Objective To investigate the effect of DHA on proliferation and killing activity of γδT cells against SW1990 cells in vitro.Methods γδT cells were generated in vitro by stimulating peripheral blood mononuclear cells of healthy donors in RPMI 1640 completed medium containing IPP and IL-2 for 8 d,and then co-cultured with different concentrations of DHA for 48 h.Proliferation rates of γδT cells for each group were detected by MTT method.The perforin,granzyme B and CD107a expression in γδT cells were verified by flow cytometer.The cytotoxic activity of γδT cells against SW1990 cells were analyzed by CCK-8 kit.Results The purity of γδT cells in each group reached 75.46％ ±5.32％ after 8 d of culture.Compared with the control group,the proliferative capability of γδT cells were enhanced significantly after treated with 50-100 μmol/ml DHA for 48 h,moreover,cytotoxicity against SW1990 cells and perforin,granzyme B and CD107a expression of the γδT cells treated with DHA were higher than the control group.Conclusion DHA could enhance the antitumor activity of γδT cells,which may be associated with the upregulation of perforin and granzyme B expression in γδT cells.

Objective To investigate the early changes of CD4 + T cells and the relevant cytokines in blood of patients with severe trauma.Methods Sixty-one consecutive patients with trauma admitted into the 97th Hospital of PLA from September 2009 to November 2011 were enrolled in this study.The exclusion criteria included:① Patients were younger than 18 or older than 75 years.②Patients received blood transfusion.③Those suffered from immune system disorders,tumors or diabetes,or recent history of virus,bacteria or parasites infections.④ Those had current or recent treatment with corticosteroids or immunosuppressive drugs.According to ISS score ≥ 16,there were 61 traumatic patients divided into mild trauma group and severe trauma group.Seventeen healthy volunteers were taken as control subjects.At admission,the peripheral venous blood samples of patients were taken to count the number of CD3+,CD4 +,CD8 + T cells by flow cytometry and to detect TNF-α,INF-γ,IL-1,IL4,IL-6 and IL-12 levels in plasma by enzyme linked immunosorbent assay,meanwhile the ratio of Th1 / Th2 type cytokine were calculated.Data were analyzed with t test and ANOVA or Kruskal-Wallis test by using SPSS version 16.0package.P value ＜ 0.05 was considered to be statistical significance.Results Compared with control group,mild trauma and severe trauma groups showed a similar decrease in number of CD3 + T cells,and severe trauma group showed a most significant decrease in number of CD4 + T cells.Severe trauma group had a lower INF-γ level compared with control group; IL-1 level in both mild trauma and severe trauma groups were lower than that in the control group; INF-γ/ IL-6 in severe group was significantly lower than that in the mild group.However,INF-γ IL-6 in mild group was higher than that in the control group.Conclusions The early stage of severe trauma exhibits a significant decrease in number of both CD3 + and CD4 +T cells,accompanied by a significant reduction in most of cytokines,and has a tendency of shift to Th2 type cytokine.Therefore,the changes of immune cells should be promptly and successively monitored after severe trauma.

BACKGROUND:Animal models of osteoporosis play an important role in the research of the pathogenesis, occurrence and development of osteoporosis, as wel as in the clinical diagnosis, prevention and treatment of osteoporosis. OBJECTIVE: To summarize and discuss the establishment and research ideas of osteoporosis models, explore the current situation and advance of osteoporosis models, compare the advantages and disadvantages of various methods, and provide evidence for clinical investigation. METHODS: A computer-based online search was conducted in SinoMed, VIP, Wanfang and PubMed databases by using the key words of “animal model, osteoporosis” from January 1969 to October 2015. The language was limited to both Chinese and English. Relevant articles were screened according to inclusion and exclusion criteria. The documents about the methods of osteoporosis model preparation, method improvement as wel as their advantage and disadvantage were summarized. RESULTS AND CONCLUSION:A total of 576 articles were included. Among them, articles published earlier, duplicated, and similarly were excluded, and 53 articles were finaly included. Various animal models of osteoporosis may only focus on the certain causes, certain stage, some of the main symptoms and some pathophysiological changes of disease. Accordingly, appropriate modeling methods and experimental animals should be selected based on research objective. Rat undergoing castration is the most commonly used model in the modeling of osteoporosis. Among drug methods for constructing osteoporosis model, glucocorticoids is the most commonly used one. Disuse method and nutritional method have limitations, and always combined with castration and drug methods. The effects of gene transfer, gene mutation and brain-derived model deserve

Objective:To investigate the anti-tumor effect of Hyperoside.Methods: Human γδT cells were amplified by isopentenyl pyrophosphate from peripheral blood cells.The proliferation capacity of γδT cells was measured with CCK-8 assay after treated with different concentrations of Hyperoside.Cytotoxicity of γδT cells was detected with LDH assay , and the expression of granzyme,perforin CD107a and IFN-γonγδT cells were measured by flow cytometry before and after treatment.Results: Hyperoside could significantly stimulate the proliferation of γδT cells at the concentration of 3.13-12.5 μg/ml.Cytotoxicity and expression of granzyme,perforin and IFN-γofγδT cells were increased after treatment.Conclusion:Hyperoside could enhance cytotoxicity of humanγδT cells through up-regulation of granzyme ,perforin CD107 a and IFN-γexpression.

Objective To investigate the effects of hydrocortisone (HC) on proliferation and killing activity of NK cells against pancreatic cancer SW1990 cells in vitro.Methods Peripheral blood mononuclear cells of healthy people were isolated and cultured with NK cells medium containing IL-1S.When the purity of NK cells reached above 70％,different concentrations of HC (10-6,10-5,10-4,10-3 μmol/L) were added and co-cultured with NK cells for 7 days.And NK cells without HC were used as control.CD3-CD56 + NK cell numbers of each group were countered by trypan blue staining.Perforin,granzyme B and IFN-γ expression of CD3-CD56+ + NK cells were verified by flow cytometry.NK cells and SW1990 cells were co-cultured with a 20∶1effector to target ratio,then the cytotoxic activity of NK cells against SW1990 cells were analyzed by CCK-8 kit.Results After treatment with different concentration of HC for 7 days,NK cells purity of each group reached 70.72％ ～ 76.39％,and it was not significantly different with that in control group [(72.61 ± 3.76) ％].The proliferation folds of NK cells treated with 10-6,10-5,10-4,10-3 μmol/L HC were (9.13 ± 0.94),(9.67 ±1.51),(10.33±1.07),(8.40±1.47) times,respectively,while it was (4.23 ±0.82) times in control group (all P ＜0.01).The killing effects of NK cells on SW1990 cells were (58.58 ± 4.89) ％,(62.27 ± 5.63) ％,(64.02 ± 5.79) ％,(63.88 ± 3.61) ％,which were higher than that in control group [(57.46 ± 5.11) ％],moreover,the difference between NK cells of 10-4 μmol/L HC treatment group and control group was statistically significant(P ＜ 0.05).The expressions of perforins of 10-4,10-3 μmol/L HC treatment group were (96.71 ± 3.04) ％,(97.56 ± 2.18) ％,which were significantly higher than that in control group [(92.40 ±3.53)％,P ＜0.05 or 0.01].The expression of granzyme B in 10-5 μmol/L HC treatment group was (78.23 ±2.94)％,which were significantly higher than that in control group [(73.68 ±3.52) ％,P ＜0.05].The expressions of IFN-γ in 10-5,10-4,10-3 μmol/L HC treatment group were (96.61 ±2.04)％,(97.58 ± 2.17)％,(98.00 ± 1.77)％,which were significantly higher than that in control group [(92.44 ± 2.74)％,P＜0.01].Conclusions HC can promote IL-15 activated NK cells proliferation and enhance NK cells mediated killing activity against SW1990 cells with proper concentration,and up-regulation of perforin,granzyme B and IFN-γ expression may be the main mechanisms.

Objective To observe the effect of pituitary adenylate cyclase activiting polypeptide (PACAP) on traumatic brain injury (TBI) and on tbeCD4+/CD8+ T cell number in blood and spleen of rats.MethodsThe male SD rats were randomly (random number) divided into sham operation group ( n =6),normal saline + TBI group ( n =6) and PACAP + TBI group ( n =6).Right parietal cortical contusion was produced by a weight-dropping method.PACAP was administered intra-cerebroventricularly in a dose of 1 μg/5 μl 20 min before TBI.Brain tissue samples were taken 24 h after trauma.The injured brain tissue of rats was examined by HE stain.The numbers of CD4+/CD8+ T cells in blood and spleen were deteced with flow cytometry.Results Edema,hemorrhage,inflammatory cell infiltration,swollen,degenerated neurons and neurons arrayed disorderly around the injured cortex in hippocampus were found under light microscope.The average numbers of CD4 + T lymphocytes counts in blood and spleen were lower ( P =0.000,P =0.005 ),and number of CD8 + T lymphocytes was higher ( P =0.01 ) in TBI rats group than those in the sham operation group.Micro-injection of PACAP into cerebroventricular attenuated the injury,significantly increased the number of CD4 + T lymphocytes in blood and spleen ( P =0.019,P =0.839),and decreased the number of CD8 + T lymphocytes.ConclusionsPretreatment with PACAP may protect against TBI via influencing periphery T cellular immune function.

ObjectiveTo observe the effect of betulinic acid(BetA) on the growth of human cytokine induced killer(CIK) cells and the killing activity of CIK cells on the gastric cancer cells in vitro before and after induced by betulinic acid,explore its mechanism.MethodsPeripheral blood mononuclear cell (PBMC) were separated form the healthy and were induced with various of cytokine to become CIK cells in vitro.CIK cells were collected on the tenth day and were induced with betulinic acid in different concentrations,followed by 48 h,the colorimetric methyl thiazolyl tetrazolium(MTT) method assay the proliferation rate of human CIK cells.Flow cytometry (FCM) was used to detect the expression changes of perforin,granzyme B and CD107a of human CIK cells before and after betulinic acid-induced.Lactate dehydrogenase (LDH) release assay was used to measure the influence on cytotoxic activity of CIK cells induced by betulinic acid against gastric cancer cell line SGC-7901 in vitro.Western blot assay was used to measure the extracellular signal-regulated kinase1/2 (ERK1/2),and adapter proteins SH2-domain containing leukocyte protein of 76KD(SLP-76) and linker for activative of T cells(LAT) expression changes of human CIK cells before and after drug-induced.ResultsBetulinic acid can promote CIK cells growth when the concentration were in 0.08-10 μg/ml,the expression of perforin,granzyme B and CD107a of CIK cells were significantly higher than control group(P＜0.05) when the concentration of betulinic acid were in 0.3 μg/ml.In the meanwhile,the cytotoxic activity of CIK cells in vitro against gastric cancer cell line SGC-7901 were also remarkably higher than the control group (P＜0.05).The expression of SLP-76,LAT and ERK1/2 were significantly increased to a certain extent than the control group( P＜0.05 ),when CIK cells were treated with betulinic acid.ConclusionThese results suggest that betulinic acid can promote CIK cells growth in some concentrations and increase the cytotoxic activity of CIK cells against gastric cancer cell line SGC-7901,its mechanism may related with two factors,on the one hand,enhancing the activity of SLP-76,LAT and ERK1/2,on the other hand,increasing the expression of perforin,granzyme B and CD107a on the surface of CIK cells.

Objective To investigate the in vitro effects of quercitrin on the proliferation and the cytotoxicity of human γδT cells.Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy subjects and cultured with isopentenyl pyrophosphate and IL -2 to induce human γδT cells.The hu-manγδT cells were cultured with quercitrin at various concentrations for 48 hours.CCK-8 kits were used to analyze the in vitro proliferation and cytotoxic activities of γδT cells.Flow cytometry was performed to meas-ure the expression of granzyme B and perforin in γδT cells.The expression of p-ERK, p-Akt and Bcl-2 at protein level were detected by Western blot .Results The percentage of human γδT cells in PBMCs was in-creased from (2.96±1.83)%to (88.94±2.36)%after 10 days of culture.The quercitrin at concentrations of 10 to 80 μg/ml could promote the growth of γδT cells and up-regulate the expression of granzyme B , per-forin, p-ERK, p-Akt and Bcl-2 in a dose dependent manner .The cytolytic activities of γδT cells against co-lonic carcinoma cells ( HCT116 ) were enhanced by quercitrin .Conclusion Quercitrin could promote the proliferation of γδT cells and enhance the expression of granzyme B and perforin at certain concentrations in vitro.ERK1/2 and Akt signal transduction systems might be involved in the process .

Objective To observe the local immue response and changes of angiogenic factors of tumor cells in Heps-bearing mice after mesenchymal stem cell (MSC) are administrated. And to explore the feasibility and safety of MSC for liver tumors therapy. Methods MSC were obtained through adherent culture method. Phe-notypes of MSC were analyzed by flow cytometry. MSC were labeled with DAPI in vitro. 54 Mice of 8 weeks of age with subcutaneously transplanted liver carcinomas were developed randomly. When the maximal diameters of the tumor reached 0.5 - 0.8cm, they were divided into three groups randomly: MSC group, DAPI group and NS control group. 2 × 10~6 MSC and MSC marked by DAPI were administrated into the mice right rear back tumor tissue. The survival time of the tumor-bearing mice was recorded and the mean survival time was calculated. Immunohistochemical staining was performed to count CD4~+ T cells and CD8~+ T cells in the local tumor,as well as to examine the expression of vascular endothelial growth factor ( VEGF) in tumor cells. Results In the MSC group,the mean survival time was 45 d (95%CI;33 ～56 d) ,in the NS control group, the mean survival time was 33 d ( 95%CI : 28 ～ 37 d). There was a statistical significance in the difference between them ( P < 0.05). Immunohistochemical staining results showed as follow: the number of CD4~+ T cells and CD8~+ T cells in the MSC group decreased significantly in comparison with the NS control group at early stage. The expression of VEGF also decreased obviously in comparison with the NS control group and induced tumor cells necrosis at late stage. The survival time of MSC group was prolonged. Conclusion MSC can engraft in Heps-bearing tumor tissue, and inhibit T lymphocyte cellular immunity at early stage. It can reduce the number of CD4~+ T cells and CD8~+ T cells and promote tumor growth. MSC can down regulate VEGF expression and induce tumor cells necrosis at late stage. By this way,it can prolong the survival time of Heps-bearing mice.

Objective:To investigate the mechanisms of TWS119 induced CCR5 expression in hunman γδT cells. Methods:After treatment with various concentrations of TWS119 for 48h, the expression of CCR5 in γδT cells were detected by flow cytometry. The p-STAT3 and GAPDH expression were examined by Western blot analysis. Results: TWS119 could upregulate the expression of CCR5 in dose dependent manner. Western blot analysis revealed that TWS119 inhibit phosphorylation of STAT3,but had no significant impact on GAPDH. In addition, pretreatment of γδT cells with 0. 5 μmol/L STAT3 specific phosphorylation inhibitor Stattic could upregulate the expression of CCR5 and enhance the TWS119 induced CCR5 expression. Conclusion: TWS119 could upregulate CCR5 expression of γδT cells by inhibiting STAT3 phosphorylation in vitro.