摘要
据WHO统计,全球每年新发脑卒中1500万例,其中500万人死亡,500万人永久性残废。我国每年发生脑卒中的病人达200万,其中120万人死亡。脑卒中发病后果严重,病人非死即残,存活病人致残率高达75%,且易复发。中国政府每年因脑卒中直接经济支出374亿元。
然而目前对于预防脑卒中的发生,除了降压外,尚无有效措施。高血压是脑卒中最主要的独立危险因素,超过60%的脑卒中患者有高血压病史。但是却并非所有高血压患者都会发生脑卒中。这就说明高血压不是脑卒中发生的唯一致病因素。那么,不易发生脑卒中的高血压患者体内是否存在着某些保护因素,或者易发生脑卒中的高血压患者体内是否存在着某些非高血压的其他危险因素?
为了寻找除高血压以外与脑卒中发病相关的保护或危险因素,提出新的防治脑卒中的策略,进而更好地预防脑卒中的发生,大幅度降低脑卒中对人类的危害的需求显得异常迫切。
脑卒中倾向的自发性高血压大鼠(Stroke-Prone Spontaneously Hypertensive Rats,SHR-SP)是目前研究脑卒中最常用的动物模型。它是从自发性高血压大鼠(Spontaneously Hypertensive Rats,SHR)中选择交配,再经数代近亲繁殖培育而成的品系。SHR-SP具有自发性脑卒中倾向,几乎全部死于脑卒中,是研究脑卒中的理想模型。
与人类相似,SHR与SHR-SP同为高血压动物,但SHR-SP更易发生脑卒中,而SHR发生脑卒中的几率则极低。因此本实验设计通过SHR-SP与SHR脑组织的基因表达谱对比分析,试图寻找脑卒中发生中除高血压以外的其他因素,阐明脑卒中发生的病理机制,为脑卒中的防治提供新的靶点和思路。
本实验利用基因芯片技术对SHR和SHR-SP脑组织的全基因表达谱进行了对比分析。结果发现与SHR相比,SHR-SP中核黄素激酶(riboflavin kinase,rfk)相关的功能可能有所变化。rfk是游离核黄素转化成体内活性形式FMN(flavin mononucleotide)的催化剂,FMN再经进一步转化成为核黄素在体内的另一活性形式FAD(flavin adenine dinucleotide)。目前,对于核黄素相关功能与脑卒中的直接关系报道较少,更没有rfk与脑卒中的关系的报道,对rfk的相关研究也仅仅局限于植物或菌类中。
综上所述,本课题主要拟进行以下几个方面的研究:
基因芯片技术检测SHR-SP和SHR脑组织基因表达谱;
生物信息学分析SHR-SP与SHR脑组织差异表达基因;
SHR-SP与SHR脑内rfk差异表达验证;
核黄素及其相关物质对脑卒中发生的影响。
【研究目的】:试图寻找脑卒中发病原因中除高血压之外的重要影响因素;探索rfk表达水平对脑卒中发生的影响;阐述核黄素相关物质对脑卒中发病的影响及其机制。
【研究方法和结果】:
1、SHR-SP与SHR脑组织差异基因表达谱分析
基因组学分析:通过全基因组芯片技术筛选出SHR-SP与SHR的脑组织差异表达基因。这些基因可能是参与抑制或促进脑卒中发生的重要基因。实验样本经质检合格。通过对芯片扫描转化为相应信号值,并对信号值做分析比对。结果显示实验样本均一性良好,实验成功。按照P≤0.05且Ratio≥2或≤-2,共筛选出106个差异表达基因,其中55个在SHR-SP中高表达,51个在SHR中高表达;
生物信息学分析:将基因芯片导出的基因原始数据信息用生物信息学方法进行分析,筛选出抑制或促进脑卒中发生的相对重要的基因或通路。对芯片数据均一化处理后,按照P≤0.05且Fold Change≥2或≤-2筛选,结果显示有76个差异表达基因,其中41个在SHR-SP中高表达,35个在SHR中高表达;17个Pathway差异具有统计学意义(P≤0.05),252个GO差异具有统计学意义(P≤0.05)。通过差异GO的分析结果,我们认为SHR-SP脑内rfk水平的下调(P<0.01)可能与脑卒中的发生有密切关系。
2、SHR-SP与SHR脑内rfk差别验证
对于筛选出来的差异表达基因rfk进行不同水平的验证,包括real-time PCR验证rfk mRNA表达水平差异、Western Blot(WB)验证rfk蛋白质表达水平差异。
Real-time PCR:结果显示与SHR相比,SHR-SP脑内rfk的mRNA表达水平下降至SHR脑内表达量的73 %,差异具有统计学意义(P<0.01)。这一结果验证了基因芯片的检测结果;
WB:结果显示与SHR相比,SHR-SP脑内rfk的蛋白表达水平明显下降。这一结果与基因芯片和PCR检测结果相一致,进一步说明了SHR-SP脑内rfk的下调可能与脑卒中的发生有重要关系。
3、黄素类物质对脑卒中发病的影响
VB2(核黄素)、FMN、FAD统称为黄素类物质。基于前期工作的基础上,我们认为rfk的下调可能与脑卒中的发生有密切的关系。rfk主要催化体内核黄素向活性形式FMN的转化,rfk水平下调,势必会降低核黄素在体内的生物利用,影响核黄素功能的正常发挥。那么核黄素相关物质对脑卒中的发生和发展过程是否起到保护或改善的作用为解决这一问题,我们进行了如下实验。
MCAO实验:实验动物为40只雄性C57BL小鼠,随机分为四组:对照组、VB2组、FMN组、FAD组。每只小鼠均以3.5 mmol/kg腹腔注射相应黄素类物质,对照组注射同体积生理盐水。注射一周后,于第8天进行大脑中动脉栓塞(Middle Cerebral Artery Occlusion,MCAO)手术,术后及术后12h分别腹腔注射一次相应黄素类物质。于24h取脑组织,进行TTC染色。结果显示与对照组脑梗死率16.6±3.3%相比,VB2、FMN、FAD均使MCAO所导致的脑梗死面积明显下降,差别具有统计学意义,梗死率分别为11.5±3.9%(P<0.01),9.5±3.2%(P<0.01),11.2±4.4%(P<0.01)。这一结果说明VB2、FMN、FAD明显抑制脑卒中的发生和发展过程,起到保护性作用。
流式细胞术实验:取SHR-SP胎鼠,培养原代大脑皮层神经元,于种板后第7 ~ 10天进行实验。神经元缺糖缺氧模型(Oxygen and Glucose Deprivation,OGD)建立主要是模拟脑卒中发病时的缺血缺氧状态。神经元首先以分别加入不同浓度VB2、FMN、FAD的高糖Krebs培养基处理2 h。2 h后进行缺糖缺氧模型建立,神经元以分别加入不同浓度VB2、FMN、FAD的无糖Krebs培养基中,放入三气培养箱,设定为94% N2,5%CO2,1% O2作用1.5 h。缺糖缺氧1.5h后,立即将神经元消化收集到eppendorf管中,应用流式细胞术测定神经元死亡和凋亡状况。结果显示VB2、FMN、FAD在0.5μM时神经元凋亡率和死亡率明显下降,而在浓度高于2.5μM时抗死亡和抗凋亡作用不明显。这一结果说明黄素类物质在0.5μM浓度时对神经元具有明显的抗缺糖缺氧的保护作用。这提示我们黄素类物质对MCAO脑梗死的保护作用可能是通过抑制神经元凋亡产生的。
WB实验:为了探讨黄素类物质抗凋亡作用的确切机制,我们进行了如下实验。神经元培养、OGD模型的建立和药物处理过程同上。缺糖缺氧1.5h后,立即对神经元进行蛋白提取,并进行WB实验,检测凋亡相关蛋白bax和bcl-2的表达情况。结果显示抗凋亡物质bcl-2在各剂量组间没有明显变化,而bax则在VB2和FMN浓度为1.5μM时的表达量最少,在FAD中则变化不明显。说明VB2和FMN在1.5μM剂量时对OGD神经元具有很好的抗凋亡保护作用。这一结果证明黄素类物质抗OGD神经元凋亡的机制可能是通过抑制促凋亡蛋白bax的表达从而抑制线粒体凋亡实现的。
【结论】:
SHR-SP与SHR相比,共有76个基因表达发生变化,其中35个基因下调,41个基因上调。
rfk相关功能的变化可能与脑卒中发生有密切关系。rfk水平下调,可能影响核黄素在体内的吸收和利用而失去对脑组织的保护作用,导致脑卒中发生。
黄素类物质能明显抑制MCAO脑梗死的发生和发展;黄素类物质能明显抑制SHR-SP大脑皮层神经元在OGD条件下的凋亡和死亡的发生,这可能是其对MCAO脑梗死具有保护作用的重要原因;黄素类物质抗凋亡的机制可能是通过抑制促凋亡蛋白bax的表达从而抑制线粒体凋亡实现的。
According to the report of WHO, 15 million people suffered form stroke each year, in which 5 million people died and 5 million people became disabled. In China, there are 2 million people suffered from stroke including 1.2 million peoples died. The consequences of stroke are severe. Patients die or become disabling, and the rate of disability among the survival patients is high to 75%. China government has to cost 37.4 billion Yuan on stroke per year.
While, except decreasing blood pressure, there is no effective method to prevent stroke. Hypertension has been confirmed to be the first risk factor to induce stroke. More than 60% stroke patients had the history of hypertension. But, not all the patients with hypertension suffered stroke and not all the patients with stroke had the history of hypertension. It is to say that hypertension isn’t the only risk to induce stroke. Therefore, is it possible that some protective factors exist in patients with hypertension who didn’t suffer from stroke or that some other risk factors exist except hypertension to induce stroke
It’s an urgent demand to find out more protective or risk factors related to stroke except hypertension.
Stroke-Prone Spontaneously Hypertensive Rats (SHR-SP) is the most frequently used animal model on stroke study. SHR-SP is preferential mating from Spontaneously Hypertensive Rats (SHR) and endogamy through generations. Adult SHR-SP spontaneously suffers from stroke. Therefore, SHR-SP is believed to be ideal animal model on stroke study.
Both SHR and SHR-SP are hypetensive animals, whereas all SHR-SP are prone to suffer from stroke. Therefore, we compared the gene expression profiling of brain from SHR-SP with brain form SHR, through genechips, on the purpose of finding the other factors related to stroke except hypertension.
In our experiment, genechips were used to comparatively analyse the gene expression spectrum of brain from SHR-SP and SHR. The result showed that compared with the level of riboflavin kinase (rfk) in SHR, it is down regulated in SHR-SP. Till now, no relationship between rfk and stroke has been reported.
Therefore, our study was designed as follows:
The gene expression spectrums of brain from SHR-SP and SHR were comparatively analysed by genechips;
The differently expressed genes detected by genechips between SHR-SP and SHR were analysed by bioinformatics;
The rfk level in SHR-SP and SHR were analysed by real-time PCR and Western Blot (WB);
The relationship between riboflavin kinase and stroke were studied.
【PURPOSE】: Our purpose was to find the risk factors inducing stroke excluding hypertension and to explore the relationship of riboflavin kinase with stroke.
【METHODS and RESULTS】:
1、Comparative analysis of gene expression spectrums of SHR-SP and SHR by genechips
Gene expression profiling: Genechips were used to analyse the different expressed genes between SHR-SP and SHR. These genes may be the factors inhibit or prompt the occurrence of stroke. All the brain samples were qualified after quality monitoring. The gray scales of genechips were scaned and transformed to digital signals. The digital signals were used to analyse. With the condition of P≤0.05 and Ratio≥2 or≤-2, 106 genes were screened, in which 55 genes highly expressed in SHR-SP and 51 genes highly expressed in SHR.
Bioinformatics analysis: The raw data obtained from genechips were performed bioinformatics analysis, to screen the important genes or pathways related to stroke. The data were uniformly treated and screened with the condition P≤0.05 and Fold Change≥2 or≤-2. The result showed 76 genes differently expressed in SHR-SP and SHR, in which 41genes highly expressed in SHR-SP and 35 genes highly expressed in SHR. 17 Pathways and 252 Gene ontology (GO) significantly changed (P≤0.05). According to the GO analysis, we deduced the down regulation of rfk (P<0.01) might highly related to the occurrence of stroke.
2、The rfk level in SHR-SP and SHR was comparatively analysed
The level of rfk, which was doubted to down regulation in SHR-SP based on the genechip analysis and bioinformatics analysis, was comparatively analysed by real-time PCR and WB.
Real-time PCR: compared with the mRNA level of rfk in SHR, it was obviously decreased to 73% in SHR-SP (P<0.01). This confirmed the result of genechips.
WB: compared with the protein level of rfk in SHR, it was obviously decreased in SHR-SP. This confirmed the result of both genechips and real-time PCR. The results further indicated that the down regulation of rfk might highly relate to the occurrence of stroke.
3、Effect of flavins on stroke
VB2, FMN and FAD are called flavins together. Based on the above results, we believe the down regulation of rfk may highly relate to the occurrence of stroke. Flavokinase is an enzyme that catalyzes the phosphorylation of riboflavin (VB2) to form
FMN, which is an obligatory step in VB2 utilization and flavin cofactor synthesis. Down regulation of rfk will affect the bioavailability of riboflavin in vivo and therefore affect the function of riboflavin. To study whether flavins avoid or alleviate stroke, we designed the experiment as follows:
MCAO: Forty C57BL mouse were randomly assigned into four groups: the controls, VB2 group, FMN group and FAD group. All animals were intraperitoneally injected 3.5 mmol/kg corresponding falvins. The controls were injected the same volume of physiological saline. After a week administration, the operation of Middle Cerebral Artery Occlusion (MCAO) was performed on animals on day 8. Additional injections of corresponding flavins were performed both 0h and 12h after MCAO. 24h after MCAO,brains of all the animals were dissected and proceeded TTC stain. From the result, compared with the infarction rate of the controls with 16.6±3.3%, VB2, FMN and FAD all decreased the infarction rate with 11.5±3.9% (P<0.01), 9.5±3.2% (P<0.01) and 11.2±4.4% (P<0.01), respectively. This indicated that VB2, FMN and FAD could obviously protect brain from stroke.
Flow cytometry: Fetuses of SHR-SP were used to cultivate primary neurons of cerebral cortex. Between d7 ~ 10 after mother blank, neurons could be used to perform experiment. The model of Oxygen and glucose deprivation (OGD) on neurons was established to simulate the ischemia and anerobic environment of stroke. Neurons were treated with high glucose Krebs solution, in which flavins were added respectively, for 2h before OGD. Then neurons were treated with no glucose Krebs solution, in which flavins were added respectively, and were moved into incubator with 94% N2, 5%CO2 and 1% O2 for 1.5h. After1.5h OGD, neurons were immediately digested and collected into eppendorf tubes and the flow cytometry related operating was performed. The results showed VB2, FMN and FAD decreased the apoptosis rate and the death rate statisticly on the concentration of 0.5μM, but these protective effect diappeared when the concentration was up to 2.5μM. This suggesting that flavins protect neurons against OGD on the concentration of 0.5μM.
WB: to explore the accurate mechnisums of protective effect of flavins on neurons against apoptosis, we designed the experiment as follows. The neurons cultivation, OGD model establishment and the flavins treating were the same as described in Folw cytometry. After OGD for 1.5h, the neurons were immediately treated with protein extraction. Protein level of bcl-2 and bax among groups were detected by WB. The results showed the level of bcl-2 did not alter among different doses of flavins. Whereas, the level of bax had the lowest expression at the concentration of 1.5μM VB2 and FMN, but not FAD. These suggest that VB2 and FMN at the concentration of 1.5μM displayed the strongest protection of neurons against apoptosis induced by OGD.
【CONCLUSIONS】:
Between SHR-SP and SHR, 76 genes differently expressed, of which 35 genes highly expressed in SHR and 41 genes highly expressed in SHR-SP.
Down regulation of rfk might highly relate to the occurrence of stroke. The level of rfk may affect the absorbance and utility of riboflavin. That’s the reason why brain loses the protection from riboflavin and increases the risk of stroke.
Brain infarction induced by MCAO could be obviously inhibited by flavins. The apoptosis rates and death rates of neurons induced by OGD could be obviously inhibited by flavins. Flavins protect brain from MCAO through anti-apoptosis. The mechenisum of flavins anti-apoptosis may mediated by depressing the expression of bax protein, who paly an important role in inducing mitochondrial apoptosis.
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