脊髓康含药血清联合SOX-2对星形胶质细胞重编程诱导为神经元细胞的影响  

Effect of Jisuikang-containing Serum Combined with SOX-2 on Astrocyte Being Reprogrammed and Induced into Neurons

作　　者：王浩阗 王建伟[2] 吴毛[2] 汪国澎 李绍烁 戎毅[1] 邵阳[2] WANG Haotian;WANG Jianwei;WU Mao;WANG Guopeng;LI Shaoshuo;RONG Yi;SHAO Yang(Nanjing University of Chinese Medicine,Nanjing,210023;Wuxi Traditional Chinese Medicine Hospital,Jiangsu Province)

机构地区：[1]南京中医药大学,江苏省南京市210023 [2]江苏省无锡市中医医院

出　　处：《中医杂志》2023年第3期288-294,共7页Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(82174400);江苏省中医药科技发展计划项目(YB2020041,YB2020042);无锡市科学技术局医疗卫生指导性项目(SKJJZD19);无锡市卫生健康委科研项目(Q201945)。

摘　　要：目的研究脊髓康联合SRY转录盒因子2(SOX-2)治疗脊髓损伤的作用机制。方法成年SD大鼠随机分为空白组和给药组各5只。给药组大鼠以生药量20 g/(kg·d)脊髓康灌胃,空白组给予16 ml/(kg·d)生理盐水灌胃,均连续灌胃3天后腹主动脉采血制备含药血清和空白血清。体外培养1~4日龄大鼠幼仔皮层脑组织三代星形胶质细胞,分为空白血清组、SOX-2组、SOX-2+脊髓康血清组。SOX-2组加入10 mmol/L SOX-2;SOX-2+脊髓康血清组加入2.5%脊髓康含药血清和10 mmol/L SOX-2,使培养基中血清终浓度为10%;空白血清组加入10%空白血清。诱导培养21天后,光镜下观察3组神经元样细胞形态改变,细胞免疫荧光检测神经元轴突标志物微管相关蛋白2(MAP2)的表达,Western-Blot检测细胞中下游代谢产物S腺苷甲硫氨酸(SAM)、乙酰辅酶A(acetyl-CoA)表达,PCR定量分析星形胶质细胞中糖酵解相关基因己糖激酶1(HK1)、己糖激酶2(HK2)、磷酸果糖激酶1(PFK1)、磷酸果糖激酶2(PFK2)、M2型丙酮酸激酶同工酶(PKM2)基因表达。细胞能量代谢分析仪比较SOX-2组、SOX-2+脊髓康血清组细胞外酸化率(EACR)和耗氧率(OCR)。结果光镜下见SOX-2+脊髓康血清组大量具有两极且长轴突的神经元样细胞,较SOX-2组明显增多;空白血清组见星形胶质细胞,未见神经元样细胞。与空白血清组和SOX-2组比较,SOX-2+脊髓康血清组MAP2、SAM、acetyl-CoA表达均升高,HK1、HK2、PFK1、PFK2、PKM2基因表达亦显著升高(P<0.05或P<0.01)。SOX-2+脊髓康血清组ECAR和OCR亦高于SOX-2组。空白血清组和SOX-2组MAP2、SAM、acetyl-CoA表达差异无统计学意义(P>0.05)。结论脊髓康含药血清联合SOX-2可以将星形胶质细胞重编程诱导为神经元细胞,其机制可能是通过提高糖酵解能力从而提升星形胶质细胞重编程。Objective To study the effect and machanism of Jisuikang(脊髓康)combined with SRY transcription box factor 2(SOX-2)on spinal cordinjury(SCI).Methods Adult SD rats were randomly divided into blank group and medication group with five rats in each.Rats in the medication group were given 20 g/(kg·d)of Jisuikang by gavage,while those in the control group received 18 ml/(kg·d)of normal saline by gavage,both for three days.After that,blood was collected from the abdominal aorta to prepare Jisuikang-containing serum and blank serum.Three generations of astrocytes were cultured in vitro in the cerebral cortex of 1~4 day newborn rats,and were divided into three groups including blank serum group,SOX-2 group,and combination of Jisuikang-containing serum plus SOX-2 group(SOX-2+JSK group).Add 10 mmol/L of SOX-2 to the SOX-2 group,and 2.5%Jisuikang-containing serum plus 10 mmol/L of SOX-2 to the SOX-2+JSK group,making the final serum concentration in the medium to 10%;blank serum group was added with 10%of blank serum.After 21 days of induction and culture,the morphology of neuron-like cells in the three groups were observed under light microscope.The expression of neuronal axon marker microtubule-associated protein 2(MAP2)was detected by cellular immunofluorescence,and the expression of downstream metabolites S-adenosylmethionine(SAM)and acetyl-CoA(acetyl-CoA)were detected by Western Blot.Quantitative PCR analysis was performed for the glycolysis-related genes expression including hexokinase 1(HK1),hexokinase 2(HK2),phosphofructokinase 1(PFK1),phosphofructokinase 2(PFK2),and M2-type pyruvate kinase isozyme(PKM2).The cell energy metabolism analyzer was used to compare the extracellular acidification rate(EACR)and oxygen consumption rate(OCR)of the SOX-2 group and the SOX-2+JSK serum group.Results Under the light microscope,a large number of neuron-like cells with bipolar and long axons were found in the SOX-2+JSK group,which was significantly more than that in the SOX-2 group;in the blank serum group,astrocytes were foun

关 键 词：脊髓损伤 星形胶质细胞 重编程神经元 有氧糖酵解 表观遗传学 脊髓康 

分 类 号：R651.2[医药卫生—外科学]