BJAB细胞在转导6A8 α-甘露糖苷酶的反义DNA后发生oncosis样死亡

目的 采用形态学和分子生物学方法 ,分析转导 6A8α 甘露糖苷酶的反义DNA导致的B细胞株BJAB死亡的性质。方法 用Giemsa染色、annexin Ⅴ荧光染色、梯形DNA电泳以及透射电子显微镜等方法 ,分析细胞死亡的性质 ;应用ConA结合试验 ,检测转基因细胞 6A8α 甘露糖苷酶活性的改变 ;应用Fluo 3探针测定细胞胞浆 [Ca2 +]i。结果 用重组腺相关病毒 (rAAV)颗粒成功地将反义 6A8DNA转导入了EB病毒转化的B细胞株BJAB。在克隆中 ,转导反义 6A8DNA的BJAB细胞在生长到 3～ 1 0个细胞时死亡 ,而野生型、转导空载或转导正义 6A8DNA的BJAB细胞生长良好。转导反义 6A8DNA的BJAB细胞呈聚集状生长 ,且有大量细胞肿胀 ,最终死亡。Giemsa染色光镜观察、an nexin Ⅴ荧光染色、梯形DNA电泳及透射电镜观察结果表明 ,这种死亡是一种oncosis(肿胀死 )样死亡。ConA结合试验表明 ,BJAB细胞在转导反义 6A8DNA后的结合率升高 ,而转导正义 6A8DNA后的结合率则下降。另外 ,转导反义 6A8DNA的细胞胞浆 [Ca2 +]i升高。结论 转导反义 6A8DNA引起BJAB细胞发生oncosis样死亡 ,这种死亡可能与 6A8α 甘露糖苷酶表达被抑制有关。

Oncosis in human esophageal squamous cell carcinoma and its relationship with apoptosis and microvessel density

Background Previous studies have shown that oncosis in malignant tumors might be related to cellular energy supply. The aim of this study was to detect oncosis in human esophageal squamous cell carcinoma （ESCC）, and to investigate its relationship with apoptosis and microvessel density （MVD）. Methods ESCC specimens were obtained from 30 patients with ESCC after surgery. Transmission electron microscopy TUNEL, and immunohistochemistry were used to detect oncosis, apoptosis, and MVD. The relation of oncosis to apoptosis and MVD was analyzed by ANOVA, t test, and q test using SPSS 10.0. Results Transmission electron microscopy revealed both oncosis and apoptosis in the ECSS tissues. About 10% of the TUNEL-positive cells, which were considered apoptotic cells, showed the characteristics of oncosis. In the areas, where oncotic cells accumulated, apoptotic cells were rare; contrarily, where apoptotic cells gathered, oncotic cells were sparse. Compared with the tissues with a high MVD, the number of oncotic cells was increased and that of apoptotic cells was decreased in the tissues with a low MVD. Conclusions Cellular oncosis can be detected in human ESCC tissues. The distribution of oncotic cells presents a close relationship with cellular apoptosis and MVD. Oncosis might be induced by poor blood supply.

A neuron-specific Isca1 knockout rat developments multiple mitochondrial dysfunction syndromes

Background:Multiple mitochondrial dysfunction syndromes(MMDS)are rare mitochondrial diseases caused by mutation of mitochondrial iron–sulfur cluster synthesis proteins.This study established a rat model simulating MMDS5 disease in the nervous system to investigate its pathological features and neuronal death.Methods:We generated neuron-specific Isca1 knockout rat(Isca1 flox/flox-NeuN-Cre)using CRISPR-Cas9 technology.The brain structure changes of CKO rats were studied with MRI,and the behavior abnormalities were analyzed through gait analysis and open field tests,Y maze tests and food maze tests.The pathological changes of neurons were analyzed through H&E staining,Nissl staining,and Golgi staining.Mitochondrial damage was assessed by TEM,western blot and ATP assay,and the morphology of neurons was assessed by WGA immunofluorescence to detect the death of neurons.Results:This study established the disease model of MMDS5 in the nervous system for the first time,and found that after Isca1 loss,the rats suffered from developmental retardation,epilepsy,memory impairment,massive neuronal death,reduced number of Nissl bodies and dendritic spines,mitochondrial fragmentation,cristae fracture,reduced content of respiratory chain complex protein,and reduced production of ATP.Isca1 knockout caused neuronal oncosis.Conclusions:This rat model can be used to study the pathogenesis of MMDS.In addition,compared with human MMDS5,the rat model can survive up to 8 weeks of age,effectively extending the window of clinical treatment research,and can be used for the treatment of neurological symptoms in other mitochondrial diseases.

The Influence of Nano-apatite on c-myc and p53 Gene in the Hepatocellular Carcinoma

The influence mechanism of the nano-apatite on the human hepatocellular carcinoma in vitro was investigated. Using the homogeneous precipitation method, the nano-apatite was synthesized at room temperature, and it was characterized with transmission electron microscopy (TEM) and the Zataplus. The influence on the expression of the c-myc and p53 gene in the human hepatocellular carcinoma cell lines were tested with the TEM and hybridization in situ. The TEM and the Zataplus analyses show that the nano-apatite is distributed homogenously in size and needle-shaped sizes, which ranges from 67.5 nm to 88.3 nm. It is found that the nano-apatitet increases the volume of the human hepatocellular carcinoma cells, makes extensive cytoplasmic vacuolization, the mitochondria swelling, chromatin in nucleus dispersed partially and condensed around the nuclear membranes.The interspace in nuclear membranes were separated and even the cytoplasm dissolved. It is also found that the expression of the c-myc gene is inhibited, but the p53 is enhanced. The experimental results demonstrate that the nano-apatite enables the oncosis of the human hepatocellular carcinoma cells by down-regulation of the expression of the c-myc and up-regulation of the expression of the p53 in vitro.

Intervention Timing and Effect of PJ34 on Astrocytes During Oxygen-glucose Deprivation/reperfusion and Cell Death Pathways

Poly （ADP-ribose） polymerase-I （PARP-1） plays as a double edged sword in cerebral ischemia-reperfusion, hinging on its effect on the intracellular energy storage and injury severity, and the prognosis has relationship with intervention timing. During ischemia injury, apoptosis and oncosis are the two main cell death pathway sin the ischemic core. The participation of astrocytes in ische- mia-reperfusion induced cell death has triggered more and more attention. Here, we examined the pro- tective effects and intervention timing of the PARP-1 inhibitor PJ34, by using a mixed oxygen-glucose deprivation/reperfusion （OGDR） model of primary rat astrocytes in vitro, which could mimic the ische- mia-reperfusion damage in the ＂ischemic core＂. Meanwhile, cell death pathways of various P J34 treated astrocytes were also investigated. Our results showed that P J34 incubation （10 μmol/L） did not affect release of lactate dehydrogenase （LDH） from astrocytes and cell viability or survival 1 h after OGDR. Interestingly, after 3 or 5 h OGDR, P J34 significantly reduced LDH release and percentage of PI-positive cells and increased cell viability, and simultaneously increased the caspase-dependent apop- totic rate. The intervention timing study demonstrated that an earlier and longer P J34 intervention dur- ing reperfusion was associated with more apparent protective effects. In conclusion, earlier and longer PJ34 intervention provides remarkable protective effects for astrocytes in the ＂ischaemic core＂ mainly by reducing oncosis of the astrocytes, especially following serious OGDR damage.

An iron-dependent form of non-canonical ferroptosis induced by labile iron

Ferroptosis is a recently identified iron-dependent form of nonapoptotic cell death characterized by reactive oxygen species(ROS) generation and lipid peroxidation.Here,we report a novel iron-dependent form of ferroptosis induced by labile iron and investigate the mechanism underlying this process.We find that labile iron-induced ferroptosis is distinct from canonical ferroptosis and is linked to the mitochondrial pathway.Specifically,the mitochondrial calcium uniporter mediates the ferroptosis induced by labile iron.Interestingly,cells undergoing labile iron-induced ferroptosis exhibit cytoplasmic features of oncosis and nuclear features of apoptosis.Furthermore,labile iron-induced ferroptosis involves a unique set of genes.Finally,labile ironinduced ferroptosis was observed in liver subjected to acute iron overload in vivo.Our study reveals a novel form of ferroptosis that may be implicated in diseases caused by acute injury.