肝郁证模型大鼠海马差异基因表达谱的研究
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摘要
一、研究背景
辨证论治是中医学的精华和优势,“证”是辨证论治的依据,是疾病处于一定阶段病因、病位、病性和病势的综合,能体现患者的整体性特征。证本质的研究是中医学现代化研究的核心内容。肝郁证是中医肝病的一个基本证型,是许多常见临床证候的基础,广泛存在于临床各系统疾病中。自上世纪50年代以来,肝郁证本质的研究一直是中医研究领域的热点,已初步证实肝郁证具有现代病理生理学基础,并部分地阐释了其中医理论的科学性,取得了阶段性成果,认为和机体神经、内分泌、循环、消化、免疫等多系统密切相关。但是对肝郁证的评价还处于笼统、多指标、不确定状态,有待进一步深入研究,从系统而全面的角度来阐释肝郁证的实质。
基因组学在整体上研究基因的功能,这与中医学的“整体观”概念十分相似。基因芯片是随着人类基因组研究的深入而迅速发展起来的一项重要的生物学技术,它的特点是高通量、快速、并行化采集生物信息。作为后基因组时代一项举足轻重的技术,基因芯片使生命科学与医学用全方位的整体新思维方式研究成为可能,因而广泛的应用于医学研究的各个领域。若借助基因芯片这种先进、快速的研究手段,可以尝试从基因水平探讨肝郁证的本质。可以设想,通过对同病异证和异病同证的基因表达谱差异比较中寻找证候的共同性和差异性建立一个“证候-基因表达谱”,揭示基因与证的内在联系,也会使中医辨证论治理论得到全面的检验和应用。
二、研究方法
目的:
采用慢性束缚应激的方法复制与临床接近的、有效的大鼠肝郁证模型;利用基因芯片对肝郁证大鼠的海马组织中的差异表达基因进行分析比较,在动物模型上寻找肝郁证相关的基因,尝试从基因水平对肝郁证的微观辨证进行初步探讨,也为从临床疾病的角度对肝郁证相关基因的研究提供理论支持;比较逍遥散干预大鼠与模型大鼠之间的差异基因表达,一方面从基因转录水平探讨逍遥散对肝郁证的作用机理,另一方面以方测证,方证互参,说明肝郁证的本质。
方法:
在肝郁证理论指导下,复制慢性束缚应激肝郁证大鼠模型,并通过行为学实验和检测大鼠血清激素水平验证模型的可靠性;并用中药复方逍遥散干预造模动物,验证逍遥散对肝郁证大鼠的治疗作用;采用含有27000个已知基因的大鼠全基因组芯片检测大鼠海马内差异基因表达情况,比较模型大鼠与正常大鼠、逍遥散干预大鼠与模型大鼠的差异表达基因,利用生物分子功能注释系统(MAS)对与疾病相关的基因进行分析;用实时荧光定量PCR技术验证部分与疾病相关的差异表达基因结果的可靠性。
1.慢性束缚应激肝郁证大鼠模型的复制
情志异常是肝郁证的主要病因,不良生活事件所形成的心理应激则是情志病因的实质;肝郁证是对郁怒、抑郁、焦虑等负性情绪心理应激状态下,以高级神经中枢调节机制紊乱为前提,涉及多系统、多器官的病理改变。基于上述考虑,参照文献报道,从慢性应激的角度出发,将18只同周龄雄性Wistar大鼠随机分3组:肝郁证模型组、逍遥散组、正常对照组,每组各6只,模型组与逍遥散组大鼠单笼饲养,每日将大鼠置于束缚笼内,固定其身体与尾部,使之不能随意活动,每天6小时;逍遥散组大鼠在造模的同时,以逍遥散中药煎液10g/kg.d灌胃;正常对照组不予任何干涉,时间为3周。观察大鼠的一般状况,每周对各组大鼠进行一次液体消耗实验及摄食实验,造模开始与结束时分别对各组大鼠进行一次敞箱实验,并在造模结束后取大鼠血清,检测血清的CRH含量,以验证肝郁证模型的可靠性,并且论证逍遥散对肝郁证的治疗作用。
2.利用基因芯片检测模型组与正常组大鼠海马的差异表达基因
造模结束后处死所有大鼠,分离海马组织,提取海马总RNA,在检验总RNA的质量与数量合格后,总RNA反转录得到cDNA,体外转录合成生物素标记的cRNA,再与基因芯片进行杂交反应,这里我们选用北京博奥生物公司的含有27000个已知基因的晶芯~(?)大鼠全基因组表达谱芯片(27K Rat Genome Array);用LuxScan 10KA双通道激光扫描仪对基因芯片进行扫描,得到半定量杂交结果后,采用LuxScan 3.0图像分析软件对芯片图像进行分析,把图像信号转化为数字信号,筛选出模型组与正常组大鼠海马的差异表达基因。借助MAS系统,通过搜索NCBI基因组数据库,对检测到的差异表达基因进行查询和确认,获得相关基因的信息并进行生物学注释,分析与疾病相关的基因在肝郁证的发生中可能的机制。
3.利用基因芯片检测逍遥散组与模型组大鼠海马的差异表达基因
同样采用27K Rat Genome Array芯片筛选出逍遥散组与模型组大鼠海马的差异表达基因,借助MAS系统,通过搜索NCBI基因组数据库,对差异表达基因进行查询和确认,获得相关基因的信息并进行生物学注释,尝试初步探讨逍遥散对肝郁证在基因水平上的作用机理。
4.实时荧光定量PCR方法验证部分差异表达基因
提取各组大鼠海马的总RNA,反转录得到cDNA,采用SYBR GreenⅠ荧光染料实时定量PCR方法,检测已有的基因芯片结果中部分与疾病相关的基因在各组大鼠海马中的表达情况,用以验证基因芯片结果。
结果:
1.成功复制了慢性应激肝郁证大鼠模型。造模3周后,一般状况来看,模型组大鼠主要表现为倦怠少动、喜欢贴壁、叫声尖细、反应迟缓、饮食减少,伴有毛色晦暗发黄、大便颗粒松散甚至呈水样等等肝郁证的表现。液体消耗实验结果发现,模型组大鼠造模后的糖水偏好程度显著低于正常组大鼠(P＜0.01);摄食实验中模型组大鼠的摄食量亦较正常组大鼠有显著性下降(P＜0.05);畅箱实验测得的动物行为学评分来看,造模后模型组大鼠的水平运动得分和垂直运动得分明显低于同时期的正常组大鼠(P＜0.01);此外,模型组大鼠的血清CRH浓度显著高于正常组大鼠(P＜0.01)。说明慢性束缚应激确能造成大鼠的肝郁证,能有效的模拟临床中肝郁证兴趣丧失、快感缺乏等症状。
2.大鼠海马样品的总RNA经检验质量合格,顺利进行后续实验。模型组与正常对照组大鼠海马的芯片检测结果为:共有48个差异表达基因,表达上调的基因有15个;表达下调的基因33个。搜索基因组数据库及用MAS系统进行分析,对与疾病相关的基因进行功能分析,结果发现,部分差异表达的基因可以阐释肝郁证模型以及临床病例的某些主要表现。大致分为3类:(1)与神经精神类疾病相关的基因:主要有脑源性神经营养因子(Bdnf)、色氨酸羟化酶1(Tph1)、糖原合成酶激酶-3(Gsk3b)等,它们的表达异常,说明肝郁证中存在神经内分泌系统的紊乱,神经递质的调节上的发挥失常及神经元受损等机制,可能是导致肝郁证患者情绪低落、兴趣丧失、运动迟缓、饮食减少等的原因,也在一定程度上解释了肝郁证与现代医学中抑郁症、焦虑症等疾病的密切联系;(2)与肿瘤相关的基因:中期因子(Mdk)、叶酸受体1(Folr1)、水通道蛋白1(Aqp1)、胰岛素样生长因子结合蛋白2(Igfbp2)等,此结果提示肝郁证大鼠存在某些肿瘤的易感性,可能与临床上肝郁证患者气滞血瘀,日久成积而引起肿瘤发生有一定的相关,与我们前期肝郁证大鼠蛋白质组学研究结果类似;(3)其他疾病相关基因:如血管紧张素转化酶(Ace)、细胞内氯通道(Clic6)、X联内肽酶同源性的磷酸盐调节基因(Phex)等,这些基因的表达改变与肝郁证的关系有待进一步研究。
3.逍遥散干预3周后,逍遥散组大鼠的倦怠少动、饮食减少等症状明显改善;液体消耗实验、摄食实验及畅箱实验结果发现,逍遥散组大鼠的糖水偏好程度、摄食量及行为学评分在第3周时与模型组相比,均有明显改善;与模型组大鼠比较,逍遥散组大鼠血清CRH浓度亦有升高趋势;逍遥散干预组与模型组大鼠海马的芯片检测结果为:共有22个差异表达基因,表达上调的基因有7个;表达下调的基因15个。其中与疾病关系密切的基因有:神经元钙传感器(Ncs-1)、阿片黑素促皮质激素原(Pomc)、色氨酸羟化酶1(Tph1)等,提示逍遥散对肝郁证的治疗作用可能与稳定细胞膜,防止钙超载,提高神经元抗损伤能力,双向调节HPA轴,神经内分泌功能等机制相关。另一方面从方证互参的角度也反证了肝郁证模型的可靠性。
4.实时荧光定量PCR方法检测了Bdnf、Tph1、Mdk等3个基因在各组大鼠海马中的表达情况。结果发现,RNA质量完好,PCR反应扩增效果较好,PCR产物纯度较高,目的基因Bdnf、Tph1、Midkine在各组大鼠海马的表达比较与基因芯片检测结果基本一致,验证了芯片部分检测结果的可靠性。
结论:
1.本次研究采用慢性应激束缚的方法,成功的复制了大鼠的肝郁证模型,模型可靠。
2.利用基因芯片检测出的模型组与正常组大鼠海马的差异表达基因中,部分基因的异常表达提示肝郁证可能与神经内分泌系统的紊乱,神经递质调节失常、神经元受损等机制密切相关,同时也提示肝郁证存在一定的肿瘤易感性。
3.利用基因芯片检测出的逍遥组与模型组大鼠海马的的差异表达基因中,部分差异基因的表达提示逍遥散可能通过提高神经元对应激损伤的耐受性,调节神经内分泌功能等机制来发挥治疗肝郁证的作用。
4.通过实时荧光定量PCR方法检测了部分与疾病相关基因在各组大鼠海马中的表达情况,与基因芯片的检测结果是基本吻合的,验证了部分基因芯片表达结果的可靠性。
肝郁证大鼠模型海马差异表达基因的研究,为从基因水平探讨肝郁证实质研究的深入作了有益的尝试。
Background
Syndrome differentiation and treatment are the advantage of the Traditional Chinese Medicine. Being one of features of TCM theory, syndrome was based on TCM basic theory, has been paid attention in studying of Traditional Chinese Medicine in recent years. As basic syndrome of liver diseases of Chinese medicine is concerned, liver-qi stagnation syndrome is one of common syndromes in clinical practice and highly valuable for profound research. The nature of liver-qi stagnation syndrome is the focus of TCM theory study today. Although lots of achievement was got during the pasted 50 years, they still can't evaluation the essence of liver depression syndrome integrity, more exploration was needed.
Gene chip, also called DNA chip or DNA array, is one of advanced molecular biological technology which was accompanied by development of study of human genome and is applied to many medical and biological science research areas already. As an important method in the post-genome era, gene chip gives a chance to study of liver-qi stagnation syndrome deeply. Both genomics and the study of traditional Chinese medicine syndrome integrity and dynamic generated the syndrome-genomics accordingly.
Objective
To set up the effective rat model of liver depression syndrome from chronic constraint stress to find differentially expressed genes in hippocampus between the rat model and the normal control, and between the rat model and rat treated by Xiaoyao Powder through gene chip.
Methods
Set up the rat model of liver depression syndrome from chronic constraint stress, compared with normal control group and treatment group of Xiaoyao Powder, every group 6 rats. The changes in body weight and food intake in the animals were observed. By open-field test and fluid consumption test, the behaviors of the animals were determined every week. We harvested the serum and separated hippocampus tissues of rats respectively after 3 weeks. Corticotropin releasing factor (CRF, also CRH) in serum was detected after the animals were executed.
Gene chip is used in this study. Method of one step of Trizol reagent was applied to extract the total RNA of hippocampus of every group. The total RNA was reverse transcribed to cDNA which was then transcribed to cRNA marked by cy3 and cy5 in vitro. The cRNA was hybridized with 27K Rat Genome Array, and the gene chip was scanned. After analyze the gene chip image, we screened the differentially expressed genes, and then searched the NCBI genomic databases to defined the gene by means of CapitalBio(?) Molecule Annotation System (CB-MAS). Comparing and analyzing the differentially expressed genes between the rat model and normal control, we speculated the pathogenesis of liver depression syndrome, and the differentially expressed genes between the rat treated by Xiaoyao Powder and the rat model to search for the mechanisms of Xiaoyao Powder to liver depression syndrome at the level of genome and prove reliability of liver depression syndrome model on the other hand. Some differentially expressed genes of hippocampus of rats in different groups are verified through real-time PCR.
Results
The rat model of liver depression syndrome has been established successfully at 21st day. The model rats displayed the typical appearances such as low activity, slow reaction, taper cry, reduces food and drink, like adherence, and so on. In the model group, the rats showed decreased body weight and food intake, reduced squares crossing and rearing in open-field test, a significantly reduced consumption of preference for sucrose solutions and an increased pure water consumption as compared with control group. The serum level of CRH was also increased in rat model, showing the function disorder of HPA axis.
We found that a total of 48 genes expressed differentially in hippocampus between rat model and normal control, among which 15 were up-regulated and 33 were down-regulated. After analyzing the function of genes relate to common disease, we knew the differential expression genes of liver-qi stagnation syndrome involved in three categories: (1) neurologic and psychiatric diseases genes, such as Bdnf, Tph1, Gsk3b etc; (2) tumor genes, including Midkine, Folr1, Aqp1, Igfbp2 and so on; (3) genes related to other diseases, such as Ace, Clic, Phex.
It was found that Xiaoyao Powder can improve the typical symptoms of liver depression syndrome, increasing the food intake, consumption of and preference for sucrose solutions and squares crossing and rearing in open-field test. It can also reduced the serum level of CRH in rat model. In addition, a total of 22 genes expressed differentially in hippocampus between rat treated by Xiaoyao Powder and rat model, among which 7 were up-regulated and 15 were down-regulated. We analyzed these genes closely related to diseases such as Ncs-1 and Pomc and found that Xiaoyao Powder probably can regulate the neuroendocrinology and function of HPA axis.
Expressions of Bdnf, Tph1 and Midkine of hippocampus in different groups tsested through real-time PCR are consistent with results of the gene chips. The results of real-time PCR prove part of chip datas are credible.
Conclusion
The rat model of liver depression syndrome from chronic constraint stress is reliable and credible. The abnormal expressed genes in hippocampus of rat model suggest that liver depression syndrome is probably related to disorder of neuroendocrinology and neurotransmitters, and have tumor susceptibility at level of genome, witch may be regulated by Xiaoyao Powder. The results of real-time PCR of some differentially expressed genes of hippocampus of rats in different groups prove the reliability of the datas of gene chips.
The study of gene differential expression in Rat Modal of liver depression by gene chip established a foundation from theory and technique for the deeper study of liver-qi stagnation syndrome.
辨证论治是中医学的精华和优势,“证”是辨证论治的依据,是疾病处于一定阶段病因、病位、病性和病势的综合,能体现患者的整体性特征。证本质的研究是中医学现代化研究的核心内容。肝郁证是中医肝病的一个基本证型,是许多常见临床证候的基础,广泛存在于临床各系统疾病中。自上世纪50年代以来,肝郁证本质的研究一直是中医研究领域的热点,已初步证实肝郁证具有现代病理生理学基础,并部分地阐释了其中医理论的科学性,取得了阶段性成果,认为和机体神经、内分泌、循环、消化、免疫等多系统密切相关。但是对肝郁证的评价还处于笼统、多指标、不确定状态,有待进一步深入研究,从系统而全面的角度来阐释肝郁证的实质。
基因组学在整体上研究基因的功能,这与中医学的“整体观”概念十分相似。基因芯片是随着人类基因组研究的深入而迅速发展起来的一项重要的生物学技术,它的特点是高通量、快速、并行化采集生物信息。作为后基因组时代一项举足轻重的技术,基因芯片使生命科学与医学用全方位的整体新思维方式研究成为可能,因而广泛的应用于医学研究的各个领域。若借助基因芯片这种先进、快速的研究手段,可以尝试从基因水平探讨肝郁证的本质。可以设想,通过对同病异证和异病同证的基因表达谱差异比较中寻找证候的共同性和差异性建立一个“证候-基因表达谱”,揭示基因与证的内在联系,也会使中医辨证论治理论得到全面的检验和应用。
二、研究方法
目的:
采用慢性束缚应激的方法复制与临床接近的、有效的大鼠肝郁证模型;利用基因芯片对肝郁证大鼠的海马组织中的差异表达基因进行分析比较,在动物模型上寻找肝郁证相关的基因,尝试从基因水平对肝郁证的微观辨证进行初步探讨,也为从临床疾病的角度对肝郁证相关基因的研究提供理论支持;比较逍遥散干预大鼠与模型大鼠之间的差异基因表达,一方面从基因转录水平探讨逍遥散对肝郁证的作用机理,另一方面以方测证,方证互参,说明肝郁证的本质。
方法:
在肝郁证理论指导下,复制慢性束缚应激肝郁证大鼠模型,并通过行为学实验和检测大鼠血清激素水平验证模型的可靠性;并用中药复方逍遥散干预造模动物,验证逍遥散对肝郁证大鼠的治疗作用;采用含有27000个已知基因的大鼠全基因组芯片检测大鼠海马内差异基因表达情况,比较模型大鼠与正常大鼠、逍遥散干预大鼠与模型大鼠的差异表达基因,利用生物分子功能注释系统(MAS)对与疾病相关的基因进行分析;用实时荧光定量PCR技术验证部分与疾病相关的差异表达基因结果的可靠性。
1.慢性束缚应激肝郁证大鼠模型的复制
情志异常是肝郁证的主要病因,不良生活事件所形成的心理应激则是情志病因的实质;肝郁证是对郁怒、抑郁、焦虑等负性情绪心理应激状态下,以高级神经中枢调节机制紊乱为前提,涉及多系统、多器官的病理改变。基于上述考虑,参照文献报道,从慢性应激的角度出发,将18只同周龄雄性Wistar大鼠随机分3组:肝郁证模型组、逍遥散组、正常对照组,每组各6只,模型组与逍遥散组大鼠单笼饲养,每日将大鼠置于束缚笼内,固定其身体与尾部,使之不能随意活动,每天6小时;逍遥散组大鼠在造模的同时,以逍遥散中药煎液10g/kg.d灌胃;正常对照组不予任何干涉,时间为3周。观察大鼠的一般状况,每周对各组大鼠进行一次液体消耗实验及摄食实验,造模开始与结束时分别对各组大鼠进行一次敞箱实验,并在造模结束后取大鼠血清,检测血清的CRH含量,以验证肝郁证模型的可靠性,并且论证逍遥散对肝郁证的治疗作用。
2.利用基因芯片检测模型组与正常组大鼠海马的差异表达基因
造模结束后处死所有大鼠,分离海马组织,提取海马总RNA,在检验总RNA的质量与数量合格后,总RNA反转录得到cDNA,体外转录合成生物素标记的cRNA,再与基因芯片进行杂交反应,这里我们选用北京博奥生物公司的含有27000个已知基因的晶芯~(?)大鼠全基因组表达谱芯片(27K Rat Genome Array);用LuxScan 10KA双通道激光扫描仪对基因芯片进行扫描,得到半定量杂交结果后,采用LuxScan 3.0图像分析软件对芯片图像进行分析,把图像信号转化为数字信号,筛选出模型组与正常组大鼠海马的差异表达基因。借助MAS系统,通过搜索NCBI基因组数据库,对检测到的差异表达基因进行查询和确认,获得相关基因的信息并进行生物学注释,分析与疾病相关的基因在肝郁证的发生中可能的机制。
3.利用基因芯片检测逍遥散组与模型组大鼠海马的差异表达基因
同样采用27K Rat Genome Array芯片筛选出逍遥散组与模型组大鼠海马的差异表达基因,借助MAS系统,通过搜索NCBI基因组数据库,对差异表达基因进行查询和确认,获得相关基因的信息并进行生物学注释,尝试初步探讨逍遥散对肝郁证在基因水平上的作用机理。
4.实时荧光定量PCR方法验证部分差异表达基因
提取各组大鼠海马的总RNA,反转录得到cDNA,采用SYBR GreenⅠ荧光染料实时定量PCR方法,检测已有的基因芯片结果中部分与疾病相关的基因在各组大鼠海马中的表达情况,用以验证基因芯片结果。
结果:
1.成功复制了慢性应激肝郁证大鼠模型。造模3周后,一般状况来看,模型组大鼠主要表现为倦怠少动、喜欢贴壁、叫声尖细、反应迟缓、饮食减少,伴有毛色晦暗发黄、大便颗粒松散甚至呈水样等等肝郁证的表现。液体消耗实验结果发现,模型组大鼠造模后的糖水偏好程度显著低于正常组大鼠(P＜0.01);摄食实验中模型组大鼠的摄食量亦较正常组大鼠有显著性下降(P＜0.05);畅箱实验测得的动物行为学评分来看,造模后模型组大鼠的水平运动得分和垂直运动得分明显低于同时期的正常组大鼠(P＜0.01);此外,模型组大鼠的血清CRH浓度显著高于正常组大鼠(P＜0.01)。说明慢性束缚应激确能造成大鼠的肝郁证,能有效的模拟临床中肝郁证兴趣丧失、快感缺乏等症状。
2.大鼠海马样品的总RNA经检验质量合格,顺利进行后续实验。模型组与正常对照组大鼠海马的芯片检测结果为:共有48个差异表达基因,表达上调的基因有15个;表达下调的基因33个。搜索基因组数据库及用MAS系统进行分析,对与疾病相关的基因进行功能分析,结果发现,部分差异表达的基因可以阐释肝郁证模型以及临床病例的某些主要表现。大致分为3类:(1)与神经精神类疾病相关的基因:主要有脑源性神经营养因子(Bdnf)、色氨酸羟化酶1(Tph1)、糖原合成酶激酶-3(Gsk3b)等,它们的表达异常,说明肝郁证中存在神经内分泌系统的紊乱,神经递质的调节上的发挥失常及神经元受损等机制,可能是导致肝郁证患者情绪低落、兴趣丧失、运动迟缓、饮食减少等的原因,也在一定程度上解释了肝郁证与现代医学中抑郁症、焦虑症等疾病的密切联系;(2)与肿瘤相关的基因:中期因子(Mdk)、叶酸受体1(Folr1)、水通道蛋白1(Aqp1)、胰岛素样生长因子结合蛋白2(Igfbp2)等,此结果提示肝郁证大鼠存在某些肿瘤的易感性,可能与临床上肝郁证患者气滞血瘀,日久成积而引起肿瘤发生有一定的相关,与我们前期肝郁证大鼠蛋白质组学研究结果类似;(3)其他疾病相关基因:如血管紧张素转化酶(Ace)、细胞内氯通道(Clic6)、X联内肽酶同源性的磷酸盐调节基因(Phex)等,这些基因的表达改变与肝郁证的关系有待进一步研究。
3.逍遥散干预3周后,逍遥散组大鼠的倦怠少动、饮食减少等症状明显改善;液体消耗实验、摄食实验及畅箱实验结果发现,逍遥散组大鼠的糖水偏好程度、摄食量及行为学评分在第3周时与模型组相比,均有明显改善;与模型组大鼠比较,逍遥散组大鼠血清CRH浓度亦有升高趋势;逍遥散干预组与模型组大鼠海马的芯片检测结果为:共有22个差异表达基因,表达上调的基因有7个;表达下调的基因15个。其中与疾病关系密切的基因有:神经元钙传感器(Ncs-1)、阿片黑素促皮质激素原(Pomc)、色氨酸羟化酶1(Tph1)等,提示逍遥散对肝郁证的治疗作用可能与稳定细胞膜,防止钙超载,提高神经元抗损伤能力,双向调节HPA轴,神经内分泌功能等机制相关。另一方面从方证互参的角度也反证了肝郁证模型的可靠性。
4.实时荧光定量PCR方法检测了Bdnf、Tph1、Mdk等3个基因在各组大鼠海马中的表达情况。结果发现,RNA质量完好,PCR反应扩增效果较好,PCR产物纯度较高,目的基因Bdnf、Tph1、Midkine在各组大鼠海马的表达比较与基因芯片检测结果基本一致,验证了芯片部分检测结果的可靠性。
结论:
1.本次研究采用慢性应激束缚的方法,成功的复制了大鼠的肝郁证模型,模型可靠。
2.利用基因芯片检测出的模型组与正常组大鼠海马的差异表达基因中,部分基因的异常表达提示肝郁证可能与神经内分泌系统的紊乱,神经递质调节失常、神经元受损等机制密切相关,同时也提示肝郁证存在一定的肿瘤易感性。
3.利用基因芯片检测出的逍遥组与模型组大鼠海马的的差异表达基因中,部分差异基因的表达提示逍遥散可能通过提高神经元对应激损伤的耐受性,调节神经内分泌功能等机制来发挥治疗肝郁证的作用。
4.通过实时荧光定量PCR方法检测了部分与疾病相关基因在各组大鼠海马中的表达情况,与基因芯片的检测结果是基本吻合的,验证了部分基因芯片表达结果的可靠性。
肝郁证大鼠模型海马差异表达基因的研究,为从基因水平探讨肝郁证实质研究的深入作了有益的尝试。
Background
Syndrome differentiation and treatment are the advantage of the Traditional Chinese Medicine. Being one of features of TCM theory, syndrome was based on TCM basic theory, has been paid attention in studying of Traditional Chinese Medicine in recent years. As basic syndrome of liver diseases of Chinese medicine is concerned, liver-qi stagnation syndrome is one of common syndromes in clinical practice and highly valuable for profound research. The nature of liver-qi stagnation syndrome is the focus of TCM theory study today. Although lots of achievement was got during the pasted 50 years, they still can't evaluation the essence of liver depression syndrome integrity, more exploration was needed.
Gene chip, also called DNA chip or DNA array, is one of advanced molecular biological technology which was accompanied by development of study of human genome and is applied to many medical and biological science research areas already. As an important method in the post-genome era, gene chip gives a chance to study of liver-qi stagnation syndrome deeply. Both genomics and the study of traditional Chinese medicine syndrome integrity and dynamic generated the syndrome-genomics accordingly.
Objective
To set up the effective rat model of liver depression syndrome from chronic constraint stress to find differentially expressed genes in hippocampus between the rat model and the normal control, and between the rat model and rat treated by Xiaoyao Powder through gene chip.
Methods
Set up the rat model of liver depression syndrome from chronic constraint stress, compared with normal control group and treatment group of Xiaoyao Powder, every group 6 rats. The changes in body weight and food intake in the animals were observed. By open-field test and fluid consumption test, the behaviors of the animals were determined every week. We harvested the serum and separated hippocampus tissues of rats respectively after 3 weeks. Corticotropin releasing factor (CRF, also CRH) in serum was detected after the animals were executed.
Gene chip is used in this study. Method of one step of Trizol reagent was applied to extract the total RNA of hippocampus of every group. The total RNA was reverse transcribed to cDNA which was then transcribed to cRNA marked by cy3 and cy5 in vitro. The cRNA was hybridized with 27K Rat Genome Array, and the gene chip was scanned. After analyze the gene chip image, we screened the differentially expressed genes, and then searched the NCBI genomic databases to defined the gene by means of CapitalBio(?) Molecule Annotation System (CB-MAS). Comparing and analyzing the differentially expressed genes between the rat model and normal control, we speculated the pathogenesis of liver depression syndrome, and the differentially expressed genes between the rat treated by Xiaoyao Powder and the rat model to search for the mechanisms of Xiaoyao Powder to liver depression syndrome at the level of genome and prove reliability of liver depression syndrome model on the other hand. Some differentially expressed genes of hippocampus of rats in different groups are verified through real-time PCR.
Results
The rat model of liver depression syndrome has been established successfully at 21st day. The model rats displayed the typical appearances such as low activity, slow reaction, taper cry, reduces food and drink, like adherence, and so on. In the model group, the rats showed decreased body weight and food intake, reduced squares crossing and rearing in open-field test, a significantly reduced consumption of preference for sucrose solutions and an increased pure water consumption as compared with control group. The serum level of CRH was also increased in rat model, showing the function disorder of HPA axis.
We found that a total of 48 genes expressed differentially in hippocampus between rat model and normal control, among which 15 were up-regulated and 33 were down-regulated. After analyzing the function of genes relate to common disease, we knew the differential expression genes of liver-qi stagnation syndrome involved in three categories: (1) neurologic and psychiatric diseases genes, such as Bdnf, Tph1, Gsk3b etc; (2) tumor genes, including Midkine, Folr1, Aqp1, Igfbp2 and so on; (3) genes related to other diseases, such as Ace, Clic, Phex.
It was found that Xiaoyao Powder can improve the typical symptoms of liver depression syndrome, increasing the food intake, consumption of and preference for sucrose solutions and squares crossing and rearing in open-field test. It can also reduced the serum level of CRH in rat model. In addition, a total of 22 genes expressed differentially in hippocampus between rat treated by Xiaoyao Powder and rat model, among which 7 were up-regulated and 15 were down-regulated. We analyzed these genes closely related to diseases such as Ncs-1 and Pomc and found that Xiaoyao Powder probably can regulate the neuroendocrinology and function of HPA axis.
Expressions of Bdnf, Tph1 and Midkine of hippocampus in different groups tsested through real-time PCR are consistent with results of the gene chips. The results of real-time PCR prove part of chip datas are credible.
Conclusion
The rat model of liver depression syndrome from chronic constraint stress is reliable and credible. The abnormal expressed genes in hippocampus of rat model suggest that liver depression syndrome is probably related to disorder of neuroendocrinology and neurotransmitters, and have tumor susceptibility at level of genome, witch may be regulated by Xiaoyao Powder. The results of real-time PCR of some differentially expressed genes of hippocampus of rats in different groups prove the reliability of the datas of gene chips.
The study of gene differential expression in Rat Modal of liver depression by gene chip established a foundation from theory and technique for the deeper study of liver-qi stagnation syndrome.
引文
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