bFGF- pcDNA3.1
3. 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 -PCR
Results
1.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 obtained
Conclusion :
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.
引文
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10. Zlokovic BV, Apuzzo MLJ. Cellular and molecular neurosurgery: Pathways from concept to reality[J]. Neurosurgery, 1997,40(4):789-813.
11. Zhang Y, Dijkhuizen PA, Anderson PN, et al. NT-3 delivered by an adenoviral vector induces injured dorsal root axons to regenerate into the spinal cord of adultrats[J]. J Neurosci Res,1998,54(4):554-562.
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2. Nakata N, Kato H, Kogure K. Protective effects of basic fibroblast growth factor against hippocampal neuronal damage following cerebral ischemia in the gerbil[J]. Brain Res, 1993,605(2):354-356.
3. Kerekes N, Landry M, Rydh-Rinder M. The effect of NGF, BDNF and bFGF on expression of galanin in cultured rat dorsal root ganglia[J]. Brain Res, 1997,754(1-2):131-141.
4. Lanzetta M, Gal A, Wright B, Owen E. Effect of FK506 and basic fibroblast growth factor on nerve regeneration using a polytetrafluoroethylene chamber for nerve repair[J]. Int Surg, 2003,88(1):47-51.
5. Dow JK, Devere White RW. Fibroblast growth factor-2: its structure and property, paracrine function, tumor angiogenesis, and prostate-related mitogenic and oncogenic functions[J]. Urology, 2000,55(6):800-806.
6. Follesa P, Wrathall JR, Mocchetti I. Increased basic fibroblast growth factor mRNA following contusive spinal cord injury [J]. Mol Brain Res, 1994,22(14):1-8.
7. 刘颖,叶诸榕,陆锦标,等. bFGF, bFGFR 及 Egr-1 基因蛋白在大鼠局灶性脑缺血后的表达[J].中国神经科学杂志,2002,18(3):602-606.
8. Okada-Ban M, Thiery JP, Jouanneau J. Fibroblast growth factor-2. Int J Biochem Cell Biol,2000,32(3):263-267.
9. Nakamura K, Kawaguchi H, Aoyama I, et a1. Stimulation of bone formation by intraosseous applicaton of recombinant basic fibroblast growth factor in normal and ovariectomized rabbits[J]. J Orthop Res, 1997, 15(2):307-313.
10. Zlokovic BV, Apuzzo MLJ. Cellular and molecular neurosurgery: Pathways from concept to reality[J]. Neurosurgery, 1997,40(4):789-813.
11. Zhang Y, Dijkhuizen PA, Anderson PN, et al. NT-3 delivered by an adenoviral vector induces injured dorsal root axons to regenerate into the spinal cord of adultrats[J]. J Neurosci Res,1998,54(4):554-562.
12. Tuszynski MH, Gage FH. Maintaining the neuronal phenotype after injury in the adult CNS. Neurotrophic factors, axonal growth substrates, and gene therapy. Mol Neurobiol,1995,10(2～3):151-167.
13. Tuszynski MH, Senut MC, Ray J, et al. Somatic gene transfer to the adult primate CNS: in vitro and in vivo characterization of cells genetically modified to secret NGF[J]. Neurobiol Dis,1994,1(1-2):67-78.
14. 曹文灵,李家谋,井多辉,等.成年大鼠雪旺细胞的快速扩增[J].生物物理学报,2004,22(2):150-154.
15. Keilhoff G, Fansa H, Schneider W. In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits[J]. J Neurosci Methods,1999,89(1):17~24.
16. Griffin JW, George R, Ho T. Macrophage systems in peripheral nerves[J].J Neuropathol Exp Neurol,1993,52(6):553-560.
17. Heumann R, Lindholm D, Bandtlow C, et al. Differential regulation of mRNA encoding nerve growth factor and its receptor in rat sciatic nerve during development,degeneration, and regeneration: role of macrophages[J]. Proc Natl Acad Sci USA,1987,84(23):8735-8739.
18. Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve regeneration[J]. Mol Neurobiol, 1997, 14(1-2):67-l16.
19. 李春雨,张振兴,崔树森.不同消化酶对体外培养的原代乳鼠雪旺细胞的影响[J]. Chin J Lab Diagn,2005, 9,(2):268-270.
20. Guest JD, Rao A, Olson L, et al. The ability of human Schwann cell grafts to promote regeneration in the transected nude rat spinal cord[J]. Exp Neurol, 1997,148(2):502-522.
21. Verdu E, Rodriguez FJ, Gudino-Cabrera G, et al. Expansion of adult Schwann cells from mouse predegenerated peripheral nerves[J]. J Neurosci Methods, 2000, 99(1-2):111-117.
22. Dong Z, Brennan A, Liu N, et al. Neu differentiation factor is a neuron-glia signal and regulates survival, proliferation, and maturation of rat Schwarnn cellprecursors[J].Neuron,1995,15(3):585-596.
23. Danielsen N, Kerns JM, Holmquist B, et al. Pre-degenerated nerve grafts enhance regeneration by shortening the initial delay period[J]. Brain Res, 1994, 666(2):250~254.
24. Chang JY, Browns S. Cytosine arabinoside differentially alters survival and neurite outgrowth of neuronal PC12 cells[J]. Biochem Biophys Res Commun,1996,218(3):753-758.
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