CAJ | 학술논문

目的检测再生障碍性贫血(AA)患者骨髓间充质于细胞(BMSC)过氧化物酶体增殖物激活受体-γ(PPAR γ)的表达及其对BMSC成脂的影响,探讨AA患者骨髓脂肪化的发生机制.方法分离培养16例AA患者、20例白细胞减少症患者(对照组)BMSC,显微镜下观察其形态;用Westemblot检测AA患者BMSC内PPARγ的表达;将对照组BMSC分为三组:单纯诱导组(脂肪诱导培养液)、吡格列酮组(脂肪诱导培养液+ PPARγ配体吡格列酮)及GW9662组(脂肪诱导培养液+吡格列酮+ PPARγ拮抗剂GW9662),诱导成脂21 d后,油红O染色计数脂肪细胞分化率,并用酶联免疫吸附(ELISA)法检测细胞培养上清肿瘤坏死因子α(TNF-α)含量.结果连续传至第5代AA组BMSC出现脂肪细胞分化,而对照组BMSC传至第8代仍未出现脂肪细胞分化.AA患者BMSC内PPAR γ蛋白相对表达量为1.46±0.10,高于对照组的0.86±0.06(P＜ 0.05).对照组BMSC诱导成脂中吡格列酮组脂肪细胞分化率为(87.42±0.67)％,高于单纯诱导组的(44.69±2.61)％及GW9662组的(39.29±1.59)％(P＜0.05),后两组差异无统计学意义(P＞0.05);吡格列酮组细胞培养上清中TNF-α表达量为(95.04±3.41) pg/ml,高于单纯诱导组的(30.84±3.48) pg/ml及GW9662组的(31.43±3.51)pg/ml(均P＜ 0.05),后两组间差异无统计学意义(P＞0.05).结论 AA患者BMSC出现脂肪化明显,PPARγ在AA患者BMSC中表达增高,其拮抗剂GW9662可抑制BMSC向脂肪细胞分化,提示PPARγ参与了AA患者骨髓脂肪化的病理改变.
목적 검측재생장애성빈혈(AA)환자골수간충질우세포(BMSC)과양화물매체증식물격활수체-γ(PPAR γ)적표체급기대BMSC성지적영향,탐토AA환자골수지방화적발생궤제.방법 분리배양16례AA환자、20례백세포감소증환자(대조조)BMSC,현미경하관찰기형태;용Westemblot검측AA환자BMSC내PPARγ적표체;장대조조BMSC분위삼조:단순유도조(지방유도배양액)、필격렬동조(지방유도배양액+ PPARγ배체필격렬동)급GW9662조(지방유도배양액+필격렬동+ PPARγ길항제GW9662),유도성지21 d후,유홍O염색계수지방세포분화솔,병용매련면역흡부(ELISA)법검측세포배양상청종류배사인자α(TNF-α)함량.결과 련속전지제5대AA조BMSC출현지방세포분화,이대조조BMSC전지제8대잉미출현지방세포분화.AA환자BMSC내PPAR γ단백상대표체량위1.46±0.10,고우대조조적0.86±0.06(P＜ 0.05).대조조BMSC유도성지중필격렬동조지방세포분화솔위(87.42±0.67)％,고우단순유도조적(44.69±2.61)％급GW9662조적(39.29±1.59)％(P＜0.05),후량조차이무통계학의의(P＞0.05);필격렬동조세포배양상청중TNF-α표체량위(95.04±3.41) pg/ml,고우단순유도조적(30.84±3.48) pg/ml급GW9662조적(31.43±3.51)pg/ml(균P＜ 0.05),후량조간차이무통계학의의(P＞0.05).결론 AA환자BMSC출현지방화명현,PPARγ재AA환자BMSC중표체증고,기길항제GW9662가억제BMSC향지방세포분화,제시PPARγ삼여료AA환자골수지방화적병리개변.
Objective To detect the expression of peroxisome prnliferator-activated receptor-γ (PPARγ) in mesenchymal stem cells from patients with aplastic anemia (AA) and assess the influence of PPARγ on bone marrow mesenchymal stem cells (MSCs) differentiation into adipocytes,explore the mechanisms of fatty marrow replacement in AA patients.Methods BMSCs isolated from 16 AA patients and 20 leucopenia patients (as the control group) were cultured in medium and the morphological characteristics were observed by microscopy.Western blot was used to detect the expression of PPARγ in BMSCs.BMSCs from the control group were divided into three groups:pure induction group (BMSCs in adipogenic induction medium),pioglitazone group (BMSCs in adipogenic induction medium with pioglitazone (PPARγ ligand)) and GW9662 group (BMSCs in adipogenic induction medium with pioglitazone and GW9662 (PPARγinhibitor)).Three weeks later,neutral lipid vacuoles were stained with oil-red O.The differentiation rate of BMSCs towards adipocytes was counted under a microscope and TNF-α expression in differentiated cell culture supernatant was detected using ELISA in the three groups.Results Expanded up to 5 passages,BMSCs from AA patients differentiated to adipocytes,while BMSCs of the controls were unchanged until 8 passages.The level of PPARγ protein expression in BMSCs of AA patients were significantly higher than that of controls (1.46±0.10 vs 0.86±0.06,P ＜ 0.05).The differentiation rate of adipocytes from BMSCs in the pioglitazone group (87.42±0.67) ％ was higher than that in the pure induction group (44.69±2.61) ％ and the GW9662 group (39.29 ±1.59) ％ (P ＜ 0.05).No significant difference in the percentage of adiposities was observed between the pure induction group and the GW9662 group (P ＞ 0.05).The expression of TNF-α in cell culture supernatant of the pioglitazone group (95.04±3.41) pg/ml was higher than that of the pure induction group (30.84±3.48) pg/ml and the GW9662 group (31.43±3.51) pg/ml (P ＜ 0.05),while there was no difference between the pure induction group and the GW9662 group (P ＞ 0.05).Conclusions BMSCs from AA patients differentiated to adiposities obviously.PPARγ was highly expressed in AA patients and GW9662 inhibited the process of differentiation of BMSCs towards adipocytes.It suggest that PPARγ may play an important role in replacement of hematopoietically active marrow with adipocytes in AA.