干扰Kinesin-12表达对大鼠脊髓损伤后形态和功能的影响*

赵攀峰1**,巫荣华1***,花娟1,王萍2,刘梅1***

(1南通大学神经再生重点实验室,南通226001;2江苏省南通市第一人民医院影像科)

[]目的:探讨干扰Kinesin-12表达对大鼠脊髓损伤后形态和功能的影响。方法:建立大鼠脊髓挫伤模型,通过实时定量聚合酶链式反应(quantitativereal time polymerase chain reaction, qRT-PCR)检测脊髓损伤后不同时间Kinesin-12基因表达,及胶质瘢痕相关基因硫酸软骨素蛋白聚醣(chondroitinsulfate proteoglycans, CSPGs) mRNA水平表达变化;脊髓损伤术后1周,在损伤处注射siRNA干扰Kinesin-12基因表达,并于干扰表达后不同时间进行运动功能(BassoBeattie Bresnahan, BBB)评分评估损伤后神经功能,磁共振成像(magneticresonance imaging, MRI)观察脊髓损伤修复程度,免疫组织化学法观察胶质纤维酸性蛋白(glialfibrillary acidic protein, GFAP)和CSPGs表达。结果:脊髓损伤后Kinesin-12基因表达显著升高,与胶质瘢痕CSPGs相关的基因表达也逐渐升高。在大鼠脊髓损伤模型中,与对照组相比,干扰Kinesin-12表达使大鼠脊髓损伤运动功能BBB评分增加,CSPGs表达下降,脊髓组织保存较多,空洞区减小。结论:在大鼠脊髓损伤后采用siRNA干扰Kinesin-12表达,可以减少空洞和胶质瘢痕的形成,利于脊髓形态和功能恢复。

[关键词]脊髓损伤;Kinesin-12;胶质纤维酸性蛋白;硫酸软骨素蛋白聚醣;实时定量聚合酶链式反应;大鼠

[中图分类号]R651.2[文献标志码] A[文章编号]1674-7887(2017)01-0001-06

Effectof Kinesin-12 knockdown on morphology and function after rat spinal cordinjury*

ZHAOPanfeng1**, WU Ronghua1***, HUA Juan1, WANG Ping2, LIU Mei1***(1Key Laboratory of Neuroregeneration, Nantong University,Nantong 226001; 2Department of Radiology,Nantong First People’s Hospital, Jiangsu Province)

[Abstract] Objective:To investigate the effect of Kinesin-12 depletion on morphology andfunction after rat spinal cord injury. Methods:The spinal cord contusion injury model was established by MASCIS impactor.At different time points after rat spinal cord injury(SCI), Kinesin-12, glialfibrillary acidic protein(GFAP) and chondroitin sulfate proteoglycans(CSPGs)genes mRNA level were detected by quantitative real time ploymerase chainreaction(qRT-PCR). In addition, we injected Kinesin-12 siRNA to the injuryregion one week later after SCI. Then, at different time points afterKinesin-12 knockdown, the Basso Beattie Bresnahan(BBB) score and the obliqueboard test were measured to evaluate the neurological function. Immunostaingwas used to detected CSPGs and GFAP proteins level. Results: After SCI, the mRNA level of Kinesin-12 was significantlyincreased, and GFAP and CSPGs related genes mRNA level were also increased. Inthe spinal cord contusion model, compared with the control siRNA group, atdifferent time points after Kinesin-12 siRNA treatment, the score of BBB and obliqueboard test was increased. The expression of CSPGs protein level was decreased,and the spinal cord cavity was decreased and more tissues were kept afterKinesin-12 depletion. Conclusion: Kinesin-12expression knockdown in rat spinal cord injury model restrained the cavitationand glial bar, and facilitated the morphological and functional recovery.

[Key words] spinal cord injury; Kinesin-12; glialfilrillary acidic protein; chondroitin sulfate proteoglycans; quantitative realtime polymerase chain reaction; rat


胶原诱导性关节炎小鼠脾脏T细胞合成儿茶酚胺增加*

芮潇潇1**,于子页2,王小琴1,陆健花1,邱一华1***

(1南通大学医学院生理学系,南通 226001;2南通大学医学院)

[]目的:探究胶原诱导性关节炎(collagen-inducedarthritis, CIA)小鼠淋巴组织中T helper 1(Th1)细胞合成、储存、降解儿茶酚胺的变化,以进一步探讨Th1细胞来源的儿茶酚胺在类风湿关节炎(rheumatoidarthritis, RA)致病机制中所起的作用。方法:将DBA/1J小鼠随机分为正常对照组和CIA模型组。Ⅱ型胶原乳剂尾根部注射制备CIA小鼠模型。Western Blot法检测脾脏组织中Th1细胞转录因子T-bet、细胞因子干扰素-γ(interferon-γ,IFN-γ)和白细胞介素-2(interleukin-2, IL-2)的表达。免疫荧光双标法检测脾脏中T-bet分别与酪氨酸羟化酶(tyrosinehydroxylase, TH)、囊泡单胺转运体-2(vesicular monoamine transporter-2, VMAT-2)、单胺氧化酶(monoamineoxidase, MAO)的共定位。结果:与正常小鼠相比,CIA小鼠脾脏组织中T-bet、IFN-γ和IL-2的蛋白表达增加。小鼠脾脏中T-bet与TH、VMAT-2、MAO分别存在免疫荧光共定位。CIA组TH、VMAT-2及MAO单阳性细胞数以及分别的双阳性细胞数均显著增加(P<0.01)。结论:CIA小鼠Th1细胞合成、储存、降解儿茶酚胺的能力增强。Th1来源的儿茶酚胺参与了CIA的发生发展。

[关键词] 胶原诱导性关节炎;儿茶酚胺;酪氨酸羟化酶;囊泡单胺转运体-2;单胺氧化酶;Th1细胞;小鼠

[中图分类号]R684.3[文献标志码] A[文章编号] 1674-7887(2017)01-0006-05

Increased synthesis ofcatecholamines by T cells in spleens from mice with collagen-induced arthritis*

RUIXiaoxiao1**, YU Ziye2, WANG Xiaoqin1, LU Jianhua1, QIU Yihua1***(1Department ofPhysiology, Medicine Schoolof Nantong University,Nantong 226001; 2MedicineSchool of Nantong University)

[Abstract]Objective: To investigate thechanges in synthesis, storage and catabolism of catecholamines by T helper1(Th1) cells in lymphoid tissues from collagen-induced arthritis(CIA) mice, forfurther understanding of the role Th1-derived catecholamines play in thepathogenic mechanism of rheumatoid arthritis(RA). Methods: DBA/1J mice were randomly assigned to one of two groups(intact and CIA). The CIA models were induced by type Ⅱ collagen(CⅡ) injectionfrom tails. Expressions of T-bet(transcription factor of Th1 cells), cytokines,interferon-γ(IFN-γ) and interleukin-2(IL-2) in spleen tissues were detected byWestern Blot. The colocalization of T-bet with hydroxylase(TH), vesicularmonoamine transporter-2(VMAT-2), and monoamine oxidase(MAO) in spleens,respectively, was determined by immunofluorescent staining. Results: Expressions of T-bet, IFN-γand IL-2 in spleen tissues were increased in CIA mice compared with the intactmice. Colocalization of T-bet with TH, VMAT-2 and MAO was observed in bothintact and CIA mice. TH+, VMAT-2+ and MAO+ cell numbers, as well as doublepositive-cell numbers were increased in CIA mice compared with the intact mice,respectively(P<0.01). Conclusions: Synthesis,storage and catabolism of Th1-derived catecholamines were increased in spleenfrom CIA mice. Th1-derived catecholamines play a role in development andprogression of CIA.

[Key words] collagen-induced arthritis; catecholamine;tyrosinehydroxylase; vesicular monoamine transporter-2; monoamine oxidase; Th1cell; mouse