中华神经医学杂志
中華神經醫學雜誌
중화신경의학잡지
CHINESE JOURNAL OF NEUROMEDICINE
2013年
8期
757-762
,共6页
汪鑫%刘天助%郭洪波%宁瑜%廖奕宏%钟文宏%黄洁棱%颜晓慧
汪鑫%劉天助%郭洪波%寧瑜%廖奕宏%鐘文宏%黃潔稜%顏曉慧
왕흠%류천조%곽홍파%저유%료혁굉%종문굉%황길릉%안효혜
Beclin 1%自噬%神经胶质瘤%耐药%替莫唑胺
Beclin 1%自噬%神經膠質瘤%耐藥%替莫唑胺
Beclin 1%자서%신경효질류%내약%체막서알
Beclin 1%Autophagy%Glioma%Drug-resistance%Temozolomide
目的 分析Beclin 1与临床胶质瘤病理等级和胶质瘤复发间的关系,探讨其在胶质瘤耐药中的作用和机制. 方法 收集南方医科大学珠江医院和南方医院神经外科自2012年4月至2013年4月手术切除的胶质瘤标本53例(WHO分级Ⅰ级7例,复发2例；Ⅱ级9例,复发4例；Ⅲ级11例,复发6例；Ⅳ级9例,复发5例),免疫组化和Western blotting检测胶质瘤标本中Beclin1的表达；体外培养胶质瘤耐药细胞系U251AR和非耐药U251细胞,Western blotting检测细胞中Beclin 1和耐药蛋白P-gP的表达；Western blotting检测双链小分子Beclin 1干扰RNA(siBeclin l)转染U251AR细胞48h后Beclin 1蛋白的表达;CCK8法检测不同浓度替莫唑胺(TMZ)作用U251、U251AR和U251AR-siBeclin 1后细胞活性；Western blotting检测200μmol/L TMZ作用U251、U251AR和U251AR-siBeclin 1 48 h后Capase-3的表达；吖啶橙染色和Western blotting分别检测干扰Beclin 1表达和HCQ对U251AR细胞自噬小体数量和Capase-3表达的影响. 结果 免疫组化染色结果显示Ⅲ、Ⅳ级胶质瘤中Beclin 1阳性细胞数明显高于Ⅰ、Ⅱ级胶质瘤.Western blotting结果显示Ⅳ级、Ⅱ和Ⅲ级、Ⅰ级中Beclin 1的表达依次降低,Ⅲ、Ⅳ级复发肿瘤Beclin 1的表达量明显高于原发肿瘤,差异有统计学意义(P＜0.05); U251AR中Beclin 1和耐药蛋白P-gP的表达量均高于U251细胞.与干扰对照组和空白对照组比较,转染siBeclin 1 48 h后干扰组Beclin 1蛋白的水平明显下降；在含TMZ(50,100,500和1000 μmol/L)培养基中培养24 h后,U251AR细胞存活率明显高于U251和si-Bec1-U251AR细胞,差异有统计学意义(P＜0.05).200 μmol/L TMZ作用48 h后,U251细胞凋亡蛋白Caspase-3的表达量明显高于U251AR和si-Bec l-U251AR细胞.吖啶橙染色显示在TMZ培养U251AR 48 h后,HCQ和Beclin 1干扰都导致自噬小体形成明显减少,Caspase-3的表达增高,差异有统计学意义(P＜0.05). 结论 Beclin 1与胶质瘤恶性程度、复发和耐药相关,可能用于胶质瘤恶性诊断和预后评估.Beclin 1在胶质瘤中的耐药功能可能是通过自噬实现的,联合运用TMZ和自噬抑制剂或Beclin 1干扰剂有助于胶质瘤的治疗.
目的 分析Beclin 1與臨床膠質瘤病理等級和膠質瘤複髮間的關繫,探討其在膠質瘤耐藥中的作用和機製. 方法 收集南方醫科大學珠江醫院和南方醫院神經外科自2012年4月至2013年4月手術切除的膠質瘤標本53例(WHO分級Ⅰ級7例,複髮2例；Ⅱ級9例,複髮4例；Ⅲ級11例,複髮6例；Ⅳ級9例,複髮5例),免疫組化和Western blotting檢測膠質瘤標本中Beclin1的錶達；體外培養膠質瘤耐藥細胞繫U251AR和非耐藥U251細胞,Western blotting檢測細胞中Beclin 1和耐藥蛋白P-gP的錶達；Western blotting檢測雙鏈小分子Beclin 1榦擾RNA(siBeclin l)轉染U251AR細胞48h後Beclin 1蛋白的錶達;CCK8法檢測不同濃度替莫唑胺(TMZ)作用U251、U251AR和U251AR-siBeclin 1後細胞活性；Western blotting檢測200μmol/L TMZ作用U251、U251AR和U251AR-siBeclin 1 48 h後Capase-3的錶達；吖啶橙染色和Western blotting分彆檢測榦擾Beclin 1錶達和HCQ對U251AR細胞自噬小體數量和Capase-3錶達的影響. 結果 免疫組化染色結果顯示Ⅲ、Ⅳ級膠質瘤中Beclin 1暘性細胞數明顯高于Ⅰ、Ⅱ級膠質瘤.Western blotting結果顯示Ⅳ級、Ⅱ和Ⅲ級、Ⅰ級中Beclin 1的錶達依次降低,Ⅲ、Ⅳ級複髮腫瘤Beclin 1的錶達量明顯高于原髮腫瘤,差異有統計學意義(P＜0.05); U251AR中Beclin 1和耐藥蛋白P-gP的錶達量均高于U251細胞.與榦擾對照組和空白對照組比較,轉染siBeclin 1 48 h後榦擾組Beclin 1蛋白的水平明顯下降；在含TMZ(50,100,500和1000 μmol/L)培養基中培養24 h後,U251AR細胞存活率明顯高于U251和si-Bec1-U251AR細胞,差異有統計學意義(P＜0.05).200 μmol/L TMZ作用48 h後,U251細胞凋亡蛋白Caspase-3的錶達量明顯高于U251AR和si-Bec l-U251AR細胞.吖啶橙染色顯示在TMZ培養U251AR 48 h後,HCQ和Beclin 1榦擾都導緻自噬小體形成明顯減少,Caspase-3的錶達增高,差異有統計學意義(P＜0.05). 結論 Beclin 1與膠質瘤噁性程度、複髮和耐藥相關,可能用于膠質瘤噁性診斷和預後評估.Beclin 1在膠質瘤中的耐藥功能可能是通過自噬實現的,聯閤運用TMZ和自噬抑製劑或Beclin 1榦擾劑有助于膠質瘤的治療.
목적 분석Beclin 1여림상효질류병리등급화효질류복발간적관계,탐토기재효질류내약중적작용화궤제. 방법 수집남방의과대학주강의원화남방의원신경외과자2012년4월지2013년4월수술절제적효질류표본53례(WHO분급Ⅰ급7례,복발2례；Ⅱ급9례,복발4례；Ⅲ급11례,복발6례；Ⅳ급9례,복발5례),면역조화화Western blotting검측효질류표본중Beclin1적표체；체외배양효질류내약세포계U251AR화비내약U251세포,Western blotting검측세포중Beclin 1화내약단백P-gP적표체；Western blotting검측쌍련소분자Beclin 1간우RNA(siBeclin l)전염U251AR세포48h후Beclin 1단백적표체;CCK8법검측불동농도체막서알(TMZ)작용U251、U251AR화U251AR-siBeclin 1후세포활성；Western blotting검측200μmol/L TMZ작용U251、U251AR화U251AR-siBeclin 1 48 h후Capase-3적표체；아정등염색화Western blotting분별검측간우Beclin 1표체화HCQ대U251AR세포자서소체수량화Capase-3표체적영향. 결과 면역조화염색결과현시Ⅲ、Ⅳ급효질류중Beclin 1양성세포수명현고우Ⅰ、Ⅱ급효질류.Western blotting결과현시Ⅳ급、Ⅱ화Ⅲ급、Ⅰ급중Beclin 1적표체의차강저,Ⅲ、Ⅳ급복발종류Beclin 1적표체량명현고우원발종류,차이유통계학의의(P＜0.05); U251AR중Beclin 1화내약단백P-gP적표체량균고우U251세포.여간우대조조화공백대조조비교,전염siBeclin 1 48 h후간우조Beclin 1단백적수평명현하강；재함TMZ(50,100,500화1000 μmol/L)배양기중배양24 h후,U251AR세포존활솔명현고우U251화si-Bec1-U251AR세포,차이유통계학의의(P＜0.05).200 μmol/L TMZ작용48 h후,U251세포조망단백Caspase-3적표체량명현고우U251AR화si-Bec l-U251AR세포.아정등염색현시재TMZ배양U251AR 48 h후,HCQ화Beclin 1간우도도치자서소체형성명현감소,Caspase-3적표체증고,차이유통계학의의(P＜0.05). 결론 Beclin 1여효질류악성정도、복발화내약상관,가능용우효질류악성진단화예후평고.Beclin 1재효질류중적내약공능가능시통과자서실현적,연합운용TMZ화자서억제제혹Beclin 1간우제유조우효질류적치료.
Objective To analyze the relations of Beclin 1 with both clinical pathological grades of gliomas and glioma recurrence,and investigate its function and mechanism in drug-resistance of gliomas.Methods Fifty-three glioma specimens (WHO grade Ⅰ in 7,2 cases of recurrence; Ⅱ grade in 9,4 cases of recurrence; Ⅲ grade in 11,6 cases of recurrence; Ⅳ grade in 9,5 cases of recurrence),colleted in our hospitals from April 2012 to April 2013,were chosen in our study.The Beclin 1 expressions in these gliomas specimens were detected by immunohistochemistry and Westem blotting;drug resistant cell line U251AR and non-drug resistant cell line U251were cultured,and Beclin 1 and resistance protein P-gP expressions in these cell lines were detected by Western blotting; double-stranded small molecule Beclin 1 interfering RNA (siBeclin 1) was transfected into the U251AR cells and the Beclin 1 protein expression 48 h after the transfection was detected by Western blotting; the activity of U251,U251AR and U251AR-siBeclin 1 cells was detected by CCK8 after being treated with different concentrations of temozolomide (TMZ); the capase-3 expression in the U251,U251AR and U251AR-siBeclin 1 cells 48 h after being treated by 20 μm TMZ was detected by Western blotting; the capase-3 expression and the number of autophagosome in the U251AR cells after Beclin 1 being knocked down or after being treated with hydroxychloroquine (HCQ) were detected by Western blotting and acridine orange staining,respectively.Results The level of Beclin 1 was proportional to the grades of gliomas.The Beclin 1 level in recurrent gliomas of grade Ⅲ and Ⅳ was higher than that in primary tumors of grade Ⅰ and Ⅱ; the Beclin 1 and P-glycoprotein (P-gP) expression in the U251AR cells were signficantly higher than those in the U251 cells (P＜0.05).As compared with interfered control group and blank control group,the interfered group had decreased Beclin 1 protein level 48 h after siBeclin 1 transfection; U251AR cell survival rate was significantly higher than that of U251 and si-Bec1-U251AR cells 24 h after being treated with TMZ (50,100,500 and 1000 μmol/L); 48 h after being treated with 200 μmol/L TMZ,the caspase-3 expression in the U251 cells was significantly higher than that in U251AR and si-Bec1-U251AR cells; both HCQ and Beclin 1 interference can induce decreased formation of autophagosome and increased caspase-3 expression after U251AR cells being treated by TMZ for 48 h (P＜0.05).Conclusion The level ofBeclin 1 is correlated to grades,recurrence and drug resistance of gliomas,which may be used for malignancy diagnosis and prognostic evaluation; the drug-resistance role of Beclin 1 may stem from autophagy,and combined use of temozolomide and inhibitors of autophagy or Beclin 1 disrupters may help the therapy.
