CN108627638A - Sirtuin 1经Bmi1介导调控老年性牙槽骨丢失作用机制的研究方法 - Google Patents
Sirtuin 1经Bmi1介导调控老年性牙槽骨丢失作用机制的研究方法 Download PDFInfo
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- CN108627638A CN108627638A CN201810435680.8A CN201810435680A CN108627638A CN 108627638 A CN108627638 A CN 108627638A CN 201810435680 A CN201810435680 A CN 201810435680A CN 108627638 A CN108627638 A CN 108627638A
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Abstract
本发明公开了一种Sirtuin 1经Bmi1介导调控老年性牙槽骨丢失作用机制的研究方法,包括以下步骤:a.实验动物培育及基因型鉴定,选用4周龄分别带有WT、Sirt1TG、Bmi1KO、Sirt1TG/Bmi1KO基因的小鼠;b.牙槽骨表型分析相关指标检测;c.小鼠颌骨M‑MSCs培养和体外实验;d.氧化应激相关指标检测;e.Sirt1激活剂处理自然衰老小鼠;f.临床牙槽骨样本采集、颌骨M‑MSCs培养及用Sirt1激活剂处理。本研究方法有望阐明Sirt1在预防牙槽骨丢失中的作用及机制,并为Sirt1激活剂白黎芦醇临床应用于防治老年性牙槽骨丢失提供理论和实验依据。
Description
技术领域
本发明涉及生物基因技术领域,具体涉及一种Sirtuin 1经Bmi1介导调控老年性牙槽骨丢失作用机制的研究方法。
背景技术
口腔疾病已被世界卫生组织和我国卫生部列为严重威胁人类健康的三大非传染性疾病之一。口腔疾病中,牙和颌骨相关疾病的发病率高达80%以上。据美国NIH统计:94%的65岁以上妇女患有牙齿松动脱落;而存在骨丢失者,牙齿脱落发生率较非骨丢失者高三倍。另据2005年全国第三次口腔疾病流行病学调查,我国50岁以上人群平均缺失12颗牙,65岁以上人群全部牙缺失患者高达30%,缺牙严重的影响患者健康和生活质量。老年性牙缺失与牙槽骨丢失密切相关,老年性牙槽骨丢失是每个人在衰老过程中必须面对的健康问题。因此,防治老年性牙槽骨丢失对于有效防治口腔疾病、促进口腔健康、提高生活质量具有重要意义。
近年来,Sirtuin蛋白家族引起科学界的广泛关注,它们对细胞的生存、凋亡、衰老等生理活动起着十分重要的调节作用1。Sirtuin1(Sirt1)为Sirtuin蛋白家族中的成员,其编码的基因被人们称作“长寿基因”,是所有生物共同的寿命控制基因之一;其编码的蛋白为去乙酰化酶,参与许多如肿瘤、糖尿病、心血管疾病等衰老相关疾病的调节3。因此,Sirt1作为抗衰老的新靶点逐渐被人们所重视。
通过对Sirt1基因敲除(Sirt1-/-)小鼠的研究发现:大多数Sirt1-/-小鼠在出生前后即发生死亡,并且伴随着骨、肾、心血管发育缺陷4。随着研究的深入,Sirt1在脑、肝、小肠、肾、心和骨等组织脏器中特定的生理功能逐步被揭示。对Sirt1在骨中的研究发现:12周龄雌性Sirt1杂合子(Sirt1+/-)小鼠较同窝野生型小鼠长骨和椎骨骨量显著降低;利用Prx1(Paired-related homeobox gene 1)启动子在间充质细胞(Mesenchymal stemcells,MSC)特异性敲除Sirt1,发现2.2年龄老年鼠长骨和椎骨的骨量丢失明显,仅为同年龄对照鼠的一半6;利用Collagen I启动子在成骨细胞特异性敲除Sirt1,发现成骨细胞骨形成减少7;利用LysozymeM启动子在破骨细胞特异性敲除Sirt1,发现破骨细胞骨吸收增加7,这些研究结果提示Sirt1在调控成骨细胞骨形成与破骨细胞骨吸收的动态平衡中发挥重要作用。
目前对于Sirt1在骨骼系统中的报道多集中在长骨和椎骨,那么,Sirt1在牙槽骨中的作用是怎样的呢?少有的几篇文献提到:Sirt1-/-小鼠颅面骨结构异常、矿化延迟,但并未阐明具体的异常及机制;人牙髓细胞有Sirt1表达,如过表达Sirt1可促进矿化结节的形成,但缺乏在体研究,且并未阐明机制;Sirt1可能介导了牙周软组织炎症发生发展的炎症通路和氧化应激通路,但缺乏针对牙槽骨的研究。牙槽骨的成骨方式与长骨和椎骨并不相同,牙槽骨发生属于膜内成骨,其成骨细胞来源于MSC。为了在体研究Sirt1在牙槽骨形成和衰老中的作用,构建了以Prx1为启动子,在MSC过表达Sirt1的转基因小鼠模型。转录因子Prx1作为MSC的标示物,已被广泛应用在间充质细胞系(Mesenchymallineage)特异性敲除或过表达靶标基因,颌骨的发生同样受Prx1的调节。MSC过表达Sirt1小鼠的牙槽骨量较同年龄野生型小鼠显著增加,并且能够纠正自然衰老小鼠的牙槽骨丢失。那么,MSC过表达Sirt1是如何调控牙槽骨量的呢?
发明内容
本发明要解决的技术问题是克服现有技术的不足,提供了一种Sirtuin 1经Bmi1介导调控老年性牙槽骨丢失作用机制的研究方法,分别从整体、细胞与分子水平出发,系统研究Sirt1在调控老年性牙槽骨丢失中的作用及机制,包括:
1)分别比较3、9和18月龄MSC过表达Sirt1的转基因(Sirt1TG)小鼠与同窝野生型(WT)小鼠下颌牙槽骨的表型差异,观察MSC过表达Sirt1能否防止老年性牙槽骨丢失;
2)通过建立MSC过表达Sirt1的Bmi1KO(Sirt1TG/Bmi1KO)小鼠模型,比较它们与Sirt1TG小鼠下颌牙槽骨的表型差异,观察Bmi1基因缺失能否阻断Sirt1过表达所引起的牙槽骨量增加;
3)将培养WT和Sirt1TG小鼠颌骨来源的MSCs(M-MSCs)22,并用Sirt1激活剂及抑制剂(或RNA干扰特异性敲低Sirt1)处理,检测Bmi1乙酰化水平、Bmi1核转位及其下游靶标分子表达水平的变化,同时检测MSC增殖、分化、凋亡和衰老相关指标的变化,观察Sirt1是否通过去乙酰化Bmi1而促进其核转位,抑制p16/p19和p53-ROS信号通路,促进MSC增殖和向成骨细胞分化,抑制其凋亡和衰老;
4)将用Sirt1的激活剂白黎芦醇分别处理自然衰老WT小鼠(16月龄)及老龄人群(≥60岁)的M-MSCs,观察上调Sirt1在治疗牙槽骨丢失中的作用。本研究方法有望阐明Sirt1在预防牙槽骨丢失中的作用及机制,并为Sirt1激活剂白黎芦醇临床应用于防治老年性牙槽骨丢失提供理论和实验依据。
为达到上述目的,本发明采用的技术方案是:Sirtuin 1经Bmi1介导调控老年性牙槽骨丢失作用机制的研究方法,包括以下步骤:
a.实验动物培育及基因型鉴定,选用4周龄分别带有WT、Sirt1TG、Bmi1KO、Sirt1TG/Bmi1KO基因的小鼠;
b.牙槽骨表型分析相关指标检测;
c.小鼠颌骨M-MSCs培养和体外实验;
d.氧化应激相关指标检测;
e.Sirt1激活剂处理自然衰老小鼠;
f.临床牙槽骨样本采集、颌骨M-MSCs培养及用Sirt1激活剂处理。
所述步骤b包括以下子步骤:
1)取材:小鼠麻醉后脱颈处死。取下颌骨,使用能更好保存酶活性和组织抗性的PLP(2%副醛,75mM赖氨酸,10mM过碘酸钠)固定液固定,用于X-线摄影、微-CT扫描和三维重建;经脱钙、脱水、浸蜡、石蜡包埋、切片,用于HE、组化和免疫组化及TUNEL染色;取新鲜牙槽骨组织,提取RNA用于Real-time RT-PCR检测;提取蛋白质用于Western blot分析;
2)X-线摄影、微-CT扫描和三维重建:固定后的下颌骨进行X线摄影,观察牙槽骨形态和密度的改变;使用先前报道的方法进行微-CT扫描和三维重建观察牙槽骨丢失和矿化程度的改变;
3)组织化学染色:进行HE、总胶原、碱性磷酸酶(ALP,成骨细胞特异性指标)染色、抗酒石酸酸性磷酸酶(TRAP,破骨细胞特异性指标)染色;
4)免疫组织化学染色:取下颌骨的石蜡切片,进行免疫组织化学染色;检测指标包括成骨细胞分化指标:I型胶原(Col I)、骨钙素(OCN);破骨细胞分化指标:核因子κB受体活化因子配体(RANKL);
5)Real-time RT-PCR:取下颌牙槽骨组织提取的RNA,做Real-time RT-PCR分析,观察骨分化相关基因表达的变化,检测指标如下:核心结合因子(Runx2):主导成骨细胞分化的基因;
6)Western blot:取牙槽骨组织提取的蛋白质,进行Western blot分析,成骨相关检测指标:Runx2、OCN、骨桥蛋白(OPN);
7)流式细胞分析:获得牙槽骨组织单细胞悬液用于流式细胞分析;处死小鼠24小时前,腹腔注射100mg BrdU/kg体重;取牙槽骨组织并用3mg/mL I型胶原酶和4mg/mL II型中性蛋白酶于37℃消化60分钟,获得单细胞悬液;用PBS/1%BSA调整细胞浓度为1×106个/100ul,加入不同荧光标记的抗体(细胞表面抗体),4度避光染30分钟,用1mlPBS/1%BSA洗涤细胞两次后,流式细胞仪检测;
所述步骤c包括以下子步骤:
1)免疫共沉淀:为了检测Bmi1与Sirt1的结合及Bmi1乙酰化水平的改变,用文献报道的方法提取细胞总蛋白21,取300ug总蛋白和2ug Bmi1抗体于4度孵育2小时;12000rpm离心后的上清与50ul argarose beads于4度孵育4小时,利用与argarose beads结合的Bmi1沉淀Sirt1和乙酰化赖氨酸,Westernblot检测沉淀物中Sirt1和乙酰化赖氨酸(AcetylatedLysine antibody,ChIPGrade,#21623,Abcam)的蛋白表达;反之,利用Sirt1抗体免疫沉淀Bmi1,Western blot检测沉淀物中Bmi1的蛋白表达;
2)Sirt1激活剂及拮抗剂处理:首先用剂量浓度梯度摸索处理M-MSCs的最适浓度,Sirt1激活剂白藜芦醇处理M-MSCs剂量梯度为0,12.5,25,50,100μM,Sirt1抑制剂烟酰胺处理M-BMSCs剂量梯度为0,50,100,250,500μM,提取细胞总蛋白,Western blot检测Sirt1蛋白表达水平的改变,筛选出最适剂量用于后续细胞学实验;
3)细胞化学染色和免疫细胞化学染色:进行Bmi1,BrdU(细胞培养终止前2小时掺入50μM BrdU)免疫细胞化学染色,进行TUNEL细胞化学染色;
4)Western blot:提取细胞的蛋白质,进行细胞周期蛋白相关指标检测:Bmi1、Cyclin D、Cyclin E、CDK2/4/6、Rb、p16、p19、p21、p53、PTEN等,细胞凋亡调控蛋白:Caspase-3、Bcl-2、Bax、Bcl-xl;
5)成骨诱导及染色:成骨诱导培养基为:生长培养基,加50mg/mL L-维生素C,10mM磷酸甘油;成骨诱导后进行ALP、von Kossa染色;
6)衰老诱导及半乳糖苷酶染色:待M-MSCs细胞生长至50%融合,更换衰老诱导培养基(生长培养基加100、200、400μM H2O2)于培养箱中培养2小时;然后用无血清培养基冲洗细胞,去掉残留的H2O2,然后更换为生长培养基±Sirt1激活剂/拮抗剂继续培养;当细胞长至80%-90%融合时,进行半乳糖苷酶染色;
7)过表达Sirt1和RNA干扰敲低Sirt1:过表达Sirt1即培养Sirt1TG小鼠的M-MSCs,RNA干扰敲低Sirt1:构建阴性以及针对小鼠Sirt1基因的shRNA干扰序列,包装成慢病毒颗粒侵染第三代M-MSCs;转染24小时后,提取RNA和蛋白质;利用Real-time RT-PCR和Westernblot检测Sirt1的表达,鉴定转染效率,筛选出沉默Sirt1的最佳shRNA干扰序列,进行相关实验;
8)流式细胞分析:0.25%胰酶-EDTA消化贴壁M-MSCs,用PBS/1%BSA调整细胞浓度为1×106个/100ul,加入不同荧光标记的抗体(细胞表面抗体),4度避光染30分钟,用1mlPBS/1%BSA洗涤细胞两次后,流式细胞仪检测;采用CD45-CD105+Sca1+为MSC。如果需检测BrdU,则应进行细胞内染色;
所述步骤d中包括以下子步骤:
1)流式细胞仪技术活性氧(ROS)水平检测:取牙槽骨组织用3mg/mL I型胶原;酶和4mg/mL II型中性蛋白酶于37℃消化60分钟获得单细胞悬液,或0.25%胰酶-EDTA消化贴壁M-MSCs,1000rpm,4℃离心5min,弃上清加PBS制得单细胞悬液,调整待测细胞的浓度为1×106-107个/ml;加入ROS敏感的荧光探针2′,7′-二氯二氢荧光素二乙酯(DCF-DA),轻轻混匀后37℃孵育30min,同上离心弃去液体,加入10%胎牛血清37℃孵育20min,同上离心弃去液体,加入适量PBS,流式仪检测细胞内荧光探针平均荧光强度;
2)Western blot:刮取贴壁M-MSCs,提取总蛋白质,进行Western blot分析,观察抗氧化蛋白prdxⅠ,prdxⅣ表达的变化;
3)Real-time RT-PCR:刮取贴壁M-MSCs,提取总RNA,作Real-time RT-PCR分析,观察氧化还原相关基因表达水平的变化;
4)Western blot:提取各组成骨成骨细胞的蛋白,作Western blot检测OPG和RANKL的表达。
所述步骤e包括以下子步骤:
1)Sirt1激活剂体外处理自然衰老小鼠的M-MSCs:培养18月龄WT小鼠的M-MSCs,利用Sirt1激活剂处理,利用6月龄年轻WT小鼠M-MSCs做为阳性对照;采用同上方法,观察细胞周期、增殖凋亡、成骨能力、衰老和氧化应激水平的变化;
2)Sirt1激活剂体内治疗自然衰老小鼠的牙槽骨丢失:16月龄自然衰老WT小鼠分成4组,每组8只,按照文献报道的给药方式、剂量和时间进行实验;第1组给予正常饮水做为阴性对照,第2-4组分别给予饮水补充不同剂量的Sirt1激活剂白藜芦醇,5、10、20mg/kg体重/天;采用多剂量梯度以保证摸索出可以增加牙槽骨量的最低药物剂量,避免药物毒性(必要时在处死小鼠时,收集小鼠心、肝、肾、脑等软组织,采用组织学及相关染色,观察有无药物毒性);以6月龄年轻WT小鼠给予正常饮水做为阳性对照;2个月后进行表型分析。
所述步骤f包括以下子步骤:
将从种植牙手术中收集牙槽骨样本,按照2015年联合国世界卫生组织确定新的年龄分段进行分组:≥60岁,纳入老龄组;≤44岁,纳入年轻对照组;
培养M-MSCs:取新鲜牙槽骨组织(含骨髓),多次冲洗去除残留的口腔内液体;
在Petri dish中用无菌小刀和注射器刮取、冲洗出骨髓细胞,细胞以1.3×104/cm2密度接种,培养基为含20%胎牛血清,100U/ml青霉素,100g/ml链霉素,2mM谷氨酰胺的,培养M-MSCs;
取第三代M-MSCs,利用Sirt1激活剂处理后,采用同上方法,观察细胞增殖凋亡、成骨能力和衰老水平的变化。
由于上述技术方案的运用,本发明与现有技术相比具有下列优点:分别从整体、细胞与分子水平出发,系统研究Sirt1在调控老年性牙槽骨丢失中的作用及机制,包括:
1)分别比较3、9和18月龄MSC过表达Sirt1的转基因(Sirt1TG)小鼠与同窝野生型(WT)小鼠下颌牙槽骨的表型差异,观察MSC过表达Sirt1能否防止老年性牙槽骨丢失;
2)通过建立MSC过表达Sirt1的Bmi1KO(Sirt1TG/Bmi1KO)小鼠模型,比较它们与Sirt1TG小鼠下颌牙槽骨的表型差异,观察Bmi1基因缺失能否阻断Sirt1过表达所引起的牙槽骨量增加;
3)将培养WT和Sirt1TG小鼠颌骨来源的MSCs(M-MSCs)22,并用Sirt1激活剂及抑制剂(或RNA干扰特异性敲低Sirt1)处理,检测Bmi1乙酰化水平、Bmi1核转位及其下游靶标分子表达水平的变化,同时检测MSC增殖、分化、凋亡和衰老相关指标的变化,观察Sirt1是否通过去乙酰化Bmi1而促进其核转位,抑制p16/p19和p53-ROS信号通路,促进MSC增殖和向成骨细胞分化,抑制其凋亡和衰老;4)将用Sirt1的激活剂白黎芦醇分别处理自然衰老WT小鼠(16月龄)及老龄人群(≥60岁)的M-MSCs,观察上调Sirt1在治疗牙槽骨丢失中的作用。本研究方法有望阐明Sirt1在预防牙槽骨丢失中的作用及机制,并为Sirt1激活剂白黎芦醇临床应用于防治老年性牙槽骨丢失提供理论和实验依据
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明。
实施例一
Sirtuin 1经Bmi1介导调控老年性牙槽骨丢失作用机制的研究方法,包括以下步骤:
a.实验动物培育及基因型鉴定,选用4周龄分别带有WT、Sirt1TG、Bmi1KO、Sirt1TG/Bmi1KO基因的小鼠;
b.牙槽骨表型分析相关指标检测;
c.小鼠颌骨M-MSCs培养和体外实验;
d.氧化应激相关指标检测;
e.Sirt1激活剂处理自然衰老小鼠;
f.临床牙槽骨样本采集、颌骨M-MSCs培养及用Sirt1激活剂处理。
所述步骤b包括以下子步骤:
1)取材:小鼠麻醉后脱颈处死。取下颌骨,使用能更好保存酶活性和组织抗性的PLP(2%副醛,75mM赖氨酸,10mM过碘酸钠)固定液固定,用于X-线摄影、微-CT扫描和三维重建;经脱钙、脱水、浸蜡、石蜡包埋、切片,用于HE、组化和免疫组化及TUNEL染色;取新鲜牙槽骨组织,提取RNA用于Real-time RT-PCR检测;提取蛋白质用于Western blot分析;
2)X-线摄影、微-CT扫描和三维重建:固定后的下颌骨进行X线摄影,观察牙槽骨形态和密度的改变;使用先前报道的方法进行微-CT扫描和三维重建观察牙槽骨丢失和矿化程度的改变;
3)组织化学染色:进行HE、总胶原、碱性磷酸酶(ALP,成骨细胞特异性指标)染色、抗酒石酸酸性磷酸酶(TRAP,破骨细胞特异性指标)染色;
4)免疫组织化学染色:取下颌骨的石蜡切片,进行免疫组织化学染色;检测指标包括成骨细胞分化指标:I型胶原(Col I)、骨钙素(OCN);破骨细胞分化指标:核因子κB受体活化因子配体(RANKL);
5)Real-time RT-PCR:取下颌牙槽骨组织提取的RNA,做Real-time RT-PCR分析,观察骨分化相关基因表达的变化,检测指标如下:核心结合因子(Runx2):主导成骨细胞分化的基因;
6)Western blot:取牙槽骨组织提取的蛋白质,进行Western blot分析,成骨相关检测指标:Runx2、OCN、骨桥蛋白(OPN);
7)流式细胞分析:获得牙槽骨组织单细胞悬液用于流式细胞分析;处死小鼠24小时前,腹腔注射100mg BrdU/kg体重;取牙槽骨组织并用3mg/mL I型胶原酶和4mg/mL II型中性蛋白酶于37℃消化60分钟,获得单细胞悬液;用PBS/1%BSA调整细胞浓度为1×106个/100ul,加入不同荧光标记的抗体(细胞表面抗体),4度避光染30分钟,用1mlPBS/1%BSA洗涤细胞两次后,流式细胞仪检测;所述步骤c包括以下子步骤:
1)免疫共沉淀:为了检测Bmi1与Sirt1的结合及Bmi1乙酰化水平的改变,用文献报道的方法提取细胞总蛋白21,取300ug总蛋白和2ug Bmi1抗体于4度孵育2小时;12000rpm离心后的上清与50ul argarose beads于4度孵育4小时,利用与argarose beads结合的Bmi1沉淀Sirt1和乙酰化赖氨酸,Westernblot检测沉淀物中Sirt1和乙酰化赖氨酸(AcetylatedLysine antibody,ChIPGrade,#21623,Abcam)的蛋白表达;反之,利用Sirt1抗体免疫沉淀Bmi1,Western blot检测沉淀物中Bmi1的蛋白表达;
2)Sirt1激活剂及拮抗剂处理:首先用剂量浓度梯度摸索处理M-MSCs的最适浓度,Sirt1激活剂白藜芦醇处理M-MSCs剂量梯度为0,12.5,25,50,100μM,Sirt1抑制剂烟酰胺处理M-BMSCs剂量梯度为0,50,100,250,500μM,提取细胞总蛋白,Western blot检测Sirt1蛋白表达水平的改变,筛选出最适剂量用于后续细胞学实验;
3)细胞化学染色和免疫细胞化学染色:进行Bmi1,BrdU(细胞培养终止前2小时掺入50μM BrdU)免疫细胞化学染色,进行TUNEL细胞化学染色;
4)Western blot:提取细胞的蛋白质,进行细胞周期蛋白相关指标检测:Bmi1、Cyclin D、Cyclin E、CDK2/4/6、Rb、p16、p19、p21、p53、PTEN等,细胞凋亡调控蛋白:Caspase-3、Bcl-2、Bax、Bcl-xl;
5)成骨诱导及染色:成骨诱导培养基为:生长培养基,加50mg/mL L-维生素C,10mM磷酸甘油;成骨诱导后进行ALP、von Kossa染色;
6)衰老诱导及半乳糖苷酶染色:待M-MSCs细胞生长至50%融合,更换衰老诱导培养基(生长培养基加100、200、400μM H2O2)于培养箱中培养2小时;然后用无血清培养基冲洗细胞,去掉残留的H2O2,然后更换为生长培养基±Sirt1激活剂/拮抗剂继续培养;当细胞长至80%-90%融合时,进行半乳糖苷酶染色;
7)过表达Sirt1和RNA干扰敲低Sirt1:过表达Sirt1即培养Sirt1TG小鼠的M-MSCs,RNA干扰敲低Sirt1:构建阴性以及针对小鼠Sirt1基因的shRNA干扰序列,包装成慢病毒颗粒侵染第三代M-MSCs;转染24小时后,提取RNA和蛋白质;利用Real-time RT-PCR和Westernblot检测Sirt1的表达,鉴定转染效率,筛选出沉默Sirt1的最佳shRNA干扰序列,进行相关实验;
8)流式细胞分析:0.25%胰酶-EDTA消化贴壁M-MSCs,用PBS/1%BSA调整细胞浓度为1×106个/100ul,加入不同荧光标记的抗体(细胞表面抗体),4度避光染30分钟,用1mlPBS/1%BSA洗涤细胞两次后,流式细胞仪检测;采用CD45-CD105+Sca1+为MSC。如果需检测BrdU,则应进行细胞内染色;
所述步骤d中包括以下子步骤:
1)流式细胞仪技术活性氧(ROS)水平检测:取牙槽骨组织用3mg/mL I型胶原;酶和4mg/mL II型中性蛋白酶于37℃消化60分钟获得单细胞悬液,或0.25%胰酶-EDTA消化贴壁M-MSCs,1000rpm,4℃离心5min,弃上清加PBS制得单细胞悬液,调整待测细胞的浓度为1×106-107个/ml;加入ROS敏感的荧光探针2′,7′-二氯二氢荧光素二乙酯(DCF-DA),轻轻混匀后37℃孵育30min,同上离心弃去液体,加入10%胎牛血清37℃孵育20min,同上离心弃去液体,加入适量PBS,流式仪检测细胞内荧光探针平均荧光强度;
2)Western blot:刮取贴壁M-MSCs,提取总蛋白质,进行Western blot分析,观察抗氧化蛋白prdxⅠ,prdxⅣ表达的变化;
3)Real-time RT-PCR:刮取贴壁M-MSCs,提取总RNA,作Real-time RT-PCR分析,观察氧化还原相关基因表达水平的变化;
4)Western blot:提取各组成骨成骨细胞的蛋白,作Western blot检测OPG和RANKL的表达。
所述步骤e包括以下子步骤:
1)Sirt1激活剂体外处理自然衰老小鼠的M-MSCs:培养18月龄WT小鼠的M-MSCs,利用Sirt1激活剂处理,利用6月龄年轻WT小鼠M-MSCs做为阳性对照;采用同上方法,观察细胞周期、增殖凋亡、成骨能力、衰老和氧化应激水平的变化;
2)Sirt1激活剂体内治疗自然衰老小鼠的牙槽骨丢失:16月龄自然衰老WT小鼠分成4组,每组8只,按照文献报道的给药方式、剂量和时间进行实验;第1组给予正常饮水做为阴性对照,第2-4组分别给予饮水补充不同剂量的Sirt1激活剂白藜芦醇,5、10、20mg/kg体重/天;采用多剂量梯度以保证摸索出可以增加牙槽骨量的最低药物剂量,避免药物毒性(必要时在处死小鼠时,收集小鼠心、肝、肾、脑等软组织,采用组织学及相关染色,观察有无药物毒性);以6月龄年轻WT小鼠给予正常饮水做为阳性对照;2个月后进行表型分析。
所述步骤f包括以下子步骤:
将从种植牙手术中收集牙槽骨样本,按照2015年联合国世界卫生组织确定新的年龄分段进行分组:≥60岁,纳入老龄组;≤44岁,纳入年轻对照组;
培养M-MSCs:取新鲜牙槽骨组织(含骨髓),多次冲洗去除残留的口腔内液体;
在Petri dish中用无菌小刀和注射器刮取、冲洗出骨髓细胞,细胞以1.3×104/cm2密度接种,培养基为含20%胎牛血清,100U/ml青霉素,100g/ml链霉素,2mM谷氨酰胺的,培养M-MSCs;
取第三代M-MSCs,利用Sirt1激活剂处理后,采用同上方法,观察细胞增殖凋亡、成骨能力和衰老水平的变化。
实施例二
首先,研究间充质细胞过表达Sirt1在防止老年性牙槽骨丢失中的作用:
1.为了明确间充质细胞(MSC)过表达Sirt1是否具有增加牙槽骨量,防止牙槽骨丢失的作用,通过影像学的方法观察3,9,18月龄同窝野生型(WT)和MSC过表达Sirt1的转基因(Sirt1TG)小鼠下颌骨形态及骨密度的变化;采用组织学和组织化学染色的方法,观察WT和Sirt1TG小鼠下颌牙槽骨量的改变。
2.为了明确MSC过表达Sirt1对小鼠下颌牙槽骨量的影响是否与成骨细胞骨形成增加,和破骨细胞骨吸收减少有关,通过组织学,组织化学染色、免疫组织化学染色及Real-time RT-PCR和Western blot的方法,分别观察WT和Sirt1TG小鼠牙槽骨成骨细胞骨形成、破骨细胞骨吸收及其相关基因、蛋白表达水平的改变。
3.为了明确MSC过表达Sirt1是否通过促进MSC增殖和向成骨细胞分化,抑制其凋亡和衰老,从而发挥防止牙槽骨丢失的作用,将取WT和Sirt1TG小鼠的牙槽骨组织并用酶消化为单细胞悬液,采用流式细胞术标记CD45-CD105+Sca1+(MSC表面标记物),分析MSC的增殖指标BrdU(5-溴脱氧尿核苷,处死小鼠前标记),凋亡指标7-AAD,衰老指标ROS等的改变;并培养小鼠颌骨来源的MSCs(M-MSCs)22,通过BrdU掺入、成骨诱导培养及衰老诱导培养等方法,采用细胞化学、免疫细胞荧光染色、Western blot和Real-time RT-PCR的方法检测MSC增殖、分化、凋亡及衰老各项指标的变化。
其次,研究Sirt1经Bmi1介导,发挥促进牙槽骨形成的作用:
1.为了明确Sirt1是否通过干细胞维持和衰老相关基因Bmi1介导,发挥抗牙槽骨丢失的作用,将建立Sirt1在MSC过表达的Bmi1KO(Sirt1TG/Bmi1KO)小鼠模型,比较其与WT,Sirt1TG和Bmi1KO小鼠牙槽骨的表型差异,利用研究内容第一部分的方法比较分析小鼠下颌牙槽骨密度、牙槽骨量、成骨细胞数、破骨细胞数,及MSC增殖、分化、凋亡和衰老的变化,观察敲除Bmi1基因能否阻断Sirt1在MSC过表达引起的牙槽骨量增加。
2.鉴于Bmi1基因缺失激活p16/p19和p53-ROS信号通路,为了观察Bmi1基因敲除是否也能阻断Sirt1对p16/p19和p53-ROS信号通路的调节作用,将培WT,Sirt1TG,Bmi1KO,Sirt1TG/Bmi1KO小鼠的M-MSCs,利用Real-time RT-PCR和Western blot的方法,检测p16/p19,p53-ROS信号分子及氧化应激相关指标的变化。
再次,研究Sirt1通过去乙酰化Bmi1,促进其核转位,抑制p16/p19和p53-ROS信号通路,从而促进MSC增殖和向成骨细胞分化,抑制其凋亡和衰老的作用由于颌骨和长骨的胚胎来源和成骨方式不同,颌骨由神经嵴发育而来,为膜内成骨;长骨由中胚层发育而来,为软骨内成骨,两者表现为不同的生物学特征。将培养Sirt1TG小鼠的M-MSCs(即Sirt1在M-MSCs过表达),以WT小鼠的M-MSCs作为对照,用于以下机制研究:
1.为了明确Sirt1通过去乙酰化Bmi1,促进其核转位,抑制p16/p19和p53-ROS信号通路,将分别用Sirt1激活剂及抑制剂(或RNA干扰特异性敲低Sirt1)处理,通过免疫共沉淀的方法,检测Sirt1与Bmi1的结合及Bmi1乙酰化水平的变化;采用免疫细胞化学染色的方法,观察Bmi1细胞定位的变化;利用Western blot和Real-time RT-PCR的方法,检测Bmi1下游靶标p16/p19和p53-ROS信号通路的改变。
2.为了明确Sirt1是否具有促进MSC增殖和向成骨细胞分化,抑制其凋亡和衰老的作用,将分别用Sirt1激活剂及抑制剂(或RNA干扰特异性敲低Sirt1)处理,采用BrdU掺入免疫荧光、TUNEL染色、流式细胞术和Western blot的方法,观察M-MSCs增殖、凋亡的改变;利用成骨诱导培养基培养细胞,观察M-MSCs成骨能力的改变;通过衰老诱导培养细胞,观察M-MSCs衰老水平的改变;通过检测氧化应激相关指标及相关信号分子,观察M-MSCs抗氧化能力的改变。
3.为了明确Sirt1是否完全或部分通过Bmi1发挥作用,将培养Bmi1KO和Sirt1TG/Bmi1KO小鼠M-MSCs,分别以WT和Sirt1TG小鼠的M-MSCs为对照,分别用Sirt1激活剂及抑制剂(或RNA干扰特异性敲低Sirt1)处理,观察Bmi1基因缺失可否阻断Sirt1上调或下调所引起的细胞表型改变。
最后,研究Sirt1的激活剂白藜芦醇在预防牙槽骨丢失中的作用:
1.为了观察Sirt1的激活剂能否改善自然衰老小鼠的细胞异常,将用Sirt1的激活剂处理自然衰老小鼠M-MSCs,以年轻小鼠M-MSCs作为对照,观察Sirt1的激活剂能否改善自然衰老小鼠的细胞异常。
2.为了观察Sirt1的激活剂能否改善自然衰老小鼠的牙槽骨量丢失,将利用16月龄野生型小鼠,饮水补充Sirt1激活剂白藜芦醇,持续2个月25。以年轻小鼠作为对照。将分析18月龄给药组WT小鼠、对照组WT小鼠以及年轻小鼠的牙槽骨表型差异,以观察Sirt1的激活剂能否改善自然衰老小鼠的牙槽骨量丢失。分析方法和指标同第一部分。
3.为了观察Sirt1的激活剂能否改善自然衰老人群的M-MSCs的异常,将从临床收集老年人的牙槽骨组织,培养人M-MSCs26,并利用Sirt1的激活剂处理该细胞,以年轻人M-MSCs作为阳性对照,观察Sirt1的激活剂能否改善老龄人群的牙槽骨细胞异常。
以上仅是本发明的具体应用范例,对本发明的保护范围不构成任何限制。凡采用等同变换或者等效替换而形成的技术方案,均落在本发明权利保护范围之内。
Claims (6)
1.Sirtuin 1经Bmi1介导调控老年性牙槽骨丢失作用机制的研究方法,其特征在于,包括以下步骤:
a.实验动物培育及基因型鉴定,选用4周龄分别带有WT、Sirt1TG、Bmi1KO、Sirt1TG/Bmi1KO基因的小鼠;
b.牙槽骨表型分析相关指标检测;
c.小鼠颌骨M-MSCs培养和体外实验;
d.氧化应激相关指标检测;
e.Sirt1激活剂处理自然衰老小鼠;
f.临床牙槽骨样本采集、颌骨M-MSCs培养及用Sirt1激活剂处理。
2.根据权利要求1所述的Sirtuin 1经Bmi1介导调控老年性牙槽骨丢失作用机制的研究方法,其特征在于,所述步骤b包括以下子步骤:
1)取材:小鼠麻醉后脱颈取下颌骨,使用能更好保存酶活性和组织抗性的PLP(2%副醛,75mM赖氨酸,10mM过碘酸钠)固定液固定,用于X-线摄影、微-CT扫描和三维重建;经脱钙、脱水、浸蜡、石蜡包埋、切片,用于HE、组化和免疫组化及TUNEL染色;取新鲜牙槽骨组织,提取RNA用于Real-time RT-PCR检测;提取蛋白质用于Western blot分析;
2)X-线摄影、微-CT扫描和三维重建:固定后的下颌骨进行X线摄影,观察牙槽骨形态和密度的改变;使用先前报道的方法进行微-CT扫描和三维重建观察牙槽骨丢失和矿化程度的改变;
3)组织化学染色:进行HE、总胶原、碱性磷酸酶(ALP,成骨细胞特异性指标)染色、抗酒石酸酸性磷酸酶(TRAP,破骨细胞特异性指标)染色;
4)免疫组织化学染色:取下颌骨的石蜡切片,进行免疫组织化学染色;检测指标包括成骨细胞分化指标:I型胶原(Col I)、骨钙素(OCN);破骨细胞分化指标:核因子κB受体活化因子配体(RANKL);
5)Real-time RT-PCR:取下颌牙槽骨组织提取的RNA,做Real-time RT-PCR分析,观察骨分化相关基因表达的变化,检测指标如下:核心结合因子(Runx2):主导成骨细胞分化的基因;
6)Western blot:取牙槽骨组织提取的蛋白质,进行Western blot分析,成骨相关检测指标:Runx2、OCN、骨桥蛋白(OPN);
7)流式细胞分析:获得牙槽骨组织单细胞悬液用于流式细胞分析;处死小鼠24小时前,腹腔注射100mg BrdU/kg体重;取牙槽骨组织并用3mg/mL I型胶原酶和4mg/mL II型中性蛋白酶于37℃消化60分钟,获得单细胞悬液;用PBS/1%BSA调整细胞浓度为1×106个/100ul,加入不同荧光标记的抗体(细胞表面抗体),4度避光染30分钟,用1mlPBS/1%BSA洗涤细胞两次后,流式细胞仪检测。
3.根据权利要求1所述的Sirtuin 1经Bmi1介导调控老年性牙槽骨丢失作用机制的研究方法,其特征在于,所述步骤c包括以下子步骤:
1)免疫共沉淀:为了检测Bmi1与Sirt1的结合及Bmi1乙酰化水平的改变,用文献报道的方法提取细胞总蛋白21,取300ug总蛋白和2ug Bmi1抗体于4度孵育2小时;12000rpm离心后的上清与50ul argarose beads于4度孵育4小时,利用与argarose beads结合的Bmi1沉淀Sirt1和乙酰化赖氨酸,Westernblot检测沉淀物中Sirt1和乙酰化赖氨酸(AcetylatedLysine antibody,ChIPGrade,#21623,Abcam)的蛋白表达;反之,利用Sirt1抗体免疫沉淀Bmi1,Western blot检测沉淀物中Bmi1的蛋白表达;
2)Sirt1激活剂及拮抗剂处理:首先用剂量浓度梯度摸索处理M-MSCs的最适浓度,Sirt1激活剂白藜芦醇处理M-MSCs剂量梯度为0,12.5,25,50,100μM,Sirt1抑制剂烟酰胺处理M-BMSCs剂量梯度为0,50,100,250,500μM,提取细胞总蛋白,Western blot检测Sirt1蛋白表达水平的改变,筛选出最适剂量用于后续细胞学实验;
3)细胞化学染色和免疫细胞化学染色:进行Bmi1,BrdU(细胞培养终止前2小时掺入50μM BrdU)免疫细胞化学染色,进行TUNEL细胞化学染色;
4)Western blot:提取细胞的蛋白质,进行细胞周期蛋白相关指标检测:Bmi1、CyclinD、Cyclin E、CDK2/4/6、Rb、p16、p19、p21、p53、PTEN等,细胞凋亡调控蛋白:Caspase-3、Bcl-2、Bax、Bcl-xl;
5)成骨诱导及染色:成骨诱导培养基为:生长培养基,加50mg/mL L-维生素C,10mM磷酸甘油;成骨诱导后进行ALP、von Kossa染色;
6)衰老诱导及半乳糖苷酶染色:待M-MSCs细胞生长至50%融合,更换衰老诱导培养基(生长培养基加100、200、400μM H2O2)于培养箱中培养2小时;然后用无血清培养基冲洗细胞,去掉残留的H2O2,然后更换为生长培养基±Sirt1激活剂/拮抗剂继续培养;当细胞长至80%-90%融合时,进行半乳糖苷酶染色;
7)过表达Sirt1和RNA干扰敲低Sirt1:过表达Sirt1即培养Sirt1TG小鼠的M-MSCs,RNA干扰敲低Sirt1:构建阴性以及针对小鼠Sirt1基因的shRNA干扰序列,包装成慢病毒颗粒侵染第三代M-MSCs;转染24小时后,提取RNA和蛋白质;利用Real-time RT-PCR和Westernblot检测Sirt1的表达,鉴定转染效率,筛选出沉默Sirt1的最佳shRNA干扰序列,进行相关实验;
8)流式细胞分析:0.25%胰酶-EDTA消化贴壁M-MSCs,用PBS/1%BSA调整细胞浓度为1×106个/100ul,加入不同荧光标记的抗体(细胞表面抗体),4度避光染30分钟,用1ml PBS/1%BSA洗涤细胞两次后,流式细胞仪检测;采用CD45-CD105+Sca1+为MSC;如果需检测BrdU,则应进行细胞内染色。
4.根据权利要求1所述的Sirtuin 1经Bmi1介导调控老年性牙槽骨丢失作用机制的研究方法,其特征在于,所述步骤d中包括以下子步骤:
1)流式细胞仪技术活性氧(ROS)水平检测:取牙槽骨组织用3mg/mL I型胶原;酶和4mg/mL II型中性蛋白酶于37℃消化60分钟获得单细胞悬液,或0.25%胰酶-EDTA消化贴壁M-MSCs,1000rpm,4℃离心5min,弃上清加PBS制得单细胞悬液,调整待测细胞的浓度为1×106-107个/ml;加入ROS敏感的荧光探针2′,7′-二氯二氢荧光素二乙酯(DCF-DA),轻轻混匀后37℃孵育30min,同上离心弃去液体,加入10%胎牛血清37℃孵育20min,同上离心弃去液体,加入适量PBS,流式仪检测细胞内荧光探针平均荧光强度;
2)Western blot:刮取贴壁M-MSCs,提取总蛋白质,进行Western blot分析,观察抗氧化蛋白prdxⅠ,prdxⅣ表达的变化;
3)Real-time RT-PCR:刮取贴壁M-MSCs,提取总RNA,作Real-time RT-PCR分析,观察氧化还原相关基因表达水平的变化;
4)Western blot:提取各组成骨成骨细胞的蛋白,作Western blot检测OPG和RANKL的表达。
5.根据权利要求1所述的Sirtuin 1经Bmi1介导调控老年性牙槽骨丢失作用机制的研究方法,其特征在于,所述步骤e包括以下子步骤:
1)Sirt1激活剂体外处理自然衰老小鼠的M-MSCs:培养18月龄WT小鼠的M-MSCs,利用Sirt1激活剂处理,利用6月龄年轻WT小鼠M-MSCs做为阳性对照;采用同上方法,观察细胞周期、增殖凋亡、成骨能力、衰老和氧化应激水平的变化;
2)Sirt1激活剂体内治疗自然衰老小鼠的牙槽骨丢失:16月龄自然衰老WT小鼠分成4组,每组8只,按照文献报道的给药方式、剂量和时间进行实验;第1组给予正常饮水做为阴性对照,第2-4组分别给予饮水补充不同剂量的Sirt1激活剂白藜芦醇,5、10、20mg/kg体重/天;采用多剂量梯度以保证摸索出可以增加牙槽骨量的最低药物剂量,避免药物毒性(必要时在处死小鼠时,收集小鼠心、肝、肾、脑等软组织,采用组织学及相关染色,观察有无药物毒性);以6月龄年轻WT小鼠给予正常饮水做为阳性对照;2个月后进行表型分析。
6.根据权利要求1所述的Sirtuin 1经Bmi1介导调控老年性牙槽骨丢失作用机制的研究方法,其特征在于,所述步骤f包括以下子步骤:
将从种植牙手术中收集牙槽骨样本,按照2015年联合国世界卫生组织确定新的年龄分段进行分组:≥60岁,纳入老龄组;≤44岁,纳入年轻对照组;
培养M-MSCs:取新鲜牙槽骨组织(含骨髓),多次冲洗去除残留的口腔内液体;
在Petri dish中用无菌小刀和注射器刮取、冲洗出骨髓细胞,细胞以1.3×104/cm2密度接种,培养基为含20%胎牛血清,100U/ml青霉素,100g/ml链霉素,2mM谷氨酰胺的,培养M-MSCs;
取第三代M-MSCs,利用Sirt1激活剂处理后,采用同上方法,观察细胞增殖凋亡、成骨能力和衰老水平的变化。
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