中华糖尿病杂志
中華糖尿病雜誌
중화당뇨병잡지
CHINES JOURNAL OF DLABETES MELLITUS
2015年
5期
316-320
,共5页
赵坤%郝好杰%臧丽%杨国庆%董亮%韩为东%母义明
趙坤%郝好傑%臧麗%楊國慶%董亮%韓為東%母義明
조곤%학호걸%장려%양국경%동량%한위동%모의명
间充质干细胞%糖脂毒性%线粒体%INS-1细胞
間充質榦細胞%糖脂毒性%線粒體%INS-1細胞
간충질간세포%당지독성%선립체%INS-1세포
Mesenchymal stem cells%Glucolipotoxicity%Mitochondria%Insulinoma-1 cells
目的:探讨骨髓间充质干细胞(BMSCs)对糖脂毒性诱导的大鼠胰岛素瘤细胞(INS?1)损伤的保护作用及其机制。方法 INS?1细胞分为正常对照组、高糖高脂组和高糖高脂+BMSCs组。高糖高脂+BMSCs组的INS?1细胞使用高糖高脂(16.7 mmol/L葡萄糖、0.4 mmol/L棕榈酸)培养基处理48 h后,与BMSCs共培养24 h,进行检测:(1)CCK8法检测INS?1细胞存活率;(2)Western blotting检测细胞内裂解的半胱氨酸?天冬氨酸蛋白酶(Cleaved Caspase?3)的表达;(3)膜连蛋白V/碘化丙啶(Annexin V/PI)检测细胞凋亡率;(4)放免法检测胰岛素分泌功能;(5) JC?1荧光探针检测线粒体膜电位(MMP);(6)二氯荧光素双醋酸盐(DCFH?DA)检测细胞内活性氧(ROS)的生成量。两组间比较采用t检验进行统计学分析。结果与正常对照组比较,高糖高脂组INS?1细胞存活率明显下降[(100.0%±0.8%)比(71.9%±3.2%), t=8.46, P<0.01],Cleaved Caspase?3表达量增加(0.248±0.033比1.01±0.066,t=10.28,P<0.01),凋亡细胞比例增加[(6.9%±0.4%)比(16.4%±1.2%), t=17.38, P<0.01],基础胰岛素分泌量(BIS)与葡萄糖刺激的胰岛素分泌量(GSIS)均减少(t=5.745、13.559,均P<0.05)。与高糖高脂组相比,高糖高脂+BMSCs组INS?1细胞的存活率上升,Cleaved Caspase?3表达量和凋亡细胞比例均减少(t=3.98、5.16、13.08,均P<0.05),BIS及GSIS均显著增加(t=5.674、10.148,均P<0.05)。高糖高脂损伤INS?1细胞内线粒体功能,表现为线粒体膜电位去极化(96±8比46±4, t=5.239, P<0.05)、ROS生成量较对照组明显增加[(13.3%±0.9%)比(34.2%±0.8%), t=17.38, P<0.01],而与高糖高脂组相比,BMSCs共培养有效促进线粒体膜电位恢复,并减少细胞内ROS含量[分别为46±4比87±14,(34.2%±0.8%)比(22.5%±0.4%),t=2.822、13.080,均P<0.05],改善了高糖高脂损伤的INS?1细胞的线粒体功能。结论 BMSCs可有效保护糖脂毒性损伤的INS?1细胞,其作用机制可能与BMSCs改善线粒体功能有关。
目的:探討骨髓間充質榦細胞(BMSCs)對糖脂毒性誘導的大鼠胰島素瘤細胞(INS?1)損傷的保護作用及其機製。方法 INS?1細胞分為正常對照組、高糖高脂組和高糖高脂+BMSCs組。高糖高脂+BMSCs組的INS?1細胞使用高糖高脂(16.7 mmol/L葡萄糖、0.4 mmol/L棕櫚痠)培養基處理48 h後,與BMSCs共培養24 h,進行檢測:(1)CCK8法檢測INS?1細胞存活率;(2)Western blotting檢測細胞內裂解的半胱氨痠?天鼕氨痠蛋白酶(Cleaved Caspase?3)的錶達;(3)膜連蛋白V/碘化丙啶(Annexin V/PI)檢測細胞凋亡率;(4)放免法檢測胰島素分泌功能;(5) JC?1熒光探針檢測線粒體膜電位(MMP);(6)二氯熒光素雙醋痠鹽(DCFH?DA)檢測細胞內活性氧(ROS)的生成量。兩組間比較採用t檢驗進行統計學分析。結果與正常對照組比較,高糖高脂組INS?1細胞存活率明顯下降[(100.0%±0.8%)比(71.9%±3.2%), t=8.46, P<0.01],Cleaved Caspase?3錶達量增加(0.248±0.033比1.01±0.066,t=10.28,P<0.01),凋亡細胞比例增加[(6.9%±0.4%)比(16.4%±1.2%), t=17.38, P<0.01],基礎胰島素分泌量(BIS)與葡萄糖刺激的胰島素分泌量(GSIS)均減少(t=5.745、13.559,均P<0.05)。與高糖高脂組相比,高糖高脂+BMSCs組INS?1細胞的存活率上升,Cleaved Caspase?3錶達量和凋亡細胞比例均減少(t=3.98、5.16、13.08,均P<0.05),BIS及GSIS均顯著增加(t=5.674、10.148,均P<0.05)。高糖高脂損傷INS?1細胞內線粒體功能,錶現為線粒體膜電位去極化(96±8比46±4, t=5.239, P<0.05)、ROS生成量較對照組明顯增加[(13.3%±0.9%)比(34.2%±0.8%), t=17.38, P<0.01],而與高糖高脂組相比,BMSCs共培養有效促進線粒體膜電位恢複,併減少細胞內ROS含量[分彆為46±4比87±14,(34.2%±0.8%)比(22.5%±0.4%),t=2.822、13.080,均P<0.05],改善瞭高糖高脂損傷的INS?1細胞的線粒體功能。結論 BMSCs可有效保護糖脂毒性損傷的INS?1細胞,其作用機製可能與BMSCs改善線粒體功能有關。
목적:탐토골수간충질간세포(BMSCs)대당지독성유도적대서이도소류세포(INS?1)손상적보호작용급기궤제。방법 INS?1세포분위정상대조조、고당고지조화고당고지+BMSCs조。고당고지+BMSCs조적INS?1세포사용고당고지(16.7 mmol/L포도당、0.4 mmol/L종려산)배양기처리48 h후,여BMSCs공배양24 h,진행검측:(1)CCK8법검측INS?1세포존활솔;(2)Western blotting검측세포내렬해적반광안산?천동안산단백매(Cleaved Caspase?3)적표체;(3)막련단백V/전화병정(Annexin V/PI)검측세포조망솔;(4)방면법검측이도소분비공능;(5) JC?1형광탐침검측선립체막전위(MMP);(6)이록형광소쌍작산염(DCFH?DA)검측세포내활성양(ROS)적생성량。량조간비교채용t검험진행통계학분석。결과여정상대조조비교,고당고지조INS?1세포존활솔명현하강[(100.0%±0.8%)비(71.9%±3.2%), t=8.46, P<0.01],Cleaved Caspase?3표체량증가(0.248±0.033비1.01±0.066,t=10.28,P<0.01),조망세포비례증가[(6.9%±0.4%)비(16.4%±1.2%), t=17.38, P<0.01],기출이도소분비량(BIS)여포도당자격적이도소분비량(GSIS)균감소(t=5.745、13.559,균P<0.05)。여고당고지조상비,고당고지+BMSCs조INS?1세포적존활솔상승,Cleaved Caspase?3표체량화조망세포비례균감소(t=3.98、5.16、13.08,균P<0.05),BIS급GSIS균현저증가(t=5.674、10.148,균P<0.05)。고당고지손상INS?1세포내선립체공능,표현위선립체막전위거겁화(96±8비46±4, t=5.239, P<0.05)、ROS생성량교대조조명현증가[(13.3%±0.9%)비(34.2%±0.8%), t=17.38, P<0.01],이여고당고지조상비,BMSCs공배양유효촉진선립체막전위회복,병감소세포내ROS함량[분별위46±4비87±14,(34.2%±0.8%)비(22.5%±0.4%),t=2.822、13.080,균P<0.05],개선료고당고지손상적INS?1세포적선립체공능。결론 BMSCs가유효보호당지독성손상적INS?1세포,기작용궤제가능여BMSCs개선선립체공능유관。
Objective To investigate the protective roles of bone marrow?derived mesenchymal stem cells in glucolipotoxicity-induced INS-1 cell damage and the possible mechanisms involved. Methods INS?1 cells were divided into three groups according to different treatment, control group, high glucose group/PA group and high glucose/PA with BMSCs co?culture group. INS-1 cells in high glucose/PA with BMSCs co?culture group were exposured to the culture medium containing 16.7 mmol/L glucose and 0.4 mmol/L palmitic acid (PA) for 48 hours, and then co?cultured with BMSCs for another 24 hours. The viability of INS?1 cells was assessed by CCK8 assay. The expression of Cleaved cysteine asparate pro?tease?3 (Caspase?3) were detected by western blot. The cell apoptosis incidence was measured using Annexin V/propidium iodinate (PI) staining by a flow cytometer. The insulin secretion of INS?1 cells were measured using a Ratio Immunity Assay (RIA) kit. The mitochondrial membrane potential (MMP) was detected with JC?1 probe by a flow cytometer. Intracellular reactive oxygen species (ROS) production was detected using dichlorofluoresceindiacetate (DCFH?DA) and quantified by a flow cytometer.Comparisons between two groups were measured using Student′s t?test. Results Compared with control group, there were a significant decrease of INS?1 cell viability((100.0%±0.8%) vs (71.9%±3.2%), t=8.46, P<0.01), upregulated expression of Cleaved Caspase?3 (0.25 ± 0.03 vs 1.01 ± 0.07, t=10.28, P<0.01) and an increase of apoptosis incidence ((6.9%± 0.4%)vs (16.4%± 1.2%), t=17.38, P<0.01), combined with reduced basal insulin secretion (BIS) and glucose stimulated insulin secretion (GSIS) (t=5.745,13.559, P<0.01) in high glucose/PA group. BMSCs co?culture improved the viability of INS?1 cells, down?regulated the expression of Cleaved Caspase?3, reduced the apoptosis incidence t=3.98,5.16,13.08, P<0.05), and significantly increased the BIS((80.743±0.012) vs (109.67±1.058) ng/mg, t=5.674, P<0.05)and GSIS ((120.0±1.3) vs (231.2±1.6)ng/mg protein, t=10.148, P<0.01), compared with chronic high glucose/PA?treated INS?1 cells. Additionally, mitochondrial function was impaired according to the glucolipotoxicity?induced mitochondrial depolarization (95.7 ± 8.5 vs 45.9 ± 4.2,t=5.239,P<0.05) and considerable ROS accumulation ((13.3%± 0.9%)vs(34.2%± 0.8%), t=17.38, P<0.01). However, compared with chronic high glucose/PA-treated INS-1 cells, BMSCs treatment effectively reversed glucolipotoxicity?induced depolarization (46±4 vs 87±14,t=2.822, P<0.05) and reduced the cellular ROS level ((34.3%±0.8%) vs (22.5%±0.4%), t=13.08, P<0.01), indicating that BMSCs could recover the injury of the mitochondrial function. Conclusion BMSCs played an important cyto?protective role in glucolipotoxicity?induced INS?1 cell damage, and the possible mechanism underlying this effect might be associated with the improvement of mitochondrial function.