Effect of axial vertical vibration on degeneration of lumbar intervertebral discs in modified bipedal rats: An in-vivo study

Objective: To assess the effects of axial vibrations on gene expression and lumbar intervertebral disc degeneration in vivo. Methods: A modified bipedal rat model was established using a brachial plexus rhizotomy approach to imitate human upright posture. The experimental animals were randomly divided into three groups: control, vertical vibration, and whole-body vibration. Gene expression in degeneration of the intervertebral discs was assessed by reverse transcription-quantitative polymerase chain reaction. Results: The expression of aggrecan, Col1α1, Col2α1, and decorin were shown to be up-regulated in 14-week-old rats in the vertical vibration and whole-body vibration groups, whereas biglycan and versican expression was down-regulated in 14-week-old rats of the two experimental groups. Furthermore, biglycan and versican expression levels were shown to be lower in the whole-body vibration group than in the vertical vibration group(P<0.05). Conclusions: This in-vivo study demonstrated that vibrations can influence the expression of anabolic genes. Furthermore, whole-body vibrations seem to have a greater effect in this regard than vertical vibrations. A new method is expected to relieve the low back pain of the patients through our research.

Analysis of Gene Expression Pattern of Lumbar Intervertebral Disc Degeneration in Human

ObjectiveTo investigate the gene expression changes in normal and degeneration lumbar intervertebral disc in humans, providing information for clinical. MethodsThe PCR products of 4096 human genes were spotted onto a kind of chemical-material-coated-glass slides. The total RNAs were isolated from the tissues. Both the mRNAs from the degeneration and normal lumbar intervertebral disc in humans were reversely transcribed to the cDNAs, which used as the hybridization probes with the incorporations of fluorescent dUTP. The mixed probes were then hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signals and analyzed with computer image analysis. ResultsAmong the 4096 targets, there were 706 genes whose expression levels differed between the degeneration and normal lumbar intervertebral disc in all cases, comprising 298 up-regulated and 358 down-regulated ones. ConclusionDNA microarray technology is an effective technique in screening for differently expressed genes between the degeneration and normal lumbar intervertebral disc. Cell apoptosis plays an important role in the process of lumbar intervertebral disc degeneration.

The study of migration of bone mesenchymal stem cells transplanted in intervertebral discs of rabbits and expression of exogenous gene

Objective： To explore the survival and migration of bone mesenchymal stem cells transplantated in intervertebral disc of rabbits and expression of the exogenic genes. Methods. Thirty-two rabbits were used, A randomized block design was used and discs in the same rabbit were one block,the lumbar discs from L2-3 to L5-6 were randomly divided into blank group, saline group, cell transplantation group Ⅰand cell transplantation group Ⅱ. The fluorescence microscopy was used to determine the fluorescence of the maker protein GFP and DNA-PCR was used to analyze the copies of DNA of neomycin-resistant gene at 1, 3, 6, months after transplantation. Results： There was fluorescence in cell transplantation group Ⅰ and Ⅱ and none in blank group, saline group at 1, 3, 6 months after transplantation. In cell transplantation groups,the fluorescent distribution was more scatter with time, but no significant difference between cell groups Ⅰ and Ⅱ. The test of neomycin resistant gene expressed in cell transplantation group Ⅰ and Ⅱ and quantitative analysis showed that there was no significant difference between the cell groups Ⅰ and Ⅱ （P〉0.05）. Conclusion： The transplanted bone mesenchymal stem cells can survive, migrate and the transfer genes can express efficiently, it suggests that the BMSC therapy may be effective to prevent and treat intervertebral disc degeneration.

Regulating the fate of stem cells for regenerating the intervertebral disc degeneration

Lower back pain is a leading cause of disability and is one of the reasons for the substantial socioeconomic burden.The etiology of intervertebral disc(IVD)degeneration is complicated,and its mechanism is still not completely understood.Factors such as aging,systemic inflammation,biochemical mediators,toxic environmental factors,physical injuries,and genetic factors are involved in the progression of its pathophysiology.Currently,no therapy for restoring degenerated IVD is available except pain management,reduced physical activities,and surgical intervention.Therefore,it is imperative to establish regenerative medicine-based approaches to heal and repair the injured disc,repopulate the cell types to retain water content,synthesize extracellular matrix,and strengthen the disc to restore normal spine flexion.Cellular therapy has gained attention for IVD management as an alternative therapeutic option.In this review,we present an overview of the anatomical and molecular structure and the surrounding pathophysiology of the IVD.Modern therapeutic approaches,including proteins and growth factors,cellular and gene therapy,and cell fate regulators are reviewed.Similarly,small molecules that modulate the fate of stem cells for their differentiation into chondrocytes and notochordal cell types are highlighted.

Transcription regulators differentiate mesenchymal stem cells into chondroprogenitors,and their in vivo implantation regenerated the intervertebral disc degeneration

BACKGROUND Intervertebral disc degeneration(IVDD)is the leading cause of lower back pain.Disc degeneration is characterized by reduced cellularity and decreased production of extracellular matrix(ECM).Mesenchymal stem cells(MSCs)have been envisioned as a promising treatment for degenerative illnesses.Cell-based therapy using ECM-producing chondrogenic derivatives of MSCs has the potential to restore the functionality of the intervertebral disc(IVD).AIM To investigate the potential of chondrogenic transcription factors to promote differentiation of human umbilical cord MSCs into chondrocytes,and to assess their therapeutic potential in IVD regeneration.METHODS MSCs were isolated and characterized morphologically and immunologically by the expression of specific markers.MSCs were then transfected with Sox-9 and Six-1 transcription factors to direct differentiation and were assessed for chondrogenic lineage based on the expression of specific markers.These differentiated MSCs were implanted in the rat model of IVDD.The regenerative potential of transplanted cells was investigated using histochemical and molecular analyses of IVDs.RESULTS Isolated cells showed fibroblast-like morphology and expressed CD105,CD90,CD73,CD29,and Vimentin but not CD45 antigens.Overexpression of Sox-9 and Six-1 greatly enhanced the gene expression of transforming growth factor beta-1 gene,BMP,Sox-9,Six-1,and Aggrecan,and protein expression of Sox-9 and Six-1.The implanted cells integrated,survived,and homed in the degenerated intervertebral disc.Histological grading showed that the transfected MSCs regenerated the IVD and restored normal architecture.CONCLUSION Genetically modified MSCs accelerate cartilage regeneration,providing a unique opportunity and impetus for stem cell-based therapeutic approach for degenerative disc diseases.

椎间盘退变伴氧化应激关键生物标志物:生物信息学和机器学习算法的识别

背景:氧化应激与椎间盘退变的发生发展息息相关,但其发病机制和有效治疗方法仍不明确。目的:运用生物信息学及3种机器学习算法识别与椎间盘退变伴氧化应激相关的关键基因及免疫浸润分析,并进行实验验证。方法:从GEO数据库获得椎间盘退变基因表达谱以及从GeneCards数据库获得氧化应激相关基因,对椎间盘退变数据集进行差异分析及加权基因共表达网络(WGCNA)分析,两者取交集并与氧化应激相关基因取交集得到候选hub基因,对候选hub基因进行GO和KEGG分析;运用机器学习(LASSO回归、SVM-RFE及随机森林)筛选最佳特征基因并进行受试者特征曲线验证,同时行相关免疫浸润分析。收集2023年7-11月就诊于山西医科大学第二医院的颈椎病患者的椎间盘样本作为椎间盘退变组,颈椎脊髓损伤患者的椎间盘样本作为对照组,采用qPCR方法验证特征基因在椎间盘退变组织中的相对表达量。结果与结论:(1)经过差异基因分析获取424个差异表达基因,WGCNA分析得到5087个基因,同时获得氧化应激基因1399个,进而得到23个候选hub基因;(2)GO分析结果显示,主要参与细菌防御反应、细菌来源分子反应等生物过程;涉及分泌颗粒腔、细胞质囊泡腔等细胞组成;涉及内肽酶活性和硫化合物结合等分子功能;(3)KEGG分析结果显示,候选hub基因与中性粒细胞胞外诱捕网形成、肾素-血管紧张素系统通路等信号通路有关;(4)运用3种机器学习和ROC验证后得到关键基因HSPA6和PKD1;(5)免疫浸润分析显示HSPA6与活化树突状细胞(r=0.88,P<0.001)、活化CD4^(+)T细胞(r=-0.72,P<0.01)等密切相关,同时PKD1与效应型记忆CD8^(+)T细胞(r=0.55,P<0.05)、活化树突状细胞(r=-0.56,P<0.05)等密切相关;(6)q PCR实验结果表明椎间盘退变组中HSPA6基因低于对照组(P<0.0001),而PKD1基因高于对照组(P<0.0001);(7)结果表明运用生物信息学及机器学习算法证实HSPA6和PKD1可作为椎间盘退变伴氧化应激的生物标志物,可能通过干预HSPA6和PKD1来改善椎间盘退变。

Transplantation of gene-modified nucleus pulposus cells reverses rabbit intervertebral disc degeneration

Background Intervertebral disc degeneration is the main cause of low back pain. The purpose of this study was to explore potential methods for reversing the degeneration of lumbar intervertebral discs by transplantation of gene-modified nucleus pulposus cells into rabbit degenerative lumbar intervertebral discs after transfecting rabbit nucleus pulposus cells with adeno-associated virus 2 （AAV2）-mediated connective tissue growth factor （CTGF） and tissue inhibitor of metalloproteinases 1 （TIMP1） genes in vitro. Methods Computer tomography （CT）-guided percutaneous annulus fibrosus injury was performed to build degenerative lumbar intervertebral disc models in 60 New Zealand white rabbits, rAAV2-CTGF-IRES-TIMPI-transfected rabbit nucleus pulposus cells were transplanted into degenerative lumbar intervertebral discs （transplantation group）, phosphate-buffered saline （PBS） was injected into degenerative lumbar intervertebral discs （degeneration control group） and normal lumbar intervertebral discs served as a blank control group. After 6, 10 and 14 weeks, the disc height index （DHI） and signal intensity in intervertebral discs were observed by X-ray and magnetic resonance imaging （MRI） analysis The expression of CTGF and TIMP1 in nucleus pulposus tissue was determined by Western blotting analysis, the synthesis efficiency of proteoglycan was determined by a 35S-sulfate incorporation assay, and the mRNA expression of type II collagen and proteoglycan was detected by RT-PCR. Results MRI confirmed that degenerative intervertebral discs appeared two weeks after percutaneous puncture. Transgenic nucleus pulposus cell transplantation could retard the rapid deterioration of the DHI. MRI indicated that degenerative intervertebral discs were relieved in the transplantation group compared with the degeneration control group. The expression of collagen II mRNA and proteoglycan mRNA was significantly higher in the transplantation group and the blank control group compared with the degeneration control group （P 〈0.05）. Conclusions CT-guided percutaneous puncture can successfully build rabbit degenerative intervertebral disc models. Both CTGF and TIMPl-transfected cell transplantation helps to maintain disc height, and promotes the biosynthesis of tvDe II collaQen and proteoalvcan in intervertebral discs, reversinq the de（：ieneration of intervertebral discs.

Vitamin D Receptor(VDR) Genetic Polymorphisms Associated with Intervertebral Disc Degeneration

Intervertebral disc degeneration （IDD） is characterized by disc dehydration and herniation, which is often associated with low back pain and lumbar radiculopathy due to nerve root compression or inflammation. The pathophysiology of IDD is not completely elucidated so far. Some researchers have indicated that disc degeneration begins as early as the second decade of life （Mayer et al., 2013）. Common risk factors are considered to associate with age, gender, smoking history, occupation, disc injury, and biomechanical factors. However, some epidemiologic studies highlighted that disc degeneration may be caused to a large degree by hereditary factors with apparently a relatively minor effects of environmental and behavioral risk factors （Videman et al., 1998; Cheung et al., 2006; Eser et al., 2010; Mayer et al., 2013; Vieira et al., 2014）, which indicated that genetic factors might play an important role in the pathogenesis of IDD.

Construction of recombinant adenoviral vector Ad-CMV-hTGFβ1 for reversion of intervertebral disc degeneration by gene transfer

To provide a highly efficient adenov iral vector Ad CMV hTGFβ1 for the study of gene therapy for reversion of the intervertebral disc degeneration.Methods: A newly developed recombinant adenoviral vector constr uction system was used in the study. The cDNA of hTGFβ1 was first subcloned into a shuttle plasmid pShuttle CMV. The resultant plasmid was linearized by d ig esting with restriction endonuclease PmeI, and subsequently transformed into E .coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy 1. Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis. Finally, the recombinant plasmid linearized by PmeI was transfected in to 293 cells. Recombinant adenoviruses were generated within 2 weeks. Results: The recombinant adenoviral plasmids were cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8 % agarose electrophoresis. The infected 293 cells showed evident cytopathic effect (CPE). The productions of PCR confirmed the presence of recombinant adenovirus. The exp ression of hTGFβ1 was verified by immunohistochemical staining. Conclusions: The successful generation of the adenoviral vector Ad CMV hTGFβ1 and the confirmation of the interest gene expression make it p ossible for the experimental study of the reversion of the intervertebral disc d egeneration by gene therapy.

Gene expression profile of degenerated cervical intervertebral disc tissues in rats

Objective: To analyze the gene expression profile of degenerated cervical intervertebral disc of Sprague Dawley rats on a large scale.Methods: Degenerated models of Sprague Dawley rats of 9 months old (degeneration group, n=9) and normal Sprague Dawley rats of 3 months old (control group, n=9) were prepared, respectively. mRNA was obtained from the cervical intervertebral disc of rats in both groups, respectively, and then labelled by Cy5 and Cy3 fluorescence respectively after reverse transcription to obtain intervertebral disc cDNA probes. cDNA probes were hybridized with BiostarR-40s gene expression profile chips and scanned by laser scanner. The results were treated with portrait analysis, standardization management, and ratio analysis with softwares.Results: Compared with the rats in the control group, (9.6)% (381 pieces in total) gene expression changed obviously in the rats in the degeneration group, among which, the gene expression quantities of 171 pieces increased significantly (r=the ratio of the degeneration group to the control group >(2.0)), 52 pieces of which had certain function. While the gene expression quantities of 211 pieces decreased significantly (r<(0.5)), 41 pieces of which had certain function.Conclusions: Gene chip technology can be used to analyze the gene expression profile of degenerated intervertebral disc of rats in parallel, in quantity and on a large scale, which helps to testify the representative genes and protein expression, and plays an important role in clarifying the pathogenesis of degenerated intervertebral disc.

转录组分析鉴定腰椎椎间盘退行性变中潜在免疫相关生物标志物

目的通过转录组分析鉴定腰椎椎间盘退行性变(IDD)中潜在的免疫相关生物标志物,寻找IDD免疫相关的关键靶点。方法从基因表达汇编数据库(GEO)的GSE67567数据集中获得基因表达谱,并筛选差异表达mRNA、长链非编码RNA(lncRNA)和微RNA(miRNA)。使用加权基因共表达网络分析(WGCNA)和基因集富集分析(GSEA)筛选免疫相关模块,并使用Cytoscape构建lncRNA-miRNA-mRNA竞争性内源RNA(ceRNA)网络,通过免疫浸润分析确定相关的免疫细胞,使用Pearson相关分析筛选免疫相关的关键靶点。通过GSEA筛选关键的生物过程和途径。使用GEO的GSE124272数据集验证关键靶基因的表达。结果WGCNA结果表明,蓝绿色模块与免疫力有关。免疫浸润分析显示,CD4幼稚T细胞可能是关键的免疫细胞。Pearson相关分析表明,4个mRNA(UHMK1、ZFP36L2、ZCCHC3、ZBTB20)和1个lncRNA(LINC00641)可能是IDD免疫相关的关键靶点,建立了一个免疫调节相关的ceRNA网络。GSEA结果显示,LINC00641可能通过TGF-β信号通路调节IDD。验证结果表明,这些关键基因在GSE124272数据集中存在差异表达。结论mRNA(UHMK1、ZFP36L2、ZCCHC3、ZBTB20)和lncRNA(LINC00641)是IDD免疫相关的关键靶点。

miR-663b对IL-1β诱导的髓核细胞炎症反应和凋亡的影响及其机制

目的 探讨miR-663b对白细胞介素-1β(IL-1β)诱导的髓核细胞(NPCs)炎症反应和凋亡的影响及其机制。方法 根据对NPCs不同处理方式分为A组(无任何处理)、B组(IL-1β诱导)、C组(IL-1β诱导+miR-663b mimic转染)、D组(IL-1β诱导+miR-633b NC转染)。采用实时荧光定量PCR(RT-qPCR)法检测4组NPCs中miR-663b及炎症因子(TNF-α、IL-6、IL-1β)、Ⅱ型胶原蛋白、多聚糖蛋白基因的表达情况,采用CCK8法和TUNEL染色法检测4组NPCs增殖和凋亡情况,采用免疫印迹法检测各组NPCs IL-1受体1(IL1R1)蛋白表达情况。使用TargetScan数据库预测miR-663b与IL1R1间的潜在结合位点,将293T细胞分E组(转染IL1R1-wt质粒+miR-663b mimic)、F组(转染IL1R1-wt质粒+miR-663b mimic NC)、G组(转染IL1R1-mut质粒+miR-663b mimic)和H组(转染IL1R1-mut质粒+miR-663b mimic NC),检测各组NPCs荧光素酶的活性。结果 RT-qPCR结果显示,C组较A、B、D组miR-663b相对表达量显著上升(t=9.41~22.93,P<0.01);与B、D组相比,C组TNF-α、IL-6、IL-1β、Ⅱ型胶原蛋白及多聚糖蛋白基因相对表达量显著改变(t=3.17~32.51,P<0.01)。CCK8法及TUNEL染色法结果显示,与B、D组相比,C组NPCs增殖显著增加(t=3.14、3.96,P<0.01),而细胞凋亡显著减少(t=4.28、168.61,P<0.01)。RT-qPCR和免疫印迹法结果显示,与B、D组相比,C组NPCs IL1R1和IL1R1蛋白相对表达量显著下降(t=6.39~12.84,P<0.01)。双荧光素酶报告基因实验显示,E组荧光素酶活性显著低于F、G、H组(t=10.62~16.27,P<0.01)。结论 miR-663b或通过与IL1R1靶向结合下调NPCs的IL1R1表达,并减缓NPCs炎症反应及凋亡进程。

Caspase-3基因多态性与腰椎间盘突出症的关系

目的探讨Caspase-3基因多态性与腰椎间盘突出症(lumbar disc herniation,LDH)的关系。方法选择2021年1月~2022年7月在本院就诊的LDH患者137例作为研究组,选择同期在本院健康体检的受试者137例作为对照组。观察两组受试者Caspase-3基因中的三个SNP位点rs4647693、rs4647610和rs12108497单核苷酸多态性,观察研究组不同rs4647693、rs4647610和rs12108497基因型患者的临床资料差异。结果研究组和对照组Caspase-3 rs4647693基因型观察值和期望值吻合度良好,符合H-W平衡定律(x^(2)_(研究组)=3.153,P=0.076;x^(2)_(对照组)=2.902,P=0.088)。两组受试者基因型和等位基因频率差异均存在统计学意义(P<0.05)。_(研究组)和_(对照组)Caspase-3 rs4647610基因型和期望值吻合度良好,符合H-W平衡定律(x^(2)_(对照组)=2.058,P=0.151;x^(2)_(研究组)=3.614,P=0.057)。两组受试者基因型和等位基因差异均存在统计学意义(P<0.05)。_(研究组)和_(对照组)Caspase-3 rs12108497基因型和期望值吻合度良好,符合H-W平衡定律(x^(2)_(对照组)=3.367,P=0.067;x^(2)_(研究组)=2.243,P=0.134)。两组受试者基因型和等位基因差异均存在统计学意义(P<0.05)。与rs4647693 GG基因型比较,rs4647693 GA+AA基因型患者脊柱负荷1~2级患者比例显著升高,差异有统计学意义(P<0.05)。与rs4647610 AA基因型比较,rs4647610 AG+GG基因型患者LDH家族史和脊柱负荷3~4级比例明显升高(P<0.05);与rs12108497 TT基因型比较,TC+CC基因型患者LDH家族史和脊柱负荷3~4级患者比例显著升高(P<0.05)。结论Caspase-3 rs4647693、rs4647610和rs12108497 SNP与LDH的遗传易感性密切相关,Caspase-3基因多态性与LDH风险的影响可能也受脊柱负荷等级和家族史影响。

长链非编码SNHG1/miR-145/ADAM17轴在椎间盘髓核退变中的作用及其调控机制

目的 :研究长链非编码RNA小核仁RNA宿主基因1(SNHG1)、miR-145及解整合素-金属蛋白酶17(ADAM17)在椎间盘退变(IDD)组织中的表达,探讨SNHG1调控miR-145/ADAM17轴对椎间盘髓核细胞退变的影响及机制。方法 :采用qRT-PCR、免疫印迹检测30例IDD患者经手术摘除的髓核组织SNHG1、miR-145与ADAM17的表达;双荧光素酶报告基因、RNA免疫共沉淀等确定SNHG1、miR-145与ADAM17的关系;白介素(IL)-1β诱导髓核细胞建立细胞退化模型,SNHG1 siRNA或miR-145模拟物等转染后,CCK-8、流式细胞术等检测细胞增殖、凋亡以及相关分子指标的变化。结果 :相比于对照髓核组织,IDD髓核组织中SNHG1与ADAM17表达上调,miR-145表达下调,均与Pfirrmann分级显著相关。SNHG1可通过海绵作用靶向miR-145,miR-145也可靶向ADAM17的3’-非编码区(3’-UTR)抑制ADAM17蛋白表达。SNHG1沉默逆转了IL-1β诱导的髓核细胞凋亡及基质酶(基质金属蛋白酶13、ADAMTS4)合成,上调了collagenⅡ和aggrecan表达;而SNHG1过表达增强了IL-1β诱导的上述效应。结论 :SNHG1调控miR-145/ADAM17分子轴,促进髓核细胞凋亡和细胞外基质降解,参与椎间盘退变的发展变化。

大鼠退变颈椎间盘组织基因表达谱的研究

目的 :研究正常和退变模型大鼠颈椎间盘的基因表达谱 ,分析大鼠颈椎间盘基因表达水平的变化 ,用于指导诊断和治疗。 方法 :建立动静力失衡性大鼠颈椎间盘退变模型 ,从造模后 5个月对照组和模型组大鼠颈椎间盘中抽提 m RNA ,经逆转录分别用 cy3、cy5荧光标记 ,获得两组动物椎间盘 c DNA的探针 ,再与 5 12 c DNA基因表达谱芯片杂交 ,结果由激光扫描仪扫描并用软件进行图像分析、标准化处理、ratio值分析、聚类分析。结果 :共筛选出 18条表达有差异的基因 ,11条基因表达量明显下降 (ratio<0 .4 ) ,其中细胞信号转导类有 4条 ;7条基因表达量明显上升 (ratio>2 .5 )。这些基因在颈椎间盘退变的表达模式与其他的报道类似。 结论 :退变大鼠椎间盘基因表达的调节发生变化 。

退变腰椎间盘组织中细胞凋亡及相关基因bcl-2和bax表达的研究

目的:探讨腰椎间盘退变的发病机制。方法:采用原位DNA片段末端标记和免疫组织化学方法,检测40例退变腰椎间盘组织及10例正常腰椎间盘组织细胞凋亡状态及凋亡相关基因bcl-2和bax蛋白的表达。结果:40例退变腰椎间盘组织凋亡指数(AI)均值为70.51,bcl-2蛋白阳性细胞百分比为10.12％,bax蛋白阳性细胞百分比为54.56％。10例正常腰椎间盘组织AI均值为20.02,bcl-2蛋白阳性细胞百分比为53.46％,bax蛋白细胞百分比为10.54％。两组间AI均值、bcl-2蛋白表达、bax蛋白表达差异均有显著性意义(P均<0.05)。bcl-2蛋白表达越弱,bax蛋白表达越强,凋亡细胞数越多。结论:bcl-2和bax蛋白均参与了腰椎间盘组织中细胞凋亡的调节,并在腰椎间盘退行性变发生和发展中发挥着重要作用。

益气化瘀方调控颈椎间盘细胞基因表达谱的研究

目的 :分析正常、退变SD大鼠颈椎间盘基因表达水平的变化 ,研究益气化瘀方对大鼠退变颈椎间盘基因表达谱的调节作用。方法 :建立动、静力失衡性大鼠颈椎间盘退变模型 ,分别从正常、退变和益气化瘀方组大鼠颈椎间盘中抽提mRNA ,经反转录后分别用Cy3、Cy5荧光标记 ,获得动物椎间盘cDNA的探针 ,再与 5 12点cDNA基因表达谱芯片杂交 ,激光扫描仪扫描并进行图像分析、标准化处理、ratio值分析、聚类分析。结果 :模型组与对照组比较 ,共筛选出 18条表达有差异的基因 ,11条基因表达量明显下降 (ratio <0 4 ) ,7条基因表达量明显上升 (ratio >2 5 )。益气化瘀方与模型组比较 ,共筛选出 2 2条表达有差异的基因 ,益气化瘀方上调 18条基因 (ratio >2 5 ) ,下调 4条基因 (ratio <0 4 )。其中在上述检测中表达最明显的是信号传导类基因 ,益气化瘀方能够上调多数细胞信号传导类基因 (如PTK等 )。结论 :初步筛选出大鼠退变椎间盘中相关基因 ,与已报道基因在颈椎间盘退变的表达模式类似 ,说明大鼠椎间盘退变受到各种基因的调控 ,初步阐明了颈椎病在基因水平上的发病机理。益气化瘀方参与了椎间盘细胞内相关基因的调控 ,参与椎间盘细胞内外的信号传导过程 ,从基因表达角度解释了中药治疗颈椎病的药效机理 ,丰富了中医?

人退变椎间盘组织的基因表达谱

目的 :研究人类退变椎间盘的基因表达谱 ,分析人退变椎间盘基因表达水平的变化。方法 :按条件优化的一步法抽提人退变及正常椎间盘组织的总RNA各 3例 ,分别用cy3 ,cy5荧光标记 ,获得 2组椎间盘cDNA的探针 ,与含有 40 96条人类全长基因的cDNA表达谱芯片杂交 ,扫描芯片荧光信号图像 ,对所获得的基因进行生物信息学分析。结果 :在 40 96条基因中 ,有差异表达的基因 70 6条 ,3 5 8条基因表达量明显下降 ,2 98条基因表达量明显上升。在有差异表达的基因中 ,细胞凋亡相关类蛋白 8条 ,其中上皮细胞膜蛋白 (EMP 1 )基因的表达上调明显。结论 :退变椎间盘的基因表达发生变化 ,细胞凋亡增加可能是椎间盘退变发生和发展的因素之一。

益气化瘀中药对大鼠椎间盘软骨细胞基因表达的影响

目的:观察正常培养、诱导凋亡以及益气化瘀中药干预后的大鼠椎间盘软骨细胞基因表达水平的变 化,研究益气化瘀中药对椎间盘凋亡软骨细胞基因表达谱的调节作用。方法:建立大鼠颈椎间盘软骨细胞培 养体系,使用抗Fas抗体诱导凋亡,分别从正常组、诱导凋亡组和益气化瘀中药组的大鼠颈椎间盘软骨细胞 中抽提mRNA,经反转录后分别用Cy3、Cy5荧光标记,获得椎间盘软骨细胞cDNA的探针,再与 BiostarR 40s的基因表达谱芯片杂交,ScanArray4000激光扫描仪扫描,GenePixPro3.0图像处理软件进 行图像分析、标准化处理和ratio值分析。结果:诱导凋亡组与正常组比较,共筛选出30条表达有差异的已 知基因,20条基因表达量明显下降(ratio<0.5),10条基因表达量明显上升(ratio>2);中药组与凋亡组比 较,共筛选出97条表达有差异的已知基因,中药上调44条基因(ratio>2),下调53条基因(ratio<0.5),涉 及到丝分裂素激活蛋白激酶(mitogen activatedproteinkinase,MAPK)和信号转导和激活因子(signal transducerandactivatoroftranscription,STAT)。结论:益气化瘀中药对椎间盘凋亡软骨细胞的调控涉及 多个方面,其对MAPK和JAK STAT通路的影响可提示益气化瘀中药对信号转导通路的调控作用。这从 一个侧面探索了中医气血理论与信号转导的关系。

骨形态发生蛋白-2与椎间盘细胞Sox9和Ⅱ型胶原基因的调控关系

目的:探讨骨形态发生蛋白-2(BMP-2)对椎间盘细胞Sox9和Ⅱ型胶原基因表达的调节作用。方法:应用逆转录聚合酶链反应和Western印迹检测不同浓度BMP-2对培养的人椎间盘细胞中软骨特异性基因Sox9和Ⅱ型胶原基因表达的调节作用。结果:BMP-2浓度为100ng/ml和1000ng/ml时,椎间盘细胞中Sox9和Ⅱ型胶原基因mRNA表达与0ng/ml组(对照组)相比明显增加(P<0.05);在蛋白水平的检测中,也得到了一致的结果。结论:BMP-2可以按照剂量依赖方式正向调节椎间盘细胞Sox9和Ⅱ型胶原基因的表达,提示BMP-2可能对退变早期的椎间盘具有修复功能。