转录因子组合诱导成纤维细胞直接重编程为汗腺样细胞的实验研究
发布时间：2018-08-11 11:04
【摘要】：目的：前期,我们团队已经确证了建立一定的共培养条件可以诱导间充质干细胞转分化为汗腺样细胞,并经裸鼠的脚掌创面模型试验和初步的人体试验证实,经过诱导的自体骨髓间充质干细胞移植于切除瘢痕的创面,可以生长出汗腺样结构并且具有一定的发汗功能。本研究的主要目的是在前期工作的基础上,通过对成纤维细胞重编程进一步识别控制其分化为汗腺样细胞的关键重编程转录因子(NF-κB和Lef-1)及其介导的信号调控通路,进一步阐明成纤维细胞可能分化为汗腺样细胞的重编程机制,为利用其它多种类型细胞作为汗腺再生种子细胞,为临床大面积汗腺损伤的患者实现汗腺再生带来希望。方法：前期研究表明,调控成纤维细胞重编程为汗腺样细胞关键信号通路涉及NF-KB和Lef-1等,结合文献报道的参与调控胚胎发育过程汗腺细胞发生、发展过程蛋白的变化,寻找出主要的参与调控关键转录因子NF-κB和Lef-1的基因组合,分析其参与的信号通路网络,并进一步验证其生物功能。1.实验分组与操作程序：待细胞培养至对数生长期,生长密度达到80%以上时,按106个/孔的密度将细胞接种于6孔板后采用完全随机(完全随机设计也叫组间设计,被试被分成若干组,每组分别接受一种实验处理,有几种实验处理被试也相应的被分为几组,各实验组的被试之间相互独立,因而又叫“独立组”设计)将其分为3组(每组的样本量为5),①目的基因转染组：将所构建的含有NF-κB和Lef-1基因的真核表达载体转染入细胞并筛选出稳定表达的单克隆,待后续实验使用；②空载体组：将pcDNA3.1(+)空载体转染细胞并筛选出稳定表达的单克隆,待后续实验使用;③空白对照组：不转染目的基因,相同实验条件下培养。2. NF-κB和Lef-1基因对人成纤维细胞的重编程：2.1真核表达载体的构建：在美国国立生物技术信息中心(National Center of Biotechnology Information. NCB1)上查找实验所需人NF-κB和Lef-1的基因序列,根据所查序列和实验需要分别设计NF-κB和Lef-1基因开放阅读框(open reading frame, ORF)PCR引物,用总RNA提取试剂盒提取人成纤维细胞总RNA,进行逆转录酶的作用合成目的cDNA,将cDNA和pcDNA3.1(+)载体分别用HindⅢ/SalⅠ及HindⅢ/PstⅠ双酶切并连接转化大肠杆菌DH-5a扩增,为了验证NF-κB和Lef-1目的基因序列是否有突变,将含有重组子的菌液送华大基因测序,选取无突变的单克隆菌液保种,经测序正确后提取质粒,并检测所提取质粒浓度。2.2转染细胞并筛选稳定表达细胞系：将所培养的细胞以5000个/孔的密度接种于96孔板,用不同浓度的G418培养基培养细胞,以10～14 d杀死全部细胞的浓度为基本浓度,并以此确定筛选浓度和维持浓度。转染前一天按106个/孔的密度将所培养细胞接种于6孔板,按脂质体转染的标准程序转染细胞,转染48 h后用含G418的培养基按800 mg/L的浓度筛选转染成功的细胞,2周后以200 mg/L的浓度维持筛选压力,挑选并在96孔板中培养成阳性单克隆,扩大培养将稳定转染的细胞用于后续实验。利用Realtime-PCR检测3组细胞NF-κB和Lef-1的mRNA表达情况。3. NF-κB和Lef-1重编程人成纤维细胞诱导培养后变化：EDA-A1能促进正常和外胚层发育缺陷小鼠的汗腺、毛囊等外胚层组织器官的发育,EGF能提高MSCs分化为SGLCs的诱导率。因此,本实验采用1μg/ml EDA-A1、50 ng/ml EGF加入培养基诱导重编程后的人成纤维细胞分化为SGLCs.3.1汗腺标志物的检测：应用免疫荧光法鉴定NF-κB和Lef-1重编程人成纤维细胞的汗腺相关分子如CEA,CK7,CK14和CK19等的表达；应用Western blot技术从蛋白层面检测NF-κB和Lef-1重编程人成纤维细胞的汗腺相关分子CEA,CK7,CK14和CK19等的表达变化；应用Realtime-PCR定量检测NF-κB和Lef-1重编程人成纤维细胞的汗腺相关分子CEA,CK7,CK14和CK19等的表达变化。3.2 NF-κB和Lef-1通路下游基因的检测：应用Realtime-PCR定量检测NF-κB和Lef-1重编程人成纤维细胞的NF-κB和Lef-1通路关键下游基因Shh和Cyclin D1的表达变化.3.3裸鼠脚掌移植实验：应用前期实验所建立的裸鼠模型探究重编程汗腺样细胞的功能以及汗腺再生修复作用。将转染了pcDNA3.1(+)-NF-KB和pcDNA3.1(+)-Lef-1的实验组成纤维细胞和另外两组对照组的成纤维细胞以1×106个细胞制成的150μl混悬液用注射器植入裸鼠烫伤后的脚掌,20天后进行组织学检查和发汗实验观察重编程汗腺样细胞的生物学功能,并进行安全性评价。结果：1. NF-κB和Lef-1真核表达载体构建：NF-κB和Lef-1基因的基因开放阅读框(open reading frame, ORF)克隆进pcDNA3.1(+)载体,经Hindlll/SalⅠ及Hindlll/PstⅠ双酶切后琼脂糖电泳分别可见相对分子质量约为3000 bp和1200 bp的条带,经测序鉴定碱基对无突变,表明成功构建了pcDNA3.1(+)-NF-κB和pcDNA3.1(+)-Lef-1真核表达载体。2.G418筛选及NF-κB和Lef-1表达检测：G418培养液递增筛选2周后未转染成功的细胞全部死亡,维持筛选20 d后形成阳性克隆。Realtime-PCR结果显示,目的基因转染组、空载体组、空白对照组的NF-κB mRNA的相对表达量分别为3.651±0.062、0.987±0.098、1.118±0.024,Lef-1 mRNA的相对表达量分别为2.451±0.032、0.997±0.078、1.158±0.043,表明稳定转染的人成纤维细胞的NF-κB和Lef-1 mRNA的表达也显著增加,与空白对照组相比差异有统计学意义(P0.01),与空载体组相比差异也有统计学意义(P0.01),可认为重组载体在细胞内已有较高转录水平。3.汗腺标志物的检测：Western-blot结果显示,pcDNA3.1(+)-NF-KB和pcDNA3.1(+)-Lef-1转染组中CEA,CK7,CK14和CK19蛋白均有明显地表达,而空载体组、空白对照组中均未看见有表达。Realtime-PCR结果显示,pcDNA3.1(+)-NF-κB和pcDNA3.1(+)-Lef-1转染组中CEA,CK7,CK14和CK19 mRNA均有明显地表达,而空载体组、空白对照组中均未看见有表达。免疫荧光染色结果显示,pcDNA3.1(+)-NF-KB和pcDNA3.1(+)-Lef-1转染组中CEA,CK7,CK14和CK19荧光亮度很强,而其他两个对照组相同蛋白的荧光基本上看不见。4. NF-κB和Lef-1通路下游基因的检测：Realtime-PCR结果显示,目的基因转染组、空载体组、空白对照组的ShhmRNA的相对表达量分别为3.151±0.052、0.997±0.038、1.098±0.074,Cyclin D1mRNA的相对表达量分别为2.653±0.045、0.997±0.048、1.118±0.053,表明稳定转染的人成纤维细胞的Shh和Cyclin D1 mRNA的表达也显著增加,与空白对照组相比差异有统计学意义(P0.01),与空载体组相比差异也有统计学意义(P0.01),可认为被公认参加汗腺再生过程中很重要的Shh和Cyclin D1在细胞内已有较高转录水平。5.裸鼠脚掌移植实验：20天后进行的碘淀粉发汗实验结果显示,大约7/10的实验组脚掌为阳性结果,在脚掌中间可以明显的看到蓝黑色的区域,而两组对照则没有看到相应的阳性结果。组织学检查发现,目的基因转染组的裸鼠脚掌HE染色切片中可以看到有汗腺的再生,基底层汗腺导管相连接,而对照组则没有发现汗腺结构的再生。冰冻切片中可看见ck7和ck19的红色荧光。移植瘤实验证明了汗腺样细胞的安全性.结论：NF-κB和Lef-1的基因组合可以直接重编程人成纤维细胞分化成汗腺样细胞,在诱导成汗腺样细胞的过程中汗腺特异性标志物CEA,CK7,CK14和CK19的表达有明显的表达,并且通过检测通路下游基因Shh和Cyclin D1证实了NF-κB和Lef-1这两条通路在直接重编程过程中的重要作用.直接重编程后的人成纤维细胞在裸鼠模型中得到了生物学功能的验证,为利用其它多种类型细胞进行汗腺样细胞重编程、实现汗腺再生奠定理论基础,为临床大面积汗腺损伤的患者实现汗腺再生带来希望.
[Abstract]:OBJECTIVE: Previously, our team has confirmed that the establishment of certain co-culture conditions can induce mesenchymal stem cells to transdifferentiate into sweat gland-like cells, and through the paw wound model test in nude mice and preliminary human experiments, it has been proved that the induced autologous bone marrow mesenchymal stem cells transplanted into scar-resected wounds can produce sweat glands. The main purpose of this study is to further identify the key reprogramming transcription factors (NF-kappa B and Lef-1) controlling the differentiation of fibroblasts into sweat-like cells and their mediated signaling pathways by reprogramming fibroblasts. The reprogramming mechanism of apocrine sweat-like cells provides hope for the use of other types of cells as seed cells for sweat gland regeneration and for the realization of sweat gland regeneration in patients with extensive clinical sweat gland injury. METHODS: Previous studies have shown that the key signaling pathways involved in regulating fibroblast reprogramming into sweat-like cells involve NF-KB and Lef-1, and so on. Documented involvement in regulating the development of sweat gland cells during embryonic development, changes in proteins in the development process, to identify the major transcription factors involved in the regulation of NF-kappa B and Lef-1 gene combinations, analysis of its involved signal pathway network, and further verify its biological function. 1. Experimental grouping and operating procedures: cells to be cultured to logarithmic. At the growth stage, when the growth density is above 80%, the cells are inoculated into 6-well plates according to the density of 106 holes. The cells are randomly divided into several groups. Independent, so called "independent group" design) will be divided into three groups (each group sample size is 5). 1) target gene transfection group: the construction of eukaryotic expression vector containing NF-kappa B and Lef-1 gene into cells and screened out stable expression of monoclonal, for subsequent experimental use; 2 empty vector group: pcDNA3.1 (+) empty vector transfection fine. Blank control group: no transfection of the target gene, cultured under the same experimental conditions. 2. Reprogramming of human fibroblasts with NF-kappa B and Lef-1 genes: Construction of eukaryotic expression vector 2.1: National Center of Biotechnology I Find the gene sequences of human NF-kappa B and Lef-1 on nformation.NCB1, design the open reading frame (ORF) PCR primers of NF-kappa B and Lef-1 genes according to the sequences and the experimental needs, extract the total RNA of human fibroblasts with the total RNA extraction kit, synthesize the target cDNA by the action of reverse transcriptase, and synthesize the cDNA and pcDN. A3.1 (+) vectors were digested with Hind III/Sal I and Hind III/Pst I and linked with DH-5a respectively. In order to verify the mutation of the target gene sequences of NF-kappa B and Lef-1, the recombinant bacterial fluid was sent to Huada gene for sequencing, the mutant-free monoclonal bacterial fluid was selected for preservation, and the plasmid was extracted correctly after sequencing. Plasmid concentration 2.2 was used to transfect the cells and select stable expression cell lines. The cultured cells were inoculated on 96-well plate with 5000 cells per hole density. The cells were cultured on G418 medium of different concentrations. The concentration of killing all the cells from 10 to 14 days was used as the basic concentration to determine the screening concentration and maintain the concentration. The cultured cells were inoculated into 6-well plate and transfected according to the standard procedure of liposome transfection. After 48 hours of transfection, the transfected cells were screened by the medium containing G418 at 800 mg/L concentration. After 2 weeks, the screening pressure was maintained at 200 mg/L concentration. Positive monoclones were selected and cultured in 96-well plate. The stable transfected cells were expanded to culture. Realtime-PCR was used to detect the mRNA expression of NF-kappa B and Lef-1 in three groups of cells. Therefore, we used 1 ug/ml EDA-A1, 50 ng/ml EGF to induce reprogrammed human fibroblasts to differentiate into SGLCs.3.1 sweat gland markers. The expression of sweat gland-related molecules such as CEA, CK7, CK14 and CK19 in NF-kappa B and Lef-1 reprogrammed human fibroblasts was identified by immunofluorescence assay. The expression of sweat gland-related molecules CEA, CK7, CK14 and CK19 in human fibroblasts reprogrammed with NF-kappa B and Lef-1 were detected at protein level, and the expression of sweat gland-related molecules CEA, CK7, CK14 and CK19 in human fibroblasts reprogrammed with NF-kappa B and Lef-1 were quantitatively detected by Realtime-PCR. Realtime-PCR was used to quantitatively detect the expression of Shh and Cyclin D 1, the key downstream genes of NF-kappa B and Lef-1 pathways in human fibroblasts reprogrammed with NF-kappa B and Lef-1. 3.3 Nude mouse paw transplantation experiment: The function of reprogrammed sweat-like cells and the regeneration and repair of sweat glands were explored by using a nude mouse model established in previous experiments. Fibroblasts made up of pcDNA3.1 (+) - NF-KB and pcDNA3.1 (+) - Lef-1 and fibroblasts from the other two groups of control group were implanted into the paws of scalded nude mice with a 150 ml suspension made of 1 *106 cells by injector. The biological functions of reprogrammed sweat-like cells were observed by histological examination and sweat test 20 days later. Results: 1. Construction of eukaryotic expression vectors of NF-kappa B and Lef-1: The open reading frame (ORF) of NF-kappa B and Lef-1 genes were cloned into pcDNA3.1 (+) vector. After digestion by Hindlll/Sal I and Hindlll/Pst I, the bands with relative molecular weight of 3000 BP and 1200 BP were detected by agarose electrophoresis. Eukaryotic expression vectors pcDNA3.1 (+) - NF-kappa B and pcDNA3.1 (+) - Lef-1 were successfully constructed. 2. G418 screening and detection of NF-kappa B and Lef-1 expression showed that all the non-transfected cells died after 2 weeks of incremental screening in G418 medium, and positive clones were formed after 20 days of screening. The relative expression levels of NF-kappa B mRNA in blank vector group and blank control group were 3.651+0.062,0.987+0.098,1.118+0.024,2.451+0.032,0.997+0.078,1.158+0.043 respectively,indicating that the expression of NF-kappa B and Lef-1 mRNA in stably transfected human fibroblasts also increased significantly compared with blank control group. There was significant difference between the two groups (P 0.01), and there was also significant difference between the two groups (P 0.01). The recombinant vector had a higher transcription level in the cells. 3. Sweat gland markers detection: Western blot results showed that the expression of CEA, CK7, CK14 and CK19 proteins in pcDNA3.1 (+) -NF-KB and pcDNA3.1 (+) -Lef-1 transfection group were significantly higher than that in the empty vector group. The expression of CEA, CK7, CK14 and CK19 mRNA in pcDNA3.1 (+) -NF-kappa B and pcDNA3.1 (+) -Lef-1 transfection groups were significantly higher than that in blank control group and blank control group. Immunofluorescence staining showed that pcDNA3.1 (+) -NF-KB and pcDNA3.1 (+) -Lef-1 were transfected. The fluorescence intensity of CEA, CK7, CK14 and CK19 was very strong in the infection group, but the fluorescence intensity of the same protein in the other two control groups was basically invisible. The relative expression of Cyclin D1 mRNA was 2.653+0.045, 0.997+0.048, 1.118+0.053 respectively, indicating that the expression of SHH and Cyclin D1 mRNA in stably transfected human fibroblasts was also significantly increased compared with the blank control group (P 0.01), and the difference was also statistically significant compared with the empty carrier group (P 0.01). Shh and Cyclin D1, which are considered to be important in the process of sweat gland regeneration, have higher transcription levels in cells. 5. Nude mice plantar transplantation test: iodine starch sweating test 20 days later showed that about 7/10 of the experimental group's plantar was positive, and blue-black areas were visible in the middle of the plantar, whereas the two control groups showed that No corresponding positive results were found. Histological examination showed that the regeneration of sweat glands and the connection of sweat gland ducts in basal layer were seen in HE stained sections of the soles of the nude mice in the target gene transfection group, while no regeneration of sweat gland structures was found in the control group. Conclusion: The gene combination of NF-kappa B and Lef-1 can directly reprogram human fibroblasts to differentiate into sweat-like cells, and the expression of sweat-gland specific markers CEA, CK7, CK14 and CK19 in the process of inducing sweat-like cells is obvious, and the downstream genes Shh and Cyclin D1 confirm the expression of NF-kappa B and Lef-1. These two pathways, kappa B and Lef-1, play an important role in the process of direct reprogramming. The reprogrammed human fibroblasts have been validated in a nude mouse model, which lays a theoretical foundation for the reprogramming of sweat gland-like cells with other types of cells and the regeneration of sweat gland. People bring hope to the regeneration of sweat glands.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R329.2
本文编号：2176816
[Abstract]:OBJECTIVE: Previously, our team has confirmed that the establishment of certain co-culture conditions can induce mesenchymal stem cells to transdifferentiate into sweat gland-like cells, and through the paw wound model test in nude mice and preliminary human experiments, it has been proved that the induced autologous bone marrow mesenchymal stem cells transplanted into scar-resected wounds can produce sweat glands. The main purpose of this study is to further identify the key reprogramming transcription factors (NF-kappa B and Lef-1) controlling the differentiation of fibroblasts into sweat-like cells and their mediated signaling pathways by reprogramming fibroblasts. The reprogramming mechanism of apocrine sweat-like cells provides hope for the use of other types of cells as seed cells for sweat gland regeneration and for the realization of sweat gland regeneration in patients with extensive clinical sweat gland injury. METHODS: Previous studies have shown that the key signaling pathways involved in regulating fibroblast reprogramming into sweat-like cells involve NF-KB and Lef-1, and so on. Documented involvement in regulating the development of sweat gland cells during embryonic development, changes in proteins in the development process, to identify the major transcription factors involved in the regulation of NF-kappa B and Lef-1 gene combinations, analysis of its involved signal pathway network, and further verify its biological function. 1. Experimental grouping and operating procedures: cells to be cultured to logarithmic. At the growth stage, when the growth density is above 80%, the cells are inoculated into 6-well plates according to the density of 106 holes. The cells are randomly divided into several groups. Independent, so called "independent group" design) will be divided into three groups (each group sample size is 5). 1) target gene transfection group: the construction of eukaryotic expression vector containing NF-kappa B and Lef-1 gene into cells and screened out stable expression of monoclonal, for subsequent experimental use; 2 empty vector group: pcDNA3.1 (+) empty vector transfection fine. Blank control group: no transfection of the target gene, cultured under the same experimental conditions. 2. Reprogramming of human fibroblasts with NF-kappa B and Lef-1 genes: Construction of eukaryotic expression vector 2.1: National Center of Biotechnology I Find the gene sequences of human NF-kappa B and Lef-1 on nformation.NCB1, design the open reading frame (ORF) PCR primers of NF-kappa B and Lef-1 genes according to the sequences and the experimental needs, extract the total RNA of human fibroblasts with the total RNA extraction kit, synthesize the target cDNA by the action of reverse transcriptase, and synthesize the cDNA and pcDN. A3.1 (+) vectors were digested with Hind III/Sal I and Hind III/Pst I and linked with DH-5a respectively. In order to verify the mutation of the target gene sequences of NF-kappa B and Lef-1, the recombinant bacterial fluid was sent to Huada gene for sequencing, the mutant-free monoclonal bacterial fluid was selected for preservation, and the plasmid was extracted correctly after sequencing. Plasmid concentration 2.2 was used to transfect the cells and select stable expression cell lines. The cultured cells were inoculated on 96-well plate with 5000 cells per hole density. The cells were cultured on G418 medium of different concentrations. The concentration of killing all the cells from 10 to 14 days was used as the basic concentration to determine the screening concentration and maintain the concentration. The cultured cells were inoculated into 6-well plate and transfected according to the standard procedure of liposome transfection. After 48 hours of transfection, the transfected cells were screened by the medium containing G418 at 800 mg/L concentration. After 2 weeks, the screening pressure was maintained at 200 mg/L concentration. Positive monoclones were selected and cultured in 96-well plate. The stable transfected cells were expanded to culture. Realtime-PCR was used to detect the mRNA expression of NF-kappa B and Lef-1 in three groups of cells. Therefore, we used 1 ug/ml EDA-A1, 50 ng/ml EGF to induce reprogrammed human fibroblasts to differentiate into SGLCs.3.1 sweat gland markers. The expression of sweat gland-related molecules such as CEA, CK7, CK14 and CK19 in NF-kappa B and Lef-1 reprogrammed human fibroblasts was identified by immunofluorescence assay. The expression of sweat gland-related molecules CEA, CK7, CK14 and CK19 in human fibroblasts reprogrammed with NF-kappa B and Lef-1 were detected at protein level, and the expression of sweat gland-related molecules CEA, CK7, CK14 and CK19 in human fibroblasts reprogrammed with NF-kappa B and Lef-1 were quantitatively detected by Realtime-PCR. Realtime-PCR was used to quantitatively detect the expression of Shh and Cyclin D 1, the key downstream genes of NF-kappa B and Lef-1 pathways in human fibroblasts reprogrammed with NF-kappa B and Lef-1. 3.3 Nude mouse paw transplantation experiment: The function of reprogrammed sweat-like cells and the regeneration and repair of sweat glands were explored by using a nude mouse model established in previous experiments. Fibroblasts made up of pcDNA3.1 (+) - NF-KB and pcDNA3.1 (+) - Lef-1 and fibroblasts from the other two groups of control group were implanted into the paws of scalded nude mice with a 150 ml suspension made of 1 *106 cells by injector. The biological functions of reprogrammed sweat-like cells were observed by histological examination and sweat test 20 days later. Results: 1. Construction of eukaryotic expression vectors of NF-kappa B and Lef-1: The open reading frame (ORF) of NF-kappa B and Lef-1 genes were cloned into pcDNA3.1 (+) vector. After digestion by Hindlll/Sal I and Hindlll/Pst I, the bands with relative molecular weight of 3000 BP and 1200 BP were detected by agarose electrophoresis. Eukaryotic expression vectors pcDNA3.1 (+) - NF-kappa B and pcDNA3.1 (+) - Lef-1 were successfully constructed. 2. G418 screening and detection of NF-kappa B and Lef-1 expression showed that all the non-transfected cells died after 2 weeks of incremental screening in G418 medium, and positive clones were formed after 20 days of screening. The relative expression levels of NF-kappa B mRNA in blank vector group and blank control group were 3.651+0.062,0.987+0.098,1.118+0.024,2.451+0.032,0.997+0.078,1.158+0.043 respectively,indicating that the expression of NF-kappa B and Lef-1 mRNA in stably transfected human fibroblasts also increased significantly compared with blank control group. There was significant difference between the two groups (P 0.01), and there was also significant difference between the two groups (P 0.01). The recombinant vector had a higher transcription level in the cells. 3. Sweat gland markers detection: Western blot results showed that the expression of CEA, CK7, CK14 and CK19 proteins in pcDNA3.1 (+) -NF-KB and pcDNA3.1 (+) -Lef-1 transfection group were significantly higher than that in the empty vector group. The expression of CEA, CK7, CK14 and CK19 mRNA in pcDNA3.1 (+) -NF-kappa B and pcDNA3.1 (+) -Lef-1 transfection groups were significantly higher than that in blank control group and blank control group. Immunofluorescence staining showed that pcDNA3.1 (+) -NF-KB and pcDNA3.1 (+) -Lef-1 were transfected. The fluorescence intensity of CEA, CK7, CK14 and CK19 was very strong in the infection group, but the fluorescence intensity of the same protein in the other two control groups was basically invisible. The relative expression of Cyclin D1 mRNA was 2.653+0.045, 0.997+0.048, 1.118+0.053 respectively, indicating that the expression of SHH and Cyclin D1 mRNA in stably transfected human fibroblasts was also significantly increased compared with the blank control group (P 0.01), and the difference was also statistically significant compared with the empty carrier group (P 0.01). Shh and Cyclin D1, which are considered to be important in the process of sweat gland regeneration, have higher transcription levels in cells. 5. Nude mice plantar transplantation test: iodine starch sweating test 20 days later showed that about 7/10 of the experimental group's plantar was positive, and blue-black areas were visible in the middle of the plantar, whereas the two control groups showed that No corresponding positive results were found. Histological examination showed that the regeneration of sweat glands and the connection of sweat gland ducts in basal layer were seen in HE stained sections of the soles of the nude mice in the target gene transfection group, while no regeneration of sweat gland structures was found in the control group. Conclusion: The gene combination of NF-kappa B and Lef-1 can directly reprogram human fibroblasts to differentiate into sweat-like cells, and the expression of sweat-gland specific markers CEA, CK7, CK14 and CK19 in the process of inducing sweat-like cells is obvious, and the downstream genes Shh and Cyclin D1 confirm the expression of NF-kappa B and Lef-1. These two pathways, kappa B and Lef-1, play an important role in the process of direct reprogramming. The reprogrammed human fibroblasts have been validated in a nude mouse model, which lays a theoretical foundation for the reprogramming of sweat gland-like cells with other types of cells and the regeneration of sweat gland. People bring hope to the regeneration of sweat glands.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R329.2
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