轉(zhuǎn)錄因子組合誘導成纖維細胞直接重編程為汗腺樣細胞的實驗研究
發(fā)布時間：2018-08-11 11:04
【摘要】：目的：前期,我們團隊已經(jīng)確證了建立一定的共培養(yǎng)條件可以誘導間充質(zhì)干細胞轉(zhuǎn)分化為汗腺樣細胞,并經(jīng)裸鼠的腳掌創(chuàng)面模型試驗和初步的人體試驗證實,經(jīng)過誘導的自體骨髓間充質(zhì)干細胞移植于切除瘢痕的創(chuàng)面,可以生長出汗腺樣結(jié)構(gòu)并且具有一定的發(fā)汗功能。本研究的主要目的是在前期工作的基礎上,通過對成纖維細胞重編程進一步識別控制其分化為汗腺樣細胞的關鍵重編程轉(zhuǎn)錄因子(NF-κB和Lef-1)及其介導的信號調(diào)控通路,進一步闡明成纖維細胞可能分化為汗腺樣細胞的重編程機制,為利用其它多種類型細胞作為汗腺再生種子細胞,為臨床大面積汗腺損傷的患者實現(xiàn)汗腺再生帶來希望。方法：前期研究表明,調(diào)控成纖維細胞重編程為汗腺樣細胞關鍵信號通路涉及NF-KB和Lef-1等,結(jié)合文獻報道的參與調(diào)控胚胎發(fā)育過程汗腺細胞發(fā)生、發(fā)展過程蛋白的變化,尋找出主要的參與調(diào)控關鍵轉(zhuǎn)錄因子NF-κB和Lef-1的基因組合,分析其參與的信號通路網(wǎng)絡,并進一步驗證其生物功能。1.實驗分組與操作程序：待細胞培養(yǎng)至對數(shù)生長期,生長密度達到80%以上時,按106個/孔的密度將細胞接種于6孔板后采用完全隨機(完全隨機設計也叫組間設計,被試被分成若干組,每組分別接受一種實驗處理,有幾種實驗處理被試也相應的被分為幾組,各實驗組的被試之間相互獨立,因而又叫“獨立組”設計)將其分為3組(每組的樣本量為5),①目的基因轉(zhuǎn)染組：將所構(gòu)建的含有NF-κB和Lef-1基因的真核表達載體轉(zhuǎn)染入細胞并篩選出穩(wěn)定表達的單克隆,待后續(xù)實驗使用；②空載體組：將pcDNA3.1(+)空載體轉(zhuǎn)染細胞并篩選出穩(wěn)定表達的單克隆,待后續(xù)實驗使用;③空白對照組：不轉(zhuǎn)染目的基因,相同實驗條件下培養(yǎng)。2. NF-κB和Lef-1基因?qū)θ顺衫w維細胞的重編程：2.1真核表達載體的構(gòu)建：在美國國立生物技術信息中心(National Center of Biotechnology Information. NCB1)上查找實驗所需人NF-κB和Lef-1的基因序列,根據(jù)所查序列和實驗需要分別設計NF-κB和Lef-1基因開放閱讀框(open reading frame, ORF)PCR引物,用總RNA提取試劑盒提取人成纖維細胞總RNA,進行逆轉(zhuǎn)錄酶的作用合成目的cDNA,將cDNA和pcDNA3.1(+)載體分別用HindⅢ/SalⅠ及HindⅢ/PstⅠ雙酶切并連接轉(zhuǎn)化大腸桿菌DH-5a擴增,為了驗證NF-κB和Lef-1目的基因序列是否有突變,將含有重組子的菌液送華大基因測序,選取無突變的單克隆菌液保種,經(jīng)測序正確后提取質(zhì)粒,并檢測所提取質(zhì)粒濃度。2.2轉(zhuǎn)染細胞并篩選穩(wěn)定表達細胞系：將所培養(yǎng)的細胞以5000個/孔的密度接種于96孔板,用不同濃度的G418培養(yǎng)基培養(yǎng)細胞,以10～14 d殺死全部細胞的濃度為基本濃度,并以此確定篩選濃度和維持濃度。轉(zhuǎn)染前一天按106個/孔的密度將所培養(yǎng)細胞接種于6孔板,按脂質(zhì)體轉(zhuǎn)染的標準程序轉(zhuǎn)染細胞,轉(zhuǎn)染48 h后用含G418的培養(yǎng)基按800 mg/L的濃度篩選轉(zhuǎn)染成功的細胞,2周后以200 mg/L的濃度維持篩選壓力,挑選并在96孔板中培養(yǎng)成陽性單克隆,擴大培養(yǎng)將穩(wěn)定轉(zhuǎn)染的細胞用于后續(xù)實驗。利用Realtime-PCR檢測3組細胞NF-κB和Lef-1的mRNA表達情況。3. NF-κB和Lef-1重編程人成纖維細胞誘導培養(yǎng)后變化：EDA-A1能促進正常和外胚層發(fā)育缺陷小鼠的汗腺、毛囊等外胚層組織器官的發(fā)育,EGF能提高MSCs分化為SGLCs的誘導率。因此,本實驗采用1μg/ml EDA-A1、50 ng/ml EGF加入培養(yǎng)基誘導重編程后的人成纖維細胞分化為SGLCs.3.1汗腺標志物的檢測：應用免疫熒光法鑒定NF-κB和Lef-1重編程人成纖維細胞的汗腺相關分子如CEA,CK7,CK14和CK19等的表達；應用Western blot技術從蛋白層面檢測NF-κB和Lef-1重編程人成纖維細胞的汗腺相關分子CEA,CK7,CK14和CK19等的表達變化；應用Realtime-PCR定量檢測NF-κB和Lef-1重編程人成纖維細胞的汗腺相關分子CEA,CK7,CK14和CK19等的表達變化。3.2 NF-κB和Lef-1通路下游基因的檢測：應用Realtime-PCR定量檢測NF-κB和Lef-1重編程人成纖維細胞的NF-κB和Lef-1通路關鍵下游基因Shh和Cyclin D1的表達變化.3.3裸鼠腳掌移植實驗：應用前期實驗所建立的裸鼠模型探究重編程汗腺樣細胞的功能以及汗腺再生修復作用。將轉(zhuǎn)染了pcDNA3.1(+)-NF-KB和pcDNA3.1(+)-Lef-1的實驗組成纖維細胞和另外兩組對照組的成纖維細胞以1×106個細胞制成的150μl混懸液用注射器植入裸鼠燙傷后的腳掌,20天后進行組織學檢查和發(fā)汗實驗觀察重編程汗腺樣細胞的生物學功能,并進行安全性評價。結(jié)果：1. NF-κB和Lef-1真核表達載體構(gòu)建：NF-κB和Lef-1基因的基因開放閱讀框(open reading frame, ORF)克隆進pcDNA3.1(+)載體,經(jīng)Hindlll/SalⅠ及Hindlll/PstⅠ雙酶切后瓊脂糖電泳分別可見相對分子質(zhì)量約為3000 bp和1200 bp的條帶,經(jīng)測序鑒定堿基對無突變,表明成功構(gòu)建了pcDNA3.1(+)-NF-κB和pcDNA3.1(+)-Lef-1真核表達載體。2.G418篩選及NF-κB和Lef-1表達檢測：G418培養(yǎng)液遞增篩選2周后未轉(zhuǎn)染成功的細胞全部死亡,維持篩選20 d后形成陽性克隆。Realtime-PCR結(jié)果顯示,目的基因轉(zhuǎn)染組、空載體組、空白對照組的NF-κB mRNA的相對表達量分別為3.651±0.062、0.987±0.098、1.118±0.024,Lef-1 mRNA的相對表達量分別為2.451±0.032、0.997±0.078、1.158±0.043,表明穩(wěn)定轉(zhuǎn)染的人成纖維細胞的NF-κB和Lef-1 mRNA的表達也顯著增加,與空白對照組相比差異有統(tǒng)計學意義(P0.01),與空載體組相比差異也有統(tǒng)計學意義(P0.01),可認為重組載體在細胞內(nèi)已有較高轉(zhuǎn)錄水平。3.汗腺標志物的檢測：Western-blot結(jié)果顯示,pcDNA3.1(+)-NF-KB和pcDNA3.1(+)-Lef-1轉(zhuǎn)染組中CEA,CK7,CK14和CK19蛋白均有明顯地表達,而空載體組、空白對照組中均未看見有表達。Realtime-PCR結(jié)果顯示,pcDNA3.1(+)-NF-κB和pcDNA3.1(+)-Lef-1轉(zhuǎn)染組中CEA,CK7,CK14和CK19 mRNA均有明顯地表達,而空載體組、空白對照組中均未看見有表達。免疫熒光染色結(jié)果顯示,pcDNA3.1(+)-NF-KB和pcDNA3.1(+)-Lef-1轉(zhuǎn)染組中CEA,CK7,CK14和CK19熒光亮度很強,而其他兩個對照組相同蛋白的熒光基本上看不見。4. NF-κB和Lef-1通路下游基因的檢測：Realtime-PCR結(jié)果顯示,目的基因轉(zhuǎn)染組、空載體組、空白對照組的ShhmRNA的相對表達量分別為3.151±0.052、0.997±0.038、1.098±0.074,Cyclin D1mRNA的相對表達量分別為2.653±0.045、0.997±0.048、1.118±0.053,表明穩(wěn)定轉(zhuǎn)染的人成纖維細胞的Shh和Cyclin D1 mRNA的表達也顯著增加,與空白對照組相比差異有統(tǒng)計學意義(P0.01),與空載體組相比差異也有統(tǒng)計學意義(P0.01),可認為被公認參加汗腺再生過程中很重要的Shh和Cyclin D1在細胞內(nèi)已有較高轉(zhuǎn)錄水平。5.裸鼠腳掌移植實驗：20天后進行的碘淀粉發(fā)汗實驗結(jié)果顯示,大約7/10的實驗組腳掌為陽性結(jié)果,在腳掌中間可以明顯的看到藍黑色的區(qū)域,而兩組對照則沒有看到相應的陽性結(jié)果。組織學檢查發(fā)現(xiàn),目的基因轉(zhuǎn)染組的裸鼠腳掌HE染色切片中可以看到有汗腺的再生,基底層汗腺導管相連接,而對照組則沒有發(fā)現(xiàn)汗腺結(jié)構(gòu)的再生。冰凍切片中可看見ck7和ck19的紅色熒光。移植瘤實驗證明了汗腺樣細胞的安全性.結(jié)論：NF-κB和Lef-1的基因組合可以直接重編程人成纖維細胞分化成汗腺樣細胞,在誘導成汗腺樣細胞的過程中汗腺特異性標志物CEA,CK7,CK14和CK19的表達有明顯的表達,并且通過檢測通路下游基因Shh和Cyclin D1證實了NF-κB和Lef-1這兩條通路在直接重編程過程中的重要作用.直接重編程后的人成纖維細胞在裸鼠模型中得到了生物學功能的驗證,為利用其它多種類型細胞進行汗腺樣細胞重編程、實現(xiàn)汗腺再生奠定理論基礎,為臨床大面積汗腺損傷的患者實現(xiàn)汗腺再生帶來希望.
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R329.2
本文編號：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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R329.2
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