| Background and SignificanceMorphine is the main alkaloid in opioid, possessing the forceful effect of anesthesia and analgesia. Although morphine is the most effective compound for the treatment of pain and have been used over the centuries, the side-effects limit its usage, for example, immunosuppression, hyperpathia, dependence and tolerance. What is more, drug abuse is one of the social events which threaten the survival of mankind. Therefore, research on its molecular mechanism would contribute to find the approach to prevention and treatment of addiction. The mechanisms of drug addiction is very complex. For decades, domestic and overseas research have been focused on ethology, neurotransmitter, receptor and differential expression of proteins. However, few reports investigate the mechanism from the perspective of ncRNA, especially lncRNA (Long non-coding RNA).In the present study we aimed to find lncRNAs involved in morphine-induced side-effects and to explore its molecular mechanism. lncRNA is a class of RNA longer than 200nt, which cannot encode proteins but regulates gene expression on multiple levels. The resent reports suggested IncRNA is involved in a variety of biological processes and its dysregulated expression was closely related to many diseases. lncRNA gradually become the new molecular target for disease treatment.The animal model we used was the morphine-addicted C57 BL/6 mice. We chose the prefrontal cortex (PFC) as the experimental material because PFC is one of the most sensitive part in the brain to morphine. The neuroanatomy shows there are dense μ opioid receptor in the PFC. PFC is involved in the regulation of addiction-related behavior and mood changes. To build the corresponding in vitro model, we treated the BV2 cell, a cell line of mouse microglia (MG), with 10μM morphine for different time period. MG is the smallest gliocyte in the central nervous system (CNS) and is the immunocyte of nervous system, which can protect the brain from damage or the recurrence of brain diseases. In addition, MG takes the role of maintenance, nutrition, protection and recovery in the physiological activities of the neurons, and MG is the first and foremost immune defense of the CNS.After the modeling of the morphine-addicted mice, the total RNA of PFC was extracted. And after the quality testing of RNA samples, the lncRNA expression profile was analyzed by the lncRNA microarray and verified subsequently by the method of qRT-PCR. In the meantime, the MG was treated by morphine in order to verify on a cellular level. For the significance of this study, we research the side-effects of morphine from the point of lncRNA, providing a new train of thought to investigate the mechanisms of the dependence, addiction and immunosuppression, broadening and enriching the addiction-related molecular regulation network, offering theoretical foundation and novel therapeutic targets, providing application prospect for lncRNA as the target of clinical addiction-treatment agents.Results1. The lncRNA expression profile chip screened out 493 upregulated lncRNAs and 255 downregulated lncRNAs in the PFCTo find out lncRNAs involved in the side-effects of morphine, the morphine-addicted mice model was built. After dissected the mice and fast isolated the PFC, the total RNA of the tissue was extracted. Then the RNA samples were send to company of biochip and detected by the lncRNA microarray. The number of upregulated and downregulated lncRNAs was 493 and 255 (|Fold change|> 1.5 and P<0.05), respectively.2.5 lncRNAs were verified in the PFC and none was verified in the BV2 by qRT-PCR assay10 lncRNAs (|Fold change|> 6 and P< 0.01) was picked out for further research. The results of qRT-PCR of PFC suggested that 5 lncRNAs:AK157323, AK007908, AK160450, AK018061 and AK140599, were agreed well with the results of microarray, among which AK157323 and AK140599 were upregulated observably, AK007908, AK160450 and AK018061 were downregulated markedly. Nevertheless, the changes of lncRNAs did not verified favourably in BV2 cell, which still need further verification and investigation.ConclusionWe finally confirmed that AK157323, AK007908, AK160450, AK018061 and AK 140599 changed significantly in the PFC of morphine-addicted mice by IncRNA microarray and the subsequent qRT-PCR verification. It is possible that these lncRNAs have the potential of participation in the process of dependence, immunosuppression or apoptosis induced by morphine, which will lay a solid foundation for the further research of molecular mechanisms.Background and SignificanceHeart disease is the leading killer threatening human survival and health. For the treatment of myocardial infarction (MI), the therapy of coronary artery reperfusion can inhibit the occurrence of heart failure and decrease the mortality. Although it is resultful, the process of reperfusion itself could induce myocardial injury and pathological myocardial remodeling, which is known as the’myocardial ischemia/reperfusion injury’. The most prominent characteristic is the irreversible cell death.Researchers usually conduct study in vitro, in which the cardiomyocytes bear Hypoxia/Reoxygenation (H/R) treatment to simulate the process of ischemia/ reperfusion (I/R) in vivo. Namely, the in vitro cultured cardiomyocytes bear the absence of serum from its culture medium and hypoxia, and then bear the recovery of serum and reoxygenation. H/R can induce destructive apoptosis and autophagy and H/R is an important pathological factor for tissue injury caused by myocardial infarction. Hence, it would provide potential targets for the treatment of H/R to investigate the molecular mechanism of H/R injury. Heart diseases are generally considered to be caused by the dysregulation of gene regulatory network in heart. Further more, long non-coding RNA (lncRNA) dysregulation is involed in a great variety of human disease including cardiovascular diseases.We built the research model of I/R injury in vitro using H9c2. After the extraction of the total RNA, lncRNA sequencing was conducted and the relative bioinformatics analysis was carried out. The current research would provide novel theoretical basis and molecular targets for the ischemic heart diseases from a new field of lncRNA.Results1. Hypoxia/Reoxygenation induced apoptosis of H9c2After the H/R treatment, the morphology of partial H9c2 turned into sphere and floated in the culture medium, indicating the partial cells died. The findings of Western Blot suggested that H/R induced apoptosis dramatically. This conclusion was further confirmed by the results of Flow cytometry (FCM) analysis. In addition, the cell necrosis was also increased.2. Hypoxia/Reoxygenation induced autophagy of H9c2The findings of Western Blot showed that the level of autophagy elevated distinctly after H/R.3. The results of lncRNA sequencingCompared with control group, the difference was statistically significant in the expression of 46 mRNA transcripts,2 known lncRNA transcripts,1029 new lncRNA transcripts and 100 genes in the samples of experiment group.ConclusionH/R induced apoptosis and autophagy prominently, manifesting the model in vitro we built was very successful. In the follow-up studies, we will conduct the investigation of the molecular mechanism based on the lncRNA sequencing results combined with the bioinformatics analysis results (lncRNA target prediction, interaction of lncRNA-miRNA, KEGG biological pathway enrichment analysis, GO gene function enrichment analysis and so on) and conventional experimental methods.Background and SignificanceStem cell is a category of cell with the ability of proliferation, capacity of self-renewal and the potential of differentiation. Stem cells can generate highly differentiated functioning cells. In recent years, the advanced stem cell transplantation therapy springed up based on an increasing number of studies on stem cell. Hematopoietic stem cells (HSCs) is researched most widely in aldut. HSCs belong to multipotent adult stem cell and can differentiate into all kinds of mature blood cells.Stress have a significant influence on organism. Chronic stress have great effect on immune system, leading to the occurrence and development of diseases, for example, immune disorders, infection and cancer. Restraint stress upregulates the level of glucocorticoids, giving rise to the apoptotic of lymphocyte in spleen, thymus and peripheral blood. It is reported that bone marrow mesenchymal stem cell (MSC) inhibited the apoptosis of splenic lymphocyte induced by restraint stress. While, HSC is the origin of the hematopoietic cells and immune cells. Furthermore, HSC is more specific than MSC in respect of function. The purpose of the current study is to explore whether HSC take a role in restraint stress-induced immunosuppression and even have better efficacy than MSC. The model in vivo we used was the restraint stress model of Balb/C mice. In the corresponding experiments in vitro, the splenic lymphocyte was treated with dexamethasone (Dex), a synthetic glucocorticoid steroid hormones, to simulate the impact of restraint stress.We obtained HSCs from bone marrow of normal donor mice by magnetic cell separation system, and the HSCs were treated by CFDA SE so that the HSCs could be labeled with green fluorescence. The marked HSCs were injected via the caudal vein into the recipient mice. Next, the recipient mice were subjected to chronic physical restraint. Then, we observed the distribution of HSCs labeled with green fluorescence in the spleen and bone marrow. In the experiment in vitro, splenocytes were co-cultured with HSCs in culture medium containing dexamethasone. The apoptosis and necrosis were detected by flow cytometry (FCM).Results1. HSCs homed to the spleen and bone marrow from the third day of restraint stress CFDA SE-treated HSCs were not found in the spleen and bone marrow of the recipient mice on the second day of restraint stress, but observed from the third day of restraint stress. Moreover, compared with the third day, the number of cells marked with green fluorescene increased on the forth day.2. HSCs inhibited necrosis of splenocytes rather than apoptosis in vitro The results of FCM reveled that dexamethasone induced the apoptosis and necrosis of splenocytes. However, when co-cultured with HSCs, the necrosis rate of splenocytes decreased but the apoptosis rate increased, which was inconsistent with the previous report more or less.Conclusion1. HSCs appeared in spleen and bone marrow from the third day of restraint stress. It is likely that HSCs take a recovery function in damaged location.2. HSCs prevent necrosis and promote apoptosis of splenocytes induced by dexamethasone, leading to the viability increased. |
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