Kruppel样因子4（KLF4）在小鼠表皮干细胞中的表达和功能研究
发布时间：2018-07-13 16:32
【摘要】：第一部分KLF4在小鼠表皮干细胞中的表达 目的：研究KLF4在小鼠表皮干细胞中的表达情况。 方法：使用干细胞特异性细胞表面标志CD34和CD49f,分离纯化小鼠毛囊隆突部干细胞(CD34+CD49f+细胞),通过实时定量RT-PCR方法检测CD34+CD49f+富集细胞中KLF4的表达水平。建立KLF4/EGFP小鼠模型,体内标记表达KLF4的细胞,通过流式细胞术检测表皮干细胞群中KLF4的表达情况,并通过标记滞留实验分析LRCs中有无KLF4的表达进一步验证KLF4在慢周期干细胞中的表达。建立KLF4/CreERTM小鼠模型,在KLF4/CreERTM/Rosa26RLacZ转基因小鼠中通过世系追踪实验检测KLF4在表皮干细胞以及其子代细胞中的表达情况。 结果：KLF4在CD34+CD49f+毛囊干细胞中的表达水平较分化CD34-CD49f+细胞高。CD34+CD49f+细胞中58.6%的细胞表达KLF4/EGFP, Sca-1+CD49f+细胞中73%的细胞可以检测到KLF4/EGFP信号。在纯化的小鼠表皮细胞中,14.6%的细胞能够滞留BrdU标记,在这些标记滞留细胞中,4.1%的细胞为KLF4/EGFP阳性。世系追踪实验显示,X-Gal染色后,在皮肤组织的毛囊隆突部,皮脂腺和毛囊间表皮中均能检测到蓝色LacZ+ (KLF4+)干细胞及其子代细胞。 结论：结合上述各种实验结果,我们证实KLF4在表皮干细胞中表达,表达KLF4的表皮干细胞具有标记滞留功能并能增殖分化。[关键词]KLF4；表皮干细胞；细胞表面标志；标记滞留；世系追踪 第二部分KLF4在小鼠表皮干细胞中的功能研究 目的：检测KLF4在小鼠表皮干细胞中的功能。 方法：建立他莫昔芬诱导的KLF4基因敲除小鼠模型,在成年小鼠腹腔内注射他莫昔芬诱导KLF4基因敲除。使用免疫组织化学染色方法检测KLF4敲除后小鼠皮肤组织的结构变化。分离纯化小鼠表皮细胞,通过流式细胞术检测并比较野生小鼠与基因敲除组中毛囊隆突部干细胞(CD34+CD49f+细胞)和IFE干细胞(Sca-1+CD49f+细胞)数量。通过体外克隆形成实验,比较野生小鼠与基因敲除组中表皮细胞体外克隆形成能力,并使用免疫荧光激活细胞分选术(FASC)分离两组表皮细胞中的CD34+CD49f+细胞,比较其体外克隆形成能力。 结果：与对照野生小鼠相比,KLF4敲除小鼠的表皮组织内毛囊显著增生,基底细胞从单层增殖为多层。KLF4+/+小鼠表皮细胞中CD34+CD49f+毛囊干细胞和Sca-1+CD49f+IFE干细胞的比例分别为12.8%和68.73%,而KLF4-/-小鼠表皮细胞中干细胞的比例分别降至6.26%和52.6%。体外克隆形成实验显示KLF4敲除后表皮细胞体外形成的克隆数目从22下降至9。此外,KLF4+/+小鼠表皮CD34+CD49f+细胞体外能形成33个克隆,但是在KLF4-/-小鼠中这一数目降至15。 结论：KLF4敲除后表皮干细胞的数目减少,其自我更新能力下降,因此KLF4在表皮干细胞的维持中发挥着重要的角色。 第三部分小鼠皮肤急性创伤愈合过程中表达KLF4的表皮干细胞的作用 目的：研究表达KLF4的表皮干细胞对小鼠皮肤急性创伤愈合的作用。 方法：利用KLF4基因敲除小鼠模型,在小鼠背部用活检穿孔器造成急性皮肤创伤,研究KLF4敲除是否影响小鼠皮肤急性创伤愈合过程；在KLF4/CreERTM/Rosa26RLacZ转基因小鼠中,使用他莫昔芬诱导LacZ表达从而标记表达KLF4的多潜能细胞,通过世系追踪实验在急性皮肤创伤愈合过程中研究KLF4+多潜能细胞是否参与皮肤愈合；同时建立体外KLF4稳定敲除的人正常上皮HacaT细胞系,通过划痕试验进一步研究KLF4对创伤愈合的影响以及潜在的分子机制。 结果：创伤愈合实验中,KLF4+/+小鼠背部伤口2天内开始愈合,10天之后创面几乎完全愈合。KLF4敲除使小鼠皮肤愈合过程明显推迟,第10天仍有40%的创伤面积未愈合。世系追踪试验表明,给予他莫昔芬诱导的KLF4/CreERTM/Rosa26RLacZ小鼠创伤刺激后,在伤口边缘能够检测到蓝色的KLF4+多潜能细胞及其子代细胞向创面迁移。长期世系追踪试验显示,他莫昔芬诱导八个月之后,小鼠完整的皮肤内较难观察到蓝色的长期存活KLF4+表皮干细胞,但是给予创伤刺激后,伤口边缘可以观察到激活的KLF4+表皮干细胞以及其子代细胞。此外,体外实验显示细胞划痕12小时后,对照组40%的划痕面积愈合,而KLF4敲除组仅愈合12%,并伴随着表皮生长因子受体(Epidermal growth factor receptor, EGFR)的表达及磷酸化水平下降。 结论：表达KLF4的表皮干细胞参与小鼠皮肤急性创伤愈合过程。在HacaT细胞系中敲除KLF4,上皮细胞迁移减慢并伴随EGFR表达下调。
[Abstract]:The expression of the first part KLF4 in mouse epidermal stem cells
Objective : To study the expression of KLF4 in mouse epidermal stem cells .
Methods : CD34 + CD49f + cells were isolated and purified using stem cell specific cell surface markers CD34 and CD49f . The expression of KLF4 was detected by real - time quantitative RT - PCR .
Results : The expression levels of KLF4 / EGFP and Sca - 1 + CD49f + cells in CD34 + CD49f + cells were higher than that in CD34 + CD49f + cells .
Conclusion : In combination with the above experimental results , we confirm that KLF4 is expressed in epidermal stem cells , and the epidermal stem cells expressing KLF4 have marker retention function and can proliferate and differentiate .
epidermal stem cells ;
Cell surface markers ;
Marker retention ;
descent tracking
Study on the function of the second part KLF4 in mouse epidermal stem cells
Objective : To study the function of KLF4 in mouse epidermal stem cells .
Methods : A mouse model of tamoxifen - induced KLF4 knockout mice was established .
Results : In KLF4 + / + mice , the percentage of CD34 + CD49f + hair follicle stem cells and Sca - 1 + CD49f + ife stem cells in KLF4 + / + mouse epidermal cells decreased to 6 . 26 % and 52 . 6 % , respectively .
Conclusion : KLF4 plays an important role in the maintenance of epidermal stem cells . KLF4 plays an important role in the maintenance of epidermal stem cells .
The role of KLF4 - expressing epidermal stem cells in the course of acute wound healing in the third part of mice
Objective : To study the effect of KLF4 - expressing epidermal stem cells ( KLF4 ) on acute wound healing in mice .
Methods : Using KLF4 knockout mice model , the acute skin trauma was induced by biopsy perforator on the back of mice .
In the KLF4 / CreERTM / Rosa26RAd transgenic mice , the multi - potential cells expressing KLF4 were identified by the use of tamoxifen - induced protein expression , and whether KLF4 + multi - potential cells were involved in skin healing were investigated in the course of acute skin wound healing .
At the same time , the human normal epithelial cells of human normal epithelial cells in vitro KLF4 were established , and the effects of KLF4 on wound healing and potential molecular mechanism were further investigated by scratch test .
Results : In the wound healing experiment , KLF4 + / + mice began to heal within 2 days after wound healing . After 10 days of wound healing , KLF4 + multi - potential cells and their progeny cells could not be observed .
Conclusion : The expression of KLF4 epidermal stem cells is involved in the healing process of acute wound healing in mice . KLF4 is knocked out in HHT T cell line , the migration of epithelial cells is slowed down and the expression of EGFR is downregulated .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329
本文编号：2120048
[Abstract]:The expression of the first part KLF4 in mouse epidermal stem cells
Objective : To study the expression of KLF4 in mouse epidermal stem cells .
Methods : CD34 + CD49f + cells were isolated and purified using stem cell specific cell surface markers CD34 and CD49f . The expression of KLF4 was detected by real - time quantitative RT - PCR .
Results : The expression levels of KLF4 / EGFP and Sca - 1 + CD49f + cells in CD34 + CD49f + cells were higher than that in CD34 + CD49f + cells .
Conclusion : In combination with the above experimental results , we confirm that KLF4 is expressed in epidermal stem cells , and the epidermal stem cells expressing KLF4 have marker retention function and can proliferate and differentiate .
epidermal stem cells ;
Cell surface markers ;
Marker retention ;
descent tracking
Study on the function of the second part KLF4 in mouse epidermal stem cells
Objective : To study the function of KLF4 in mouse epidermal stem cells .
Methods : A mouse model of tamoxifen - induced KLF4 knockout mice was established .
Results : In KLF4 + / + mice , the percentage of CD34 + CD49f + hair follicle stem cells and Sca - 1 + CD49f + ife stem cells in KLF4 + / + mouse epidermal cells decreased to 6 . 26 % and 52 . 6 % , respectively .
Conclusion : KLF4 plays an important role in the maintenance of epidermal stem cells . KLF4 plays an important role in the maintenance of epidermal stem cells .
The role of KLF4 - expressing epidermal stem cells in the course of acute wound healing in the third part of mice
Objective : To study the effect of KLF4 - expressing epidermal stem cells ( KLF4 ) on acute wound healing in mice .
Methods : Using KLF4 knockout mice model , the acute skin trauma was induced by biopsy perforator on the back of mice .
In the KLF4 / CreERTM / Rosa26RAd transgenic mice , the multi - potential cells expressing KLF4 were identified by the use of tamoxifen - induced protein expression , and whether KLF4 + multi - potential cells were involved in skin healing were investigated in the course of acute skin wound healing .
At the same time , the human normal epithelial cells of human normal epithelial cells in vitro KLF4 were established , and the effects of KLF4 on wound healing and potential molecular mechanism were further investigated by scratch test .
Results : In the wound healing experiment , KLF4 + / + mice began to heal within 2 days after wound healing . After 10 days of wound healing , KLF4 + multi - potential cells and their progeny cells could not be observed .
Conclusion : The expression of KLF4 epidermal stem cells is involved in the healing process of acute wound healing in mice . KLF4 is knocked out in HHT T cell line , the migration of epithelial cells is slowed down and the expression of EGFR is downregulated .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329
【共引文献】
相关期刊论文 前3条
1 石家仲;杨恬;雷明星;李进;邱伟明;连小华;;gasdermin3在小鼠同步化毛囊周期中的表达[J];第三军医大学学报;2011年05期
2 张志强;王玉杰;李佳;木拉提·热夏提;;大鼠毛囊干细胞的成骨及成脂诱导[J];医学研究生学报;2014年04期
3 张志强;王玉杰;木拉提·热夏提;李佳;;角质细胞无血清培养基促进大鼠毛囊干细胞的增殖[J];中国组织工程研究;2013年45期
相关硕士学位论文 前1条
1 赖向东;TNFR-1在小鼠毛囊周期中的表达及其作用初步探讨[D];重庆大学;2013年
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