地塞米松对支气管哮喘急性发作患者外周血T淋巴细胞亚群自噬的影响

目的 本文拟研究地塞米松对哮喘急性发作患者外周血T淋巴细胞亚群自噬的影响.方法 分离哮喘组及健康者外周血T淋巴细胞亚群(CD+4 T,CD+8 T和 CD+4CD+25 T 细胞),分别与地塞米松(10-5 mol·L-1)共培养.首先以电子显微镜及荧光显微镜观察培养后细胞的自噬形态学改变;然后丹(磺)酰戊二胺(MDC)染色后,以流式细胞术检测上述细胞的自噬水平及CD+4CD+25 T细胞的Foxp3表达.结果 ①镜下可观察到与地塞米松共培养后细胞的典型自噬形态学改变;②地塞米松可以上调哮喘组外周血CD+4 T和 CD+4CD+25 T 细胞的自噬率(P<0.05),自噬水平的升高均呈时间依赖性 (P<0.05);③哮喘组CD+4CD+25 T细胞的Foxp3表达显著低于健康对照组(P<0.05),但地塞米松预处理对Foxp3的表达无影响(P>0.05).结论 地塞米松诱导哮喘急性发作患者外周血T淋巴细胞亚群自噬水平的增高可能是糖皮质激素治疗哮喘的作用机制之一.     

自噬在高血压足细胞损伤中的作用

肾脏可表达肾素-血管紧张素系统(RAS)所有的组成成分,其中血管紧张素Ⅱ(AngⅡ)是主要的生物活性物质。高血压时,肾脏AngⅡ明显升高,使得足细胞长期处在一种高水平AngⅡ环境中。AngⅡ可诱导自噬,但是目前尚不清楚这种诱导效应在高血压足细胞损伤中起到什么作用。通过了解原发性高血压、AngⅡ、足细胞自噬三者的关系,我们可以进一步分析自噬在高血压足细胞损伤中的作用。     

姜黄素激活自噬对非酒精性脂肪性肝病大鼠线粒体途径凋亡的影响

目的探讨姜黄素对非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)大鼠的治疗作用及机制。方法油红O染色观察各组大鼠肝内细胞脂滴分布,透射电镜观察肝组织中自噬泡形成情况。蛋白印迹检测P62、微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、自噬相关蛋白(Beclin-1)和B淋巴细胞瘤-2(Bcl-2)、相关x蛋白(Bax)、线粒体内细胞色素C(mCytc)、半胱氨酸蛋白酶(Caspase-3、Caspase-9)标记蛋白的表达水平。结果与正常对照组比较,肝组织脂质沉积在模型组中明显增加;姜黄素明显抑制模型组大鼠肝组织脂质沉积,自噬抑制剂削弱姜黄素的治疗作用;透射电镜观察发现正常对照组可见散在自噬泡,模型组自噬泡数量显著减少,姜黄素可明显增加自噬体数量。蛋白印迹结果提示,模型组大鼠肝组织中Bax、Caspase-3、Caspase-9、P62蛋白表达均升高,而mCytc、Bcl-2、Beclin-1、LC3Ⅱ/LC3Ⅰ均降低;经姜黄素治疗后肝组织中Bax、P62、Caspase-3、Caspase-9蛋白表达均降低,而mCytc、Bcl-2、Beclin-1、LC3Ⅱ/LC3Ⅰ升高;自噬抑制剂可以部分抑制姜黄素对自噬和凋亡相关蛋白的影响。结论姜黄素对NAFLD大鼠模型的抗凋亡作用与诱导自噬有关。     

ATF3转录调控HSP110对低氧诱导的肺动脉平滑肌细胞自噬和增殖的影响

目的 探讨ATF3调控HSP110表达对低氧刺激下人肺动脉平滑肌细胞增殖及自噬的影响。 方法 体外培养人肺动脉平滑肌细胞，单独感染ATF3沉默腺病毒或共感染HSP110过表达腺病毒，48 h后加入自噬激活剂雷帕霉素预处理3 h，建立缺氧细胞模型，24 h后，CCK-8法检测细胞增殖能力，流式细胞术分析细胞凋亡，Real-time PCR检测ATF3、HSP110 mRNA的表达，Western blot检测ATF3、HSP110、LC3Ⅱ/Ⅰ、Beclin-1、p62蛋白的表达，免疫荧光观察LC3斑点形成，荧光素酶报告系统鉴定ATF3对HSP110的调控作用。 结果 缺氧组ATF3和HSP110的表达显著增加（P<0.01），ATF3沉默后，细胞增殖能力下降，凋亡率增加（P<0.01），同时自噬蛋白LC3 Ⅱ/Ⅰ、Beclin-1的表达及LC3荧光斑点显著减少(P<0.01)，自噬降解底物p62的表达明显增加(P<0.01)，而加入雷帕霉素或共感染HSP110过表达腺病毒后，ATF3沉默对细胞增殖和自噬的抑制作用显著减弱(P<0.01)。双荧光素酶报告基因检测结果显示，ATF3可上调HSP110启动子活性。 结论 ATF3调控HSP110表达增加而促进人肺动脉平滑肌细胞增殖，其机制可能与介导细胞自噬有关。     

血管紧张素Ⅱ诱发自噬对血管平滑肌细胞表型转换的调控作用

目的观察血管紧张素Ⅱ(AngⅡ)对小鼠主动脉血管平滑肌细胞(VSMC)自噬的影响以及自噬对细胞表型转换的调控作用。方法原代培养小鼠VSMC,用10~(-6)mol/L AngⅡ作用VSMC不同时间,采用Western blot检测微管相关蛋白轻链-3-Ⅱ(LC3-Ⅱ)的表达以观察AngⅡ对VSMC自噬的影响,透射电镜观察对照组及AngⅡ组的自噬小体。用Western blot检测自噬抑制剂3-MA和Baf-A1干预后对AngⅡ诱导自噬及细胞表型转换的影响。使用siRNA抑制自噬相关基因Atg7的表达,qRT-PCR检测转染后Atg7的表达变化,Western blot检测转染siRNA Atg7后对LC3-Ⅱ及细胞表型蛋白标志物的影响。结果 AngⅡ以时间依赖方式促进LC3-Ⅱ表达,自噬抑制剂3-MA抑制AngⅡ促LC3-Ⅱ的表达作用,而Baf-A1则增强AngⅡ促LC3-Ⅱ的表达作用,两种自噬抑制剂均可抑制AngⅡ促VSMC表型转换作用。转染siRNA Atg7后显著抑制AngⅡ促LC3-Ⅱ的表达作用,并可抑制AngⅡ促细胞表型转换作用。结论 AngⅡ促进VSMC从收缩表型转化为合成表型可能是自噬依赖性的,抑制自噬可以抑制AngⅡ诱导的VSMC表型转换。     

糖尿病加剧小鼠脑缺血后脑内自噬活性的增高

目的 评估糖尿病(DM)短暂性脑缺血引起小鼠脑内自噬活性的改变.方法 腹腔注射链脲佐菌素(STZ)诱导小鼠DM模型,通过短暂性双侧颈总动脉夹闭(CCAO)手术在DM模型上建立脑缺血模型.通过免疫印迹和透射电子显微镜(EM)检测脑内自噬活性.结果 在缺血早期,自噬活性标记物LC3-Ⅱ表达大幅度上调,至少持续72 h.与假手术(Sham)组比较,DM组LC3-Ⅱ表达的基线水平增高.DM加剧了中风诱导的LC3-Ⅱ水平,DM-脑缺血(DM-VO)组的LC3-Ⅱ表达增高最为显著.通过透射电镜观察到,缺血后实验动物神经元大量表达自噬囊泡样物质,以DM-VO组表达最为显著.结论 DM加剧了脑缺血后脑内自噬活性水平的增高,调节脑内自噬可能成为一种防治脑缺血损伤的新途径.     

自噬相关蛋白ATG5/BECLIN-1调控细胞自噬和凋亡的分子机理研究进展

ATG5和BECLIN-1(酵母ATG6同源物)是自噬体形成过程中所必需的两种自噬相关蛋白质,它们除了促进自噬体的形成,还能诱导细胞凋亡的发生,被认为是调控细胞自噬和凋亡的分子开关蛋白。前期研究揭示ATG5通过组成泛素化系统ATG5-ATG12-ATG16L介导自噬体的形成,而BECLIN-1通过组成磷脂酰肌醇3-激酶(PtdIns3KC3)复合体诱导细胞自噬,现在认为ATG5的氨基端截短分子tATG5-N和BECLIN-1的羧基端截短分子BECLIN-1-C能诱导细胞凋亡。本研究对近年来ATG5和BECLIN-1调控细胞自噬和凋亡的研究进展作一综述,为研究ATG5/ BECLIN-1调控细胞自噬和凋亡的分子机理奠定基础。     

Parkin促进HepG2细胞自噬并抑制星型孢菌素诱导的细胞凋亡

目的 观察Parkin在星型孢菌素(staurosporine, STS)诱导的HepG2细胞凋亡及细胞自噬中的作用。方法 流式细胞仪检测STS作用前后线粒体膜电位变化；Western blotting检测经STS处理后HepG2细胞caspase3、caspase7、PARP、Parkin/PINK1、LC3Ⅱ/LC3Ⅰ比值、ATg5、Vps34表达水平；免疫荧光法检测线粒体细胞色素C的释放情况；双荧光mRFP-eGFP-LC3自噬指示系统检测细胞自噬率。在HepG2细胞中过表达重组载体Parkin-flag,经STS处理后，蛋白质印记法检测凋亡蛋白表达变化，双荧光mRFP-eGFP-LC3自噬指示系统检测细胞自噬率改变。结果 与对照组相比，STS下调线粒体膜电位并促进细胞色素C释放，同时上调凋亡蛋白cleaved-caspase3、cleaved-caspase7及PARP表达水平。经STS处理后Parkin蛋白表达被抑制，LC3Ⅱ/LC3Ⅰ比值及自噬相关蛋白ATg5、Vps34的表达下降。转染mRFP-eGFP-LC3后，STS组自噬体及自噬溶酶体形成明显减少。过表达Parkin...     

急性冠状动脉综合征患者树突状细胞介导的热休克蛋白60特异性细胞毒作用

目的:探讨急性冠状动脉综合征(ACS)患者体内是否存在树突状细胞(DC)介导的热休克蛋白60(HSP60)特异性的T细胞毒反应。方法:分离ACS患者(ACS组),稳定性心绞痛(SA)患者(SA组)和正常人(对照组)外周血单核细胞,制备DC。流式细胞仪检测HSP60负载及未负载的DC CD86的表达及各组T细胞中CD45RO+细胞的百分比;二氮唑法(MTT)法检测各组DC刺激自体淋巴细胞增殖的能力,并检测各组DC诱导的细胞毒T细胞(CTL)对负载HSP60的人脐静脉内皮细胞(HUVECS)的杀伤活性;酶联免疫吸附法(ELISA)测定HSP60冲击T细胞后干扰素γ的分泌。结果:与未负载HSP60的SA组和对照组的DC比较,ACS组的DC以及负载HSP60对照组的DC CD86表达较高;ACS 组的DC及HSP60负载的DC刺激自体细胞毒T细胞增殖的能力增强;ACS组的DC及HSP60负载的DC诱导的细胞毒T 细胞对负载HSP60的人脐静脉内皮细胞有高度的杀伤作用;ACS患者体内存在HSP60致敏的记忆性T细胞。结论:ACS患者体内存在强烈的DC介导的HSP60抗原特异性T细胞毒反应。     

饥饿诱导胃癌细胞自噬发生及Beclin-1表达的变化

目的探讨饥饿诱导胃癌细胞MKN1自噬的发生及自噬相关蛋白Beclin-1表达的变化。方法培养胃癌细胞MKN1至对数生长期后,以无氨基酸培养液EBSS代替RPMI1640培养液培养细胞,培养12h后收集细胞,应用Western印迹和定量PCR(qRT-PCR)方法分别检测特异性标记自噬的微管相关蛋白1轻链3B(LC3B)以及自噬相关蛋白Beclin-1的蛋白和mRNA表达。免疫荧光检测饥饿诱导后细胞自噬体的产生。结果饥饿处理12 h后,Western印迹和qRT-PCR检测LC3B和Beclin-1蛋白和mRNA表达与对照组比较均明显增加(P0.05)。免疫荧光下可见点状自噬体。结论饥饿可以诱导胃癌细胞MKN1发生自噬,Beclin-1与饥饿诱导胃癌细胞MKN1的自噬相关,为进一步研究Beclin-1在胃癌细胞自噬中的作用机制奠定了基础。     

Heat shock pretreatment improves stem cell repair following ischemia-reperfusion injury via autophagy

AIM: To investigate whether heat shock pretreatment (HSP) improves mesenchymal stem cell (MSC) repair via autophagy following hepatic ischemia-reperfusion injury (HIRI).METHODS: Apoptosis of MSCs was induced by 250 mM hydrogen peroxide (H₂O₂) for 6 h. HSP was carried out using a 42 °C water bath for 1, 2 or 3 h. Apoptosis of MSCs was analyzed by flow cytometry, and Western blot was used to detect Bcl-2, Bax and cytochrome C expression. Autophagy of MSCs was analyzed by flow cytometry and transmission electron microscopy, and the expression of beclin I and LC3-II was detected by Western blot. MSCs were labeled in vivo with the fluorescent dye, CM-Dil, and subsequently transplanted into the portal veins of rats that had undergone HIRI. Liver levels of proliferating cell nuclear antigen (PCNA) were quantified by fluorescent microscopy. Serum aminotransferase activity and the extent of HIRI were also assessed at each time point.RESULTS: HSP for 2 h reduced apoptosis of MSCs induced by H₂O₂ as seen by a decrease in apoptotic rate, a decrease in Bax and cytochrome C expression and an increase in Bcl-2 expression (P < 0.001). In addition, HSP for 2 h induced autophagy of MSCs exposed to H₂O₂ as shown by an increase in acidic vesicular organelle-positive cells, beclin 1 and LC3-II expression, and autophagosome formation (P < 0.05). Treatment with 3-methyladenine attenuated HSP-induced autophagy and abolished the protective effects of HSP on the apoptosis of MSCs. Rapamycin failed to have additional effects on either autophagy or apoptosis compared with HSP alone. The phosphorylation of p38MAPK was significantly elevated and the phosphorylation of mTOR was downregulated in heat shock pretreated MSCs. Treatment with the p38MAPK inhibitor, SB203580, reduced HSP-induced autophagy in MSCs. In vivo studies showed that the transplantation of HSP-MSCs resulted in lower serum aminotransferase levels, lower Suzuki scores, improved histopathology and an increase in PCNA-positive cells (P < 0.05).CONCLUSION: HSP effectively induces autophagy following exposure to H₂O₂ via the p38MAPK/mTOR pathway, which leads to enhanced MSC survival and improved MSC repair following HIRI in rats.     

抗CD3单克隆抗体对支气管哮喘患者CD4＋CD25＋T细胞凋亡和自噬的影响

目的探讨抗CD3单克隆抗体对分离培养的支气管哮喘（简称哮喘）患者外周血CD4＋CD25＋T细胞凋亡和自噬及其分泌的代表性因子转化生长因子p（TGF—β）的影响。方法采用密度梯度离心法及尼龙棉柱法分离32例哮喘患者（哮喘组）及30名健康者（对照组）外周血T细胞,磁性细胞分离器分离得到CD4＋CD25＋T细胞,分别利用电镜及流式细胞仪观察、检测抗CD3单克隆抗体干预72h的细胞凋亡率、自噬率。用EI,ISA法检测细胞培养上清液中细胞因子TGF-β的水平。结果抗CD3单克隆抗体干预72h后两组外周血CD4＋CD25＋T细胞凋亡率、自噬率及TGF-β均增加（P值均〈0．01）,但哮喘组凋亡率、自噬率均低于健康对照组（P值均〈0．01）;两组间 TGF-β水平无显著差异（P〉0．01）。哮喘组外周虹CD4＋CD25＋T调节细胞在CD3单克隆抗体的干预下自噬与TGF-β的分泌呈显著负相关（r=-0．38,P〈0．01）。结论抗CD3单克隆抗体可促进CD4＋CD25＋T细胞凋亡和自噬及TGF-β分泌。     

Heat shock proteins and autophagy in rats with cerulein-induced acute pancreatitis

Background/Aims

Heat shock proteins (HSPs) protect rats from cerulein-induced acute pancreatitis (AP) by preventing the subcellular redistribution of cathepsin B and the activation of trypsinogen. Autophagy plays a critical role in the secretion of digestive enzymes and triggering of cerulein-induced AP via the colocalization of trypsinogen and lysosomes. Therefore, using a rat cerulein-induced AP model, we investigated whether HSPs prevent AP by regulating autophagy.

Methods

Twelve hours after fed standard laboratory chow and water, the experimental groups (cerulein, water-immersion [WI]-cerulein and heat-shock [HS]-cerulein) and the control groups (control, WI, and HS) received one intraperitoneal injection of cerulein (50 µg/kg) or saline, respectively. All of the rats were sacrificed at 6 hours after injection. The severity of the AP was assessed based on the serum amylase level and the histological and electron microscopy findings. Western blotting was also performed for HSP60/70 and LC3B-II.

Results

WI and HS induced HSP60 and HSP70, respectively. The induced HSP60/70 effectively prevented the development of cerulein-induced AP. Autophagy developed in the rats with cerulein-induced AP and was documented by the expression of LC3-II and electron microscopy findings. The WI-stressed rats and HS-treated rats did not develop cerulein-induced autophagy.

Conclusions

HSPs exert protective effects against cerulein-induced AP in rats by inhibiting autophagy.     

脂多糖对大鼠胰岛素瘤细胞INS-1自噬和凋亡的影响

目的研究脂多糖（LPS）对大鼠胰岛素瘤细胞INS-1自噬和凋亡的影响及其可能机制。方法取处于对数期生长的INS-1细胞用于实验，分别用0．1、1．0、10．0mg／LLPS处理细胞，24h后用磺酰罗丹明B（SRB）染色法检测LPS对INS-1细胞增殖的影响，用二氯荧光素双醋酸盐（DCFH-DA）作为荧光探针检测细胞内活性氧的水平变化，应用Western blotting法检测细胞自噬体膜上自噬标志分子微管相关蛋白1的轻链3-Ⅱ（Map1LC3-Ⅱ）和凋亡标志蛋白聚腺苷二磷酸-核糖多聚酶（PARP）切割带的变化。将INS-1细胞的自噬必需基因7（A增7）通过siRNA介导的RNA干扰手段进行沉默，然后以1．0mg／LLPS处理细胞，24h后应用Western blotting检测细胞中Atg7、Map1LC3-Ⅱ和PARP切割带的水平。先用400μmoL／L的活性氧清除剂N-乙酰半胱氨酸（NAC）处理INS-1细胞，1h后加入10．0mg／L的LPS，24h后应用Western blotting法检测细胞Map1LC3-Ⅱ蛋白和PARP切割蛋白的水平。组间数据比较采用方差分析和t检验。结果与对照组（0．755±0．030）比较，0．1、1．0、10．0mg／LLPS组INS-1存活率降低，差异有统计学意义（分别为0．658±0．042、0．658±0．015、0．634±0．029，F=30．98，P〈0．01）；与对照组（0．447±0．012）相比，0．1、1．0、10．0mg／LLPS组Map1LC3-Ⅱ蛋白水平增加，差异有统计学意义（分别为0．992±0．030、0．949±0．020、0．982±0．013，F=525．79，P〈0．01）；与对照组（0．055±0．001）相比，0．1、1．0、10．0mg／LLPS组PARP切割蛋白水平增加，差异有统计学意义（分别为0．313±0．007、0．390±0．011、0．500±0．033，F=327．09，均P〈0．01）；与对照组比较，10mg／LLPS组活性氧产生明显增多。与对照组比较，转染Atg7siRNA组Atg7、Map1LC3-Ⅱ和PARP切割蛋白水平均显著下降，差异均有统计学意义（t=156．069、123．154、103．246，均P〈0．01）。与LPS10．0mg／L组比较，LPS10．0mg／L＋NAC组Map1LC3-Ⅱ和PARP切割蛋白水平均显著下降，差异均有统计学意义（t=66．37、26．84，均P〈0．01）。结论LPS可诱导INS-1细胞的自噬和凋亡，且其所诱导的凋亡依赖于自噬的发生；活性氧参与了LPS诱导的INS-1细胞的自噬和凋亡。     

谷氨酸诱导大鼠结肠 Cajal 间质细胞构建自噬模型

目的通过谷氨酸诱导离体大鼠结肠Cajal间质细胞(ICC)构建细胞自噬模型。方法使用不同浓度的谷氨酸不同时间点作用于原代培养的大鼠结肠ICC,免疫荧光鉴定大鼠结肠ICC细胞;CCK-8法检测谷氨酸对ICCs活力的影响;Western印迹检测不同浓度谷氨酸在不同作用时点对大鼠结肠ICC自噬蛋白微管相关蛋白轻链(LC)3表达水平的影响;透射电镜观察谷氨酸干预后细胞内自噬体和超微结构的变化。结果与空白组相比,谷氨酸(5 mmol/L)24 h显著降低大鼠结肠ICC活力(P<0.01)。谷氨酸(5 mmol/L)作用6 h、24 h均可显著提高自噬蛋白LC3-Ⅱ/LC3-Ⅰ的比值(P<0.05),其中以5 mmol/L谷氨酸作用24 h的LC3-Ⅱ/LC3-Ⅰ比值最高(P<0.01)。透射电镜检测5 mmol/L谷氨酸干预大鼠结肠ICC 24 h发现,细胞内自噬空泡和自噬体数量明显增加。结论谷氨酸诱导大鼠结肠ICC自噬模型的最佳浓度和时间为5 mmol/L和24 h。     

雷帕霉素对1型糖尿病小鼠免疫功能的影响及其分子机制

目的研究雷帕霉素对1型糖尿病（T1DM）小鼠的影响及其分子机制。方法40mg／kg的STZ腹腔注射C57BL／6小鼠连续5d建立T1DM模型,正常和T1DM小鼠按2mg／kg腹腔注射RAPA连续两周。监测血糖、体重、进食量和饮水量;观测胰岛炎、主要脏器的超微结构和凋亡和自噬的发生;检测脾脏Th1／Th2分群和调节性T细胞。结果RAPA对正常小鼠一般特性及主要脏器的超微结构无明显影响。但可使T1DM小鼠血糖升高、体重下降、采食和饮水量增加（P〈0．05）,并加重其胰岛炎程度;诱导其胰腺、肾脏、脾脏和胸腺细胞自噬或凋亡,并使LC3、Beclin1、Caspase-3的表达增加;减少正常和T1DM小鼠的Th1细胞,增加Th2细胞,并上调CD4^＋CD25%＋T细胞的数量。结论RAPA既可诱导免疫耐受,发挥免疫抑制作用,又可通过自噬直接破坏胰岛从而加重T1DM代谢紊乱和并发症。     

Autophagy in anti-apoptotic effect of augmenter of liver regeneration in HepG2 cells

AIM:To investigate the role of autophagy in the antiapoptotic effect of augmenter of liver regeneration(ALR).METHODS:Autophagy was induced through serum deprivation.An ALR-expressing plasmid was transfected into HepG2 cells,and autophagic flux was determined using fluorescence microscopy,electron microscopy,Western blot and quantitative polymerase chain reaction(q PCR) assays.After ALR-expressing plasmid transfection,an autophagy inhibitor [3-methyladenine(3-MA)] was added to HepG2 cells,and apoptosis was observed using fluorescence microscopy and flow cytometry.RESULTS:Autophagy was activated in HepG2 cells,peaking at 24 h after serum deprivation.Microtubuleassociated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells,fluorescence microscopy,electron microscopy and q PCR studies showed the similar trend,and p62 levels showed the opposite trend,which indicated that ALR may play an important role in increasing autophagy flux.The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone.Therefore,the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited,indicating that the anti-apoptotic effect of ALR may be related to autophagy.CONCLUSION:ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.     

细胞内酸化对自噬的影响

目的探讨细胞内酸化对自噬的影响。方法利用pAd-miR-NHE-1病毒悬液转染人源性神经母细胞瘤细胞株,制备NHE-1基因敲低细胞模型。在透射电镜下观察阴性对照组及腺病毒转组转染成功后0.5、2、4、8 h自噬体的形成、线粒体形态及溶酶体数目的变化;Western blotting法检测自噬标记物LC3Ⅱ的表达。结果透射电镜下,阴性对照组线粒体形态及溶酶体数目正常,腺病毒转染成功后0.5～4 h可见线粒体肿胀、变形,溶酶体数目增多,自噬囊泡增多,自噬小体形成。8 h可见凋亡小体出现,线粒体数目减少,未见自噬小体,可见少量溶酶体。Western blotting显示,阴性对照组LC3Ⅱ表达较低,腺病毒转染成功后0.5 h LC3Ⅱ表达升高(P<0.01),4 h达高峰(P<0.01),8 h表达降低,且低于阴性对照组(P<0.01)。结论细胞内酸化可以激活自噬的发生。     

Differential capacity of CD90+ cells in autophagy activation following chemotherapy in hepatocellular carcinoma

Introduction and Objectives. Analysis of cancer biomarkers is an important tool in developing targeted-therapy and in modulating chemoresistance. Here, we analyze the relevance of CD90, a marker of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) and its correlation with autophagy. Materials and Methods. For in vivo study, 86 specimens were collected from 43 patients undergoing liver resections. In each patient, HCC nodule (HCC) and surrounding non-tumor (SNT) were collected. For in vitro study, HCC cells JHH6 subpopulations expressing CD90+ and CD90- were isolated using magnetic-sorter and confirmed by flow-cytometry. Upon doxorubicin treatment, autophagy turn-over was analyzed by RTqPCR for mRNA expression, Western blot for protein expression, and autophagosome staining for autophagy-flux. Cytotoxicity test was performed by MTT assay. Gene and protein analysis were performed in clinical samples together with immunohistostaining. Results. CD90 mRNA expression was higher in HCC than in SNT for 8-fold (p < 0.001). LC3-II protein was up-regulated in the HCC in comparison with the SNT (p < 0.05). In vitro model showed that CD90+ and CD90- cells had diverse expressions of autophagy-related genes. Upon doxorubicin treatment, autophagy was activated in both cells by increasing LC3-II protein expression, autophagic vacuoles, and dysregulation of autophagy-related mRNAs. A differential autophagic capacity was noticed between two subpopulations and it was correlated with cellular toxicity assay. Conclusions. We demonstrated the relevance of differential autophagy capacity of CD90+ cells in HCC. Autophagy was involved in cancer-defense mechanism against doxorubicin. Cancer promoting function of autophagy in CD90+ cells was also related to cancer environment.