CN112067821A - 四氢孕酮对pmdd肝气逆证模型大鼠关键脑区中gaba功能调控机制的研究方法 - Google Patents
四氢孕酮对pmdd肝气逆证模型大鼠关键脑区中gaba功能调控机制的研究方法 Download PDFInfo
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Abstract
本发明公开了四氢孕酮对PMDD肝气逆证模型大鼠关键脑区中GABA功能调控机制的研究方法,属于四氢孕酮对GABA功能调控机制的研究方法技术领域,包括以下步骤:S1:构建PMDD肝气逆证模型并评价;S2:检测各组大鼠中枢不同脑区、血清中关键指标;S3:进行各组大鼠中枢不同脑区GABAAR亚单位mRNA和蛋白表达检测;S4:采用膜片钳技术进行各组大鼠关键脑区中GABAAR相关CI‑离子通道检测;S5:明确PMDD肝气逆证模型大鼠关键脑区四氢孕酮及GABA水平、GABAAR表达与功能变化特征;S6:依据变化特征给予药物治疗,实现四氢孕酮对PMDD肝气逆证模型大鼠关键脑区GABA功能调控机制的阐释。
Description
技术领域
本发明涉及四氢孕酮对GABA功能调控机制的研究方法技术领域,特别涉及四氢孕酮对PMDD肝气逆证模型大鼠关键脑区中GABA功能调控机制的研究方法。
背景技术
经前烦躁障碍症(premesnstrualdysphoricdisorder,PMDD)是一种在月经周期黄体晚期周期性发作的一系列情感性和躯体性临床症状。是经前期综合征(premesnstrualsyndrome,PMS)的严重类型。大约50%-80%的育龄妇女有轻微的经前症状,高达20%的女性报告有严重的经前症状,并且有3-8%的妇女符合DSM-IV的严格标准。BorensteinJ等发现PMS的年度直接成本一般(59美元,如医疗费用),但间接成本要高得多(4333美元,如工作天数损失和生产效率丧失。Halbreich研究发现,在多个领域(工作、婚姻、社交、休闲),PMDD导致的生活能力损害和丧失,与心境恶劣障碍及重度抑郁障碍相似。
目前,已确定PMDD与神经类固醇,特别是孕激素及其代谢物四氢孕酮的相关性较强。多数报道显示,PMDD患者黄体期孕酮及其代谢产物四氢孕酮异常。有研究发现:ALLO在正常女性血清中的浓度随月经周期呈现倒“U”型分布,当血清中四氢孕酮的浓度与黄体期浓度一致时会出现负性情绪,当浓度较高或较低时情绪反应较少,孕酮/四氢孕酮的浓度低至中等时会出现焦虑情绪,PMDD是PMS的严重类型,可出现自杀倾向或攻击行为,影响家庭稳定、社会和谐,但其发病机制尚不清楚。四氢孕酮(ALLO)可通过GABA系统参与情绪调节,并导致情感障碍的易感性。
发明内容
本发明的目的在于提供四氢孕酮对PMDD肝气逆证模型大鼠关键脑区中GABA功能调控机制的研究方法,揭示PMDD病因学中ALLO对GABA功能的调控机制,肝疏泄失常发病中枢机制,进而依据变化特征给予药物治疗,实现四氢孕酮对PMDD肝气逆证模型大鼠关健脑区GABA功能调控机制的阐释,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:四氢孕酮对PMDD肝气逆证模型大鼠关键脑区中GABA功能调控机制的研究方法,包括以下步骤:
S1:构建PMDD肝气逆证模型并评价,根据行为学测试筛选分组,设置模型组、正常组和对照组,对照组喂食药剂;
S2:检测各组大鼠中枢不同脑区、血清中关键指标;
S3:进行各组大鼠中枢不同脑区GABAAR亚单位mRNA和蛋白表达检测;
S4:采用膜片钳技术进行各组大鼠关键脑区中GABAAR相关CI-离子通道检测;
S5:明确PMDD肝气逆证模型大鼠关健脑区四氢孕酮及GABA水平、GABAAR表达与功能变化特征;
S6:依据变化特征给予药物治疗,实现四氢孕酮对PMDD肝气逆证模型大鼠关健脑区GABA功能调控机制的阐释。
进一步地,PMDD肝气逆证大鼠模型构建的方法,包括以下步骤:
S101:饲养环境;
S102:动情周期规律大鼠的确定;
S103:入侵鼠卵巢摘除手术;
S104:居住入侵;
S105:模型评价;
S106:分组与给药;
S107:行为学检测;
S108:取材。
进一步地,检测各组大鼠中枢不同脑区、血清中关键指标的方法包括以下步骤:
S201:比对各组大鼠血清中关键激素的含量变化;
S202:比对各组大鼠中枢不同脑区关键激素的含量变化;
S203:比对各组大鼠血清及中枢不同脑区关键神经递质GABA、Glu的含量变化。
进一步地,检测各组大鼠中枢不同脑区GABAAR亚单位mRNA和蛋白表达的方法包括以下步骤:
S301:检查PMDD肝气逆证模型大鼠关键脑区GABAARα4亚基mRNA表达变化;
S302:检测PMDD肝气逆证模型大鼠关键脑区GABAARα4亚基蛋白水平表达变化;
S303:ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠行为学变化及关键指标变化;
S304:ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠血清及关键脑区激素和神经递质变化;
S305:ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠关键脑区GABAARα4亚基mRNA表达变化;
S306:ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠关键脑区GABAARα4亚基蛋白水平变化。
进一步地,S303包括以下步骤:
S3031:ALLO及其抑制剂干预后PMDD肝气逆证大鼠攻击行为变化;
S3032:ALLO及其抑制剂干预后PMDD肝气逆证大鼠高架十字迷宫得分变化;
S3033:ALLO及其抑制剂干预后PMDD肝气逆证大鼠旷场实验得分变化。
进一步地,S304包括以下步骤:
S3041:ALLO及其抑制剂干预后PMDD肝气逆证大鼠血清中激素含量变化;
S3042:ALLO及其抑制剂干预后PMDD肝气逆证大鼠不同脑区中激素含量变化;
S3043:ALLO及抑制剂干预后PMDD肝气逆证模型大鼠血清中神经递质含量变化。
与现有技术相比,本发明的有益效果是:本发明提出的四氢孕酮对PMDD肝气逆证模型大鼠关键脑区中GABA功能调控机制的研究方法,通过检测PMDD肝气逆证模型大鼠关键脑区ALLO及GABA功能变化,并采用孕酮受体及GABAA受体拮抗剂干预,得出PMDD肝气逆证大鼠血清中ALLO显著降低,下丘脑中ALLO也显著降低,同是,血清及下丘脑、前额叶皮质中GABA显著降低,同是下丘脑及杏仁核中GABAARα4亚基表达降低,全细胞膜片钳结果显示氯离子通道电流降低,从分子及细胞层面说明ALLO孕酮的降低,会引起外周及中枢GABA含量下降,降低GABA系统的抑制作用,是PMDD肝气逆证重要发病机制,揭示PMDD病因学中ALLO对GABA功能的调控机制,肝疏泄失常发病中枢机制,进而依据变化特征给予药物治疗,实现四氢孕酮对PMDD肝气逆证模型大鼠关健脑区GABA功能调控机制的阐释。
附图说明
图1为本发明的四氢孕酮对PMDD肝气逆证模型大鼠关键脑区中GABA功能调控机制的研究方法流程图;
图2为本发明的ALLO及其抑制剂干预前后海马神经元中Cl-通道电流变化情况图;
图3为本发明的PMDD肝气逆证大鼠模型构建的方法流程图;
图4为本发明的给药前后PMDD肝气逆证大鼠攻击行为得分情况图;
图5为本发明的检测各组大鼠中枢不同脑区、血清中关键指标的方法流程图;
图6为本发明的各组大鼠血清中关键激素的含量变化图;
图7为本发明的检测各组大鼠中枢不同脑区GABAAR亚单位mRNA和蛋白表达的方法流程图;
图8为本发明的ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠行为学变化及关键指标变化的方法流程图;
图9为本发明的ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠血清及关键脑区激素和神经递质变化的方法流程图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
参阅图1至图2,四氢孕酮对PMDD肝气逆证模型大鼠关键脑区中GABA功能调控机制的研究方法,包括以下步骤:
S1:构建PMDD肝气逆证模型并评价,根据行为学测试筛选分组,设置模型组、正常组和对照组,对照组喂食药剂;
S2:检测各组大鼠中枢不同脑区、血清中关键指标;
S3:进行各组大鼠中枢不同脑区GABAAR亚单位mRNA和蛋白表达检测;
S4:采用膜片钳技术进行各组大鼠关键脑区中GABAAR相关CI-离子通道检测;
S5:明确PMDD肝气逆证模型大鼠关健脑区四氢孕酮及GABA水平、GABAAR表达与功能变化特征;
S6:依据变化特征给予药物治疗,实现四氢孕酮对PMDD肝气逆证模型大鼠关健脑区GABA功能调控机制的阐释;
通过制备海马神经元,并采用上述实验所得血清进行干预,之后进行全细胞膜片钳检测,记录GABA电流,电压刺激方案如下:当形成全细胞封接后细胞膜电压钳制于-70mV,在Gap-free模式下进行记录给予细胞表面喷射30μMGABA,10μMStr以及300nMTTX后电流的峰值,试验数据由EPC-10放大器(HEKA)进行采集并储存于PatchMaster(HEKA)软件中;
用微电极拉制仪(P97,SutterInstruments)将毛细玻璃管(BF150-86-10.SutterInstruments)拉制成记录电极。在倒置显微镜(IX71,Olympus)下操纵微电极操纵仪(MP285,SutterInstruments)将记录电极接触到细胞上,给予负压抽吸,形成GΩ封接。形成GΩ封接后进行快速电容补偿,然后继续给予负压,吸破细胞膜,形成全细胞记录模式。然后进行慢速电容的补偿并记录膜电容及串联电阻,不给予漏电补偿,数据质量标准以下标准用来判断数据是否可以接受:
串联电阻≤20MΩ;
封接电阻≥1GΩ;
在膜电位为-70mV下无明显的漏电流(漏电流≤100pA),PMDD肝气逆证大鼠血清干预海马神经元后,Cl-通道开放的频率减少,突触负性电流减小PMDD的主要症状是由ALLO介导的,GABAA受体对其周期性波动敏感性的改变是导致PMDD患者情绪不稳定重要机制,主要集中在特定脑区海马、杏仁核、额叶等部位。
参阅图3至图4,PMDD肝气逆证大鼠模型构建的方法,包括以下步骤:
S101:饲养环境,大鼠饲养进行昼夜颠倒,每天晚上8点开灯,次日早上8点关灯,自由饮水进食,饲养大鼠房间保持21±1℃,湿度45%,大鼠运抵后,适应环境一周,每日行抓握(控制期)平行操作,熟悉环境,消除人为操作影响,适应期大鼠5只一笼饲养,且所有实验操作均为暗淡红灯(<25lux)下进行,入侵鼠进行摘卵巢处理,与实验鼠在不同的房间饲养;
S102:动情周期规律大鼠的确定,适应性饲养结束后,采用阴道电阻对大鼠的动情周期进行检测,筛选出动情周期规律的大鼠,一般检测3个周期(15天),按照体质量分组,挑选出正常组,其余的作为居住鼠进入后续实验;
S103:入侵鼠卵巢摘除手术,入侵鼠的体质量比实验鼠约小50g,手术前一天,将所有手术器械用75%乙醇溶液消毒,并用高压蒸汽灭菌锅灭菌,灭菌结束后放入烘干箱(≥60℃)备用,大鼠腹腔注射10%水合氯醛(0.3ml/100g)麻醉,待大鼠失去知觉后将其固定于大鼠板上,用剪刀将大鼠腹部毛发剪去,用脱脂棉蘸取少量碘伏擦拭去毛处皮肤消毒,用手术刀将大鼠腹腔皮肤割开(1cm宽),暴露腹腔,并采用两个止血钳分别牵拉开口两边,撑开开口,左右两手各持一个止血钳在大鼠腹中线向两侧寻找卵巢,找到后用手术线结扎大鼠卵巢与输卵管相交部位后剪下大鼠卵巢,松开牵拉皮肤的止血钳后,滴入庆大霉素,再将大鼠伤口缝合,在伤口周围再滴几滴庆大霉素,假手术组仅开其腹腔摘除部分脂肪,进行缝合即可,并不进行卵巢摘除,术后连续3天每只老鼠每天进行腹腔注射庆大霉素(2万单位/只)进行伤口消炎,防止感染,术后恢复一周后,进行阴道电阻测试,持续一周,确定摘除卵巢后没有动情周期,手术后恢复两周方可进行居住入侵实验,术前大鼠禁食禁水12h,术后12h后给食物和水,为避免大鼠进食、饮水过多,术后第一次给食物和水要适量;
经过切除卵巢手术的大鼠(入侵鼠)被饲养于另外的实验室中(与正常大鼠相同的饲养环境),以保证入侵鼠与居住鼠在攻击行为测试前是陌生的;只有攻击行为测试时,入侵鼠才被暂放于居住鼠饲养环境中。
S104:居住入侵,实验时间为下午2:00-5:30光线暗淡(相当于大鼠夜间),并且于雌性大鼠正常饲养相同的房间进行,将观察的老鼠放到需要观察的笼子内,适应15min,然后放入一只切除卵巢雌性入侵大鼠(与居住鼠大小相同)持续10分钟,对此过程进行录像,后期对其攻击行为进行打分,主要是居住鼠针对攻击鼠的攻击情况,得分标准主要有:攻击、攻击时间、撕咬、攀爬、攀爬时间和竖毛,攻击行为包含两种类型:正面攻击(当入侵鼠试图逼近时跃起)和侧旁攻击时(弓背从斜地里窜出将入侵者推出);
混和攻击行为得分计算公式:混和攻击行为得分=攻击次数+0.2攻击时间+撕咬次数+0.2攀爬时间+竖毛;
对所有居住鼠进行连续4或5天(一个完整生理周期)的居住入侵诱导实验,入侵鼠在对居住鼠进行攻击测试时,采用拉丁方设计,保证每次居住鼠面对不同的入侵鼠;
对给药前后PMDD肝气逆证大鼠攻击行为得分比较:
给药前,各组按照攻击行为得分进行分组,模型组与正常组相比,打斗得分有明显差异(P<0.01),其余给药组与模型组相比无显著差异(P≥0.05),给药后,模型组打斗得分明显高于正常组(P<0.05);与模型组相比,COCS组、逍遥丸组、白香丹组打斗得分均明显下降(P<0.01);
S105:模型评价,在大鼠的动情期和间期进行行为测试,具体为旷场、攻击行为和社交,在测试基础上对大鼠模型进行评价;
S106:分组与给药,根据行为学测试的结果,筛选出有焦虑情绪的大鼠,依据大鼠在NR期的打斗,将模型组大鼠分为为阳性对照组:COCs(屈螺酮炔雌醇)、逍遥丸、白香丹,正常组、模型组(对照组均给予等量体积生理盐水),每天8:30进行灌胃给药,给药三个动情周期;
S107:行为学检测,给药两个完整的生理周期,并第二个动情周期的接受期和非接受期进行行为学检测(打斗、旷场、高架),行为学检测结束后取材。给药30min后先进行6min的旷场,再进行5min的十字高架;
对给药前后PMDD肝气逆证大鼠高价十字迷宫得分比较得出:给药前,与正常组相比,模型组OE%和OT%均较低(P<0.01),与模型组相比COCS组OE%高于模型组,其余组别OE%和OT%均无统计学差异;给药后,模型组OE%和OT%均低于正常组(P<0.05);与模型组相比,COCS组、逍遥丸组、白香丹组OE%和OT%均有显著提高(P<0.01,P<0.01),其中进入开放臂次数百分比OE%,进入开放臂时间百分比OT%;
对给药前PMDD肝气逆证大鼠旷场实验评价比较得出:给药前,除逍遥丸组总路程高于模型组之外(P<0.05),其余组别之间总路程及中央区进入次数均无明显差异;给药后,模型组总路程与中央区进入次数均低于正常组(P<0.01);与模型组相比,COCS组、逍遥丸组、白香丹组OE%和OT%均有显著提高(P<0.01,P<0.01);
S108:取材,在居住鼠最后一次NR期测试完行为检测和攻击行为之后,进行取材,分为腹腔动脉取血,脑组织取材(前额叶皮质、顶区、下丘脑、杏仁核、左右海马);按照动物伦理学要求进行操作,取材前对所有的动物进行麻醉给药,取腹腔动脉血5ml;迅速断头后,冰上取脑组织,分离前额叶、杏仁核、下丘脑、海马并称重,放在-80℃冰箱保存。
参阅图5至图6,检测各组大鼠中枢不同脑区、血清中关键指标的方法包括以下步骤:
S201:比对各组大鼠血清中关键激素的含量变化;
PMDD肝气逆证大鼠血清中PROG、ALLO、CORT和PRL含量变化:
给药后,模型组血清中PROG含量明显高于正常组(P<0.05),逍遥丸组和白香丹组可以降低血清中PROG含量(P<0.01);模型组血清中ALLO含量比正常组较低(P<0.01),而白香丹组可以明显提升大鼠血清中的ALLO。给药后,模型组大鼠血清中CORT含量和正常组并无差异,COCS、逍遥丸、白香丹均可以提高大鼠血清中CORT的含量(P<0.05,P<0.01);给药后,各组大鼠血清中PRL含量并无统计学差异;
S202:比对各组大鼠中枢不同脑区关键激素的含量变化;
海马:给药后,模型组大鼠海马区PROG含量高于正常组((P<0.01)),与模型组相比,白香丹组海马区PROG含量减少((P<0.01);给药后,各组之间海马区ALLO并无明显差异;
下丘脑:给予药物治疗后,模型组大鼠下丘脑中PROG、ALLO含量高于正常组(P<0.05,P<0.01),与模型组相比,COCS组ALLO含量升高(P<0.05),逍遥丸组和白香丹组PORG含量降低、ALLO含量升高(P<0.05,P<0.01);
前额叶皮质:给予药物治疗后,模型组大鼠前额叶中PROG含量明显高于正常组(P<0.01),与模型组相比,COCS组、逍遥丸组和白香丹组PORG含量均降低;给予药物治疗后,所有组别之间前额叶ALLO含量均无统计学意义;
S203:比对各组大鼠血清及中枢不同脑区关键神经递质GABA、Glu的含量变化;
血清中:采用高效液相检测大鼠血清中Glu、GABA含量,与正常组相比,模型组血清中GABA升高(P<0.05),与模型组相比,COCS、逍遥丸和白香丹均能提高大鼠血清中Glu的含量(P<0.05,P<0.01),逍遥丸和白香丹组GABA含量也明显高于模型组(P<0.01);
海马中:模型组海马区中Glu含量高于正常组(P<0.05);所有组别海马区中GABA含量并无显著差异;
下丘脑中:与模型组相比,COCS、逍遥丸和白香丹均能提高大鼠血清中Glu、GABA的含量(P<0.01);
前额叶皮质中:给药后,大鼠前额叶中,模型组Glu和GABA均明显低于正常组;与模型组相比,COCS、逍遥丸和白香丹均能提高大鼠前额叶中Glu的含量(P<0.01,P<0.01),但GABA含量并无显著差异;
参阅图7,检测各组大鼠中枢不同脑区GABAAR亚单位mRNA和蛋白表达的方法包括以下步骤:
S301:检查PMDD肝气逆证模型大鼠关键脑区GABAARα4亚基mRNA表达变化;
给予药物治疗后,在前额叶中,与模型组相比,COCS、逍遥丸、白香丹GABAAR4αmRNA表达升高(P<0.01)。在下丘脑中,模型组GABAAR4αmRNA表达低于正常组(P<0.01),给予药物逍遥丸和白香丹后,GABAAR4α,mRNA表达升高(P<0.01,P<0.05),在杏仁核中,模型组GABAAR4αmRNA表达低于正常组(P<0.01),COCS、逍遥丸、和白香丹组均能提高大鼠杏仁核中GABAAR4αmRNA的表达(P<0.01,P<0.05)。在海马区中,模型组GABAAR4αmRNA的表达高于正常组,逍遥丸可以降低GABAAR4αmRNA的表达(P<0.01);
S302:检测PMDD肝气逆证模型大鼠关键脑区GABAARα4亚基蛋白水平表达变化;
与正常组相比,模型组GABAARα4亚基蛋白表达高于正常组(P<0.01),与模型组相比,COCS、逍遥丸、白香丹组GABAARα4亚基蛋白表达明显降低(P<0.01);
S303:ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠行为学变化及关键指标变化;
S304:ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠血清及关键脑区激素和神经递质变化;
S305:ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠关键脑区GABAARα4亚基mRNA表达变化;与正常组相比,模型组前额叶和杏仁核中GABAAR4αmRNA的表达低于正常组(P<0.01),ALLO组前额叶中GABAAR4αmRNA表达明显高于模型组(P<0.01);
S306:ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠关键脑区GABAARα4亚基蛋白水平变化,与正常组相比,海马中模型组GABAARα4亚基蛋白表达高于正常组(P<0.01),与模型组相比,ALLO组GABAARα4亚基蛋白表达明显降低(P<0.01);其他脑区均无显著性差异。
参阅图8,S303包括以下步骤:
S3031:ALLO及其抑制剂干预后PMDD肝气逆证大鼠攻击行为变化;给药前,除了正常组之外,所有组别之前打斗得分并无差异;与正常组相比,模型组打斗得分依然保持高水平(P<0.01);与模型组相比,给予腹腔注射ALLO后,其打斗得分降低(P<0.01),注射非那雄胺后打斗得分升高(P<0.05);
S3032:ALLO及其抑制剂干预后PMDD肝气逆证大鼠高架十字迷宫得分变化;
给药前,与正常组相比,模型组的OE%明显低于正常组(P<0.01),与模型组相比,给药组之间没有差异(P>0.05);所有组别之间的OT%无统计学差异;给药后,模型组的OE%、OT%均小于正常组(P<0.01),腹腔给予ALLO后,ALLO组的OE%、OT%较模型组有明显的上升(P<0.01);
S3033:ALLO及其抑制剂干预后PMDD肝气逆证大鼠旷场实验得分变化;
给药前,与正常组相比,其余各组别的总路程均明显低于正常组(P<0.01),所有组别之间中央区停留时间无统计学差异;给药后,ALLO组中央区总路程较模型组明显增多(P<0.01),且进入中央区的次数也有所增加(P<0.05)。
参阅图9,S304包括以下步骤:
S3041:ALLO及其抑制剂干预后PMDD肝气逆证大鼠血清中激素含量变化;给药治疗后,与正常组相比,模型组中E2明显升高(P<0.01),而ALLO组E2的含量低于模型组(P<0.01),与正常组相比,模型组血清中PROG、CORT的含量都明显升高(P<0.05),且腹腔给予非那雄胺后,血清中PROG、CORT的含量呈现断层式升高(P<0.000),与正常组相比,模型组血清中ALLO、PRL含量并无明显变化,但ALLO组血清中ALLO较模型组明显升高(P<0.01);
S3042:ALLO及其抑制剂干预后PMDD肝气逆证大鼠不同脑区中激素含量变化;
海马中,给予药物治疗后,模型组海马中PROG的含量比正常组降低(P<0.01),ALLO组海马中ALLO含量高于模型组(P<0.01),所有组别海马中ALLO的含量没有明显差异;
下丘脑中,给予药物治疗后,模型组下丘脑中PROG的含量比正常组升高(P<0.01),ALLO组下丘脑中ALLO含量低于模型组(P<0.01);而模型组下丘脑中ALLO的含量比正常组降低,ALLO组下丘脑中ALLO含量高于模型组(P<0.01);
前额叶中,给予药物治疗后,模型组前额叶中PROG的含量比正常组升高(P<0.01),ALLO组中ALLO含量低于模型组(P<0.01),而ALLO组前额叶中ALLO含量高于模型组(P<0.01);
S3043:ALLO及抑制剂干预后PMDD肝气逆证模型大鼠血清中神经递质含量变化;给予药物治疗后,模型组血清中Glu、GABA的含量比正常组降低(P<0.01),ALLO组中GABA含量高于模型组(P<0.05),血清中5-HT的浓度高于正常组(P<0.05),相比较于模型组,ALLO组血清中中5-HT含量较低(P<0.01)。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (6)
1.四氢孕酮对PMDD肝气逆证模型大鼠关键脑区中GABA功能调控机制的研究方法,其特征在于,包括以下步骤:
S1:构建PMDD肝气逆证模型并评价,根据行为学测试筛选分组,设置模型组、正常组和对照组,对照组喂食药剂;
S2:检测各组大鼠中枢不同脑区、血清中关键指标;
S3:进行各组大鼠中枢不同脑区GABAAR亚单位mRNA和蛋白表达检测;
S4:采用膜片钳技术进行各组大鼠关键脑区中GABAAR相关CI-离子通道检测;
S5:明确PMDD肝气逆证模型大鼠关健脑区四氢孕酮及GABA水平、GABAAR表达与功能变化特征;
S6:依据变化特征给予药物治疗,实现四氢孕酮对PMDD肝气逆证模型大鼠关健脑区GABA功能调控机制的阐释。
2.如权利要求1所述的四氢孕酮对PMDD肝气逆证模型大鼠关键脑区中GABA功能调控机制的研究方法,其特征在于,PMDD肝气逆证大鼠模型构建的方法,包括以下步骤:
S101:饲养环境;
S102:动情周期规律大鼠的确定;
S103:入侵鼠卵巢摘除手术;
S104:居住入侵;
S105:模型评价;
S106:分组与给药;
S107:行为学检测;
S108:取材。
3.如权利要求1所述的四氢孕酮对PMDD肝气逆证模型大鼠关键脑区中GABA功能调控机制的研究方法,其特征在于,检测各组大鼠中枢不同脑区、血清中关键指标的方法包括以下步骤:
S201:比对各组大鼠血清中关键激素的含量变化;
S202:比对各组大鼠中枢不同脑区关键激素的含量变化;
S203:比对各组大鼠血清及中枢不同脑区关键神经递质GABA、Glu的含量变化。
4.如权利要求1所述的四氢孕酮对PMDD肝气逆证模型大鼠关键脑区中GABA功能调控机制的研究方法,其特征在于,检测各组大鼠中枢不同脑区GABAAR亚单位mRNA和蛋白表达的方法包括以下步骤:
S301:检查PMDD肝气逆证模型大鼠关键脑区GABAARα4亚基mRNA表达变化;
S302:检测PMDD肝气逆证模型大鼠关键脑区GABAARα4亚基蛋白水平表达变化;
S303:ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠行为学变化及关键指标变化;
S304:ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠血清及关键脑区激素和神经递质变化;
S305:ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠关键脑区GABAARα4亚基mRNA表达变化;
S306:ALLO及其抑制剂干预后PMDD肝气逆证模型大鼠关键脑区GABAARα4亚基蛋白水平变化。
5.如权利要求4所述的四氢孕酮对PMDD肝气逆证模型大鼠关键脑区中GABA功能调控机制的研究方法,其特征在于,S303包括以下步骤:
S3031:ALLO及其抑制剂干预后PMDD肝气逆证大鼠攻击行为变化;
S3032:ALLO及其抑制剂干预后PMDD肝气逆证大鼠高架十字迷宫得分变化;
S3033:ALLO及其抑制剂干预后PMDD肝气逆证大鼠旷场实验得分变化。
6.如权利要求4所述的四氢孕酮对PMDD肝气逆证模型大鼠关键脑区中GABA功能调控机制的研究方法,其特征在于,S304包括以下步骤:
S3041:ALLO及其抑制剂干预后PMDD肝气逆证大鼠血清中激素含量变化;
S3042:ALLO及其抑制剂干预后PMDD肝气逆证大鼠不同脑区中激素含量变化;
S3043:ALLO及抑制剂干预后PMDD肝气逆证模型大鼠血清中神经递质含量变化。
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