CAJ | 학술논문

目的观察嘌呤霉素(PAN)对足细胞凋亡率、足细胞分子CD2相关蛋白(CD2AP)和磷脂酰肌醇3-激酶(PI3 K) p85表达的影响,探讨CD2AP异常表达在足细胞凋亡中的意义.方法体外培养小鼠肾小球足细胞系,设立对照组及PAN刺激组,处理后8h、24 h和48 h观察细胞形态并摄像,采用流式细胞仪检测足细胞凋亡率,实时荧光定量PCR检测CD2AP mRNA的表达,Western blot法检测CD2AP和PI3K p85蛋白的表达,间接免疫荧光标记在共聚焦显微镜下观察CD2AP与PI3K p85的共定位分布.结果 PAN刺激后足细胞胞体缩小,出现足突回缩至逐渐消失,失去细胞间连接;与对照组比较,PAN组足细胞24 h和48 h凋亡率[(7.52±1.35)％、(17.09±2.53)％比(4.32±0.81)％、(6.81 ±1.34)％]均明显升高,差异均有统计学意义(=4.98、8.79,P均＜0.05);PAN刺激组CD2AP mRNA表达各时间点均较对照组明显降低(0.34 ±0.12、0.46±0.21、0.47 ±0.10比0.62±0.23、0.97±0.14、0.96±0.23),差异均有统计学意义(t=2.64、4.95、4.79,P均＜0.05);24 h和48 h CD2AP(0.36±0.12、0.27±0.15比0.64±0.23、0.51±0.20)与PI3K p85(0.61 ±0.20、0.48±0.20比0.97±0.31、0.84±0.35)蛋白表达均显著降低,差异均有统计学意义(=2.64、2.35、2.31、2.40,P均＜0.05);CD2AP在足细胞的分布由均匀细丝状分布于胞质足突及核内转为不连续粗颗粒状集中分布于核周,CD2AP与PI3K p85的共定位关系也明显异常.结论 PAN诱导足细胞凋亡过程中CD2AP和PI3Kp85表达及分布异常,说明CD2AP可能通过PI3K通路在足细胞的凋亡中起重要作用.
목적 관찰표령매소(PAN)대족세포조망솔、족세포분자CD2상관단백(CD2AP)화린지선기순3-격매(PI3 K) p85표체적영향,탐토CD2AP이상표체재족세포조망중적의의.방법 체외배양소서신소구족세포계,설립대조조급PAN자격조,처리후8h、24 h화48 h관찰세포형태병섭상,채용류식세포의검측족세포조망솔,실시형광정량PCR검측CD2AP mRNA적표체,Western blot법검측CD2AP화PI3K p85단백적표체,간접면역형광표기재공취초현미경하관찰CD2AP여PI3K p85적공정위분포.결과 PAN자격후족세포포체축소,출현족돌회축지축점소실,실거세포간련접;여대조조비교,PAN조족세포24 h화48 h조망솔[(7.52±1.35)％、(17.09±2.53)％비(4.32±0.81)％、(6.81 ±1.34)％]균명현승고,차이균유통계학의의(=4.98、8.79,P균＜0.05);PAN자격조CD2AP mRNA표체각시간점균교대조조명현강저(0.34 ±0.12、0.46±0.21、0.47 ±0.10비0.62±0.23、0.97±0.14、0.96±0.23),차이균유통계학의의(t=2.64、4.95、4.79,P균＜0.05);24 h화48 h CD2AP(0.36±0.12、0.27±0.15비0.64±0.23、0.51±0.20)여PI3K p85(0.61 ±0.20、0.48±0.20비0.97±0.31、0.84±0.35)단백표체균현저강저,차이균유통계학의의(=2.64、2.35、2.31、2.40,P균＜0.05);CD2AP재족세포적분포유균균세사상분포우포질족돌급핵내전위불련속조과립상집중분포우핵주,CD2AP여PI3K p85적공정위관계야명현이상.결론 PAN유도족세포조망과정중CD2AP화PI3Kp85표체급분포이상,설명CD2AP가능통과PI3K통로재족세포적조망중기중요작용.
Objective To observe the effect of puromycin aminonucleoside(PAN) on the expressions of CD2associated protein (CD2AP) and phosphatidylinositol 3-kinase (PI3 K) p85 in induced podocytes,and to explore the significance of abnormal expression of CD2AP in apoptosis of podocytes in vitro.Methods Mouse podocytes were injected into the control group and the PAN treatment group (PAN group).The cells were cultured for 8 h,24 h and 48 h respectively.The podocyte morphology was observed by phase-contrast microscope.The apoptosis ratio of podocytes was determined with flow cytometry.The mRNA expression of CD2AP was detected by real time fluorescent quantitative polymerase chain reaction.The protein expressions of CD2AP and PI3K p85 were detected by Western blot,respectively.The distributions and the relationships between CD2AP and PI3K p85 in the podocytes were detected by confocal fluorescence microscopy.Results PAN treatment led to significant shrinkage of podocytes with decreasing distribution tendency,podocyte foot process retraction as well as effacement and loss of cell contact.Compared with the control group,the apoptosis rate was increased at 24 h and 48 h[(7.52 ± 1.35)％,(17.09 ±2.53)％ vs (4.32 ±0.81％,(6.81 ± 1.34)％] in PAN group and the differences were significant (t =4.98,8.79,all P ＜ 0.05) ;CD2AP mRNA expression tended to decrease at each time point,and the differences were significant (0.34 ± 0.12,0.46 ±0.21,0.47 ± 0.10 vs 0.62 ± 0.23,0.97 ± 0.14,0.96 ± 0.23),and there were statistical differences (t =2.64,4.95,4.79,all P ＜ 0.05) ;CD2AP and PI3K p85 protein expressions tended to decrease at 24 h (0.36 ± 0.12,0.61 ± 0.20 vs 0.64 ±0.23,0.97 ±0.31) and 48 h(0.27 ± 0.15,0.48 ± 0.20 vs 0.51 ± 0.20,0.84 ± 0.35),and the differences were significant(t =2.64,2.31,2.35,2.40,all P ＜ 0.05).In the control group,the distribution of CD2AP was uniformly filamentary structure in cytoplasm and nucleus of podocytes,after PAN treatment the distribution of CD2AP changed to discontinuous coarse granular concentrated in the perinuclear.Immunocyte fluorescence showed that the distribution and the relationships between CD2AP and PI3K p85 in the podocytes became abnormal.Conclusions When the apoptosis of podocytes was induced by PAN,the expression and distribution of CD2AP were abnormal.CD2AP may play an important role in the survival of podocytes though the PI3K signaling pathway.