本文選題：表皮細胞生長因子　切入點：臍帶間充質(zhì)干細胞　出處：《河北醫(yī)科大學》2010年碩士論文　論文類型：學位論文

【摘要】： 目的：本研究應用人工體外汗腺細胞損傷的微環(huán)境,誘導人臍帶間充質(zhì)干細胞(UC-MSCs)向汗腺細胞轉(zhuǎn)化,并通過檢測不同組別抗原的表達情況,探討表皮細胞生長因子(EGF)對轉(zhuǎn)化的影響,以及在轉(zhuǎn)化過程中細胞外信號調(diào)節(jié)蛋白激酶(ERK)信號通路的作用,為臨床應用人臍帶間充質(zhì)干細胞重建汗腺提供理論依據(jù)。 方法：無菌條件下獲得足月妊娠分娩胎兒的臍帶,將其用剪刀剪切成4cm左右長短的臍帶組織段,無菌生理鹽水反復沖洗干凈,除去臍帶中殘留的血液,剔除血管(一條臍靜脈,兩條臍動脈)以避免被血管內(nèi)皮細胞污染。取出臍帶中的華通膠組織(Wharton's Jelly),撕裂成條索狀,用剪刀將其剪成大小約1mm3的Wharton's Jelly組織塊,將組織塊置于培養(yǎng)瓶中,采用組織塊培養(yǎng)法得到臍帶間充質(zhì)干細胞(UC-MSCs),流式細胞術(shù)檢測其表面抗原表達情況；取人頭、面、頸部正常全層皮膚,用Ⅱ型膠原酶消化法獲得汗腺組織,培養(yǎng)汗腺細胞(h-SGCs)貼壁生長,免疫組織化學法檢測其為純化的h-SGCs；依據(jù)干細胞誘導分化的微環(huán)境理論,將h-SGCs行熱損傷處理,消化后種植于transwell小室,將第四代的UC-MSCs種植于transwell板下層,誘導間充值干細胞向汗腺細胞表型轉(zhuǎn)化,并依據(jù)處理因素不同,分為四組,組1：對照組,UC-MSCs用h-SGCs培養(yǎng)基培養(yǎng),不加熱休克h-SGCs；組2：UC-MSCs用h-SGCs培養(yǎng)基培養(yǎng),于transwell小室中放入熱損傷h-SGCs進行誘導；組3：在組2基礎(chǔ)上,加入50ng/ml的EGF；組4：在組2基礎(chǔ)上加入10ng/ml的PD98059。一周后用流式細胞術(shù)檢測共培養(yǎng)的UC-MSCs的CK7、CK19表達率,用免疫組織化學法檢測其CK19、CEA的染色情況,并應用SPSS13.0統(tǒng)計軟件進行數(shù)據(jù)統(tǒng)計分析。 結(jié)果：(1)流式細胞技術(shù)檢測顯示：①培養(yǎng)傳代的UC-MSCs CD14、CD29、CD34、CD44、CD45、CD105、CK7、CK19陽性率分別為0.6%,91.3%,0.6%,99.1%,0.9%,99.2%,0.8%,0.9%。其中CD29、CD44、CD105為干細胞表面標志物,表達率高,CD14、CD34、CD45為造血干細胞表面標志物,幾乎不表達,證明培養(yǎng)獲得的細胞為純化的間充質(zhì)干細胞。CK7、CK19在培養(yǎng)的細胞中的陽性率很低,說明UC-MSCs正常情況下不表達CK7、CK19等標志物。②對各組進行流式細胞術(shù)檢測,組1 CK7、CK19陽性率分別為：(2.20±1.51)%,(2.23±0.68)%；組2 CK7、CK19陽性率分別為(6.37±0.74)%,(5.73±0.32)%：組3 CK7、CK19陽性率分別為(14.3±0.96)%,(12.57±1.06)%：組4 CK7、CK19陽性率分別為(2.30±1.08)%,(2.13±0.61)%。CK7統(tǒng)計結(jié)果顯示,組1和組4沒有明顯統(tǒng)計學差異(P=0.999＞0.05),組2與組3的表達陽性率明顯高于這兩組,組2與組1(P=0.005＜0.05),組3與組1(P=0.000＜0.05),且組3高于組2(P=0.001＜0.05)。CK19統(tǒng)計結(jié)果顯示,組1和組4沒有明顯統(tǒng)計學差異(P=0.997＞0.05),組2與組3的表達陽性率明顯高于這兩組,組2與組1：(P=0.005＜0.05),組3與組4：(P=0.000＜0.05),且組3高于組2(P=0.001＜0.05)； (2)免疫組織化學染色結(jié)果：培養(yǎng)的人UC-MSCs的CEA和CK19免疫組織化學染色為陰性,培養(yǎng)的UC-MSCs不表達CEA和CK19,由于CEA和CK19表面抗原相結(jié)合可作為汗腺細胞的標志物,說明其不具有汗腺細胞的表型；而獲得的汗腺細胞的CEA和CK19染色均為陽性,證明為純化的汗腺細胞。不同條件下培養(yǎng)的各組細胞均有部分細胞表達CEA和CK19陽性,其中組3表達陽性細胞數(shù)最高。每組選取6個視野計算各組細胞表達CEA的陽性率,組1的CEA平均陽性率為(3.33±0.71)%；組2的CEA平均陽性率分別為(7.43±1.01)%；組3后的UC-MSCs的CEA平均陽性率分別為(17.63±2.31)%；組4的CEA平均陽性率分別為(3.17±0.35)%。統(tǒng)計結(jié)果顯示,組1和組4沒有明顯統(tǒng)計學差異(P=0.997＞0.05),組2與組3的陽性表達率明顯高于這兩組,組2與組1：(P=0.005＜0.05),組3與組1：(P=0.000＜0.05)，且組3高于組2：(P=0.001＜0.05)。 (3) Western blot:組1至組4各組的OD值分別為：組1：(0.64±0.026),組2：(0.79±0.049),組3(1.20±0.032),組4(0.62±0.042),組1與組4之間p-ERK表達相對強度無統(tǒng)計學差異(P=0.9100.05),組3表達相對強度最高,其次是組2,與組1統(tǒng)計分析后的差異分別為：(P=0.000＜0.05)、(P=0.003＜0.05),并且組3的表達強度高于組2。 結(jié)論：UC-MSCs與熱損傷的h-SGCs經(jīng)transwell板間接共同培養(yǎng)后,部分UC-MSCs表面抗原CK7、CK19、CEA表達陽性,證明已具有h-SGCs的表型,提示體外h-SGCs損傷的微環(huán)境具有促進UC-MSCs向h-SGCs分化的作用；而在培養(yǎng)基加入50ng/mlEGF的組3的細胞表面抗原陽性率明顯高于組2和對照組,加入ERK信號通路特異性抑制劑PD98059的組4與對照組陽性表達率沒有統(tǒng)計學差異,并且低于組2和組3,證明EGF可以明顯促進UC-MSCs向汗腺細胞轉(zhuǎn)化,并且ERK通路在通過共培養(yǎng)的方法誘導UC-MSCs向h-SGCs轉(zhuǎn)化以及EGF促進UC-MSCs向汗腺細胞轉(zhuǎn)化過程中發(fā)揮了重要作用。
[Abstract]:Objective: To study the in vitro artificial sweat gland cell injury microenvironment, induce human umbilical cord mesenchymal stem cells (UC-MSCs) into sweat gland cells, and the expression was detected by different groups of antigen, epidermal growth factor (EGF) effect on transformation, and the cells in the process of transformation of extracellular signal regulated protein kinase (ERK) signaling pathway, and provide a theoretical basis for the clinical application of human umbilical cord mesenchymal stem cells to rebuild the sweat glands.
Methods: fetal childbirth full-term umbilical cord under aseptic conditions, the scissors cut into about 4cm length of the umbilical cord, sterile saline rinse repeatedly, removing the residual blood from the umbilical cord, blood vessels (an umbilical vein, two umbilical arteries) in order to avoid being taken out of vascular endothelial cell contamination. Huatong Rubber Organization (Wharton's Jelly) in the umbilical cord, tearing into cords with a pair of scissors to cut into Wharton's Jelly block size of about 1mm3, the tissue into the culture bottle, obtained by using the method of tissue culture of umbilical cord mesenchymal stem cells (UC-MSCs), the detection of surface antigen expression by flow cytometry operation; take the head, face, neck skin was normal, sweat glands with type II collagenase digestion, cultured sweat gland cells (h-SGCs) adherent growth, immunohistochemical method for the purification of h-SGCs; on the basis of stem cell differentiation The micro environment theory, the h-SGCs for processing thermal damage after digestion cultivated in Transwell chamber, the fourth generation of UC-MSCs grown in Transwell in the layers, inducing mesenchymal stem cells into sweat gland cell phenotype, and according to different treatment, divided into four groups, 1 groups: control group, UC-MSCs in h-SGCs medium culture, not heat shock h-SGCs; group 2:UC-MSCs cultured in h-SGCs, Transwell cell in h-SGCs induced heat damage; group 3: in the group of 2 on the basis of adding 50ng/ml EGF; group 4: group 2 added 10ng/ml one week after PD98059. by flow cytometry UC-MSCs co culture the expression rate of CK19, CK7, CK19 detected by immunohistochemistry staining, CEA, statistics and data analysis using SPSS13.0 statistical software.
緇撴灉錛，

本文編號：1636411