Mortalin promotes ovarian cancer cell growth through MAPK-ERK signal pathway / 解剖学报

Objective By constructing mortalin stably expressing ovarian cancer cell lines in A 2780 and A2780/cis, we demonstrate the role of mortalin in the ovarian cancer cell growth .Methods CCK-8 assay was used to measure cell viability in the overexpression mortalin group compared with the control group .The flow cytometry analysis was used to understand the effect of upregulated mortalin on the ovarian cancer cell cycle .Western blotting was used to determine the expression and phosphorylation level of MAPK /ERK and JNK/SAPK signal pathways .Results The results showed that increased expression of mortalin could accelerate ovarian cancer cell proliferation and promote G 1 transition, leading to a faster restoration of normal distribution of cell cycle .We found that mortalin overexpression significantly activated p-Raf and p-ERK1/2, but not p-JNK.Conclusion The results demonstrate that mortalin effect on the ovarian cancer cell proliferation contributes to active the MAPK-ERK signaling pathway .

GRP75 overexpression inhibits apoptosis induced by glucose deprivation via Raf/Mek/Erk1/2 signaling pathway / 生理学报

The purpose of the present study is to investigate whether glucose-regulated protein 75 (GRP75) overexpression inhibits apoptosis induced by glucose deprivation through Raf/Mek/Erk1/2 signaling pathway. After pretreatment with Mek-specific inhibitor U0126, GRP75 overexpressing PC12 cells were incubated in glucose-free DMEM medium for indicated time (6, 12 and 24 h). And DMSO-treated GRP75 overexpressing PC12 cells were applied as control. Western blot was used to determine the expression and phosphorylation level of Erk1/2. MTT assay was used to measure cell viability. Hoechst 33258 staining and flow cytometry using propidium iodide (PI) staining was used to analysis apoptosis. Immunofluorescence with antibody against cytochrome c (Cyt c) was used to detect Cyt c release from mitochondrion. The results showed U0126 prevented the activation of Erk1/2 maintained by GRP75, but the total Erk1/2 expression was not affected. U0126-treated group showed lower cell viability and higher apoptotic rate compared with control group. Immunofluorescence indicated the delay in release of Cyt c was blocked by U0126. These results suggest U0126 prevents protective effect of GRP75 on PC12 cells by inhibiting Erk1/2 phosphorylation, which certifies that GRP75 can inhibit the mitochondria-dependent apoptotic pathway through Raf/Mek/Erk1/2 signaling cascade.

Effect of Grp75 on the alteration of apoptosis related gene Bax and NF-κB induced by glucose deprivation / 解剖学报

Objective To study the effect of glucose regulated protein 75(Grp75) on the alteration of Bax and NF-κB induced by glucose deprivation through the stably transfected PC12 cells with Grp75. Methods The cells of Grp75-overexpressing group and control group incubated in glucose-free DMEM medium for indicated time (6, 12, 24 and 48hours). The expression level of Grp75, Bax and the activity of NF-κB were determined by Western blotting, and the expression level of Bax was determined by semi-quantitative RT-PCR and Western blotting. Immunocytochemistry was performed using a conformation specific anti-Bax (6A7) antibody to detect the activation of Bax. Results The activation of Bax and the decline of NF-κB activity played important roles in the apoptosis of PC12 cells induced by glucose deprivation. Grp75 inhibited the apoptosis induced by glucose deprivation through inhibition of the activation of Bax and the decline of NF-κB activity. There was no change in Bax expression level under glucose deprivation in two groups. Conclusion The activation of Bax and the decline of NF-κB activity were associated with apoptosis of PC12 cells induced by glucose deprivation, and Grp75 provided protection to PC12 cells through inhibition of activation of Bax and maintaining activation of NF-κB.

Construction of eukaryotic expression vector of glucose-regulated protein 75 gene deletion mutant and its expression in PC12 cells / 生理学报

Glucose-regulated protein 75 (Grp75) binds to p53 and inhibits its nuclear translocation, and thus plays a role in cell protection. To investigate whether the binding of Grp75 and p53 would influence the viability of cells, we constructed the eukaryotic expression vector of Grp75 deletion mutant. The deletion mutant gene was obtained by SOE-PCR (gene splicing by overlap extension) and then linked to the pcDNA3.0 vector. The constructed specific expression vector, pcDNA3.0/Grp75(Δ253-282), was identified by restriction enzymes and sequencing. Then we used liposome to transfect the specific vector into PC12 cells. The stable cell strain PC12/Grp75(Δ253-282)(+) was selected by G418 (1 mg/mL). Semi-quantitative RT-PCR and Western blot showed that Grp75 mRNA and protein expressions in PC12/Grp75(Δ253-282)(+) cells were higher than those in PC12 cells. The viability of cells undergoing 0 h, 3 h, 9 h, 18 h and 36 h of glucose deprivation respectively was measured by MTT assay. The results showed that the cell viability of PC12/Grp75(+) group was significantly higher than that of the other two groups, and the cell viability of PC12/Grp75(Δ253-282)(+) group was significantly higher than that of the PC12 group (P<0.05). Hoechst33324 staining was employed to detect cell apoptosis and the results were consistent with the MTT assay results. Western blot results indicated that the expression of p53 in PC12/Grp75(+) cells was lower than those in the other two groups, which might be due to the overexpression of Grp75. These results suggest that the protective role of Grp75 is partly associated with its binding to p53. The above results suggest that Grp75 deletion mutation could to some extent reduce the viability of cells.

Local mild hypothermia therapy for neurogenic pulmonary edema / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the changes in stress hormones in neurogenic pulmonary edema (NPE) and explore the clinical value of mild hypothermia therapy for treatment of NPE.</p><p><b>METHODS</b>Fifty-two patients with cerebral hemorrhage patients and concomitant NPE were randomly divided into two groups for local mild hypothermia therapy (23 cases, LMH group) or routine treatment (29 cases, RT group). In the former group, local mild hypothermia therapy was applied in addition to the routine treatment. The changes of serum corticotrophin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), corticosteroid (Cor), arginine vasopressin (AVP) and blood sugar were observed before and 7 days after the treatment, and compared with those of 58 NPE-free patients with cerebral hemorrhage and 40 healthy individuals.</p><p><b>RESULTS</b>Serum CRH, ACTH, Cor, and AVP levels and blood sugar in NPE patients and the NPE-free patients were all significantly higher than those in the healthy individuals (P<0.01), and the levels were significantly higher in NPE patients than in the NPE-free patients (P<0.05). In the NPE patients, the mortality rate and NIHSS score were significantly lower in RT group (P<0.01); after 7 days of treatment, both LMH and RT groups showed significant reduction in serum CRH, ACTH, Cor, and AVP levels (P<0.05), and the reduction was more conspicuous in LMH group (P<0.05).</p><p><b>CONCLUSION</b>The occurrence of NPE is closely associated with stress reactions, which might be the basis of NPE. Local mild hypothermia therapy improves of the quality of life of NPE patients and also decreases the mortality of NPE possibly by inhibiting the secretion of stress hormones and stabilizing the hypothalamic-pituitary-adrenal axis.</p>

Glucose regulates protein 75 expression in PC12 cell line under glucose deprivation / 基础医学与临床

Objective To identify the effect of glucose regulating protein 75 expression in PC12 cells under glucose deprivation.Methods MTT method was used to monitor the cell viability.The leakage of LDH was represented as the index of cell injury.Flow cytometry was applied for cell cycle analysis,apoptosis and necrosis.Using RT-PCR detected and quantified mRNA.Western blot to measure relative amounts of the protein.Results Cell viability decreased and the LDH leakage increased.While the treated PC12 cells had two main forms of apoptosis and necrosis.GRP75 mRNA and protein levels were upregulated.Conclusion GRP75 has protective function during glucose deprivation.

Wnt signalling pathway regulates the growth and several phenotypes of Rat-1 cells / 生理学报

The Wnt signaling pathway is thought to be functionally conserved in vertebrates and invertebrates and plays an important role during the embryonic and postembryonic development. Recent studies indicated that this pathway may be also involved in the controlled proliferation and migration of some kinds of fibroblasts during the wound healing process. To verify this assumption in vitro, we chose Rat-1, a kind of rat fibroblasts to investigate the regulation of Wnt signaling pathway to the growth and changes of several phenotypes of this kind of cells. Full length Wnt-3a cDNA was inserted in pcDNA 3.1 vector to construct the Wnt-3a mammalian expression vector, which was stably transfected into Rat-1 cells, and then to establish a cell model in which Wnt signaling pathway was constantly activated. When Wnt signaling pathway was activated constantly, Rat-1 cells exhibited morphological changes: grew more densely as a monolayer, adopted an elongated and refractile appearance, forming cord-like bundles lined up in a uniform direction. The results of MTT assay and FCM analysis indicated that more Rat-1/Wnt-3a cells entered into G(2) phase and the proliferation rate of the Rat-1/ Wnt-3a cells increased significantly compared to the non-transfected cells. Though the migration of Rat-1/Wnt-3a cells increased slightly by the method of Transwell migration assay, there was no statistic significance compared to the non-transfected cells. The result of in vitro scrape wound healing assay showed that for Rat-1/Wnt-3a cells the time course of wound healing decreased significantly. It is therefore concluded that the activation of Wnt signaling pathway can regulate some of the phenotypes of Rat-1 cells, facilitate cell proliferation and promote the scrape wound healing in vitro.

The effects of GM1 and bFGF synergistically inducing adult rat bone marrow stromal cells to form neural progenitor cells and their differentiation / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the effects of GM1 on inducing adult rat bone marrow stromal cells (MSCs) to form neural progenitor cells and their differentiation.</p><p><b>METHODS</b>Purified MSCs were induced by different components of basic fibroblast growth factor (bFGF) alone, GM1 alone or combination of bFGF with GM1. After 3 days' incubation, fibronectin and collagen I were detected with immunocytochemistry, and nestin was detected with immunofluorescence. Neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP) and galactose cerebroside (GalC) were detected with immunocytochemistry after 7 days' incubation.</p><p><b>RESULTS</b>After induction with bFGF alone or combination of bFGF and GM1, some MSCs exhibited the phenotypes of neural progenitor cells, and then neurons and astrocytes. In these two groups, the positive cells for fibronectin and collagen I decreased markedly after 3 days' induction. At the same time, the positive cells for nestin increased markedly. After 7 days' induction, NSE and GFAP-positive cells increased significantly. Furthermore, the addition of bFGF and GM1 caused the maximal variation. However, addition of GM1 alone had no inductive effects.</p><p><b>CONCLUSIONS</b>Combination of bFGF with GM1 may synergistically promote the transformation of MSCs and differentiation into neurons and astrocyte-like cells. The results suggest a promising route for the application of MSCs.</p>

Recent advances in wnt-frizzled cascade and its relation to cardiovascular diseases / 中国病理生理杂志

Many researches have focused on the wnt-frizzled cascade in the recent years, while much work has been done in neoplastic diseases and embryology, the role of the wnt-frizzled signal transduction pathway in cardiovascular diseases has only recently begun to be explored. It plays a very important role in many physiological and pathophysiological processes, such as its transduction pathway, the healing after myocardial infarction, the proliferation, differentiation and orientation of cardiomyocytes, angiogenesis/neovascularization, cardiac hypertrophy and heart failure, the deposition of the extracellular matrix and so on. This article is aimed at its relation with myocardial infarction and the role of this pathway in cardiovascular diseases.[