表皮细胞生长因子对人脐带间充质干细胞向汗腺细胞转化的影响

发布时间：2018-03-19 23:05

  本文选题：表皮细胞生长因子　切入点：脐带间充质干细胞　出处：《河北医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的：本研究应用人工体外汗腺细胞损伤的微环境,诱导人脐带间充质干细胞(UC-MSCs)向汗腺细胞转化,并通过检测不同组别抗原的表达情况,探讨表皮细胞生长因子(EGF)对转化的影响,以及在转化过程中细胞外信号调节蛋白激酶(ERK)信号通路的作用,为临床应用人脐带间充质干细胞重建汗腺提供理论依据。 方法：无菌条件下获得足月妊娠分娩胎儿的脐带,将其用剪刀剪切成4cm左右长短的脐带组织段,无菌生理盐水反复冲洗干净,除去脐带中残留的血液,剔除血管(一条脐静脉,两条脐动脉)以避免被血管内皮细胞污染。取出脐带中的华通胶组织(Wharton's Jelly),撕裂成条索状,用剪刀将其剪成大小约1mm3的Wharton's Jelly组织块,将组织块置于培养瓶中,采用组织块培养法得到脐带间充质干细胞(UC-MSCs),流式细胞术检测其表面抗原表达情况；取人头、面、颈部正常全层皮肤,用Ⅱ型胶原酶消化法获得汗腺组织,培养汗腺细胞(h-SGCs)贴壁生长,免疫组织化学法检测其为纯化的h-SGCs；依据干细胞诱导分化的微环境理论,将h-SGCs行热损伤处理,消化后种植于transwell小室,将第四代的UC-MSCs种植于transwell板下层,诱导间充值干细胞向汗腺细胞表型转化,并依据处理因素不同,分为四组,组1：对照组,UC-MSCs用h-SGCs培养基培养,不加热休克h-SGCs；组2：UC-MSCs用h-SGCs培养基培养,于transwell小室中放入热损伤h-SGCs进行诱导；组3：在组2基础上,加入50ng/ml的EGF；组4：在组2基础上加入10ng/ml的PD98059。一周后用流式细胞术检测共培养的UC-MSCs的CK7、CK19表达率,用免疫组织化学法检测其CK19、CEA的染色情况,并应用SPSS13.0统计软件进行数据统计分析。 结果：(1)流式细胞技术检测显示：①培养传代的UC-MSCs CD14、CD29、CD34、CD44、CD45、CD105、CK7、CK19阳性率分别为0.6%,91.3%,0.6%,99.1%,0.9%,99.2%,0.8%,0.9%。其中CD29、CD44、CD105为干细胞表面标志物,表达率高,CD14、CD34、CD45为造血干细胞表面标志物,几乎不表达,证明培养获得的细胞为纯化的间充质干细胞。CK7、CK19在培养的细胞中的阳性率很低,说明UC-MSCs正常情况下不表达CK7、CK19等标志物。②对各组进行流式细胞术检测,组1 CK7、CK19阳性率分别为：(2.20±1.51)%,(2.23±0.68)%；组2 CK7、CK19阳性率分别为(6.37±0.74)%,(5.73±0.32)%：组3 CK7、CK19阳性率分别为(14.3±0.96)%,(12.57±1.06)%：组4 CK7、CK19阳性率分别为(2.30±1.08)%,(2.13±0.61)%。CK7统计结果显示,组1和组4没有明显统计学差异(P=0.999＞0.05),组2与组3的表达阳性率明显高于这两组,组2与组1(P=0.005＜0.05),组3与组1(P=0.000＜0.05),且组3高于组2(P=0.001＜0.05)。CK19统计结果显示,组1和组4没有明显统计学差异(P=0.997＞0.05),组2与组3的表达阳性率明显高于这两组,组2与组1：(P=0.005＜0.05),组3与组4：(P=0.000＜0.05),且组3高于组2(P=0.001＜0.05)； (2)免疫组织化学染色结果：培养的人UC-MSCs的CEA和CK19免疫组织化学染色为阴性,培养的UC-MSCs不表达CEA和CK19,由于CEA和CK19表面抗原相结合可作为汗腺细胞的标志物,说明其不具有汗腺细胞的表型；而获得的汗腺细胞的CEA和CK19染色均为阳性,证明为纯化的汗腺细胞。不同条件下培养的各组细胞均有部分细胞表达CEA和CK19阳性,其中组3表达阳性细胞数最高。每组选取6个视野计算各组细胞表达CEA的阳性率,组1的CEA平均阳性率为(3.33±0.71)%；组2的CEA平均阳性率分别为(7.43±1.01)%；组3后的UC-MSCs的CEA平均阳性率分别为(17.63±2.31)%；组4的CEA平均阳性率分别为(3.17±0.35)%。统计结果显示,组1和组4没有明显统计学差异(P=0.997＞0.05),组2与组3的阳性表达率明显高于这两组,组2与组1：(P=0.005＜0.05),组3与组1：(P=0.000＜0.05)，且组3高于组2：(P=0.001＜0.05)。 (3) Western blot:组1至组4各组的OD值分别为：组1：(0.64±0.026),组2：(0.79±0.049),组3(1.20±0.032),组4(0.62±0.042),组1与组4之间p-ERK表达相对强度无统计学差异(P=0.9100.05),组3表达相对强度最高,其次是组2,与组1统计分析后的差异分别为：(P=0.000＜0.05)、(P=0.003＜0.05),并且组3的表达强度高于组2。 结论：UC-MSCs与热损伤的h-SGCs经transwell板间接共同培养后,部分UC-MSCs表面抗原CK7、CK19、CEA表达阳性,证明已具有h-SGCs的表型,提示体外h-SGCs损伤的微环境具有促进UC-MSCs向h-SGCs分化的作用；而在培养基加入50ng/mlEGF的组3的细胞表面抗原阳性率明显高于组2和对照组,加入ERK信号通路特异性抑制剂PD98059的组4与对照组阳性表达率没有统计学差异,并且低于组2和组3,证明EGF可以明显促进UC-MSCs向汗腺细胞转化,并且ERK通路在通过共培养的方法诱导UC-MSCs向h-SGCs转化以及EGF促进UC-MSCs向汗腺细胞转化过程中发挥了重要作用。
[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.
缁撴灉锛，

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