针刺对备用背根节卫星细胞NGF，BDNF和NT-3表达的影响
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摘要
目的：采用免疫组织化学和原位杂交技术，探讨针刺对备用背根节卫星细胞NGF，BDNF，NT-3表达的影响。方法：成年猫5只，行双侧部分背根切断术(切除两侧L_1-L_5，L_7-S_2 DRG，保留L_6为备用根)。术后当日开始针刺一侧L_6脊神经后肢分布区的两组穴位即足三里和悬钟、伏兔和三阴交，每天一次，每次30分钟。术后7天，取非针刺侧和针刺侧的L_6 DRG制作20μm厚冰冻切片，行免疫组化及原位杂交染色。观察NGF、BDNF和NT-3免疫阳性反应(IR)在DRG卫星细胞的分布，测量各组L_6 DRG卫星细胞NGF、BDNF和NT-3的光密度值，结果用q检验进行统计分析。结果：在DRG，可见NGF、BDNF和NT-3及其mRNA阳性卫星细胞。各因子mRNA的阳性反应物定位于胞质；BDNF-IR也定位于胞质，而NGF-IR和NT-3-IR则定位于胞核。卫星细胞NGF、BDNF、NT-3及其mRNA阳性反应的光密度值针刺侧较非针刺侧明显增多(P＜0.05)。结论：背根节部分卫星细胞内有NGF、BDNF和NT-3及其mRNA表达；针刺导致备用DRG卫星细胞NGF、BDNF和NT-3表达上调。结果提示：背根节卫星细胞内NGF、BDNF和NT-3的表达变化可能与针刺促进脊髓可塑性有关。
Objective To investigate the effect of acupuncture on the expression of NGF, BDNF, NT-3 and their mRNA in satellite cells of dorsal root ganglion (DRG) in adult cat following partial dorsal root rhizotomy. Methods Five adult cats underwent partial bilateral root rhizotomy (L1-L5, L7-S2 DRG were taken and L6 DRG was spared), The cats received acupuncture for 7 days following operation (The acupuncture points including Zusanli and Xuanzhong, Futu and Sanyinjiao located in the distribution area of L6 spinal nerve in one side of hind limb were stimulated alternatively for 30min every day by electro-needle). The L6 DRGs from acupuncture side and unacupuncture side were taken and made into frozen section 20μm in thickness respectively. The sections were stained by immunohistochemistry ABC method and in situ hybridization technique. The distribution of positive satellite cells for NGF, BDNF, NT3 was observed under microscope, The 0. D value of positive satellite cells for NGF, BDNF and NT3 was measured and analyzed statistically
by q-test. Results NGF, BDNF, NT-3 and their mRNA were seen in some satellite cells of DRG. The positive signal of mRNA for NGF, BDNF and NT3 was observed in cytoplasm , BDNF-IR was located in cytoplasm, while the NGF-IR and NT3-IR were distributed in nucleus.The O.D value of satellite cells for NGF, BDNF and NT3 after operation following acupuncture increased apparently more than that of operation without acupuncture. Conclussion: NGF, BDNF and NT3 are expressed in satellite cells of DRG. And the expression for three factors was upregulated in satellite cells following acupuncture.The results indicated that the change of NGF, BDNF and NT3 in satellite cells of DRG may be involved in the plasticity of spinal cord which is promoted by acupuncture.
Objective To investigate the effect of acupuncture on the expression of NGF, BDNF, NT-3 and their mRNA in satellite cells of dorsal root ganglion (DRG) in adult cat following partial dorsal root rhizotomy. Methods Five adult cats underwent partial bilateral root rhizotomy (L1-L5, L7-S2 DRG were taken and L6 DRG was spared), The cats received acupuncture for 7 days following operation (The acupuncture points including Zusanli and Xuanzhong, Futu and Sanyinjiao located in the distribution area of L6 spinal nerve in one side of hind limb were stimulated alternatively for 30min every day by electro-needle). The L6 DRGs from acupuncture side and unacupuncture side were taken and made into frozen section 20μm in thickness respectively. The sections were stained by immunohistochemistry ABC method and in situ hybridization technique. The distribution of positive satellite cells for NGF, BDNF, NT3 was observed under microscope, The 0. D value of positive satellite cells for NGF, BDNF and NT3 was measured and analyzed statistically
by q-test. Results NGF, BDNF, NT-3 and their mRNA were seen in some satellite cells of DRG. The positive signal of mRNA for NGF, BDNF and NT3 was observed in cytoplasm , BDNF-IR was located in cytoplasm, while the NGF-IR and NT3-IR were distributed in nucleus.The O.D value of satellite cells for NGF, BDNF and NT3 after operation following acupuncture increased apparently more than that of operation without acupuncture. Conclussion: NGF, BDNF and NT3 are expressed in satellite cells of DRG. And the expression for three factors was upregulated in satellite cells following acupuncture.The results indicated that the change of NGF, BDNF and NT3 in satellite cells of DRG may be involved in the plasticity of spinal cord which is promoted by acupuncture.
引文
1.Angeletti RH, Brad shaw PA. Nerve growth factor from the mouse submaxillary glands: amino acid sequence. Proc Natl Acad Sci USA, 1971; 68(10): 2417-2420
2.Leibrock J, Lottspeich F, Hohn A: Molecular cloning and expression of BDNF. Nature, 1998; 341: 149-152
3.Maisonpierre PC: Human and rat hrain-derived neurotrophic factor and neurotrophin-3: gene structure, distribution and chromosomal localization Geromics 1991; 10: 558-568.
4.Hallbook F, Ibanez CF, Persson H: Evolutionary studies of the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary. Neuron 1991; 6: 845-858
5.Koliatsos VE, et al. Neurotrophin-4/5 is a trophic factor for mammalian facial motor neurons. Proc Natl Acad Aci USA 1994; 91: 3304-3308
6.Gotz R, Koster R, Winlder C, et al: Neurotrophin-6 is a new member of the nerve growth factor family. Nature, 1994; 372(17): 266-269.
7．王廷华，冯忠堂主编．多肽生长因子与脊髓损伤[M]．新疆：科技卫生出版社，云南：科技出版社．2003；46-51
8. Fackelman K: Protein protects, restores neurons. Science news 1995; 147(4): 52.
9. Adler R, Landa KB, Manthorpe M: Cholinergic neurotrophic factors: intraocular distribution of trophic activity for ciliary neurons.Science 1979; 204: 1434-1436
10. Barde YA, Edger D, Thoenen H. Purification of a new neurotrophic factor from mammalian brain. EMBO, 1982, 1: 549-557
11. Hatai S. Number and size of the spinal ganglion cells and dorsal root fibers in the white rat at different ages. J Comp Neurol, 1902,12: 107-124
12. Hunter T. Increase levels of trkB mRNA and trkB protein-like inmmunoreactivity in the injured rat and cat spinal cord. Proc Natl Acad Sci USA, 1992, 89: 11282-11286
13. Meyer M, Matsuoka I. Enhanced synthesis of brain derived neurotrophic factor(BDNF) in the lesioned peripheral nerve different mechanisms are responsible for the regulation of BDNF and NGF mRNA, J Cell Biol, 1992, 119: 45-54
14．廖德阳，吴良芳，保天然，等．备用背根猫脊髓Ⅱ板层组织对鸡胚背根节神经突起生长的影响．神经解剖学杂志，1996；12(1)：10-14
15．薛庆善，吴良芳，保天然，等．脊髓源性感觉神经元诱向因子初探。神经解剖学杂志，1996，12：126-130
16．周雪，吴良芳，保天然，等．脊髓源性感觉神经元诱向因子初探．神经解剖学杂志，1996；12(2)：126-130
17. Cho HJ, Kim JK, Zhou XF, et al. Increased brain-derived neurotrophic factor immunoreactivity in rat dorsal root ganglia and spinal cord following peripheral inflammation. Brain Res, 1997;
764(1-2): 269-272
18. Cho HJ, Kim SY, Park MJ, et al. Expression of mRNA for brain-derived neurotrophic factor in the dorsal root ganglion following peripheral inflammation. Brain Res, 1997; 749(2): 358-362
19．王廷华，陈彦红，冯忠堂．NGF，BDNF，NT-3和GDNF在背根节不同神经元的配布的免疫组织化学证据．神经解剖学杂志，2001；17(1)：53-56
20. R. Douglas Fields. et al. New insights into neuron-glia communication. Science, 2002, 593(298): 556-562
21. Carmignoto. G. Reciproral communication systems between astrocytes and neurons. Prog Neurobiol 2000 Dec; 62(6): 561-581
22. Wetmore C, Olson L. Neuronal and nonneuronal expression of neurotrophins and their receptors in sensory and sympathetic ganglia suggest new intercellular trophic interactions. J of comp neurol, 1995; 353: 143
23. Taniuchi M, Clark HB. Induction of nerve growth factor receptor in Schwann cells after axotomy. Plast Reconstr Surg.1984, 74(2): 173-181
24．王廷华，陈彦红，冯忠堂．BDNF，NT-3在猫背根神经元的自分泌和旁分泌作用初探—原位杂交和免疫组织化学研究．中国组织化学与细胞化学杂志，2000，9(4)：408-410
25. Mu X, Silos SI. Neurotrophin receptor genes are expressed in distinct patterns in developing dorsal root ganglia. J Neurosci, 1993;13: 4029
26. Rubio N. Mouse astrocytes store and deliver brain-derived neurotrophic factor using the non-catalytic gpqs trkB receptor. Neuroseienee, 1997; 9(9): 1847
27. Klein R, silos SI. Disruption of the neurotrophin 3 receptor gene trkC eliminates Ic muscle afferents and results in abnormal movements. Nature, 1994; 368: 249
28. Zhou XY, Chie E, et al. Satellite-cell-derived nerve growth factor and neurotrophin-3 are involved in noradrenergic sprouting in the dorsal root ganglia following peripheral nerve injury in the rat. Eur J Neurosci, 1999, 11(5): 1711-1722
2.Leibrock J, Lottspeich F, Hohn A: Molecular cloning and expression of BDNF. Nature, 1998; 341: 149-152
3.Maisonpierre PC: Human and rat hrain-derived neurotrophic factor and neurotrophin-3: gene structure, distribution and chromosomal localization Geromics 1991; 10: 558-568.
4.Hallbook F, Ibanez CF, Persson H: Evolutionary studies of the nerve growth factor family reveal a novel member abundantly expressed in Xenopus ovary. Neuron 1991; 6: 845-858
5.Koliatsos VE, et al. Neurotrophin-4/5 is a trophic factor for mammalian facial motor neurons. Proc Natl Acad Aci USA 1994; 91: 3304-3308
6.Gotz R, Koster R, Winlder C, et al: Neurotrophin-6 is a new member of the nerve growth factor family. Nature, 1994; 372(17): 266-269.
7．王廷华，冯忠堂主编．多肽生长因子与脊髓损伤[M]．新疆：科技卫生出版社，云南：科技出版社．2003；46-51
8. Fackelman K: Protein protects, restores neurons. Science news 1995; 147(4): 52.
9. Adler R, Landa KB, Manthorpe M: Cholinergic neurotrophic factors: intraocular distribution of trophic activity for ciliary neurons.Science 1979; 204: 1434-1436
10. Barde YA, Edger D, Thoenen H. Purification of a new neurotrophic factor from mammalian brain. EMBO, 1982, 1: 549-557
11. Hatai S. Number and size of the spinal ganglion cells and dorsal root fibers in the white rat at different ages. J Comp Neurol, 1902,12: 107-124
12. Hunter T. Increase levels of trkB mRNA and trkB protein-like inmmunoreactivity in the injured rat and cat spinal cord. Proc Natl Acad Sci USA, 1992, 89: 11282-11286
13. Meyer M, Matsuoka I. Enhanced synthesis of brain derived neurotrophic factor(BDNF) in the lesioned peripheral nerve different mechanisms are responsible for the regulation of BDNF and NGF mRNA, J Cell Biol, 1992, 119: 45-54
14．廖德阳，吴良芳，保天然，等．备用背根猫脊髓Ⅱ板层组织对鸡胚背根节神经突起生长的影响．神经解剖学杂志，1996；12(1)：10-14
15．薛庆善，吴良芳，保天然，等．脊髓源性感觉神经元诱向因子初探。神经解剖学杂志，1996，12：126-130
16．周雪，吴良芳，保天然，等．脊髓源性感觉神经元诱向因子初探．神经解剖学杂志，1996；12(2)：126-130
17. Cho HJ, Kim JK, Zhou XF, et al. Increased brain-derived neurotrophic factor immunoreactivity in rat dorsal root ganglia and spinal cord following peripheral inflammation. Brain Res, 1997;
764(1-2): 269-272
18. Cho HJ, Kim SY, Park MJ, et al. Expression of mRNA for brain-derived neurotrophic factor in the dorsal root ganglion following peripheral inflammation. Brain Res, 1997; 749(2): 358-362
19．王廷华，陈彦红，冯忠堂．NGF，BDNF，NT-3和GDNF在背根节不同神经元的配布的免疫组织化学证据．神经解剖学杂志，2001；17(1)：53-56
20. R. Douglas Fields. et al. New insights into neuron-glia communication. Science, 2002, 593(298): 556-562
21. Carmignoto. G. Reciproral communication systems between astrocytes and neurons. Prog Neurobiol 2000 Dec; 62(6): 561-581
22. Wetmore C, Olson L. Neuronal and nonneuronal expression of neurotrophins and their receptors in sensory and sympathetic ganglia suggest new intercellular trophic interactions. J of comp neurol, 1995; 353: 143
23. Taniuchi M, Clark HB. Induction of nerve growth factor receptor in Schwann cells after axotomy. Plast Reconstr Surg.1984, 74(2): 173-181
24．王廷华，陈彦红，冯忠堂．BDNF，NT-3在猫背根神经元的自分泌和旁分泌作用初探—原位杂交和免疫组织化学研究．中国组织化学与细胞化学杂志，2000，9(4)：408-410
25. Mu X, Silos SI. Neurotrophin receptor genes are expressed in distinct patterns in developing dorsal root ganglia. J Neurosci, 1993;13: 4029
26. Rubio N. Mouse astrocytes store and deliver brain-derived neurotrophic factor using the non-catalytic gpqs trkB receptor. Neuroseienee, 1997; 9(9): 1847
27. Klein R, silos SI. Disruption of the neurotrophin 3 receptor gene trkC eliminates Ic muscle afferents and results in abnormal movements. Nature, 1994; 368: 249
28. Zhou XY, Chie E, et al. Satellite-cell-derived nerve growth factor and neurotrophin-3 are involved in noradrenergic sprouting in the dorsal root ganglia following peripheral nerve injury in the rat. Eur J Neurosci, 1999, 11(5): 1711-1722