BAG2对蛋白酶体抑制剂诱导人甲状腺癌细胞凋亡作用影响的观察

背景与目的：蛋白酶体抑制剂对不同组织来源的肿瘤均有抑制其增长和促进细胞凋亡的作用，其作用机制可能与Bcl-2相关抗凋亡基因2（Bcl-2-associated athanogene 2，BAG2）有关，本研究探讨BAG2在蛋白酶体抑制剂诱导甲状腺癌细胞凋亡中的作用。方法：选取人甲状腺未分化癌细胞系ARO、FRO、KTC1、KTC2、KTC3、8305C和8505C，分别设培养液处理空白对照组、蛋白酶体抑制剂MG132处理组；利用实时定量逆转录聚合酶链反应（quantitative real-time polymerase chain reaction，qRT-PCR）检测各组细胞BAG2 mRNA表达及MG132诱导其表达的时效性；利用蛋白质印迹法（Western blot）检测各组细胞BAG2蛋白表达。结果：MTT结果显示，FRO和KTC2细胞系对蛋白酶体抑制剂最为敏感，与空白对照组相比，MG132能不同程度的增加各种细胞BAG2 mRNA及蛋白的表达水平（P〈0.01），在FRO和KTC2细胞中，BAG2 mRNA水平较对照组增加20-25倍，蛋白质的表达水平也显著增加；时间效应实验中，敏感的FRO细胞BAG2 mRNA水平增加快，没有延迟期；并且增加的最高水平显著高于非敏感的ARO细胞（P〈0.01）。结论：BAG2是由蛋白酶体抑制作用诱导产生的新型分子，在蛋白酶体抑制剂诱导的甲状腺癌细胞的死亡中起着促凋亡作用。

Regulatory Effects of Zuogui Pill on Apoptosis of Follicles in Rats Injured by 60Co-γRays Based on PI3K/Akt/m TOR Signaling Pathway

[Objectives]To explore the protective effects of Zuogui Pill on ^(60)Co-γ-ray-induced premature aging of rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.[Methods]Sixty sexually mature female SD rats were irradiated with ^(60)Co-γ-ray(6.0 Gy,LD 40)for 24 h at one time.These rats were randomly divided into model group,Progynova group[0.18(g·kg)/d],Progynova[0.09(g·kg)/d]+Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill medium dose[9.45(g·kg)/d)]group and Zuogui Pill low dose[4.725(g·kg)/d]group.The administration(once a day)lasted 21 d.The rat serum[follicle-stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E_(2))]were detected by Enzyme-linked immunosorbent assay(ELISA).The morphological changes of ovary were observed by hematoxylin-eosin(HE)staining.The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL).The protein expression of phosphorylated(p)-PI3K,p-Akt,p-mTOR,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)in ovarian tissues were detected by Western blot.[Results]Compared with the normal group,the model group showed significant increase in the serum FSH(P<0.01),significant decrease in serum E_(2)(P<0.05),and decrease in the number of early follicles and luteum in the ovary(P<0.01).Besides,the apoptosis rate of granulosa cells increased significantly(P<0.01);the expression of p-PI3K,p-Akt,p-mTOR and Bcl-2 in ovarian tissue decreased significantly,while the expression of Bax increased significantly(P<0.01).Compared with the model group,the number of early follicles in the ovary increased and the apoptosis rate of granulosa cells decreased after intervention in each administration group.In addition,the protein expressions of p-PI3K,p-Akt,p-mTOR and Bcl-2 increased,while the expression of Bax decreased,especially in Progynova+Zuogui Pill high dose group,the differences were statistically significant(P<0.05,P<0.01).[Conclusions]Zuogui Pill may protect the radiation-injured ovary through activating the expression of PI3K/Akt/mTOR protein in ovarian tissue,increasing the amount of Bcl-2 protein and inhibiting the expression of Bax protein.

大黄酚脂质体对脑缺血再灌注损伤小鼠海马神经元凋亡的影响

目的:观察脑缺血再灌注损伤后,大黄酚脂质体对小鼠海马神经元凋亡的影响,探讨大黄酚脂质体对脑缺血再灌注损伤小鼠海马的保护作用及其机制。方法:采用暂时性阻断颈总动脉的方法建立小鼠脑缺血再灌注损伤模型,腹腔注射(ip)大黄酚脂质体10.0,1.0,0.1 mg.kg-1,观察小鼠脑缺血再灌注后神经功能学和病理形态学的改变,并采用免疫组化方法检测海马神经元凋亡相关蛋白半胱氨酰天冬氨酸蛋白酶-3(caspase-3),Bcl-2和Bcl-2-Associated X(Bax)的表达。结果:大黄酚脂质体可明显抑制脑缺血再灌注引起的神经元的丢失,提高Bcl-2的表达,降低caspase-3和Bax的表达,提高神经功能评分,减少病理形态改变,减轻海马神经元损伤,其中以10.0 mg.kg-1组大黄酚脂质体的作用最为明显(P<0.05)。结论:脑缺血再灌注损伤后应用大黄酚脂质体对海马神经元有明显的保护作用,其机制可能与上调Bcl-2的表达,下调caspase-3和Bax的表达有关。

EGCG诱导人视网膜色素上皮细胞凋亡作用的研究

目的:探讨表没食子儿茶素没食子酸酯(EGCG)对人视网膜色素上皮细胞(ARPE-19)凋亡的影响及其机制。方法:体外培养ARPE-19,分别采用0、40、80、160μg/mL EGCG处理。处理预定时间后分别用hoechst 33258染色法检测细胞凋亡形态学变化;流式细胞仪检测细胞凋亡率;实时荧光定量RT-PCR和Western blotting检测细胞凋亡相关因子B淋巴细胞瘤-2基因(bcl-2)、BCL2-Associated X的蛋白质(Bax)、胱天蛋白酶-3(caspase-3)和p53的表达。结果:hoechst 33258染色结果发现ARPE-19随着EGCG药物浓度的增加,凋亡细胞数量逐渐增多,可见明显的凋亡小体;流式细胞仪结果显示随着EGCG药物浓度的升高,凋亡率逐渐增高,40、80、160μg/mL凋亡率分别为4.95%±0.071%、11.75%±0.075%和21.25%±0.919%与对照组(2.8%±1.556%)相比有差异(P<0.01),呈现出药物浓度依赖性;实时荧光定量PCR和Western blot结果表明EGCG能明显上调凋亡促进因子Bax、caspase-3和p53的mRNA和蛋白表达,同时下调凋亡抑制因子bcl-2的表达,均呈现浓度依赖性。结论:EGCG能明显诱导ARPE-19发生凋亡,其机制与抑制bcl-2的表达,增强Bax、caspase-3和p53的表达有关。

Functional Characterization of the Group Ⅰ Alphabaculovirus Specific Gene ac73

Baculoviridae is a family of large DNA viruses that specifically infect insects. It contains four genera, Alpha-, Beta-,Gamma-, and Deltabaculovirus. Alphabaculovirus is further divided into Group Ⅰ and Ⅱ, and Group Ⅰ appears to be emerged most recently among all baculoviruses. Interestingly, there are 12 Group Ⅰ specific genes that are only found in this lineage. Studying these genes is helpful to understand how baculoviruses evolved. Here, we reported the functional analyzing results of ac73, a function unknown Group Ⅰ specific gene of Autographa californica multiple nucleopolyhedrovirus(Ac MNPV) which is the type species of baculovirus. The AC73 protein encoded by ac73 was found to be expressed during the late stage of infection and incorporated into the nucleocapsids of budded virus(BV) and occlusionderived virus(ODV). In infected cells, AC73 resided mainly in the ring zone region of the nucleus, and appeared to be assembled into occlusion bodies(OBs). The ac73 knockout and repaired viruses were constructed and studied by in vitro and in vivo infection. Although ac73 was not essential for BV and ODV or OB formation, the BV titer and viral infectivity in insect larvae of ac73 knockout Ac MNPV decreased by about 5–8 and 3–4 fold compared to those of wild type virus,respectively, suggesting ac73 contributed to infectious BV production and viral infectivity in vivo. This research provides new insight into the function of this Group I specific gene.

Inhibitory effect of oridonin on proliferation of RPMI8226 cells and the possible underlying mechanism

OBJECTIVE:To observe the effects of oridonin on proliferation and apoptosis of myeloma RPMI8226cells and to investigate the potential underlying mechanisms.METHODS:RPMI8226 cells were treated with various concentrations of oridonin.Cell proliferation was analyzed using the thiazolyl blue tetrazolium bromide method.Ultramicrostructure was observed by transmission electron microscopy.Annexin-V/PI staining and flow cytometry was performed to determine cell apoptosis.Expression of apoptosis-related proteins was evaluated by western blot analysis.RESULTS:Oridonin suppressed the proliferation of RPMI8226 cells and induced apoptosis in a timeand dose-dependent manner.Transmission electron microscopy confirmed apoptotic morphologyupon treatment with 20μmol/L oridonin and western blot revealed decreased expressions of the apoptosis suppressors survivin,Bcl-2 and pro-caspase-3 proteins,and the increased expression of the apoptosis inducer Bax.CONCLUSION:Our results show that oridonin exhibits an inhibitory effect on the proliferation of RPMI8226 cells and induces apoptosis.This is associated with altering the balance between Bcl-2 and Bax protein expressions and decreased survivin and pro-caspase-3 expressions.

Spinal cord decompression reduces rat neural cell apoptosis secondary to spinal cord injury

Objective： To determine whether spinal cord decompression plays a role in neural cell apoptosis after spinal cord injury. Study design： We used an animal model of compressive spinal cord injury with incomplete paraparesis to evaluate neural cell apoptosis after decompression. Apoptosis and cellular damage were assessed by staining with terminal deoxynucleotidyl transferase （TdT）-mediated deoxyuridine triphosphate nick-end labelling （TUNEL） and immunostaining for caspase-3, Bcl-2 and Bax. Methods： Experiments were conducted in male Sprague-Dawley rats （n-78） weighing 300-400 g. The spinal cord was compressed posteriorly at T10 level using a custom-made screw for 6 h, 24 h or continuously, followed by decompression by removal of the screw. The rats were sacrificed on Day I or 3 or in Week 1 or 4 post-decompression. The spinal cord was removed en bloc and examined at lesion site, rostral site and caudal site （7.5 mm away from the lesion）. Results： The numbers of TUNEL-positive cells were significantly lower at the site of decompression on Day 1, and also at the rostral and caudal sites between Day 3 and Week 4 post-decompression, compared with the persistently compressed group. The numbers of cells between Day 1 and Week 4 were immunoreactive to caspase-3 and B-cell lymphoma-2 （Bcl-2）-associated X-protein （Bax）, but not to Bcl-2, correlated with those of TUNEL-positive cells. Conclusion： Our results suggest that decompression reduces neural cell apoptosis following spinal cord injury.

Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication

Human adenovirus B7(HAdV-B7)causes severe acute lower respiratory tract infections in children.However,neither the child-specific antivirals or vaccines are available,nor the pathogenesis is clear.Autophagy,as part of innate immunity,plays an important role in resistance to viral infection by degrading the virus and promoting the development of innate and adaptive immunity.This study provided evidence that HAdV-B7 infection induced complete autophagic flux,and the pharmacological induction of autophagy decreased HAdV-B7 replication.In this process,the host protein Bcl2-associated athanogene 3(BAG3)mediated autophagy to inhibit the replication of HAdV-B7 by binding to the PPSY structural domain of viral protein pVI through its WW structural domain.These findings further our understanding of the host immune response during viral infection and will help to develop broad anti-HAdV therapies.