目的：建立人骨髓间充质干细胞(hMSCs)与视网膜色素上皮细胞(RPE)低氧高糖培养的细胞共培养模型，研究在与hMSCs共培养条件下低氧及高糖时RPE细胞增殖、凋亡和移行等生物学特征的影响，对糖尿病刺激脉络膜新生血管(CNV)的机制进行初步探讨。

方法：使用Transwell间接共培养模型。将共培养模型按照培养氧浓度及糖浓度培养基类型分为4组：常氧常糖组(21% O₂加5.56mmol/L葡萄糖培养液，A组)、常氧高糖组(21% O₂加30mmol/L葡萄糖培养液，B组)、低氧常糖组(5% O₂加5.56mmol/L葡萄糖培养液，C组)和低氧高糖组(5% O₂加30mmol/L葡萄糖培养液，D组)。通过CCK-8检测间接共培养模型在12，24，48h时RPE细胞的增殖能力、Transwell检测间接共培养模型在24h时RPE细胞的移行情况、流式细胞仪检测间接共培养模型中24h时RPE细胞的凋亡情况。

结果：与对照组相比，12，24和48h时，B组(1.61±0.41，1.80±0.34，1.91±0.35)、C组(1.34±0.46，1.94±0.40，2.14±0.41)及D组(1.98±0.47，2.26±0.42，2.55±0.40)中的细胞增殖能力均较对照组(0.92±0.45，1.27±0.32，1.59±0.41)增强(P<0.05)，并且低氧高糖联合作用组增殖能力最强。在24h时，B组(149.5±9.19)、C组(140±9.90)、D组(170.5±7.78)较对照组(114.5±7.78)的RPE细胞移行能力增强(P<0.05)。而在24h时各组间的凋亡情况无明显差异(P>0.05)。

结论：与hMSCs共培养条件下，低氧及高糖环境促进RPE细胞的增殖和移行，而对其凋亡并无影响。

AIM:To evaluate cell proliferation, apoptosis and migration of human retinal pigment epithelial cells(RPE)when co-cultured with human marrow mesenchymal stem cells(hMSCs)in condition of hypoxia and hyperglycemia so as to explore possible mechanisms of diabetes aggravating choroidal neovascularization(CNV)preliminarily.

METHODS:Both hMSCs and RPE cells were co-cultured in a transwell system. The experiment was divided into four groups: 21% O₂ with 5.56mmol/L glucose(control group, A), 21% O₂ with 30mmol/L glucose(hyperglycemia and normoxia group, B), 5% O₂ with 5.56mmol/L glucose(normoglycemia and hypoxia group, C)and 5% O₂ with 30mmol/L glucose(hyperglycemia and hypoxia group, D). Cell Counting Kit-8(CCK-8)was used to detect the proliferation of RPE cells in each group at 12, 24 and 48h respectively. Flow cytometry was performed to observe apoptosis of RPE cells at 24h. Additionally, we assessed migration capabilities of RPE via transwell assay under the condition of hyperglycemia and hypoxia by co-culturing of hMSCs.

RESULTS:In this co-culturing system, at 12, 24 and 48h, group B(1.61±0.41, 1.80±0.34; 1.91±0.35), C(1.34±0.46, 1.94±0.40, 2.14±0.41)and D(1.98±0.47, 2.26±0.42, 2.55±0.40)showed significantly higher proliferation rate than group A(0.92±0.45, 1.27±0.32, 1.59±0.41, P<0.05). The migration capabilities of RPE in group B(149.5±9.19), C(140±9.90)and D(170.5±7.78)increased dramatically compared with group A(114.5±7.78, P<0.05)at 24h, whereas there was no significant difference of apoptosis ratio among four groups(P>0.05).

CONCLUSION:By coexistence with hMSCs, the synergy of hyperglycemia and hypoxia can improve migration and proliferation of RPE cells, and have no effect on apoptosis of RPE cells within short period.

国家自然科学基金(No. 81300770); 国家重点基础研究发展计划资助(No.2011CB510200)