CN115697347B - 一种调节musashi1在细胞中表达水平的方法 - Google Patents
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Abstract
一种调节Musashi1在细胞中表达水平的方法。具体而言,涉及一种细胞调节组合物,其能够调节选自以下任一项在细胞中的表达或活性:K19、α2β1整合素、Musashi1。该调节组合物能够促进表皮细胞的增殖、促进表皮细胞的生长、促进表皮细胞的迁移、促进血管内皮细胞的增殖、促进血管内皮细胞的生长、促进血管内皮细胞的迁移、促进成纤维细胞的增殖、促进成纤维细胞的生长、促进成纤维细胞的迁移。
Description
本申请要求2020年7月8日提交的专利申请(申请号为2020106497032)的优先权。
技术领域
本申请涉及细胞生物学领域。具体而言,涉及细胞调节组合物促进Musashi1在细胞中表达水平的用途。
背景技术
Musashi家族是一类进化保守的RNA结合蛋白家族,可选择性的表达于神经系统干细胞、祖细胞,包括Musashi1和Musashi2成员。Musashi1是第1个最早发现于果蝇的Musashi家族成员。Musashi1与Musashi2蛋白通过抑制其目标蛋白Numb+mRNA的翻译过程,协同激活Notch信号通路,参与干细胞非对称分裂。
Musashi1简称MSI1,是一种分子量为39KD的RNA结合蛋白。通常在CNS(中枢神经系统)干细胞和祖细胞上表达;而在分化后的细胞表达下调。Musashi1是一种转录抑制因子,其可以直接调节靶蛋白Numb和P21(CIP-1)的表达。有文献报道Musashi1在肠、乳腺和毛囊等一系列组织的干细胞有表达。此外,也有研究发现Musashi1在肺腺癌和大、小细胞癌症中有表达。最近研究发现,Musashi1在缺血性神经损伤中发挥调节细胞凋亡的作用。Musashi1目前作为一个低分化细胞的候选基因,在参与肿瘤有关信号通路、细胞增殖与凋亡等很多方面都起着重要作用。神经胶质瘤、食管癌、胃癌、结肠癌、乳腺癌等实体肿瘤中均出现Musashi1基因的高表达,Musashi的研究将对临床肿瘤疾病基因层面的深入研究和诊断治疗提供新途径。
考虑到Musashi1的重要生物学意义,实验室中需要建立Musashi1阳性表达的细胞系以供研究。鉴于此,本领域需要提供一种促进Musashi1表达的培养方法和试剂。
发明内容
本申请提供了用于调节细胞的活性成分及其应用。
根据本申请的一些实施方案,提供了一种调节组合物,所述调节组合物能够调节选自以下任一项在细胞中的表达或活性:K19、α2β1整合素、Musashi1、或其组合。
在一些实施方案中,所述调节组合物用于选自以下的一项或其组合:促进表皮细胞的生长、促进表皮细胞的增殖、促进表皮细胞的迁移;促进成纤维细胞的生长、促进成纤维细胞的增殖、促进成纤维细胞的迁移;促进血管内皮细胞的生长、促进血管内皮细胞的增殖、促进血管内皮细胞的迁移。
在一些实施方案中,所述调节组合物包含:
0.5重量%-20重量%的甾醇、
0.1重量%-2重量%的黄芩甙、和
1重量%-20重量%的蜂蜡。
在一些实施方案中,所述调节组合物还包含植物油或动物油。
在一些实施方案中,植物油选自:玉米油、花生油、棉籽油、红花油、茶树油、芝麻油、橄榄油、豆油。
在一些实施方案中,所述甾醇选自:动物甾醇、植物甾醇。本申请中使用的甾醇从各种天然来源中获取。例如,植物甾醇类可以从加工过的植物油中获取,如玉米油、小麦籽油、大豆提取物、大米提取物、米糠油、油菜籽油、芝麻油。甾醇还有其它来源如海洋动物。
在一些实施方案中,所述甾醇选自:天然胆固醇、合成胆固醇、及其异构体或其衍生物。
在一些实施方案中,所述甾醇选自:豆甾醇、β-谷甾醇、角甾醇、γ-谷甾醇、菜籽甾醇、α-菠菜甾醇、24-去氢胆固醇、多孔甾醇、胡萝卜甙、及其异构体或其衍生物;最优选豆甾醇、β-谷甾醇、菜籽甾醇的组合。
在一些实施方案中,所述甾醇的量为1重量%-10重量%,优选2重量%至6重量%。
在一些实施方案中,所述调节组合物还包含2重量%-10重量%的蜂蜡;优选2%至10%,最优选3%至6%。
蜂蜡被用作赋形剂来生产外用药。蜂蜡的组成可以被分为4类即脂类、游离酸类、游离醇类和烃类。蜂蜡也含有微量的挥发油和色素。
在本申请中,蜂蜡在调节组合物中为甾醇提供支持结构。蜂蜡能形成三维结构,其中包含溶有甾醇的油。
调节组合物可以含有少量的水,按重量计算含少于0.5%水,最好是少于0.1%。
在一些实施方案中,所述调节组合物还包含0.1重量%-30重量%的蜂胶;优选1%至20%,最优选5%至10%。
在一些实施方案中,所述黄芩甙的量为0.2重量%-1重量%,优选0.2%至1重量%,更优选0.5%至1重量%。黄芩甙可以从黄芩(Scutellaria baicalensis Georgi)中提取(《中国中药大词典》,上海科技出版社,1986,2017-2021页)。可以使用油、乙醇或其它有机溶剂提取;优选使用100℃的油(更优选的温度是在120-200℃之间,最优选的温度是在160-180℃)。
在一些实施方案中,所述调节组合物还包含0.1重量%-2重量%的黄柏内酯,优选0.2%至1重量%,更优选0.5%至1重量%。黄柏内酯可以从黄柏中提取(Phellodendronamurense Rupr)(《中国中药大词典》,上海科技出版社,1986,2031-2035页)。可以使用油、乙醇或其它有机溶剂提取;优选使用100℃的油(更优选的温度是在120-200℃之间,最优选的温度是在160-180℃)。
在一些实施方案中,所述调节组合物还包含0.001重量%-2重量%的小蘖碱(obabenine),优选0.002%至0.5重量%更优选0.003%至0.1重量%。黄小蘖碱可以从黄芩、黄柏和/或黄连(Coptis chinensis Franch)中提取(《中国中药大词典》,上海科技出版社,1986,2022-2030页)。可以使用油、乙醇或其它有机溶剂提取;优选使用100℃的油(更优选的温度是在120-200℃之间,最优选的温度是在160-180℃)。
在一些实施方案中,所述调节组合物还包含0.001重量%-2重量%的黄连素,优选0.002%至0.5重量%,更优选0.003%至0.1重量%。
在一些实施方案中,所述调节组合物还包含0.001重量%-2重量%的罂粟壳碱,优选0.002%至0.5重量%,更优选0.003%至0.1重量%。
在一些实施方案中,所述调节组合物还包含0.001重量%-2重量%的地龙,优选0.002%至0.5重量%,更优选0.003%至0.1重量%。
在一些具体的实施方案中,提供了一种调节组合物,其包含以下或由以下组成:
2重量%-6重量%的甾醇、
0.5重量%-1重量%的黄芩甙、
3重量%-6重量%的蜂蜡、
5重量%-10重量%的蜂胶、
0.5重量%-1重量%的黄柏内酯、
0.003重量%-0.1重量%的小蘖碱、
0.003重量%-0.1重量%的黄连素、
0.003重量%-0.1重量%的罂粟壳碱、
0.003重量%-0.1重量%的地龙、和
植物油或动物油。
根据一些实施方案,提供了一种原位、离体或体外调节细胞的方法,包括使细胞接触本申请的调节组合物的步骤。
根据一些实施方案,提供了一种原位、离体或体外提高细胞中Musashi1表达水平的方法,包括使细胞接触本申请的调节组合物的步骤。
在一些实施方案中,细胞是哺乳动物细胞,其选自:机械损伤的皮肤细胞、化学损伤的皮肤细胞、热损伤的皮肤细胞、糖尿病患者的皮肤细胞。
在一些实施方案中,所述哺乳动物细胞选自:表皮细胞、肉芽组织的细胞、或血管内皮细胞。
在一些实施方案中,提供了一种原位、离体或体外提高细胞中Musashi1表达水平的方法,包括:
a)任选地,从哺乳动物分离得到哺乳动物细胞;
b)使所述哺乳动物细胞接触本申请的调节组合物,接触持续至少10天,优选10至60天。
在一些实施方案中,接触维持10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60天、或以上。
在一些实施方案中,接触在30至40摄氏度进行,优选35、36、37、38摄氏度。
在本申请中,对维持细胞的条件没有特殊的限制,本领域中适于维持表皮细胞、肉芽组织的细胞、或血管内皮细胞的常规方法也适用于本申请的方法。
在一些实施方案中,所述调节是指选自以下的一项或其组合:促进表皮细胞的生长、促进表皮细胞的增殖、促进表皮细胞的迁移;促进成纤维细胞的生长、促进成纤维细胞的增殖、促进成纤维细胞的迁移;促进血管内皮细胞的生长、促进血管内皮细胞的增殖、促进血管内皮细胞的迁移。
在一些实施方案中,所述调节组合物提高表皮细胞中K19的表达。
在一些实施方案中,所述调节组合物提高肉芽组织中α2β1整合素的表达。
在一些实施方案中,所述调节组合物提高肉芽组织的细胞中Musashi1的表达。
在一些实施方案中,所述调节组合物提高血管内皮细胞中Musashi1的表达。
根据一些实施方案,还提供了一种细胞培养介质,其包含根据本申请的用于调节Musashi1表达水平的组合物。
根据一些实施方案,本申请的培养介质适用作体外细胞生长的培养介质、组织和/或器官的体外重建。
在一些实施方案中,细胞培养介质可选择地还包含各种氨基酸,例如18种天然氨基酸,从而为细胞生长提供营养支持。氨基酸可以是化学合成的,也可以是来自天然的。
在一些实施方案中,细胞培养介质可选择地还包含核苷酸或碱基,如腺嘌呤、胞啶、鸟嘌呤、胸腺嘧啶和尿苷。
在一些实施方案中,细胞培养介质可选择地还包含酶或细胞因子,从而支持细胞生长和维持所需的平衡。
根据一些实施方案,提供根据本申请的调节组合物用于构建Musashi1阳性细胞的用途。在一些实施方案中,所述细胞选自:表皮细胞、肉芽组织的细胞、或血管内皮细胞。
根据一些实施方案,提供根据本申请的调节组合物用于构建K19+、α2β1整合素+、Musashi1+三阳性细胞的用途。在一些实施方案中,所述细胞选自:表皮细胞、肉芽组织的细胞、或血管内皮细胞。
附图说明
图1A至图1D:HE染色结果(10×,患者样本No.1)。
图2A至图2D:HE染色结果(40×,患者样本No.1)。
图3:K19的表达水平(患者样本No.1)。
图4A至图4C:α2β1整合素的表达水平(患者样本No.1)。
图5A至图5B:HE染色结果(10×,患者样本No.2)。
图6A至图6B:HE染色结果(40×,患者样本No.2)。
图7A至图7B:K19的表达水平(患者样本No.2)。
图8A至图8B:α2β1整合素的表达(患者样本No.2)。
图9A至图9D:HE染色结果(10×,患者样本No.3)。
图10A至图10D:HE染色结果(40×,患者样本No.3)。
图11A至图11D:K19的表达水平(患者样本No.3)。
图12A至图12D:α2β1整合素的表达水平(患者样本No.3)。
图13A至图13C:Musashi 1免疫荧光染色结果。蓝色为DAPI(细胞核),红色为Musashi 1表达部位,分布在细胞核和细胞质(患者样本No.3)。
具体实施方式
实施例.调节组合物的制备
在植物油(如豆油、芝麻油和玉米油)中溶解2重量%-6重量%的甾醇、0.5重量%-1重量%的黄芩甙、3重量%-6重量%的蜂蜡、5重量%-10重量%的蜂胶、0.5重量%-1重量%的黄柏内酯、0.003重量%-0.1重量%的小蘖碱、0.003重量%-0.1重量%的黄连素、0.003重量%-0.1重量%的罂粟壳碱、和0.003重量%-0.1重量%的地龙。
将蜂蜡加热70-80℃时溶化;将溶化的蜂蜡与前述包含活性成分的植物油混合;逐渐冷却至环境温度(也就是20-25℃),而获得本申请的调节组合物。因为蜂蜡比油冷却得快,蜂蜡形成小“巢”样三维框架结构(其中包裹油滴)。巢的尺寸在5至50μm,例如10至30μm或15至20μm(“巢”样三维框架结构的检测方法可见于CN1827766A)。
测试例
1.样本收集:
收集Wagner分级为3级的糖尿病足患者(3例),来源于宁夏回族自治区人民医院烧伤科;3例患者均签订了科研知情同意书。
2.研究的分子:K19、α2β1整合素、Musashi1。
3.样本预处理:
使用皮肤病理取样钳,采集患处皮肤与溃疡交接处的组织标本。每次采集标本3份:
(1)1份用福尔马林固定制作成蜡块,进行形态学研究;
(2)2份液氮保存,用于分子生物学研究。
4.分组和处理方法:
(1)对照组:患者采用经典的治疗方法(清洗溃疡、切开、开放创面。用5%磺胺嘧啶锌软膏,涂抹创面,一天两次)。住院治疗时间30-60天。
(2)实验组:患者采用实施例中制备的调节组合物进行处理(清洗溃疡、切开、开放创面。然后使用本申请的调节组合物涂抹创面,一天两次)。住院治疗时间30-60天。
测试例1.HE染色结果
(1)患者No.1:
在实验组中,治疗后5、15、20天取皮肤组织,观察到表皮和少量真皮。表皮各层结构基本完整,真皮层观察到乳头层、结缔组织。表皮存在角化不全的表现。所示结果与表皮增殖的更新速度加快有关。
治疗后12天取肉芽组织,观察到大量成纤维细胞、炎性细胞和毛细血管(图1A至图1D)。与治疗后5天比较,在15天和20天时角化不全的现象减弱,表皮组织趋于成熟(图2A至图2D)。
(2)患者No.2:
治疗后40和55天取皮肤组织,观察到表皮和少量真皮。表皮各层结构基本完整,真皮观察到乳头层,结缔组织。表皮存在角化不全的表现,推测与表皮增殖更新速度加快有关。
治疗后40天、55天,颗粒层分化完全,推测表皮修复完成(图5A至图5B、图6A至图6B)。
(3)患者No.3:
治疗后5天、15天和20天取皮肤组织,观察到表皮和少量真皮。表皮各层结构基本完整,真皮观察到乳头层,结缔组织。表皮存在角化不全的表现,推测与表皮增殖更新速度加快有关(图9A至图9D)。
治疗后5天、15天时,表皮结构基本完整;治疗20天时,表皮未见角质层;治疗后10天时,取肉芽组织,观察到丰富成纤维细胞和毛细血管(图10A至图10D)。
测试例2.表皮细胞标记物K19的表达(免疫荧光法)
(1)患者No.1:治疗后12天和15天,K19的表达要高于治疗后5天和20天的水平(图3)。
(2)患者No.2:治疗后40天和55天时,K19的表达弱。在真皮乳头层,细胞外基质中见少量K19表达,表皮细胞没有K19表达(图7A至图7B)。
(3)患者No.3:治疗5天、10天和20天,K19的表达微弱,而治疗15天,观察到表皮基底层细胞有较强的K19表达(图11A至图11D)。
测试例3.表皮细胞标记物α2β1整合素的表达(免疫荧光法)
(1)患者No.1:治疗后12天,肉芽组织中见散在表达的α2β1整合素;治疗后15天和20天,表皮细胞中无α2β1整合素阳性表达。在20天时,真皮的细胞外基质中见微弱的α2β1整合素表达(图4A至4C)。
(2)患者No.2:治疗后40天、55天,可见表皮内基本无α2β1整合素的表达,真皮细胞外基质中有少量的表达(图8A至图8B)。
(3)患者No.3:治疗后5天、10天、15天、20天时,表皮细胞中基本不表达α2β1整合素,而肉芽组织(10天)中有散在的α2β1整合素表达(图12A至图12D)。
测试例4.Musashi1分子在肉芽组织和血管内皮中的表达情况(免疫荧光染色)
患者治疗10天时,肉芽组织中的Musashi1表达比较广泛(图13)。患者治疗15天时Musashi 1阳性细胞为肉芽组织中的细胞(未见明显的血管),Musashi 1表达广泛(图13B)。患者治疗30天时,Musashi1阳性细胞为血管内皮细胞、肉芽组织中的细胞,Musashi 1表达广泛且提高(图13C)。对照样本中,Musashi 1表达微弱。
综上,表皮细胞K19和α2β1整合素在损伤部位的表达存在差异。K19主要表达在表皮,在治疗后12-15天,K19的表达要高于施用本申请组合物之前的表达。α2β1整合素在表皮中基本没有表达,但在创面的肉芽组织中有表达,在治疗10-12天表达明显。在治疗后10-30天后,Musashi1在肉芽组织和血管内皮细胞中的表达提高。
Claims (6)
1.一种调节Musashi1在细胞中表达水平的方法,包括步骤:
a)任选地,从糖尿病患者分离得到皮肤组织的细胞;
b)使所述细胞接触调节组合物,接触持续至少10天;
所述细胞选自:肉芽组织的细胞、和血管内皮细胞;
所述调节组合物,按调节组合物总重量计,包含:
2重量%-6重量%的甾醇、
0.5重量%-1重量%的黄芩甙、
3重量%-6重量%的蜂蜡、
5重量%-10重量%的蜂胶、
0.5重量%-1重量%的黄柏内酯、
0.003重量%-0.1重量%的小蘖碱、
0.003重量%-0.1重量%的黄连素、
0.003重量%-0.1重量%的罂粟壳碱、
0.003重量%-0.1重量%的地龙、和
植物油或动物油;
所述植物油选自:玉米油、花生油、棉籽油、红花油、茶树油、芝麻油、橄榄油、豆油;
所述甾醇选自:豆甾醇、β-谷甾醇、角甾醇、γ-谷甾醇、菜籽甾醇、α-菠菜甾醇、24-去氢胆固醇、多孔甾醇、胡萝卜甙;
所述调节是指提高或促进;
所述方法在体外或离体进行。
2.根据权利要求1所述的方法,其中:
步骤b)中,使所述细胞接触调节组合物,接触10至60天。
3.根据权利要求1-2任一项所述的方法,其中:
所述甾醇是豆甾醇、β-谷甾醇、菜籽甾醇的组合。
4.根据权利要求1所述的方法,其中所述调节组合物能够实现选自以下的任一项或组合:
提高表皮细胞中K19的表达;
提高肉芽组织的细胞中α2β1整合素的表达;
提高肉芽组织的细胞中Musashi1的表达;
提高血管内皮细胞中Musashi1的表达。
5.组合物用于构建Musashi1阳性细胞的用途,其中:
所述细胞来自糖尿病患者分离得到的皮肤组织;
所述细胞选自:肉芽组织的细胞和血管内皮细胞;
所述组合物包含:
2重量%-6重量%的甾醇、
0.5重量%-1重量%的黄芩甙、
3重量%-6重量%的蜂蜡、
5重量%-10重量%的蜂胶、
0.5重量%-1重量%的黄柏内酯、
0.003重量%-0.1重量%的小蘖碱、
0.003重量%-0.1重量%的黄连素、
0.003重量%-0.1重量%的罂粟壳碱、
0.003重量%-0.1重量%的地龙、和
植物油或动物油;
所述植物油选自:玉米油、花生油、棉籽油、红花油、茶树油、芝麻油、橄榄油、豆油;
所述甾醇选自:豆甾醇、β-谷甾醇、角甾醇、γ-谷甾醇、菜籽甾醇、α-菠菜甾醇、24-去氢胆固醇、多孔甾醇、胡萝卜甙。
6.根据权利要求5所述的用途,所述甾醇是豆甾醇、β-谷甾醇、菜籽甾醇的组合。
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