高糖减弱肾组织干细胞条件培养液对缺氧损伤肾小管上皮细胞的修复作用

High glucose reduced the repair function of kidney stem cells conditional medium to the hypoxia-injured renal tubular epithelium cells

目的:评估高糖对肾组织干细胞(kidney stem cells,KSC)条件培养液修复缺氧损伤肾小管上皮细胞(renal tubular epithelium cells,RTEC)作用的影响。方法:分离肾乳头处的KSC,分别用正常糖浓度(简称"正糖",5.6 mmol/L)和高糖(30 mmol/L)培养基对KSC进行预处理后制备KSC条件培养液。建立大鼠RTEC缺氧/复氧模型,比较高糖与正糖刺激后KSC条件培养液对缺氧/复氧RTEC修复作用的差异。结果:(1)缺氧4 h/复氧2 h为RTEC缺氧/复氧模型的最佳时间。(2)缺氧后,RTEC早期凋亡率和晚期凋亡率均升高,采用KSC条件培养液干预12 h和24 h后,与缺氧/复氧对照组相比,正糖缺氧/复氧组和高糖缺氧/复氧组RTEC的凋亡率均明显降低(P<0.01)。正糖组和高糖组比较,干预24 h后,正糖组RTEC的总体凋亡率显著低于高糖组(P=0.02)。(3)缺氧后,RTEC上清液的乳酸脱氢酶(lactic dehydrogenase,LDH)和丙二醛(malondialdehyde,MDA)水平明显升高(P<0.01),超氧化物歧化酶(superoxide dismutase,SOD)水平明显降低(P<0.01)。采用KSC条件培养液干预12 h和24 h后,与缺氧/复氧对照组相比,正糖缺氧/复氧组和高糖缺氧/复氧组的LDH和MDA水平均显著降低(P<0.01),SOD水平均显著升高(P<0.01)。正糖组和高糖组比较,高糖组的LDH和MDA水平要显著高于正糖组(P<0.05),SOD水平要显著低于正糖组(P<0.01)。结论:KSC条件培养液对缺氧损伤的RTEC有修复作用,这种作用主要是通过减少氧化应激、抑制细胞凋亡来实现的,而经高糖预处理的KSC条件培养液的抗氧化应激、抗凋亡作用减弱。

Objective： To evaluate the impacts of high glucose on the repair function of kidney stem cells（ KSC） conditional medium to the hypoxia-injured renal tubular epithelium cells（ RTEC）. Methods： KSC were isolated from the renal papilla in 4-week-Sprague-Dawley rats. The KSC were pretreated in media with high glucose（ 30 mmol/L） or with normal glucose（ 5. 6 mmol/L）,respectively. The supernatants of the pre-treated KSC were collected as the conditional media. The hypoxia/reoxygenation（ H/R） model of rat RTEC was established using the NRK-52 E cell line. The effects of KSC conditional media on the H/R RTEC were investigated. Results：（ 1） The best H/R model of RTEC was established using hypoxia for 4 h and reoxygenation 2 h.（ 2） After hypoxia,the early and late cell apoptosis rates of the H/R RTEC were increased. The H/R RTEC were co-cultured with KSC conditional media for 12 h and 24 h,respectively. The H/R RTEC were co-cultured with DMEM/F12 as a control group. The cell apoptosis rate of H/R RTEC was lower after co-cultured with KSC conditional media（ P 〈0. 01）,and the cell apoptosis rate of H/R RTEC in high glucose group was much higher than that in normal glucose group after co-cultured 24 h（ P = 0. 02）.（ 3） After hypoxia,the lactic dehydrogenase（ LDH） and malondialdehyde（ MDA） levels of the H/R RTEC supernatant were increased,and the superoxide dismutase（ SOD） level decreased. The LDH and MDA levels were lower and the SOD level was higher after co-cultured with KSC conditional media for 12 h and 24 h,respectively（ P 〈0. 01）. The LDH and MDA levels of H/R RTEC supernatant were much higher in the high glucose group than in the normal glucose group（ P 〈0. 05）,and the SOD level of H/R RTEC supernatant was much lower in the high glucose group than in the normal glucose group（ P 〈0. 01）. Conclusion： KSC conditional media could repair the H/R injury of RTEC. The effects were mainly by inhibiting cell apoptosis,and reducing oxidative stress; the anti-cell apoptosis ability and the anti-oxidative stress capacity of the conditional medium were reduced after KSC were pre-treated with high glucose.