TNF-α和IFN-γ影响许旺细胞生长及髓鞘相关糖蛋白mRNA表达水平的研究

Effects of TNF-α and IFN-γ on thegrowth of Schwann cells and expression of myelin-associated glycoprotein mRNA

目的研究肿瘤坏死因子-α(TNF-α)和干扰素-γ(IFN-γ)对体外培养的许旺细胞(SCs)生长和髓鞘相关糖蛋白(MAG)mRNA表达水平的影响。方法建立许旺细胞与神经细胞(NCs)联合培养模型,绘制许旺细胞生长曲线,采用半定量逆转录-聚合酶链反应(RT-PCR)检测TNF-α和INF-γ对MAGmRNA表达水平的影响。结果许旺细胞培养液中加入TNF-α和IFN-γ后,许旺细咆增殖数目明显增加且高于单纯许旺细胞培养组(q=7.476,P=0.000;q=6.631,P=0.000);而TNF-α组与IFN-γ组之间差异无统计学意义(q=0.846,P=0.411)。无神经细胞存在时,许旺细胞不表达MAG mRNA;经神经细胞诱导后,许旺细胞MAC mRNA表达阳性,且具有时间性;加入TNF-α和IFN-γ后,许旺细胞MAG mRNA表达水平明显升高,且高于单纯许旺细胞与神经细胞共培养组(q=8.128,P=0.000;q=6.614,P=0.000),而加人TNF-α和IFN-γ的许旺细胞与神经细胞共培养组之间差异无统计学意义(q=1.515,P=0.156)。结论 TNF-α和IFN-γ具有促进许旺细胞增殖作用,且可提高髓鞘特异性基因MAG mRNA表达水平。提示,这两种炎性干扰因子可通过影响许旺细胞及MAC mRNA表达水平而间接影响髓鞘修复,为研究炎性脱髓鞘性神经疾病慢性期髓鞘修复障碍的发病机制提供了新的思路。

Objective To observe the effects of tumor necrosis factor-α （TNF-α） and interferon-γ （IFN-γ） on the growth of Schwann cells （SCs） and the expression of myelin-associated glycoprotein （MAG） mRNA in vitro. Methods 1） SCs were excised from sciatic nerve and nerve ceils （NCs） were dissected from cerebral cortex in 3-5 d neonatal Sprague-Dawley （SD） rats. The cells were purified with D-Glucose. 2） The co-cultured model of SCs and NCs was established. TNF-α （0.01 mg/ml） or IFN-γ （0.01 U/ml） were added during the cell culture. The characteristics of SCs and NCs were identified by immunohistochemistry and electronmicroscopy. 3） SCs were cultured and divided into 3 groups： SC group, TNF-α group （adding TNF-α 0.01 mg/ml） and IFN-γ, group （adding IFN-γ 0.01 U/ml）. Cell growth curve was drawn by methyl thiazolyl tetrazolium （MTT） method with OD value at 1, 3, 5, 7 and 9 d in 3 groups. 4） Semi-quantitative reverse transcriptase-polymerase chain reaction （RT-PCR） was used to examine MAG mRNA expression. SCs and NCs were divided into 5 groups： NC group, SC group, SC ＋ NC group （co-cultured SCs and NCs）, TNF-α ＋ SC ＋ NC group （co-cultured group adding TNF-ct 0.01 mg/ml） and IFN-5＇ ＋ SC ＋ NC group （cocultured group adding IFN-γ 0.01 U/ml）. The gray value of MAG mRNA and 13-actin electrophoretic bands in 5 groups was detected at 1 and 5 d. Results MTT showed that the OD value in TNF-α group and IFN-γ group was all significantly higher than that in SC group （q = 7.476, P = 0.000; q = 6.631, P = 0.000,respectively）, but there no significant difference between TNF-α group and IFN-γ group （q = 0.846, P = 0.411）. MAG mRNA expression was negative in SC group and NC group, but was positive in SC ＋ NC group, TNF -α ＋ SC ＋ NC group and IFN-γ ＋ SC ＋NC group, and presented time-sensitive. MAG mRNA expression in TNF-ct ＋ SC ＋ NC group and IFN-γ ＋ SC ＋ NC group was all significantly higher than that in SC ＋ NC group at 5 d （q = 8.128, P = 0.000; q = 6.614, P = 0.000, respectively）, but there was no significant difference between the groups treated with TNF-α and IFN-3, （q = 1.515, P = 0.156）. Conclusion TNF-α and IFN-γ not only promote the proliferation of Schwann ceils, but also improve the expression of MAG mRNA in vitro, and may effect remyelination. This study may provide a new idea in researching the pathogenesis of myelination disturbance at the chronic stage of inflammatory demyelinating neuropathy.