本文選題：Ins2Akita糖尿病小鼠 + 角膜上皮干細胞　； 參考：《青島大學》2012年碩士論文

【摘要】：目的 利用角膜緣干細胞體外模型和Ins2Akita糖尿病小鼠模型,研究高糖環(huán)境對角膜緣上皮王細胞活性以及角膜上皮修復的影響。 方法 1.采用小鼠角膜緣上皮干／祖細胞系(TKE2)作為體外細胞模型,在培養(yǎng)基中添加終濃度為30mM的葡萄糖進行處理,模擬糖尿病角膜病變的高糖微環(huán)境,通過相差顯微鏡觀察、MTT、干細胞克隆形成、Annexin V-PI和8-OHdG (DNA單鏈斷裂標記)免疫染色等實驗,觀察高糖環(huán)境對角膜上皮干細胞形態(tài)變化、克隆形成能力、細胞增殖、凋亡、DNA穩(wěn)定性等方面的影響。 2.選擇同齡成年雄性C57BL/6小鼠(正常對照組)和Ins2Akita小鼠(糖尿病組)各10只,比較2組小鼠在體重、血糖水平的差異；刮除小鼠角膜中央2mm的上皮,觀察并比較角膜上皮在損傷后0h、24h、48h、64h、76h、88h、100h、136、172h的修復情況,并通過透射電鏡、HE一染色、TUNEL (凋亡)標記染色、8-OHdG對角膜上皮的形態(tài)改變、細胞凋亡及DNA穩(wěn)定性進行檢測分析。 結果 1.體外細胞模型的結果顯示,高糖處理后的TKE2細胞呈長梭形,隨著高糖處理時間的延長,細胞狀態(tài)變差；高糖組干細胞克隆直徑較小,克隆形成率明顯降低(正常組為28.333±5.508,高糖處理3代TKE2克隆形成率為6.333±1.155),比較有統(tǒng)計學意義(P0.05)；MTT檢測發(fā)現(xiàn),正常組和高糖處理1代細胞間增殖能力有差異(P72h=0.013),高糖處理3代后差異更為明顯(P72h=0.000)；細胞凋亡檢測示正常組早期凋亡細胞比例為8.02-3.14,高糖組早期凋亡細胞比例為33.13-2.63,差異有統(tǒng)計學意義(P0.001)；高糖處理3代后的細胞8-OHdG陽性染色較正常細胞明顯增強。 2.Ins2Akita糖尿病小鼠在8周的檢測周期內,其體重增加與正常小鼠相比明顯減少,血糖維持在相對穩(wěn)定的高水平；糖尿病組小鼠的角膜上皮愈合時問為刮除后172h,愈合速度較正常對照組(48h)明顯延遲,并且在88h出現(xiàn)缺損面積的擴大；電鏡觀察顯示,糖尿病小鼠角膜上皮細胞表面微絨毛和微皺褶明顯減少,角膜基底層上皮細胞排列尚規(guī)則,但細胞體積增大,基底細胞空泡化明顯,細胞問橋粒連接較少,角膜上皮與基底膜問的半橋粒明顯減少；HE..染色示糖尿病小鼠角膜上皮細胞表面不平整,形態(tài)不規(guī)則,細胞間連接疏松,細胞層數(shù)減少,分界不清,角膜上皮層基底細胞空泡形成,基質層明顯水腫；TUNEL結果示角膜上皮基底層細胞TUNEL標記染色較正常組明顯增多；8-OHdG的染色結果示角膜上皮基底層細胞8-OHdG標記染色陽性細胞較正常對照組明顯增多。 結論 1.體外高糖處理條件下,角膜上皮干細胞的形態(tài)發(fā)生改變,角膜上皮干細胞的克隆形成能力和增殖明顯受到抑制；早期凋亡細胞明顯增多；基因組穩(wěn)定性下降。 2. Ins2Akita糖尿病小鼠角膜上皮修復速度明顯減慢,并出現(xiàn)反復剝脫現(xiàn)象；角膜基底層的上皮細胞形態(tài)變化明顯、角膜上皮基底層細胞的凋亡增加及其DNA穩(wěn)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 鹵5.508 in normal group and 6.333 鹵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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R772.2

【參考文獻】

相關期刊論文 前2條

1 劉曉燕;朱學軍;;糖尿病性角膜神經(jīng)病變的研究進展[J];國際眼科雜志;2008年07期

2 季迅達;朱皓皓;李洪;許速;;2型糖尿病角膜知覺減退及其與眼表異常的相關性研究[J];眼視光學雜志;2007年02期

，

本文編號：1920075