Studies On The Function Of KIAA0280 In The Hypoxia-induced Apoptotic Neurons

Stroke and cerebral infarction as a result of occlusion of cerebral arteries have long been a major health problem worldwide because they are major causes of disability and intellectual impairment in elderly. The number of patients suffering from these disorders will increase further in the future, as the life span of man is becoming longer. Recently, considerable advances have been made in our understanding of the molecular and biochemical mechanisms of ischemia-induced brain damage and a growing number of promising drugs with powerful cerebroprotective effects have been reported in basic pharmacology using experimental animals. However, the therapeutic efficiency of the drugs for patients with ischemic stroke is still limited at present. During the past years, a considerable amount of experimental studies have been devoted to elucidate the mechanism of ischemic neuronal death mediated by glutamate, calcium, altered gene expression after ischemia, neuronal death by apoptosis, inflammatory reactions induced in postischemic brain. In 1990 Kitagawa reported that transient ischemia preconditioning (IPC) could induce ischemic tolerance, which might produce neuroprotective effect.Therefore LinQ, Zang screened the differential expressed gene between acute focal ischemia and non-ischemia region in rat brain, and cloned the novel ischemia associated gene KIAA0280. And found that it over-expressed in acute focal ischemia. The function of this gene has not been reported based on literatures. It obviously over-expressed in ischemia region demonstrated that KIAA0280 had a critical function in the ischemia pathology.Many experiments showed that nerve cells death in brain occurred partly by apoptosis. The process of apoptosis was related to the expression of many genes and proteins and regulated by some genes, such as: caspase, bcl-2 et al. It can produce some new genes to regulate apoptosis in the process of apoptosis. Therefore we studied the expression of KIAA0280 in the apoptotic neurons induced by different factor, and to understand the function of KIAA0280, to contribute foundation for the study of mechanism of action in apoptosis and apoptotic theory, to provide new method for apoptotic disease such as stroke, Myocardial Infarction, renal failure and so on.ObjectiveTo establish the separation and culture of primary neuron in vitro; To establish the model of apoptotic neurons induced by different factor; To study the KIAA0280 expression in the apoptotic neurons; To study the KIAA0280 expression after silencing KIAA0280 using the technology of RNAi, and to observe the effect on the normal neurons after silencing KIAA0280; To observe the effect of apoptotic neurons after silencing KIAA0280.Methods(1) We adopted the methods of primary separation and culture to establish neuron culture in vitro.(2) We established the model of neuronal apoptosis induced by hypoxia applied airtight container to evacuate and fill with 95%N₂, 5%CO₂, and then put in the cell incubator at 37℃, hypoxia at different time, then detected apoptotic neurons with Hoechst33258, LDH leakage, SRB.(3) Established cell model of the toxic effect of excitatory amino acid in cerebral ischemia with glutamic-induced apoptotic neurons, and then we detected apoptotic neurons with Hoechst33258, LDH leakage, SRB.(4) We established the cell model of cerebral ischemia-reperfusion with H₂O₂ inducing neuronal apoptosis, and then detected apoptotic neurons with Hoechst33258, LDH leakage, SRB. (5) Adopted RT-PCR to specificity amplify the expression of KIAA0280 in the apoptotic neurons induced by different factor, and then we observed the expression of KIAA0280 in the different apoptotic neurons.(6) Transfered KIAA0280 specificity siRNA into neurons with liposome lipofectamine2000, then we observed the differences of normal and apoptotic neurons after transfection.Results(1) We have successfully established the technology of primary neuron culture. The results showed that the neurodendrons and neuraxons, the feature of neuron shape, is obvious and the neurons can survive over 30d.(2) We adopted airtight container to evacuate and filled with 95%N₂, 5%CO₂ can reduce interference factor in the experiment, and make the result more confidence and reproducibility. The results of Hoechst33258staining, SRB, and LDH approved the neuronal apoptotic model induced by hypoxia is success. The result of RT-PCR revealed that KIAA0280 obviously over-expressed in the hypoxia-induced apoptotic neurons. And the result showed that the apoptotic neurons were aggravated after silencing KIAA0280.(3) We established the cell model of excitatory amino acid toxity effect of cerebral ischemia with glutamate-induced neuronal apoptosis, and the results showed that the model is success. The result of RT-PCR showed that KIAA0280 was not obviously expressed in the apoptotic neurons induced by glutamate. And the result showed that the apoptotic neurons after transferring KIAA0280 specificity siRNA were not different from those after transferring negative control siRNA.(4) We added H₂O₂ into medium to induce neuronal apoptosis, and established the cell model of the oxygen free radical injury in cerebral ischemia-reperfusion. The results showed that the model is success. The results of RT-PCR showed that KIAA0280 was not obviously expressed in apoptotic neurons induced by H₂O₂. And the result showed that the apoptotic neurons after transferring KIAA0280 specificity siRNA were not different from those after transferring negative control siRNA. ConclusionWe successfully established the methods of primary neuron separation and culture in vitro and the model of neuronal apoptosis induced by different factor. The result from Hoechst33258, LDH, SRB and RT-PCR revealed that KIAA0280 had support effect on the normal neurons growth and played a positive role in protecting hypoxia-induced apoptosis neurons, rather than in the glutamic acid and H₂O₂ induced apoptotic neurons.