Study of Evodiamine in Inducing G2/M Phase Arrest in Renal Carcinoma 786-0 Cells and Its Molecular Mechanism / 广州中医药大学学报

Objective To investigate the growth inhibition effect of evodiamine (Evo) on renal carcinoma 786-0 cells and to explore its molecular mechanism. Methods After treated with Evo, methyl thiazolyl tetrazolium ( MTT) assay was used to detect the vitality of 786-0 cells, flow cytometry was employed to examine the cell cycle distribution in 786-0 cells, and immunoblotting was utilized to determine the expression levels of target proteins related to cell cycle progression. Results Evo remarkably inhibited 786-0 cells vitality in dose-dependent manner. Cell cycle analysis indicated that 786-0 cells were arrested in G2/M phase followed by Evo treatment. Furthermore, the results of immunoblotting showed that Evo up-regulated the protein expression levels of P53, P21 and its downstream target gene CyclinB1 in 786-0 cells. Conclusion Evo treatment can induce 786-0 cell cycle G2/M arrest, and its underlying mechanism might be dependent on the P53/P21 signal pathway.

Effect of different cult ure conditions on differentiation of Treg and Th17 in PB-MC / 中国免疫学杂志

Objectiv e:To investigate the effect of different culture conditions on the differentiation of Treg and Th17 to lay a foundation for exploring the methods to reverse the immune tolerance induced by tumor microenvironment.Methods:The IL-6 gene was cloned and stablely transferred into the tumor cell line expressing TGF-β.The conditioned mediums ( CM) were prepared by collecting the culture supernatants of tumor cell lines with or without IL-6 expression and used in the in vitro culture of peripheral blood mononuclear cells ( PBMC ) .The changes of Treg and Th17 in PBMC treated with different CM were detected with flow cytometry ( FCM) .Results:The expression of TGF-βin BEL-7402 was higher than that in HepG2.Thus the BEL-7402 was selected for preparation of cell line stablely transfected with IL -6 gene.ELISA detection confirmed the effective expression of IL -6 by the identified cell lines.It was showed that the Treg increased in PBMC treated with culture supernatants of tumor cells .However,the presence of IL-6 reversed the increase of Treg and promoted the differentiation of Th 17.Conclusion: The culture supernatants of tumor cells increases the proportion of Treg.However,the presence of IL-6 in this CM can reverse the increase of Treg and raise the proportion of Th 17.

Development of a novel assay for cytotoxicity test by Calcein-AM release / 中国免疫学杂志

Objective:To develop and optimize a novel assay for determination of cytotoxicity based Calcein -AM release.Methods:The target cells stained by Calcein-AM dye,then effectors and targets were incubated at E/T ratios from 30∶1-1∶1 for 4 h at 37℃,and the supernatant of reactions were detected by Fluorescence-Measurement to analyze specific cytotoxity.Results:The optimal excitation and emission wave lengths of Calcein were 485 nm and 515 nm.Dilutions of target cells stained by Calcein-AM had a linear relationship with measured fluorescence values.The Calcein-AM dye used to stain the living cells was shown to have a low spontaneous leakage rate-less than 15% in 4 hours at 37℃.Cytotoxicity activity of CIK showed a significant and positive correlation with E/T ratio when incubated at 4 h.Conclusion:The developed cytotoxicity test by Calcein-AM release is accurate and can avoid the application of radioactive reagents.

Specific TCRαβgene modification promotes anti-tumor reactivity of T cells / 中国免疫学杂志

Objective:To investigate effects of tumor specific TCR gene Vα12.2-Vβ7.1 modification on recognition of tumor antigen and activation of anti-tumor reactivity of T cells.Methods: T cells were transduced using recombinant Ad 5F35-TRAV-TRBV adenovirus ,and multiplicity of infection was optimized.Specific lysis of T cells was evaluated by calcein release assay.The frequency of apoptotic cells in target cells was detected by Annexin V /PI double-labeled FACS.The expression of FasL on T cells was analyzed by FACS.The secretion of cytokine IFN-γand IL-2 of T cells was determined by ELISA assays.Results: The highest tranduce efficiency was obtained at MOI 100 by recombinant Ad5F35-TRAV-TRBV adenovirus.The frequency of TCRVα12+Vβ7+cells reached above 25%3 days after transduction.TCR gene modification enhanced the ability of T cells to lyse HLA-A2+AFP+target cells(P<0.001), the ability of T cells to induce HepG-2 apoptosis(P<0.001),and expression of FasL on T cells(P<0.001).TCR gene modification also enhanced T cells to secret IFN-γafter coculture with antigen positive tumor cells ( P<0.001 ).Conclusion: Specific TCR gene modification by recombinant adeno virus effectively promotes T cells to recognize antigen positive tumor cell and exert anti -tumor reac-tivity.

Human umbilical cord-derived mesenchymal stem cells inhibit proliferation but maintain survival of Jurkat leukemia cells in vitro by activating Notch signaling / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on the proliferation and survival of Jurkat leukemia cells in vitro and explore the possible mechanism.</p><p><b>METHODS</b>Jurkat leukemia cells were co-cultured with hUC-MSCs isolated from human umbilical cord tissues by plastic adherence at a ratio of 10:1. The proliferation and survival of the co-cultured Jurkat cells, separated by immunomagnetic bead cell sorting on day 4, were evaluated by flow cytometry. Western blotting was performed to evaluate the activation of Notch signaling in the co-cultured Jurkat cells.</p><p><b>RESULTS</b>Jurkat leukemia cells co-cultured with hUC-MSCs for 4 days showed a lowered proliferation rate and cell cycle arrest at G0/G1 phase with a reduction in the cell apoptotic rate. Notch signaling pathway was activated in the co-cultured Jurkat cells as evidenced by an increased cellular expression of HES-1.</p><p><b>CONCLUSION</b>Co-culture with hUC-MSCs can inhibit the proliferation of Jurkat leukemia cells in vitro and protect the cells from apoptosis by activating Notch signaling, indicating a potential shielding effect of MSCs on leukemia cells.</p>

Progress in infection pathway and intracellular trafficking of adenovirus / 生物工程学报

The research on intracellular trafficking of adenovirus has been described mainly through observations of subgroup C adenoviruses in transformed cell lines. The basic elements of the trafficking pathway include binding to receptors at the cell surface, internalization by endocytosis, lysis of the endosomal membrane, escape to the cytosol, intracellular trafficking along microtubules, nuclear pore docking, and viral genome translocation into the nucleus. More than 80% of the adenovirus genome is delivered to the nucleus in a highly efficient manner in approximately 1 h. However, exceptions to this trafficking pattern have been noted, including: variations based on target cell type, cell physiology, and adenovirus serotype. This review summarizes mechanism of adenovirus infection pathway and intracellular trafficking, providinging a foundation for the development of clinical adenoviral vector.

Survivin-2B induces apoptosis of human breast cancer cells / 中国病理生理杂志

AIM:To explore the role of survivin-2B in the process of tumor cell apoptosis .METHODS:The survivin-2B gene was cloned into pcDNA3.1 vector and the recombinant plasmid pcDNA3.1-survivin-2B was obtained.Hu-man breast cancer MCF7 cells were transfected with pcDNA3.1 and pcDNA3.1-survivin-2B using Lipofectamine 2000.The cell cycle was determined by propidium iodide staining , and the apoptosis was detected by annexin V/7-AAD staining 48 h after transfection.Meanwhile, tatal RNA was extrated and multiplex polymerase chain reaction based on GenomeLab GeXP Genetic Analysis System was performed to detect the expression of 21 tumor-related genes .RESULTS: Flow cytometry analysis indicated that over-expression of survivin-2B promoted the apoptosis and cell cycle arrest of MCF 7 cells.Compared with control group , totally 10 differential expressed genes were related to the over-expressed survivin-2B, among which 2 were up-regulated and 8 were down-regulated. The expression of aldehyde dehydrogenase 4 family member A1 (ALDH4A1) was 48%down-regulated, and the expression of protein regulator of cytokinesis 1 (PRC1) was 1.08 folds up-regulated.CONCLUSION:Survivin-2B induces the expression changes of some tumor-related genes, which results in the apoptosis and G 2/M arrest of MCF7 cells.

Influence of supernatant from human umbilical cord-derived mesenchymal stem cells on proportions of each human lymphoid subgroup / 中国免疫学杂志

Objective:To investigate the impact of human umbilical cord-derived mesenchymal stem cells on the activation ,the survival of human peripheral blood mononuclear cell ( hPBMC) and the proportions of each human lymphoid subgroup .Methods:PB-MC were isolated from healthy donors by density gradient centrifugation , then cultured in MSC-CM as treatment group after being acti-vated by OKT3.Each lymphoid subgroup proportion was analyzed by flow cytometry to observe the difference between treatment and control group .The effect of MSC-CM on activated PBMC for the production of IFN-γand IL-10 were tested by ELISA .The level of ap-optosis was assessed by flow cytometry with Annexin-V/PI as fluorescent marker .Results:Compared with the control group , MSC-CM down-regulated the ratio of CD4 +T cell to CD8 +T cell, and increased the proportion of CD4 +CD25 +CD127low Treg cell, thus other subgroup had no significant difference .MSC-CM inhibited the production of IFN-γby PBMC, but promoted the secretion of IL-10, and protected PBMCs from apoptosis when activated with OKT 3.Conclusion:hUC-MSC may play a role of immunosuppression by promo-ting the proliferation and activation of Treg cell .This kind of inhibitory activity is neither relied direct or indirect contact with the lym -phocytes , nor influenced by inducing immune cells apoptosis .

HLA-A2-restricted point-mutated Survivin peptide elicited specific cytotoxic T lymphocyte reaction against hepatocarcinoma / 中华微生物学和免疫学杂志

Objective To investigate the anti-hepatocarcinoma(anti-HCC) function of HLA-A2-restricted point-mutated Survivin peptide induced CTLs.Methods The HLA-A2-restricted Survivin nonapeptides were evaluated using bioinformatics software.The binding affinity of Survivin peptide to HLA-A2 molecular was determined with flow cytometry analysis.After peptide-induced CTLs were generated in vitro,flow cytometry and ELISA were performed to detect the levels of IFN-γ,which were secreted by reactive CTLs.Peptide-induced CTLs were co-cultured with hepatoma cell lines HepG2 and BEL-7402.The rates of tumor cells lysis were assayed using CytoTox 96(R) and the morphological changes of tumor cells were observed with inverted phase contrast microscope.Results Point-mutated Survivin nonapeptide Sur79M2 (KMSSGCAFL) was filtered out,which was shown higher scores compared with the wild-type peptide Sur79.Consistent with the results of software analysis,Sur79M2 showed higher binding ability in T2 binding assays.At the same time,Sur79M2-induced CTLs could release a large number of IFN-γ after incubated with target cells rather than Sur79.When co-cultured with HCC cell lines HepG2 and BEL-7402,Sur79M2-induced CTLs effectively lysis HepG2 on HLA-A2-restricted manner without killing effect on BEL-7402 that do not express HLA-A2 molecules.Conclusion Sur79M2 could elicited specific cytotoxic T lymphocytes in vitro,which were able to specifically kill HCC cell lines on HLA-A2-restricted manner.

Effects of Ulinastatin on the function of splenic lymphocytes from rats with severe acute pancreatitis / 中华急诊医学杂志

Objective To investigate the effects of Ulinastatin(UTI)on the function of splenic lymphocytes from rats with severe acute pancreatitis(SAP).Method Twenty-eight Wister rats(clean grade)were randomly divided into control,sham operation,SAP,and ulinastatin group.No operation was performed in control group.And rats with sham-operation received laparotomy and catheterization into choledocho-pancreatic duct without injection of sodium deoxycholic.Rats in ulinastatin group received ulinastatin injection(50000 U/kg)via tail vein 30 minutes after pancreatitis induced with DCA injected into pancreatic duct.Rats ofother groups were given equal volume of saline.At 2,4 hours after operation,all animals were killed by neck dislocation,and splenocytes were isolated and cultured in RPMI 1640 medium containing 10%fetal calf serum.Proliferation of splenecytes was determined with MIT cellular proliferation assay.Levels of Th1 cytokines(IL-2,IFN-γ)and Th2 cytokine(IL-10)in supematants of splenoeytesweremeasured by ELISA.Quantitative data were expressed as mean±SE.Statistical analyses were performed by Student's t test with SPSS software(version 10.0 for Windows).A P value less than 0.05 Was considered statistically significant. Results The concentration of IL-2, IL-10 and IFN-γ and proliferative activity of splenocytes in SAP group were significantly lower than that in sham operation group.In contrast,the proliferative as well as the eytokine-releasing capacities of the solenecms from rats treated with UTI were significantly increased compared with those from rats with SAP.Conclusions The deficiencies in proliferation and cytokine release in response to antigen stimulation inaplys an anergic state of splenocytes during SAP.Treatment with UTI contributed to the recovery of the immune function by improving proliferative responses and cytokine release of splenocytes.

The mechanism by which taxol induces apoptosis of pancreatic acinar cells during pancreatitis in rats / 中国普通外科杂志

Objective To investigate the mechanism by which taxol induces apoptosis of pancreatic acinar cells during experimental acute pancreatitis in rats. Methods Intrapancreatobiliary duct injection of sodium deoxycholate was carried out to establish acute pancreatitis model in Wistar rats. Taxol was injected intraperitonealy to induce pancreatic acinar cell apoptosis. The expression of apoptosis associated protein Bcl-2,Bax,Fas,FasL and p53 was detected by immunohistochemistry. Results The expression of Bax(2 506?942),Fas(279?150) and p53(180?56) increased,while Bcl-2(79?42) remained unchanged in pancreatic acinar cells during acute pancreatitis,and the expression of FasL could only be detected in infiltrative inflammatory cells. Conclusions During acute pancreatitis,the acinar cell apoptosis induced by taxol is associated with activity regulation of Bax,Fas/FasL system and p53.

A study on the relationship between acute pancreatitis and apoptosis of the acinar cells / 中国普通外科杂志

ObjectiveTo investigate the relationship between the severity of acute pancreatitis and apoptosis of acinar cells. MethodsThe apoptotic ratio of acinar cells was measured in rat acute pancreatitis model by methods of in situ end labeling.ResultsThere was almost no apoptotic acinar cells in the model of acute edematous pancreatitis. With the increase in the severity of the pancreatitis apoptotic cells were more and more common until to the late stage of hemorrhagic and necrotic pancreatitis when the apoptotic ratio of acinar cells dwindled, meanwhile the necrotic acinar cell increased continuously.ConclusionsIn acute simple edematous pancreatitis apoptotic acinar cells is infrequently seen. In acute moderate hemorrhagic and necrotic pancreatitis the ratio is correlated positively with the severity of pancreatitis.In the late stage the ratio was correlated negatively with the severity of pancreatitis.