角膜缘干细胞培养上清液对血管内皮细胞增殖的影响

Effect of limbal stem cell culture medium on vascular endothelial cell proliferation

摘要:

目的：观察角膜缘干细胞培养液对血管内皮细胞增殖的影响。

方法：分别培养角膜缘干细胞及球结膜上皮细胞，并通过免疫组化检测AE-5蛋白鉴别角膜缘干细胞。搜集两组培养细胞的上清液加入培养的人脐静脉血管内皮细胞中，并设立对照组。培养24h后通过MTT法检测细胞增殖情况并进行统计学分析。

结果：角膜缘干细胞AE-5染色呈阴性，而球结膜上皮细胞为阳性。加入角膜缘干细球结膜上皮细胞和对照组培养液的血管内皮细胞MTT值分别为2.097±0.079，1.981±0.034和1.990±0.044。三组间有显著性差异(F=9.169，P=0.000)。加入角膜缘干细胞培养上清液的血管内皮细胞增殖活性明显高于其他两组(P=0.005和P=0.007)。

结论：角膜缘干细胞培养液能够促进血管内皮细胞的增殖，这可能是角膜缘干细胞的特征。本研究从功能学角度为角膜缘干细胞理论提供更多的依据。

Abstract:

AIM: To investigate the effect of cultured limbal stem cells on proliferating of vascular endothelial cells and validate the theory of limbal stem cells.

METHODS: The limbal stem cells and epithelium cells of bulbar conjunctiva were cultured and determined by immunohistochemistry staining with AE-5. The supernatant was collected and added into the cultured human umbilical vein endothelium cells(ECV-304). The negative control was set up. After 24h, MTT was done to detect endothelium cell proliferation. The statistical analysis was done by analysis of Variance.

RESULTS: Negative stain of AE-5 can be seen in limbal stem cells and positive stain can be seen in conjunctiva cells. The ratio of MTT added by limbal stem cell supernatant, conjunctiva epithelium cell and negative control were 2.097±0.079, 1.981±0.034 and 1.990±0.044, respectively. There were significant difference among the three groups(F=9.169, P=0.000). The proliferate activity of vascular endothelial cells added by supernatant of limbal stem cells was higher than the other two groups(P=0.005 and P=0.007, respectively).

CONCLUSION:The supernatant of limbal stem cells can improve the proliferation of vascular endothelium cell. This may be the unique characteristics of limbal stem cells. This study provides more evidences for the theory of limbal stem cells by determining the functional methods.