高糖环境对角膜上皮修复和角膜缘干细胞活性影响的实验研究

发布时间：2018-05-21 17:18

本文选题：Ins2Akita糖尿病小鼠 + 角膜上皮干细胞　；参考：《青岛大学》2012年硕士论文

【摘要】：目的利用角膜缘干细胞体外模型和Ins2Akita糖尿病小鼠模型,研究高糖环境对角膜缘上皮王细胞活性以及角膜上皮修复的影响。方法 1.采用小鼠角膜缘上皮干／祖细胞系(TKE2)作为体外细胞模型,在培养基中添加终浓度为30mM的葡萄糖进行处理,模拟糖尿病角膜病变的高糖微环境,通过相差显微镜观察、MTT、干细胞克隆形成、Annexin V-PI和8-OHdG (DNA单链断裂标记)免疫染色等实验,观察高糖环境对角膜上皮干细胞形态变化、克隆形成能力、细胞增殖、凋亡、DNA稳定性等方面的影响。 2.选择同龄成年雄性C57BL/6小鼠(正常对照组)和Ins2Akita小鼠(糖尿病组)各10只,比较2组小鼠在体重、血糖水平的差异；刮除小鼠角膜中央2mm的上皮,观察并比较角膜上皮在损伤后0h、24h、48h、64h、76h、88h、100h、136、172h的修复情况,并通过透射电镜、HE一染色、TUNEL (凋亡)标记染色、8-OHdG对角膜上皮的形态改变、细胞凋亡及DNA稳定性进行检测分析。结果 1.体外细胞模型的结果显示,高糖处理后的TKE2细胞呈长梭形,随着高糖处理时间的延长,细胞状态变差；高糖组干细胞克隆直径较小,克隆形成率明显降低(正常组为28.333±5.508,高糖处理3代TKE2克隆形成率为6.333±1.155),比较有统计学意义(P0.05)；MTT检测发现,正常组和高糖处理1代细胞间增殖能力有差异(P72h=0.013),高糖处理3代后差异更为明显(P72h=0.000)；细胞凋亡检测示正常组早期凋亡细胞比例为8.02-3.14,高糖组早期凋亡细胞比例为33.13-2.63,差异有统计学意义(P0.001)；高糖处理3代后的细胞8-OHdG阳性染色较正常细胞明显增强。 2.Ins2Akita糖尿病小鼠在8周的检测周期内,其体重增加与正常小鼠相比明显减少,血糖维持在相对稳定的高水平；糖尿病组小鼠的角膜上皮愈合时问为刮除后172h,愈合速度较正常对照组(48h)明显延迟,并且在88h出现缺损面积的扩大；电镜观察显示,糖尿病小鼠角膜上皮细胞表面微绒毛和微皱褶明显减少,角膜基底层上皮细胞排列尚规则,但细胞体积增大,基底细胞空泡化明显,细胞问桥粒连接较少,角膜上皮与基底膜问的半桥粒明显减少；HE..染色示糖尿病小鼠角膜上皮细胞表面不平整,形态不规则,细胞间连接疏松,细胞层数减少,分界不清,角膜上皮层基底细胞空泡形成,基质层明显水肿；TUNEL结果示角膜上皮基底层细胞TUNEL标记染色较正常组明显增多；8-OHdG的染色结果示角膜上皮基底层细胞8-OHdG标记染色阳性细胞较正常对照组明显增多。结论 1.体外高糖处理条件下,角膜上皮干细胞的形态发生改变,角膜上皮干细胞的克隆形成能力和增殖明显受到抑制；早期凋亡细胞明显增多；基因组稳定性下降。 2. Ins2Akita糖尿病小鼠角膜上皮修复速度明显减慢,并出现反复剥脱现象；角膜基底层的上皮细胞形态变化明显、角膜上皮基底层细胞的凋亡增加及其DNA稳定性下降。
[Abstract]:Purpose Using limbal stem cell model and Ins2Akita diabetic mice model in vitro, the effects of high glucose environment on corneal limbal epithelial king cell activity and corneal epithelial repair were studied. Method 1. The mouse limbal epithelial stem / progenitor cell line (TKE2) was used as a cell model in vitro. Glucose with final concentration of 30mM was added to the culture medium to simulate the hyperglycemic microenvironment of diabetic keratopathy. The morphologic changes, clone forming ability and cell proliferation of corneal epithelial stem cells were observed by phase contrast microscope, Annexin V-PI and 8-OHdG DNA single strand break labeling. Effects of apoptosis on DNA stability. 2. Ten male C57BL/6 mice of the same age (normal control group) and 10 Ins2Akita mice (diabetic group) were selected to compare the difference of body weight and blood sugar level between the two groups. To observe and compare the repair of corneal epithelium at 0 h, 24 h, 48 h, 64 h, 66 h, 88 h and 100 h, 136172 h after injury, and to detect and analyze the morphological changes, apoptosis and DNA stability of corneal epithelium by TUNEL-8 OHdG staining under transmission electron microscope (TEM). Result 1. The results of cell model in vitro showed that the TKE2 cells treated with high glucose had long spindle shape, and the cell status became worse with the prolongation of high glucose treatment time, and the clone diameter of stem cells in high glucose group was smaller than that in high glucose group. The clone formation rate was significantly decreased (28.333 卤5.508 in normal group and 6.333 卤1.155 in high glucose treatment group, respectively). There were significant differences in proliferation ability between normal group and high glucose treatment group (P 72h ~ 0.013), and after high glucose treatment for 3 generations, the difference was more obvious (P 72 h ~ 0.000), and the percentage of early apoptotic cells was 8.02-3.14 in normal group and 33.13-2.63 in high glucose group. The 8-OHdG positive staining of cells treated with high glucose for 3 passages was significantly higher than that of normal cells. The weight gain of 2.Ins2Akita diabetic mice was significantly lower than that of normal mice during the 8-week detection cycle, and the blood glucose was maintained at a relatively stable high level. The healing rate of corneal epithelium in diabetic group was significantly delayed at 172 h after curettage, and the defect area was enlarged at 88 h after curettage. In diabetic mice, the microvilli and microfolds of corneal epithelial cells decreased significantly, and the basal epithelial cells arranged regularly, but the cell volume increased, the basal cells vacuolated obviously, and the desmosome connections were less. The half desmosome between corneal epithelium and basement membrane decreased significantly. The results showed that the corneal epithelial cells in diabetic mice were not smooth, irregular in shape, loose in intercellular junctions, decreased in the number of cell layers, unclear in boundary, and vacuoles formed in basal cells of corneal epithelium. The results of Tunel showed that the number of 8-OHdG positive cells in the basal layer of corneal epithelium was significantly higher than that in the control group. The results showed that the number of 8-OHdG staining positive cells in the basal layer of corneal epithelium was significantly higher than that in the control group. Conclusion 1. Under the condition of high glucose treatment in vitro, the morphology of corneal epithelial stem cells changed, the clone formation and proliferation of corneal epithelial stem cells were inhibited obviously, the number of early apoptotic cells increased obviously, and the stability of genome decreased. 2. The rate of corneal epithelium repair in Ins2Akita diabetic mice was significantly decreased and repeated exfoliation was observed. The morphological changes of corneal basal epithelial cells and the increase of corneal epithelial basal layer apoptosis and the decrease of DNA stability were observed in Ins2Akita diabetic mice.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R772.2

【参考文献】