糖尿病大鼠表皮干细胞β-catenin和cyclinD1的表达及其在创面修复中的作用研究
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摘要
糖尿病(diabetic mellitus, DM)患者皮肤易受损伤、且损伤后创面愈合延迟或不愈合,目前仍是临床常见难题。国内外研究认为糖尿病创面难愈主要与糖代谢紊乱及晚期糖基化终末产物增多、血管病变及内皮细胞异常、神经病变、生长因子受体等改变、基质金属蛋白酶的表达异常、表皮细胞增殖障碍等因素有关。针对这些靶点衍生出来的治疗策略取得了一定的成效,但仍然不尽如人意。近期研究证实,表皮干细胞(Epidermal Stem Cells, ESCs)作为皮肽组织的特异性干细胞,是皮肤及其附属器发生、修复、改建的源泉,在正常皮肤再生和创面愈合中发挥重要作用；糖尿病创面表皮干细胞数量显著减少、活性明显降低,存在局部特有的“干细胞库干涸”现象。由此提示,表皮干细胞增殖分化特性的改变可能是糖尿病创面难愈的重要原因之一
p-连环素(β-catenin)是一种细胞骨架蛋白,由位于染色体3p21的CTNNB1基因编码。它作为一种重要的上皮细胞分化调节因子,主要通过与上皮细胞钙黏蛋白结合,参与细胞-细胞黏附和细胞与细胞外基质的连接,维持上皮组织结构的完整性,同时它又是调控细胞生长、发育和分化的Wnt信号通路的关键信号传递子,在通路中发挥枢纽作用。β-catenin水平增高时,Wnt信号通路激活,激活下游靶基因细胞周期蛋白D1(cyclin Dl)等,促进细胞增殖与分化。业已研究证实β-catenin能够促进肠道上皮干细胞增殖分化,并参与肠黏膜受损后的修复过程,表明β-catenin与成体干细胞的增殖分化密切相关。Wnt和β-catenin表达增强,表皮细胞的增殖分化和迁移能力增强,创面愈合加快。但β-catenin和Cyclin Dl在糖尿病皮肤表皮干细胞中的表达及其在创面修复中的作用尚不清楚。本实验采用糖尿病大鼠模型,观察糖尿病大鼠皮肤与正常大鼠皮肤组织表皮干细胞定位、β-catenin和cyclinDl的表达差异；采用表皮干细胞体外培养模型,研究糖尿病大鼠皮肤表皮干细胞β-catenin和cyclinD1基因及其蛋白的表达特征；采用糖尿病大鼠创面模型,探究表皮干细胞移植对糖尿病创面愈合的影响以及β-catenin和cyclin D1的表达变化,为其在创面修复中的进一步应用奠定实验基础和理论依据。
第一部分糖尿病大鼠皮肤组织表皮干细胞定位及β-catenin和cyclinD1表达检测
目的：观察糖尿病大鼠皮肤组织表皮干细胞定位和β-catenin、cyclinD1及其相关蛋白的表达检测,探讨其在糖尿病皮肤难愈创面修复中的意义。
方法：20只SD大鼠随机分为DM组和正常对照组。DM组大鼠采用一次性腹腔注射链脲佐菌素(Streptozocin, STZ,65 mg/kg)制备DM大鼠模型,成模第四周取大鼠背部全层皮肤,取两组大鼠胰腺组织,HE染色观察胰岛组织学变化。并取正常皮肤标本作为对照,行HE染色及β-catenin、cyclinD1、Wnt1、角蛋白19(keratin19,K19)、β1整合素(β1-integrin)和增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)免疫组织化学染色,图像分析软件测量阳性细胞积分吸光度(integral absorbance,IA)。
结果：DM大鼠连续4周血糖监测均>16.7 mmol/L且血糖波动小,DM模型具良好稳定性,建模成功率为90%。DM组胰腺HE染色可见胰岛细胞数目明显减少,出现变性坏死；对照组胰岛细胞结构清楚,无变性坏死。表皮基底层均可见表皮干细胞的标志物K19和β1-integrin阳性表达,β-catenin、cyclinD1、Wnt1、PCNA阳性细胞集中在表皮基底层,但DM组皮肤组织表皮β-catenin、cyclinD1、Wnt1、PCNA、K19和β1-integrin的表达均显著低于正常对照组皮肤(P<0.01)。DM组与正常对照组大鼠皮肤中β-catenin [(169.78±37.29), (217.88±23.51),P<0.01]、cyclinD1[(156.37±32.46),(198.26±22.15),P< 0.01]、Wnt1[(138.63±23.45),(175.82±25.12),P<0.01],PCNA[(143.17±19.82), (175.05±20.84),P<0.01]、K19[(139.54±20.69),(168.96±17.97),P<0.01]和β1-integrin[(150.58±9.98),(181.79±15.94),P<0.01]的阳性细胞积分吸光度相比差异显著,有统计学意义。
结论：糖尿病大鼠皮肤表皮干细胞主要位于表皮基底层,β-catenin和cyclinD1阳性表达区域和信号强度与表皮干细胞定位分布及阳性表达强度相一致。提示糖尿病大鼠皮肤中表皮干细胞数量减少和β-catenin、cyclinD1的表达减弱可能与糖尿病皮肤修复能力的降低密切相关。
第二部分体外培养观察糖尿病大鼠表皮干细胞β-catenin和cyclinD1的表达特征
目的：在体外培养条件下,观察糖尿病皮肤表皮干细胞β-catenin、cyclinD1及其增殖分化相关蛋白的表达特征,以探究DM皮肤创面难愈的潜在机制。
方法：40只SD雄性大鼠随机分为DM组和正常对照组。DM组大鼠一次性腹腔注射链脲佐菌素65mg/kg制备DM大鼠模型,正常对照组不作处理。注射后4周取两组背部全层皮肤,分离培养ESCs,流式细胞仪检测细胞周期,细胞接种1周后检测两组细胞克隆形成率。分别取两组ESCs行免疫细胞化学染色鉴定β-catenin、cyclinD1、Wnt1、PCNA、K19和β1-integrin阳性表达；采用RT-PCR和Western blot检测β-catenin、cyclinD1、Wnt1mRNA和蛋白表达。
结果：倒置相差显微镜下观察,DM组与正常对照组相比,表皮干细胞数量明显减少,生长速度缓慢,DM组ESCs克隆形成率为6.43%(12.87+2.03/200%)明显低于正常对照组的11.37%(22.75±3.24/200%),比较差异有统计学意义(P<0.01)。流式细胞仪分析DM组ESCs88.89%处于静息期/DNA合成前期(G0/G1期),凋亡细胞数3.98%；正常对照组ESCs91.50%处于G0/G1期,无凋亡细胞。DM组大鼠ESCs的K19、β1-integrin、β-catenin、cyclinD1、Wnt1及PCNA阳性表达的积分吸光度(IA)值分别为82.63±14.77、21.59±4.71、76.49±6.58、69.35±5.82、32.57±6.13、90.77±12.44,均低于正常对照组(分别为151.24±42.83、54.48±17.43、116.39±9.26、105.47±7.52、71.29±8.25、110.62±20.67),比较差异有统计学意义(P<0.01)。DM组表皮干细胞β-catenin[(0.556±0.017), (0.850±0.059),P<0.01]和cyclinD1[(0.682±0.023),(0.887±0.038),P<0.01] mRNA表达水平均显著低于正常对照组。Western blot检测β-catenin和cyclinD1在两组均有表达,但DM组的表达强度(0.583±0.054,0.840±0.007)低于正常对照组(1.128±0.077,1.307±0.072)(P<0.01)。
结论：1.两组体外分离培养的表皮干细胞呈克隆性生长,G0/G1期细胞比例高,β1-integrin和K19免疫细胞化学染色均呈阳性表达,表明在本实验培养条件下分离培养的表皮干细胞具有干细胞的生物学特性。但DM组ESCs生长速度缓慢,克隆形成能力减弱。
2.糖尿病大鼠皮肤表皮干细胞P-catenin和cyclinDlmRNA及其蛋白表达均明显降低。提示β-catenin和cyclinD1表达减弱引起糖尿病大鼠皮肤表皮干细胞增殖能力明显下降和数量显著减少,可能是导致糖尿病创面难愈的重要机制之一。
第三部分表皮干细胞移植对糖尿病大鼠创面愈合的影响及β-catenin和cyclinDl的表达变化
目的：观察表皮干细胞移植对糖尿病大鼠创面愈合的影响及β-catenin和cyclin D1的表达变化,为糖尿病难愈创面的治疗提供新思路。
方法：雄性SD大鼠20只用于建立糖尿病模型,雄性SD大鼠10只用于制备表皮干细胞。分离培养及鉴定SD大鼠ESCs,并用5-溴脱氧尿嘧啶(BrdU)标记。建立糖尿病SD大鼠创面模型,随机分为A、B、C三组,A组创面移植羊膜负载BrdU标记的ESCs; B组创面移植羊膜；C组为空白对照组,创面未给予干预。观察创面愈合情况、计算创面愈合率,HE及免疫组织化学SP法检测创面愈合组织中BrdU、K19、β1-integrin、β-catenin、cyclinD1、Wnt1及PCNA的表达。用图像分析软件测量阳性细胞积分吸光度。
结果：与另两组相比,表皮干细胞移植组新生表皮下胶原分布均匀,排列较整齐,在不同观察时期的创面愈合率明显增高且组间比较差异均有显著统计学意义(P<0.01)。表皮干细胞移植组创面及新生表皮中可见BrdU阳性细胞,而另两组皮肤创面组织中始终未见BrdU阳性细胞。各组创面组织中可见K19、β1-integrin、β-catenin、cyclinD1、Wnt1和PCNA阳性细胞表达,但A组的阳性细胞积分吸光度与B、C组比较差异显著(P<0.01)。
结论：表皮干细胞移植可有效促进糖尿病大鼠创面的愈合,且在新生表皮基底层表皮干细胞标志物K19、β1-integrin与β-catenin、CyclinD1的表达均增强。提示P-catenin和CyclinD1活性表达的变化可能是表皮干细胞促进糖尿病大鼠创面愈合的重要机制之一。
Skin wounds can happen easily in diabetic patients and their healing process takes much longer time than in non-DM (diabetic mellitus, DM) population and may even never heal completely.Currently, it is still a big clinical challenge to treat chronic wounds in diabetic patients. Domestic and international studies suggested that impaired wound healing in DM were due to glycometabolic disorders and advanced glycation end products increase, vasculopathy and endothelial cell abnormalities, neuropathy, growth factor receptor and other changes, abnormal expression of matrix metalloproteinases, epidermal cell proliferation barriers and other factors. Therapeutic strategies derived from these targets achieved some results, but still not satisfactory. Recent studies have confirmed that epidermal stem cells (Epidermal Stem Cells, ESCs) as the specific stem cells in skin tissues, are considered as the key source of the occurrence, repairing, rebuilding of skin and its appendages, and play an important role in regeneration and wound healing of normal skin; the amount and activity of epidermal stem cells in diabetic wounds significantly decreased, there is the local specific "stem cell bank dried up" phenomenon. Thus suggest that characteristics of proliferation and differentiation in epidermal stem cell may be one of the important reasons in diabetic impaired wound healing.
β-catenin is a cytoskeletal protein that is coded by the gene CTNNB1 located on chromosome 3p21. As an important differentiation regulator of epithelial cell, it maintains the structural integrity of epithelial tissue,mainly through the epithelial cadherin binding, involved in cell-cell adhesion and connection of cell to extracellular matrix. At the same time it is a critical part of the signal transduction, and plays a pivotal role in the Wnt signaling pathway which regulate the growth, development and differentiation of cell. When levels ofβ-catenin increased, Wnt signal pathway was activated, then activation of the downstream target gene cyclin D1, etc.,which promoted cell proliferation and differentiation. Research has confirmed thatβ-catenin can promote the proliferation and differentiation of intestinal epithelial stem cells and involve in repairing intestinal mucosa after injury. It indicates thatβ-catenin is closely related to proliferation and differentiation of adult stem cell. High expression of Wnt andβ-catenin enhanced proliferation, differentiation and migration of epidermal cell,and accelerated wound healing. However, expression ofβ-catenin and CyclinDl in epidermal stem cells and their role in diabetic skin wound repair is not clear.
In this study, we investigate localization of epidermal stem cells and the expression ofβ-catenin and cyclinDl between diabetic rats model and normal rats skin; in vitro, study gene and protein expression ofβ-catenin and cyclinD1 in epidermal stem cells of diabetic rat skin; explore the effects and expression ofβ-catenin and cyclin Dl after transplanting the epidermal stem cell on diabetic wound healing, it will provide experimental basis and theoretical foundation in its further application to diatetic wound healing.
Part I Localization of epidermal stem cell and and expression ofβ-catenin and cyclinDl in diabetic rats skin
OBJECTIVE:To study localization of epidermal stem cell and expression ofβ-catenin, cyclinDl and its related proteins in diabetic rats skin, and their role in diabetic impaired wound healing.
METHODS:Twenty SD rats were divided into DM group and control group randomly.The DM rats were induced by intraperitoneal injected 65 mg/kg streptozocin (STZ),4 weeks after injection, pancreatic tissue were taken for HE staining in two groups, then full-thickness skins were taken from the back of diabetic rats, and normal skin samples taken as controls, they were obtained for hematoxylin and eosin (HE) staining and immunohistochemical staining ofβ-catenin, cyclinDl, Wnt1, proliferating cell nuclear antigen (PCNA),keratinl9 (K19) andβ1 integrin. Then integral absorbance(IA) of positive cells of basal layer were measured with image analysis software.
RESULTS:DM rats were monitored for 4 weeks.their blood glucose were more than 16.7 mmol/L, and little fluctuations in blood glucose. The achievement ratio of diabetic rat model was 90% and the models have good stability. HE staining analysis showed the amount of pancreas islet cells in DM group significantly reduced and appeared necrotic,but in control group islet cells have intact structure and no necrosis. Expression of K19 and (31-integrin,markers of epidermal stem cells, were visible in the basal layer of two groups, and positive cells ofβ-catenin, cyclinDl, Wntl, PCNA concentrated in the basal layer. The expression ofβ-catenin, cyclinD1, Wntl, PCNA,K19 andβ1 integrin was higher in DM skin than in normal skin (P<0. 01).There was significant difference in the epidermal thickness and integral absorbance of the positive cells ofβ-catenin, cyclinD1, Wntl, PCNA, K19 andβ1 integrin between DM rats and normal rats.
CONCLUSION:Epidermal stem cells were mainly located in the basal layer of diabetic rat skin, expression region and signal intensity ofβ-catenin and cyclinDl is consistent with localization and positive expression of epidermal stem cells. It suggests that the reduced amount of epidermal stem cells and the decreased expression of (3-catenin, cyclinDl in diabetic rats skin may be closely related to the low repair capacity of the diabetic wound skin.
PartⅡIn vitro characterization ofβ-catenin and cyclinDl in epidermal stem cells of diabetic rats
OBJECTIVE:In vitro, to explore characterization ofβ-catenin and cyclinDl and its related proteins in epidermal stem cells of diabetic rats, then to study the potential mechanism of difficult recovering wounds in diabetic skin.
METHODS:Forty SD rats were divided into DM group and normal control group randomly (n= 20).The DM rats were induced by intraperitoneal injected 65 mg/kg streptozocin (STZ), The normal control group without treatment. after modeling of 4 weeks, full-thickness skins were taken from the back of two group rats for isolation,cultivation and identification of its epidermal stem cells, the cell cycle were measured by flow cytometry. Cell colony formation rate of the two groups were detected after 1 week. Meanwhile, they were obtained for immunocytochemical staining of K19,β1 integrin,β-catenin, cyclinDl, Wnt1 and PCNA. mRNA and protein expression ofβ-catenin, cyclinDl and Wnt1 were detected by RT-PCR and Western blot.
RESULTS:Under the inverted phase contrast microscope, the amount of epidermal stem cells in DM group significantly reduced, and cell growth is slower, compared with control group. The clone forming efficiency of ESCs in DM group (6.43%,12.87±2.03/200%) was significantly lower than that of ESCs in the controls(11.37%,22.75±3.24/200%)(P<0.01). Flow cytometry indicated that 3.98% apoptotic cells and 88.89% cultured ESCs of the DM group were in resting state/pre-DNA-synthetic gap(G0/G1), as the controls were 91.50% and no apoptotic cells. Integral absorbance of positive expression of K19,β1 integrin, (3-catenin, cyclinD1, Wntl and PCNA in ESCs of DM skin were respectively 82.63±14.77, 21.59±4.71,76.49±6.58,69.35±5.82,32.57±6.13,90.77±12.44 than in that of normal skin, these in ESCs of the controls were respectively 151.24±42.83,54.48±17.43, 116.39±9.26,105.47±7.52,71.29±8.25,110.62±20.67), it showed significant difference in two groups(P<0.01). the mRNA expression level ofβ-catenin[(0.556±0.017),(0.850±0.059),P<0.01] and cyclin D1[(0.682±0.023), (0.887±0.038),P<0.01] were lower in DM group than in control group. Analysis of western blotting revealed thatβ-catenin and cyclin D1 were expressed in both groups, whereas the DM group showed weaker expression ofβ-catenin[(0.583±0.054) (1.128±0.077),P<0.01] and cyclinD1 [(0.843±0.037),(1.307±0.072), P<0.01] in comparison with control group.
CONCLUSION:1. In vitro epidermal stem cells of the two groups showed clonal growth, high proportion of G0/G1 phase cells, the positive expression ofβ-integrin and K19. It indicated epidermal stem cells isolated and cultured in this experiment conditions have the biological characteristics of stem cells. However, ESCs in DM group showed slow growth rate and lower colony formation ability.
2. mRNA and protein expression of (3-catenin and cyclinDl were significantly reduced in epidermal stem cells of diabetic rat skin. the less expression ofβ-catenin and CyclinD1 which causes the decreased amount and the reduced proliferation and differentiation capacity of ESCs may be one of the important mechanisms in diabetic impaired wound healing.
PartⅢExpression ofβ-catenin and cyclinDl and its effct on epidermal stem cell transplanting in diabetic wound healing
OBJECTIVE:To investigate expression changes of P-catenin and cyclin Dl and its effct on epidermal stem cell transplanting to wound healing of diabetic rats, and provide a new idea for clinical treatment of difficult healing wound in diabetic skin.
METHODS:Twenty male SD rats were randomly selected to establish diabetic model. Ten male SD rats were selected to prepare epidermal stem cells.ESCs of SD rats were isolated, cultured, identified and labled with 5-bromo-2'-deoxyuridine (BrdU) in vitro. The wound model of diabetic SD rats were established,then divided into A,B,C group. Human amniotic membrane (HAM) loading labled BrdU ESCs were implanted to A group, Human amniotic membrane (HAM) were implanted to B group, but control group not. At 7,14 days after ESCs transplantation, general situation of wound healing, the healing rate of wound, hematoxylin and eosin(HE) staining, immunohistochemical staining of Brdu, K19,β1-integrin,β-catenin, cyclinD1,Wntl and PCNA in wounds of every group were investigated. Integral absorbance of positive cells were measured with image analysis software.
RESULTS:Compared with the other two groups, collagen under the newborn epidermis evenly distributed, arranged in neat in epidermal stem cell trratment group(A), the wound healing rate of A group was significantly higher at different observation times (P<0.01). BrdU-positive cells in the wounds and newborn epidermis of A group were visible, while wound tissue of the other two groups had no BrdU-positive cells. K19.β1-integrin,β-catenin, cyclinDl,Wntl and PCNA-positive cells in wound tissue of each group could be seen, but integral absorbance of positive cells were significantly different in A group compared to that in B, C group (P<0. 01).
CONCLUSION:Epidermal stem cells may accelerate wound healing of diabetic skin. And expression of K19,β1-integrin,β-catenin and CyclinDl were higher in the basal layer of newborn skin of epidermal stem cell treatment group.It suggested expression activity changes ofβ-catenin and CyclinDl may be one of the important mechanism in epidermal stem cells promoting diabetic wound healing.
p-连环素(β-catenin)是一种细胞骨架蛋白,由位于染色体3p21的CTNNB1基因编码。它作为一种重要的上皮细胞分化调节因子,主要通过与上皮细胞钙黏蛋白结合,参与细胞-细胞黏附和细胞与细胞外基质的连接,维持上皮组织结构的完整性,同时它又是调控细胞生长、发育和分化的Wnt信号通路的关键信号传递子,在通路中发挥枢纽作用。β-catenin水平增高时,Wnt信号通路激活,激活下游靶基因细胞周期蛋白D1(cyclin Dl)等,促进细胞增殖与分化。业已研究证实β-catenin能够促进肠道上皮干细胞增殖分化,并参与肠黏膜受损后的修复过程,表明β-catenin与成体干细胞的增殖分化密切相关。Wnt和β-catenin表达增强,表皮细胞的增殖分化和迁移能力增强,创面愈合加快。但β-catenin和Cyclin Dl在糖尿病皮肤表皮干细胞中的表达及其在创面修复中的作用尚不清楚。本实验采用糖尿病大鼠模型,观察糖尿病大鼠皮肤与正常大鼠皮肤组织表皮干细胞定位、β-catenin和cyclinDl的表达差异；采用表皮干细胞体外培养模型,研究糖尿病大鼠皮肤表皮干细胞β-catenin和cyclinD1基因及其蛋白的表达特征；采用糖尿病大鼠创面模型,探究表皮干细胞移植对糖尿病创面愈合的影响以及β-catenin和cyclin D1的表达变化,为其在创面修复中的进一步应用奠定实验基础和理论依据。
第一部分糖尿病大鼠皮肤组织表皮干细胞定位及β-catenin和cyclinD1表达检测
目的：观察糖尿病大鼠皮肤组织表皮干细胞定位和β-catenin、cyclinD1及其相关蛋白的表达检测,探讨其在糖尿病皮肤难愈创面修复中的意义。
方法：20只SD大鼠随机分为DM组和正常对照组。DM组大鼠采用一次性腹腔注射链脲佐菌素(Streptozocin, STZ,65 mg/kg)制备DM大鼠模型,成模第四周取大鼠背部全层皮肤,取两组大鼠胰腺组织,HE染色观察胰岛组织学变化。并取正常皮肤标本作为对照,行HE染色及β-catenin、cyclinD1、Wnt1、角蛋白19(keratin19,K19)、β1整合素(β1-integrin)和增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)免疫组织化学染色,图像分析软件测量阳性细胞积分吸光度(integral absorbance,IA)。
结果：DM大鼠连续4周血糖监测均>16.7 mmol/L且血糖波动小,DM模型具良好稳定性,建模成功率为90%。DM组胰腺HE染色可见胰岛细胞数目明显减少,出现变性坏死；对照组胰岛细胞结构清楚,无变性坏死。表皮基底层均可见表皮干细胞的标志物K19和β1-integrin阳性表达,β-catenin、cyclinD1、Wnt1、PCNA阳性细胞集中在表皮基底层,但DM组皮肤组织表皮β-catenin、cyclinD1、Wnt1、PCNA、K19和β1-integrin的表达均显著低于正常对照组皮肤(P<0.01)。DM组与正常对照组大鼠皮肤中β-catenin [(169.78±37.29), (217.88±23.51),P<0.01]、cyclinD1[(156.37±32.46),(198.26±22.15),P< 0.01]、Wnt1[(138.63±23.45),(175.82±25.12),P<0.01],PCNA[(143.17±19.82), (175.05±20.84),P<0.01]、K19[(139.54±20.69),(168.96±17.97),P<0.01]和β1-integrin[(150.58±9.98),(181.79±15.94),P<0.01]的阳性细胞积分吸光度相比差异显著,有统计学意义。
结论：糖尿病大鼠皮肤表皮干细胞主要位于表皮基底层,β-catenin和cyclinD1阳性表达区域和信号强度与表皮干细胞定位分布及阳性表达强度相一致。提示糖尿病大鼠皮肤中表皮干细胞数量减少和β-catenin、cyclinD1的表达减弱可能与糖尿病皮肤修复能力的降低密切相关。
第二部分体外培养观察糖尿病大鼠表皮干细胞β-catenin和cyclinD1的表达特征
目的：在体外培养条件下,观察糖尿病皮肤表皮干细胞β-catenin、cyclinD1及其增殖分化相关蛋白的表达特征,以探究DM皮肤创面难愈的潜在机制。
方法：40只SD雄性大鼠随机分为DM组和正常对照组。DM组大鼠一次性腹腔注射链脲佐菌素65mg/kg制备DM大鼠模型,正常对照组不作处理。注射后4周取两组背部全层皮肤,分离培养ESCs,流式细胞仪检测细胞周期,细胞接种1周后检测两组细胞克隆形成率。分别取两组ESCs行免疫细胞化学染色鉴定β-catenin、cyclinD1、Wnt1、PCNA、K19和β1-integrin阳性表达；采用RT-PCR和Western blot检测β-catenin、cyclinD1、Wnt1mRNA和蛋白表达。
结果：倒置相差显微镜下观察,DM组与正常对照组相比,表皮干细胞数量明显减少,生长速度缓慢,DM组ESCs克隆形成率为6.43%(12.87+2.03/200%)明显低于正常对照组的11.37%(22.75±3.24/200%),比较差异有统计学意义(P<0.01)。流式细胞仪分析DM组ESCs88.89%处于静息期/DNA合成前期(G0/G1期),凋亡细胞数3.98%；正常对照组ESCs91.50%处于G0/G1期,无凋亡细胞。DM组大鼠ESCs的K19、β1-integrin、β-catenin、cyclinD1、Wnt1及PCNA阳性表达的积分吸光度(IA)值分别为82.63±14.77、21.59±4.71、76.49±6.58、69.35±5.82、32.57±6.13、90.77±12.44,均低于正常对照组(分别为151.24±42.83、54.48±17.43、116.39±9.26、105.47±7.52、71.29±8.25、110.62±20.67),比较差异有统计学意义(P<0.01)。DM组表皮干细胞β-catenin[(0.556±0.017), (0.850±0.059),P<0.01]和cyclinD1[(0.682±0.023),(0.887±0.038),P<0.01] mRNA表达水平均显著低于正常对照组。Western blot检测β-catenin和cyclinD1在两组均有表达,但DM组的表达强度(0.583±0.054,0.840±0.007)低于正常对照组(1.128±0.077,1.307±0.072)(P<0.01)。
结论：1.两组体外分离培养的表皮干细胞呈克隆性生长,G0/G1期细胞比例高,β1-integrin和K19免疫细胞化学染色均呈阳性表达,表明在本实验培养条件下分离培养的表皮干细胞具有干细胞的生物学特性。但DM组ESCs生长速度缓慢,克隆形成能力减弱。
2.糖尿病大鼠皮肤表皮干细胞P-catenin和cyclinDlmRNA及其蛋白表达均明显降低。提示β-catenin和cyclinD1表达减弱引起糖尿病大鼠皮肤表皮干细胞增殖能力明显下降和数量显著减少,可能是导致糖尿病创面难愈的重要机制之一。
第三部分表皮干细胞移植对糖尿病大鼠创面愈合的影响及β-catenin和cyclinDl的表达变化
目的：观察表皮干细胞移植对糖尿病大鼠创面愈合的影响及β-catenin和cyclin D1的表达变化,为糖尿病难愈创面的治疗提供新思路。
方法：雄性SD大鼠20只用于建立糖尿病模型,雄性SD大鼠10只用于制备表皮干细胞。分离培养及鉴定SD大鼠ESCs,并用5-溴脱氧尿嘧啶(BrdU)标记。建立糖尿病SD大鼠创面模型,随机分为A、B、C三组,A组创面移植羊膜负载BrdU标记的ESCs; B组创面移植羊膜；C组为空白对照组,创面未给予干预。观察创面愈合情况、计算创面愈合率,HE及免疫组织化学SP法检测创面愈合组织中BrdU、K19、β1-integrin、β-catenin、cyclinD1、Wnt1及PCNA的表达。用图像分析软件测量阳性细胞积分吸光度。
结果：与另两组相比,表皮干细胞移植组新生表皮下胶原分布均匀,排列较整齐,在不同观察时期的创面愈合率明显增高且组间比较差异均有显著统计学意义(P<0.01)。表皮干细胞移植组创面及新生表皮中可见BrdU阳性细胞,而另两组皮肤创面组织中始终未见BrdU阳性细胞。各组创面组织中可见K19、β1-integrin、β-catenin、cyclinD1、Wnt1和PCNA阳性细胞表达,但A组的阳性细胞积分吸光度与B、C组比较差异显著(P<0.01)。
结论：表皮干细胞移植可有效促进糖尿病大鼠创面的愈合,且在新生表皮基底层表皮干细胞标志物K19、β1-integrin与β-catenin、CyclinD1的表达均增强。提示P-catenin和CyclinD1活性表达的变化可能是表皮干细胞促进糖尿病大鼠创面愈合的重要机制之一。
Skin wounds can happen easily in diabetic patients and their healing process takes much longer time than in non-DM (diabetic mellitus, DM) population and may even never heal completely.Currently, it is still a big clinical challenge to treat chronic wounds in diabetic patients. Domestic and international studies suggested that impaired wound healing in DM were due to glycometabolic disorders and advanced glycation end products increase, vasculopathy and endothelial cell abnormalities, neuropathy, growth factor receptor and other changes, abnormal expression of matrix metalloproteinases, epidermal cell proliferation barriers and other factors. Therapeutic strategies derived from these targets achieved some results, but still not satisfactory. Recent studies have confirmed that epidermal stem cells (Epidermal Stem Cells, ESCs) as the specific stem cells in skin tissues, are considered as the key source of the occurrence, repairing, rebuilding of skin and its appendages, and play an important role in regeneration and wound healing of normal skin; the amount and activity of epidermal stem cells in diabetic wounds significantly decreased, there is the local specific "stem cell bank dried up" phenomenon. Thus suggest that characteristics of proliferation and differentiation in epidermal stem cell may be one of the important reasons in diabetic impaired wound healing.
β-catenin is a cytoskeletal protein that is coded by the gene CTNNB1 located on chromosome 3p21. As an important differentiation regulator of epithelial cell, it maintains the structural integrity of epithelial tissue,mainly through the epithelial cadherin binding, involved in cell-cell adhesion and connection of cell to extracellular matrix. At the same time it is a critical part of the signal transduction, and plays a pivotal role in the Wnt signaling pathway which regulate the growth, development and differentiation of cell. When levels ofβ-catenin increased, Wnt signal pathway was activated, then activation of the downstream target gene cyclin D1, etc.,which promoted cell proliferation and differentiation. Research has confirmed thatβ-catenin can promote the proliferation and differentiation of intestinal epithelial stem cells and involve in repairing intestinal mucosa after injury. It indicates thatβ-catenin is closely related to proliferation and differentiation of adult stem cell. High expression of Wnt andβ-catenin enhanced proliferation, differentiation and migration of epidermal cell,and accelerated wound healing. However, expression ofβ-catenin and CyclinDl in epidermal stem cells and their role in diabetic skin wound repair is not clear.
In this study, we investigate localization of epidermal stem cells and the expression ofβ-catenin and cyclinDl between diabetic rats model and normal rats skin; in vitro, study gene and protein expression ofβ-catenin and cyclinD1 in epidermal stem cells of diabetic rat skin; explore the effects and expression ofβ-catenin and cyclin Dl after transplanting the epidermal stem cell on diabetic wound healing, it will provide experimental basis and theoretical foundation in its further application to diatetic wound healing.
Part I Localization of epidermal stem cell and and expression ofβ-catenin and cyclinDl in diabetic rats skin
OBJECTIVE:To study localization of epidermal stem cell and expression ofβ-catenin, cyclinDl and its related proteins in diabetic rats skin, and their role in diabetic impaired wound healing.
METHODS:Twenty SD rats were divided into DM group and control group randomly.The DM rats were induced by intraperitoneal injected 65 mg/kg streptozocin (STZ),4 weeks after injection, pancreatic tissue were taken for HE staining in two groups, then full-thickness skins were taken from the back of diabetic rats, and normal skin samples taken as controls, they were obtained for hematoxylin and eosin (HE) staining and immunohistochemical staining ofβ-catenin, cyclinDl, Wnt1, proliferating cell nuclear antigen (PCNA),keratinl9 (K19) andβ1 integrin. Then integral absorbance(IA) of positive cells of basal layer were measured with image analysis software.
RESULTS:DM rats were monitored for 4 weeks.their blood glucose were more than 16.7 mmol/L, and little fluctuations in blood glucose. The achievement ratio of diabetic rat model was 90% and the models have good stability. HE staining analysis showed the amount of pancreas islet cells in DM group significantly reduced and appeared necrotic,but in control group islet cells have intact structure and no necrosis. Expression of K19 and (31-integrin,markers of epidermal stem cells, were visible in the basal layer of two groups, and positive cells ofβ-catenin, cyclinDl, Wntl, PCNA concentrated in the basal layer. The expression ofβ-catenin, cyclinD1, Wntl, PCNA,K19 andβ1 integrin was higher in DM skin than in normal skin (P<0. 01).There was significant difference in the epidermal thickness and integral absorbance of the positive cells ofβ-catenin, cyclinD1, Wntl, PCNA, K19 andβ1 integrin between DM rats and normal rats.
CONCLUSION:Epidermal stem cells were mainly located in the basal layer of diabetic rat skin, expression region and signal intensity ofβ-catenin and cyclinDl is consistent with localization and positive expression of epidermal stem cells. It suggests that the reduced amount of epidermal stem cells and the decreased expression of (3-catenin, cyclinDl in diabetic rats skin may be closely related to the low repair capacity of the diabetic wound skin.
PartⅡIn vitro characterization ofβ-catenin and cyclinDl in epidermal stem cells of diabetic rats
OBJECTIVE:In vitro, to explore characterization ofβ-catenin and cyclinDl and its related proteins in epidermal stem cells of diabetic rats, then to study the potential mechanism of difficult recovering wounds in diabetic skin.
METHODS:Forty SD rats were divided into DM group and normal control group randomly (n= 20).The DM rats were induced by intraperitoneal injected 65 mg/kg streptozocin (STZ), The normal control group without treatment. after modeling of 4 weeks, full-thickness skins were taken from the back of two group rats for isolation,cultivation and identification of its epidermal stem cells, the cell cycle were measured by flow cytometry. Cell colony formation rate of the two groups were detected after 1 week. Meanwhile, they were obtained for immunocytochemical staining of K19,β1 integrin,β-catenin, cyclinDl, Wnt1 and PCNA. mRNA and protein expression ofβ-catenin, cyclinDl and Wnt1 were detected by RT-PCR and Western blot.
RESULTS:Under the inverted phase contrast microscope, the amount of epidermal stem cells in DM group significantly reduced, and cell growth is slower, compared with control group. The clone forming efficiency of ESCs in DM group (6.43%,12.87±2.03/200%) was significantly lower than that of ESCs in the controls(11.37%,22.75±3.24/200%)(P<0.01). Flow cytometry indicated that 3.98% apoptotic cells and 88.89% cultured ESCs of the DM group were in resting state/pre-DNA-synthetic gap(G0/G1), as the controls were 91.50% and no apoptotic cells. Integral absorbance of positive expression of K19,β1 integrin, (3-catenin, cyclinD1, Wntl and PCNA in ESCs of DM skin were respectively 82.63±14.77, 21.59±4.71,76.49±6.58,69.35±5.82,32.57±6.13,90.77±12.44 than in that of normal skin, these in ESCs of the controls were respectively 151.24±42.83,54.48±17.43, 116.39±9.26,105.47±7.52,71.29±8.25,110.62±20.67), it showed significant difference in two groups(P<0.01). the mRNA expression level ofβ-catenin[(0.556±0.017),(0.850±0.059),P<0.01] and cyclin D1[(0.682±0.023), (0.887±0.038),P<0.01] were lower in DM group than in control group. Analysis of western blotting revealed thatβ-catenin and cyclin D1 were expressed in both groups, whereas the DM group showed weaker expression ofβ-catenin[(0.583±0.054) (1.128±0.077),P<0.01] and cyclinD1 [(0.843±0.037),(1.307±0.072), P<0.01] in comparison with control group.
CONCLUSION:1. In vitro epidermal stem cells of the two groups showed clonal growth, high proportion of G0/G1 phase cells, the positive expression ofβ-integrin and K19. It indicated epidermal stem cells isolated and cultured in this experiment conditions have the biological characteristics of stem cells. However, ESCs in DM group showed slow growth rate and lower colony formation ability.
2. mRNA and protein expression of (3-catenin and cyclinDl were significantly reduced in epidermal stem cells of diabetic rat skin. the less expression ofβ-catenin and CyclinD1 which causes the decreased amount and the reduced proliferation and differentiation capacity of ESCs may be one of the important mechanisms in diabetic impaired wound healing.
PartⅢExpression ofβ-catenin and cyclinDl and its effct on epidermal stem cell transplanting in diabetic wound healing
OBJECTIVE:To investigate expression changes of P-catenin and cyclin Dl and its effct on epidermal stem cell transplanting to wound healing of diabetic rats, and provide a new idea for clinical treatment of difficult healing wound in diabetic skin.
METHODS:Twenty male SD rats were randomly selected to establish diabetic model. Ten male SD rats were selected to prepare epidermal stem cells.ESCs of SD rats were isolated, cultured, identified and labled with 5-bromo-2'-deoxyuridine (BrdU) in vitro. The wound model of diabetic SD rats were established,then divided into A,B,C group. Human amniotic membrane (HAM) loading labled BrdU ESCs were implanted to A group, Human amniotic membrane (HAM) were implanted to B group, but control group not. At 7,14 days after ESCs transplantation, general situation of wound healing, the healing rate of wound, hematoxylin and eosin(HE) staining, immunohistochemical staining of Brdu, K19,β1-integrin,β-catenin, cyclinD1,Wntl and PCNA in wounds of every group were investigated. Integral absorbance of positive cells were measured with image analysis software.
RESULTS:Compared with the other two groups, collagen under the newborn epidermis evenly distributed, arranged in neat in epidermal stem cell trratment group(A), the wound healing rate of A group was significantly higher at different observation times (P<0.01). BrdU-positive cells in the wounds and newborn epidermis of A group were visible, while wound tissue of the other two groups had no BrdU-positive cells. K19.β1-integrin,β-catenin, cyclinDl,Wntl and PCNA-positive cells in wound tissue of each group could be seen, but integral absorbance of positive cells were significantly different in A group compared to that in B, C group (P<0. 01).
CONCLUSION:Epidermal stem cells may accelerate wound healing of diabetic skin. And expression of K19,β1-integrin,β-catenin and CyclinDl were higher in the basal layer of newborn skin of epidermal stem cell treatment group.It suggested expression activity changes ofβ-catenin and CyclinDl may be one of the important mechanism in epidermal stem cells promoting diabetic wound healing.
引文
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