CN112601810A - 美容方法 - Google Patents
美容方法 Download PDFInfo
- Publication number
- CN112601810A CN112601810A CN201980055864.4A CN201980055864A CN112601810A CN 112601810 A CN112601810 A CN 112601810A CN 201980055864 A CN201980055864 A CN 201980055864A CN 112601810 A CN112601810 A CN 112601810A
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- Prior art keywords
- skin
- cells
- stem cells
- sebaceous glands
- mechanical stimulation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
课题是提供使干细胞及其诱导细胞增殖的方法、以及由此解决皱纹、松弛等美容上的问题。通过对包含皮脂腺的皮肤施与机械刺激,可以使干细胞及其诱导细胞增殖。
Description
技术领域
本发明涉及用于使干细胞及其诱导细胞增加的方法、以及利用了该方法的美容方法。
背景技术
真皮主要由间质成分和细胞成分构成,进一步配置有血管、神经。真皮经常受温度变化、紫外线照射、物理刺激等外部因素、压力、年龄增长等内部因素的影响。由此,真皮层所包含的真皮成纤维细胞的细胞活性发生变化。真皮成纤维细胞由于产生与皮肤的弹力、弹性有关的间质成分,因此由于细胞活性降低,从而真皮层变薄、丧失弹力,引起皱纹、松弛,成为美容上的大问题。迄今为止,作为改善皱纹、松弛、弹性等的药剂,对抑制分解间质成分的酶例如乙酰肝素酶、基质金属蛋白酶等的活性的成分进行了研究(专利文献1:日本特开2016-169238号,专利文献2:日本特开2012-144499号)。此外,对于可以使真皮成纤维细胞的细胞活性增加的成分等进行了研究,开发出了各种真皮成纤维细胞活化剂(专利文献3:日本特开2006-262806号公报)。
现有技术文献
专利文献
专利文献1:日本特开2016-169238号公报
专利文献2:日本特开2012-144499号公报
专利文献3:日本特开2006-262806号公报
发明内容
发明所要解决的课题
通过本发明者们的研究可知,真皮成纤维细胞如果通过老化等而细胞活性降低,则难以恢复细胞活性。因此,可以认为与对于细胞活性已经降低了的真皮成纤维细胞增强细胞活性相比,使新的真皮成纤维细胞分化诱导对于解决皱纹、松弛等美容上的问题是有效的。
用于解决课题的方法
本发明者们锐意尝试了诱导从干细胞向真皮成纤维细胞的分化的手段,结果令人惊讶地发现通过对干细胞大量存在的皮脂腺周围施与机械刺激,从而干细胞的***被促进,诱导出新的真皮成纤维细胞。
因此,本发明涉及以下方案:
[1]一种使皮脂腺周围的干细胞或其诱导细胞增殖的方法,所述方法包括:在将从皮肤采取的包含皮脂腺的皮肤试样进行器官培养时,对皮肤试样施加机械刺激。
[2]根据项目1所述的方法,上述机械刺激为伸展刺激。
[3]一种美容方法,包括:对担心松弛、皱纹、弹性的皮肤施与机械刺激,由此使皮脂腺周围的干细胞或其诱导细胞增殖。
[4]根据项目3所述的美容方法,在上述皮肤中,皮脂腺密度为占皮肤区域的10~80%的密度。
[5]根据项目3或4所述的美容方法,上述机械刺激为对皮肤的伸展、推压、或吸引。
[6]一种美容设备,包含:
测定皮脂腺密度的测定部、和
机械刺激施加部,
所述美容设备对皮脂腺密度高的皮肤区域施与机械刺激,由此使皮脂腺周围的干细胞或其诱导细胞增殖。
附图说明
图1是将年轻对象和年老对象的真皮中的成纤维细胞用3维电子显微镜(SBF-SEM)进行观察并重建而显示的图。
图2是对年轻受检者(20多岁)的脸颊的皮肤、和年老受检者(80多岁)的脸颊的皮肤试样进行组织染色并拍摄而得的照片。
图3是在年老受检者(80多岁)的脸颊的皮肤试样中,在通常的真皮部(Majordermal layer)、与皮脂腺周围部(Around sebaceous gland)中,将干细胞染色并拍摄而得的照片。
图4中的图4A显示在对皮肤试样不施加伸展刺激而进行了器官培养的情况(control)、和施加伸展刺激而进行了器官培养的情况下,将CD54阳性的干细胞染色并拍摄而得的照片。图4B显示对皮肤试样不施加伸展刺激而进行了器官培养的情况、和施加伸展刺激而进行了器官培养的情况下,将胶原进行染色并拍摄而得的照片。
图5中的图5A显示将向脸颊施与伸展刺激前后的鼻唇沟的照片。图5B是显示通过伸展刺激而鼻唇沟的皱纹改善了的图。
图6显示添加以0.25×104细胞/ml、0.5×104细胞/ml、1.0×104细胞/ml、2.0×104细胞/ml、和4.0×104细胞/ml制备的细胞悬浮液2.5ml进行2天培养后的显微镜照片,并显示各浓度下的接触度。
图7是将各接触度下的I型胶原的表达量进行了比较的图。
图8显示由针对各基因的siRNA产生的基因表达的抑制效果。
图9显示通过针对各基因的siRNA而抑制了基因表达的情况下的I型胶原的表达量变化。
图10显示在培养真皮成纤维细胞中抑制了钙黏着蛋白2的基因表达的情况下的细胞增殖率(A)、和p21的基因表达(B)的变化。
图11显示在培养真皮成纤维细胞中抑制了钙黏着蛋白2的基因表达的情况下的形状变化。通过钙黏着蛋白2的基因表达抑制(Knockout,敲除),从而观察不到细胞彼此的粘接,在形状带圆形的同时,观察到作为年龄标志物的β-Gal的染色。
具体实施方式
本发明涉及使干细胞或其诱导细胞增殖的方法。该方法包括:在将从皮肤采取的包含皮脂腺的皮肤试样进行器官培养时,对皮肤试样施加机械刺激的工序。
在皮脂腺的周围存在能够向真皮成纤维细胞的分化的干细胞。该干细胞以CD54表达作为特征。在将包含皮脂腺的皮肤试样进行器官培养时,通过对皮肤试样施加机械刺激而进行培养,从而皮脂腺周围的干细胞增加。这样增加的细胞可以维持干细胞性,也可以是由干细胞分化成的诱导细胞。作为由干细胞分化成的诱导细胞,可举出真皮前体细胞、真皮成纤维细胞等。这样存在于真皮的干细胞由于能够向真皮成纤维细胞分化,因此也被称为真皮干细胞。
对皮肤试样、皮肤施加的机械刺激(mechanical stress)是指带来力学作用的物理刺激。机械刺激不限于通过探针、执行器等机械而施与的刺激,也可以通过带来力学作用的任意手段施与。作为机械刺激,作为一例,为推压、吸引、压缩、伸展之中的至少1种。可以沿相对于皮肤试样、皮肤的表面平行的方向、即横向施加机械刺激,也可以沿相对于表面垂直的方向、即纵向施加机械刺激。机械刺激的强度可以在皮肤试样、皮肤不被损伤、裂断或破坏的范围任意设定。作为一例,优选以在皮肤试样中产生10%~50%左右的变形的方式向皮肤试样施与压缩或伸展刺激。变形优选为20~40%,更优选发生30%左右的变形。
皮脂腺是产生皮脂的器官,跟随于毛囊而存在。皮脂腺分布在除手掌、脚掌以外的几乎全身。因此,本发明中的皮肤试样可以由皮肤的任意位置获得,特别也可以由皮脂腺发达的脂溢部位的皮肤获得。作为脂溢部位,可举出前额、鼻翼、鼻唇沟、有头发的头部、胸骨部、腋下、腹部、外***。在其它方案中,可以以表皮中所占的皮脂腺区域的比例,来选定应获得试样的位置。作为一例,可以在皮脂腺相对于皮肤区域占10~80%的皮肤中获得试样。皮脂腺区域优选为30~80%,更优选为50~80%。皮肤试样的大小可以任意选择,可以使用例如表面积为100mm2~10000mm2、并具有到达真皮层的深度的切片。从使侵袭性降低的观点考虑,优选为500mm2以下,更优选为300mm2以下。另一方面,从获得充分量的干细胞或其诱导细胞的观点考虑,优选为100mm2以上,进一步优选为500mm2以上。
皮肤试样的器官培养只要基于常规方法进行即可,可以通过例如Journal ofDermatological Science 74(2014)236-241所记载的方法进行。机械刺激、特别是伸展刺激可以在培养前施与,也可以在培养中施与。机械刺激的强度可以根据皮肤试样的大小等来适当选择。作为一例,伸展刺激通过将皮肤试样的一端、和另一端分别用镊子等固定并进行拉伸来施与。机械刺激可以在器官培养的全部期间或一部分期间施与。
在器官培养工序中被增殖了的干细胞或其诱导细胞可以通过将被培养的器官进行切断、酶处理,从而进行分离或分取。被分取的细胞也可以进一步供于培养,或不培养而直接注入到原来的对象的真皮层。
在其它方案中,本发明涉及经由了通过对皮肤施与机械刺激而使皮脂腺周围的干细胞或其诱导细胞增殖而进行的美容方法。在这样的美容方法中,皮脂腺周围的干细胞或其诱导细胞增殖,这些细胞分化成真皮前体细胞、真皮成纤维细胞。因此,根据本发明的美容方法,可以促进真皮中的真皮成纤维细胞的增加,由此能够促进间质成分的产生。因此,本发明的美容方法也可以称为间质成分产生促进方法,皮肤的皱纹、松弛、弹性的改善方法。
应用本发明的美容方法的对象可举出担心皱纹、松弛的对象,弹性减少了的对象。松弛可以通过使用视觉判定来确定。优选对在采用视觉判定的测量中为6以下、优选为5以下、进一步优选为4以下的对象应用本发明的美容方法,和/或对在使用了Cutometer(皮肤弹性测试仪)的测量中Ur/Uf为0.8以下、优选为0.7以下、进一步优选为0.6以下的对象应用本发明的美容方法。
关于对皮肤施加的机械刺激,作为一例,为推压、吸引、压缩、伸展之中的至少1种。对皮肤的机械刺激可以沿相对于皮肤表面平行的方向、即横向施加刺激,也可以沿相对于皮肤表面垂直的方向、即纵向施加刺激。机械刺激通常可以应用1分钟以上,更优选可以应用3分钟以上,进一步更优选应用5分钟以上。上限没有特别限定,但从确保方法的简便性的观点考虑,优选为1小时以内,更优选为30分钟以内,进一步更优选为15分钟以内。可以使用具备为了施与机械刺激而应用的探针、执行器等构件的设备,对皮肤施与机械刺激。作为这样的设备,作为一例,也可以使用日本特表2011-505897号所记载的执行器。进一步在一个方案中,本美容方法也能够包含面部的练习、按摩。作为面部的练习,可以进行使脸颊鼓起、使眼睛睁开等。作为面部的按摩,可举出使用了手、滚筒的按摩。
在本发明的美容方法中,可以促进皮脂腺周围的干细胞或其诱导细胞的增殖。因此,在机械刺激的应用前后,可以进一步包含观察皮脂腺周围的干细胞或其诱导细胞的工序。优选在机械刺激的应用后,经过24小时以上,优选为48小时以上之后,在免疫组织学上观察干细胞或其诱导细胞。
代替干细胞或其诱导细胞的观察,可以包含确认皱纹、松弛、弹性的改善的工序。关于皱纹、松弛的改善,可以使用公知的测量器(日本特开2017-064391号等)来测量。此外,关于皮肤的弹性,可以使用Derma转矩测量仪、Cutometer(皮肤弹性测试仪)来测量。
在本发明中,机械刺激优选向皮脂腺密度高的区域施加。向皮脂腺密度优选为200个/cm2以上,更优选为400个/cm2以上的区域施加机械刺激在使皮脂腺周围的干细胞或其诱导细胞增殖方面是优选的。
在本发明的进一步方案中,也涉及一种美容设备,包含:
皮脂腺检测部、和
机械刺激施加部,
该美容设备对皮脂腺密度高的皮肤区域施与机械刺激,由此使皮脂腺周围的干细胞或其诱导细胞增殖。
皮脂腺检测部例如为显微镜,可以检测皮脂腺的位置和数。通过对皮脂腺的密度高的区域,由机械刺激施加部施与机械刺激,从而可以促进皮脂腺周围的干细胞或其诱导细胞的增殖。
作为机械刺激,作为一例,为推压、吸引、伸展之中的至少1种。机械刺激施加部具备为了施与这些机械刺激而被应用的探针、执行器等构件。推压探针可以通过沿皮肤的垂直方向压入皮肤,从而向皮肤施与机械刺激。吸引探针可以通过与皮肤接触而附加负压,从而向皮肤施与机械刺激。伸展探针可以通过1个以上与皮肤接触的接触部沿相对于皮肤面水平的方向移动,从而对皮肤施与机械刺激。
真皮成纤维细胞是在真皮中存在的成纤维细胞,是产生真皮中的间质成分的细胞。因此,从干细胞诱导的真皮成纤维细胞有助于补充由于外部因素、内部因素而减少了的真皮的间质成分。实际上,在通过机械刺激而增大了的皮脂腺周围的干细胞的附近,观察到胶原产生增强(图4B)。
另一方面,通过本发明者们的研究发现,在老年人的真皮中存在的真皮成纤维细胞、与在年轻人的真皮中存在的真皮成纤维细胞形态不同,与迄今为止的认知不同,真皮成纤维细胞彼此形成了粘接(图1)。具体而言,年轻人的真皮成纤维细胞具有突起,与其它成纤维细胞粘接,与此相对,老年人的成纤维细胞为球状,突起少,与其它成纤维细胞的粘接少,或完全没有。对于老年人的真皮成纤维细胞,可以认为由于丧失细胞彼此的粘接,从而细胞活性、即间质成分的产生量降低。通过本发明者们的研究显示,根据真皮成纤维细胞的细胞间粘接,胶原的产生量变化(图7),进一步钙黏着蛋白2参与这样的细胞间粘接(图9),细胞间粘接有助于以胶原产生为代表的间质成分的生产能力、即细胞活性。虽然不意图在理论上进行限定,但可以认为通过本发明的机械刺激而增大了的皮脂腺周围的干细胞增殖并且分化成真皮成纤维细胞。可以认为在该情况下,由于在周围大量存在真皮成纤维细胞,因此细胞间粘接也多,由此细胞活性高的状态的真皮成纤维细胞在皮脂腺周围增加,由此胶原产生增加(图4B),此外实际上在体内皱纹改善了(图5)。
以往,在美容的目的下,特别是以皱纹、松弛、弹性的改善作为目的,进行了能够将真皮成纤维细胞活化的药剂的探索,以提取物为代表而选择出了各种成分。另一方面,在一旦失去突起而细胞粘接消失了的成纤维细胞中,可能难以达到再次粘接。因此,本发明者们想到不是将已经存在的真皮成纤维细胞活化,而是刺激作为真皮成纤维细胞的供应源而存在的干细胞,从而发现通过机械刺激能够增加皮脂腺周围的干细胞或其诱导细胞(图4)。由干细胞诱导出的新的真皮成纤维细胞具有突起,能够与其它成纤维细胞形成粘接。由干细胞诱导出的新的真皮成纤维细胞通过与其它成纤维细胞粘接,从而以胶原为代表的间质成分的生产能力优异(图7)。
真皮的间质成分主要由胶原纤维、弹性纤维、和基质构成。因此,通过真皮成纤维细胞的细胞活性增强,从而胶原纤维、弹性纤维、和基质之中的至少1种、优选为2种、进一步优选为全部成分的产生量增大。
胶原纤维由胶原构成。胶原分子根据α链的分子结构的不同,已知20种左右。在本发明中,胶原可以为任意的胶原,但优选为主要存在于真皮的I型胶原、III型胶原、V型胶原、IV型、VII型、17型胶原,更优选为I型胶原、III型胶原、V型胶原,最优选为在真皮中占约80%的I型胶原。胶原纤维的产生量能够通过测定胶原的产生量或表达量来确定。根据真皮成纤维细胞的粘接度而胶原的产生量变化。因此,即使对于老年人的皮肤,也因为由干细胞诱导出的新的成纤维细胞维持细胞间的粘接,因此以胶原产生量为代表而间质成分的产生量高。
弹性纤维的主成分为弹性蛋白,进一步原纤蛋白围绕弹性蛋白的周围而构成。弹性纤维的产生量能够通过测定弹性蛋白或原纤蛋白的至少1种的产生量或表达量来确定。
关于基质,主要作为细胞外基质,由糖蛋白、蛋白聚糖等而构成。作为糖蛋白,在包含糖的蛋白质中,在真皮中可举出纤连蛋白等,纤连蛋白与细胞表面蛋白质结合,作为细胞支架发挥功能,并且也能够与胶原等其它高分子结合。蛋白聚糖是糖胺聚糖与轴蛋白质结合而成的巨大分子,在真皮中,作为糖胺聚糖,主要包含透明质酸、硫酸皮肤素。蛋白聚糖主要发挥真皮中的水分保持的功能。
在本说明书中提及的全部文献其整体通过引用而并入到本说明书中。
以下说明的本发明的实施例仅以例示作为目的,不限定本发明的技术范围。本发明的技术范围仅通过权利要求书的记载来限定。以不超出本发明的宗旨作为条件,可以进行本发明的变更,例如本发明的构成要件的追加、删除和置换。
实施例
采用3维显微镜的观察
将年轻对象(20岁)和年老对象(80岁)的皮肤切片用导电性树脂进行处理,供于3维电子显微镜(SBF-SEM)观察。显示3维重建而得的图(图1)。采用SBF-SEM的样品制备和观察方法如下所述。
串行块面扫描电子显微镜(SBF-SEM)
将人皮肤样品在2%戊二醛和2%低聚甲醛的缓冲溶液中在4℃下进行了固定数天。将样品用2%四氧化锇(Nisshin EM,Tokyo,Japan)和1.5%亚铁***(Wako PureChemical Industries,Ltd.)的磷酸缓冲生理盐水在4℃下处理1小时,用1%硫代碳酰肼(Sigma,St.Louis,Mo,USA)在室温下处理20分钟,用2%四氧化锇水溶液在室温下处理30分钟,用2%乙酸双氧铀溶液在4℃下处理12小时以上,进而在室温下处理1小时。将样品在0.67%硝酸铅(pH5.0~5.5,TAAB,Berkshire,UK)、0.03ML-天冬氨酸(Nacalai Tesque,Kyoto,Japan)的溶液中在65℃下孵育30分钟。接下来将样品用阶段性乙醇系列脱水,用脱水丙酮进行处理,在35℃下使Quetol812环氧树脂(Nisshin EM,Tokyo,Japan)渗透,然后包埋于含有Kejen black powder的Quetol812(Nguyen et al.Sci Rep.2016)中。将树脂在70℃下孵育超过7夜,确实地进行聚合化。使用具备3View室内超薄切片机***(Gatan,Pleasanton,CA)的场发射扫描型电子显微镜(Merlin,Carl Zeiss,Oberkochen,Germany)进行SBF-SEM下的观察。以80~90μm×80~90μm宽度(11~12nm/像素),达到60μm的深度以80nm的步长获得连续图像序列。将连续图像使用FIJI(https://fiji.sc/)进行了处理。使用Amira(Maxnet Co.,Ltd,Tokyo,Japan)进行了分割和3维重建。
皮肤切片的观察
对从年轻的脸颊获得的皮肤切片、和从年老的脸颊获得的皮肤切片分别进行范吉逊(Van Gieson)染色,在显微镜下进行了观察(图2)。对于包含皮脂腺的区域,拍摄照片(图2)。对于年轻女性,染色强度高,显示在真皮层中胶原大量存在,另一方面,对于年老女性,真皮层中的染色强度低,显示胶原量少。另一方面,显示在皮脂腺的周围的区域胶原量多(白色箭头)。
对于从老年人的脸颊获得的皮肤切片,用丙酮固定,使抗CD54抗体反应,接着使用Emvisoin(DAKO)特定了干细胞。分成真皮整个区域、和皮脂腺周围而进行了拍摄。显示虽然在老年人的皮肤也存在干细胞,但在真皮整个区域其量极其少,另一方面,在皮脂腺周围存在大量干细胞(图3:图中的黑色虚线表示皮脂腺的边界线,箭头表示干细胞。)。
通过以上实验提示,对于老年人,存在于真皮的真皮成纤维细胞失去与其它细胞的粘接,与此相伴,细胞活性(胶原产生)降低。另一方面,即使是老年人的皮肤,也在皮脂腺周围大量存在干细胞,成为真皮成纤维细胞的供应源,有助于皮脂腺周围的增强了的胶原量。
器官培养实验
获得2个10mm见方的人的皮肤试样切片。对于一个,不施加压缩刺激而培养,对于另一个,以沿纵向变形30%左右的方式施加垂直的力,同时浸渍在包含10%FBS的DMEM培养基中,在5%CO2、37℃气氛下培养7天。
将培养后的皮肤试样切片用丙酮固定,使用抗CD54抗体,将干细胞使用Emvisoin(DAKO)进行可视化。将结果示于图4A中。在施与了压缩刺激的皮肤试样中,观察到干细胞、或由干细胞诱导出的细胞增殖了。进一步,将培养后的皮肤试样切片用丙酮固定,使抗I型胶原抗体反应而使用Emvisoin(DAKO)进行可视化。将结果示于图4B中。显示出在施加了压缩刺激的皮肤试样中,在皮脂腺周围胶原产生提高了。
机械刺激的效果
在8名女性受检者(40多岁)中,1天1次进行使脸颊鼓起的运动10分钟,向脸颊施与伸展刺激。进行2个月实验,在实验前、和1个月的实验后,拍照片(图5A),视觉评价了脸颊的松弛的程度(图5B)。在实验的前后,观察到了改善。
由以上实验,显示出如果对包含皮脂腺的皮肤施与伸展刺激、压缩刺激等机械刺激,则干细胞或来自干细胞的诱导细胞增殖,实际上即使在体内也显示出伸展刺激有助于皱纹的改善。
培养实验
从人的皮肤试样按照常规方法获得人初代培养成纤维细胞。测量细胞的数,在(包含10%FBS)DMEM培养基中,制备成相对于每1ml为0.25×104细胞(无接触)、0.5×104细胞(轻度接触)、1.0×104细胞(中度接触)、2.0×104细胞(高度接触)、和4.0×104细胞(过度接触)。将这样的细胞悬浮液2.5ml滴加到6孔板(フアルコン社制)中,在37℃5%CO2气氛下培养2天。将培养后的细胞的显微镜照片示于图6中。
伴随粘接的程度的不同的基因表达的变化
从接触度不同的培养物回收细胞,供于微阵列解析,结果发现根据细胞接触度,I型胶原的表达变化(数据未刊登)。因此,通过使用了下述引物的实时PCR,确定了I型胶原的表达量(图7)。表达量的确定使用了GAPDH基因的表达作为内标。
[表1]
引物 | 序列 |
Col1A1正向引物 | AGCAGGCAAACCTGGTGAAC(序列号1) |
Col1A1反向引物 | AACCTCTCTCGCCTCTTGCT(序列号2) |
GAPDH正向引物 | GAAGGTGAAGGTCGGAGT(序列号5) |
GAPDH反向引物 | GAAGATGGTGATGGGATTTC(序列号6) |
通过以上实验显示,通过粘接度的变化,作为间质成分之一的胶原的表达根据细胞间粘接而变化。
参与粘接、影响胶原表达的粘接因子的鉴定
以确定有助于I型胶原的表达的因子作为目的,在培养成纤维细胞中,通过siRNA法抑制了细胞粘接蛋白质的表达。由キアゲン社获得了CDH2、CDH11、和CDH13的siRNA。将这些siRNA按照常规方法使用而在培养真皮成纤维细胞中抑制了各基因的表达。将1×104细胞/ml的密度的细胞悬浮液0.5ml接种于24孔板(面积:2cm2)的各孔,在37℃5%CO2气氛下培养2天。采取培养成纤维细胞,将各细胞粘接蛋白质的基因表达通过实时PCR确定,确认了目的蛋白质的表达抑制(图8)。接下来,在采取的细胞中,测定了I型胶原的表达,结果显示,在抑制了CDH2的基因表达时,I型胶原的表达量降低(图9)。
对于抑制了钙黏着蛋白2表达的真皮成纤维细胞、和对照的真皮成纤维细胞,分别将1×104细胞/ml的密度的细胞悬浮液0.5ml接种于24孔板(面积:2cm2)的各孔,在37℃5%CO2气氛下培养2天。计数培养细胞,对细胞的增殖性进行了比较(图10A)。此外,对于各培养细胞,将作为CDK家族的抑制蛋白质的p21的基因表达使用下述引物通过RT-PCR进行了测定(图10B)。进一步,使用细胞老化检测试剂盒(Biovision社),通过X-Gal根据β半乳糖苷酶活性将细胞进行染色,在显微镜下拍摄(图11)。在对照细胞中,培养真皮成纤维细胞为扁平的形状,观察到细胞彼此的粘接,但通过钙黏着蛋白2的基因表达的抑制(Knockout,敲除),细胞彼此的粘接减少,此外与此相伴细胞形状与对照相比显示球形。此外,在抑制了钙黏着蛋白2的基因表达的真皮成纤维细胞中,成为细胞老化的指标的细胞内β半乳糖苷酶活性高。
[表2]
引物 | 序列 |
p21正向引物 | AGCAGCTGCCGAAGTCAGTTCCT(序列号3) |
p21反向引物 | GTTCTGACATGGCGCCTCCTCTGA(序列号4) |
GAPDH正向引物 | GAAGGTGAAGGTCGGAGT(序列号5) |
GAPDH反向引物 | GAAGATGGTGATGGGATTTC(序列号6) |
在抑制了钙黏着蛋白2的基因表达的皮成纤维细胞中,胶原基因的表达和细胞增殖都被抑制了。此外,在抑制了钙黏着蛋白2的基因表达的真皮成纤维细胞中,显示通过细胞周期蛋白的抑制而引起细胞周期的停止的p21的基因表达增加,进一步也显示作为细胞的老化的指标的细胞内β半乳糖苷酶活性高。由以上结果显示,经由作为细胞粘接蛋白质的钙黏着蛋白2,真皮成纤维细胞形成细胞间的接触,由于失去这样的接触,从而细胞老化,细胞活性降低。
Claims (6)
1.一种使皮脂腺周围的干细胞或其诱导细胞增殖的方法,所述方法包括:在将从皮肤采取的包含皮脂腺的皮肤试样进行器官培养时,对皮肤试样施加机械刺激。
2.根据权利要求1所述的方法,所述机械刺激为伸展刺激。
3.一种美容方法,包括:对担心松弛、皱纹、弹性的皮肤施与机械刺激,由此使皮脂腺周围的干细胞或其诱导细胞增殖。
4.根据权利要求3所述的美容方法,在所述皮肤中,皮脂腺密度为10~80%。
5.根据权利要求3或4所述的美容方法,所述机械刺激为对皮肤的伸展、推压或吸引。
6.一种美容设备,包含:
测定皮脂腺密度的测定部、和
机械刺激施加部,
所述美容设备对皮脂腺密度高的皮肤区域施与机械刺激,由此使皮脂腺周围的干细胞或其诱导细胞增殖。
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