Screening for Anti-tumor Activity of Fractions from Buddleja officinalis Maxim

[Objectives]The anti-tumor activity of fractions from Buddleja officinalis Maxim.by petroleum ether,ethyl acetate,n-butanol and water solvent was studied.[Methods]The ethanol extract from B.officinalis Maxim.was extracted and then concentrated with petroleum ether,ethyl acetate,n-butanol and water,respectively,and the extracts were obtained.The inhibitory effects of the four different fractions on the growth of three tumor cell lines in vitro were detected by CCK-8 method,and the median inhibitory concentration(IC 50 value)was calculated.[Results]The four fractions inhibited the growth of the three tumor cell lines in vitro,among which the n-butanol fraction had the best anti-tumor activity.The IC 50 values of the n-butanol fraction on human gastric cancer(SGC-7901),human breast cancer(MCF-7)and human liver cancer(BEL-7404)cell lines were 0.08,1.58 and 0.12 mg/mL,respectively.[Conclusions]Petroleum ether,ethyl acetate,n-butanol and water fractions from the ethanol extract of B.officinalis Maxim.had certain anti-tumor effects,and the n-butanol fraction had the best anti-tumor activity.

Preparation of kakkatin derivatives and their anti-tumor activity

BACKGROUND Modern pharmacological studies have confirmed that plant-derived compounds from Puerariae flos(PF)has significant biological activities against liver damage,tumors and inflammation.Kakkatin is an isoflavone polyphenolic compound isolated from PF flower.However,the effect of kakkatin and its derivatives on anti-tumor has not been well explored.AIM To design and synthesize a kakkatin derivative[6-(hept-6-yn-1-yloxy)-3-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one(HK)]to explore its anti-tumor biological activity.METHODS Hept-6-yn-1-yl ethanesulfonate was introduced to replace hydrogen at the hydroxyl position of kakkatin phenol,and the derivative of kakkatin was prepared;the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to detect cell viability,a clone formation assay was adopted to detect cell proliferation,apoptosis,necrosis,and cell cycles were analyzed by Annexin V/propidium iodide staining and flow cytometry.Cell migration and invasion ability were evaluated by cell scratch assay and transwell assay.The potential mechanism of HK on hepatocellular carcinoma(HCC)SMMC-7721 cells was explored through network pharmacology and molecular docking,and finally real-time PCR assays was used to verify the potential targets and evaluate the biological activity of HK.RESULTS Compared with kakkatin,the modified HK did not significantly increase the inhibitory activity of gastric cancer MGC803 cells,but the inhibitory activity of HCC SMMC-7721 cells was increased by about 30 times,with an IC50 value of 2.5μM,and the tumor inhibition effect was better than cisplatin,which could significantly inhibit the cloning,invasion and metastasis of HCC SMMC-7721 cells,and induce apoptosis and G2/M cycle arrest.Its mechanism of action is mainly related to the upregulation of PDE3B and NFKB1 target proteins in the cAMP pathway.CONCLUSION HK have a significant inhibitory effect on HCC SMMC-7721 cells,and the targets of their action may be PDE3B and NFKB1 proteins in the cAMP pathway,making it a good lead drug for the treatment of HCC.

Galectin 2 regulates JAK/STAT3 signaling activity to modulate oral squamous cell carcinoma proliferation and migration in vitro

Background:Galectin 2(LGALS2)is a protein previously reported to serve as a mediator of disease progression in a range of cancers.The function of LGALS2 in oral squamous cell carcinoma(OSCC),however,has yet to be explored,prompting the present study to address this literature gap.Methods:Overall,144 paired malignant tumor tissues and paracancerous OSCC patient samples were harvested and the LGALS2 expression levels were examined through qPCR and western immunoblotting.The LGALS2 coding sequence was introduced into the pcDNA3.0 vector,to enable the overexpression of this gene,while an LGALS2-specific shRNA and corresponding controls were also obtained.The functionality of LGALS2 as a regulator of the ability of OSCC cells to grow and undergo apoptotic death in vitro was assessed through EdU uptake and CCK-8 assays,and flow cytometer,whereas a Transwell system was used to assess migratory activity and invasivity.An agonist of the Janus Kinase 2(JAK2)/Signal Transducer and Activator of Transcription 3(STAT3)pathway was also used to assess the role of this pathway in the context of LGALS2 signaling.Results:Here,we found that lower LGALS2 protein and mRNA expression were evident in OSCC tumor tissue samples,and these expression levels were associated with clinicopathological characteristics and patient survival outcomes.Silencing LGALS2 enhanced proliferation in OSCC cells while rendering these cells better able to resist apoptosis.The opposite was instead observed after LGALS2 was overexpressed.Mechanistically,the ability of LGALS2 to suppress the progression of OSCC was related to its ability to activate the JAK/STAT3 signaling axis.Conclusion:Those results suggest a role for LGALS2 as a suppressor of OSCC progression through its ability to modulate JAK/STAT3 signaling,supporting the potential utility of LGALS2 as a target for efforts aimed at treating OSCC patients.

Research on the biological activity and anti-tumor effect against lymphoma cells of DC-CIK cells

Objective： To investigate the proliferation capabilities, immunophenotype changes, level of secreted cytokines and activities against lymphoma cells under the condition that cytokine-induced killer （CIK） cells co-cultured with dendritic cells （DC） in vitro. Methods： DC and CIK cells were induced from peripheral blood mononuclear cells of healthy volunteers. They were co-cultured meanwhile CIK cells were cultured alone as controls. Increased number of cells were counted by tapan-blue staining, killing activities were detected by MTT assay, immunophenotype changes were analyzed by flow cytometry, the IL-12 and INF-y levels of the cultured supernatants were detected by ELISA kits. Results： The proliferation capabilities of DC-CIK cells were significantly higher than that of CIK cells （P 〈 0.05）. Under the same condition, the ratio of double positive cells such as CD3^＋ CD8^＋, CD3^＋ CD56^＋ in CIK cells was significantly enhanced by co-cultured with DC cells （P 〈 0.05）. The level of IL-12 and INF-y secreted in supernatants was increased noticeably by co-cultured DC-CIK cells on day 3 compared to CIK cells which were cultured alone （P 〈 0.01 and P 〈 0.05）. Within the effector-target ratio range between 5：1 to 40：1, the activities against lymphoma cells of DC-CIK cells were much higher than that of CIK cells （P 〈 0.05）, and this effect was showed a positive correlation with the effector-target ratio. Conclusion： The proliferation capabilities, the level of secreted cytokines and the activities against lymphoma cells of DC-CIK cells were significantly higher than those of CIK cells. The research might provides theoretical and experimental basis for clinical immunotherapy of DC-CIK cells.

Effect of anti-asthma Chinese medicine Chuankezhi on the anti-tumor activity of cytokine-induced killer cells

Chuankezhi(CKZ),a new Chinese medicine,plays an important role in immunoregulation.Cytokineinduced killer(CIK)cells have been commonly used for immunotherapy in recent years.In this study,we aimed to investigate the immunoregulatory effect of CKZ on CIK cells.Peripheral blood monocytes were isolated from healthy donors,and CIK cells were generated by culturing monocytes with interferon-gamma(IFN-γ)and interleukin 2.Different concentrations of CKZ were added on day 2.After incubation for 14days in culture,the antitumor effects of CIK cells were measured by cytotoxicity assay.Flow cytometry was used to explore the effect of CKZ on CIK cell immunophenotype,intracellular cytokine production,and apoptosis.The effect of CKZ on the antitumor activity of CIK cells in nude mice was also investigated.CKZ increased the percentage of CD3+CD56+CIK cells but did not significantly change the percentage of CD4+,CD8+,or CD4+CD25+CIK cells.CKZ-conditioned CIK cells showed a greater ability to kill tumor cells,as well as a higher frequency of IFN-γand TNF-αproduction,compared with the CIK cells in the control group.CKZ also suppressed the apoptosis of CIK cells in vitro.Furthermore,CKZ combined with CIK cells had a stronger suppressive effect on tumor growth in vivo than the CIK,CKZ,or normal saline control groups.Our results indicate that CKZ enhances the antitumor activity of CIK cells and is a potential medicine for tumor immunotherapy.

Study of Multi-directional Derivation of Cord Blood Mononuclear Cells and Observation of Killing Activity to MGC-803 Gastric Cancer Cell Strain In Vitro

Objective： To study multi-directional derivation of cord blood mononuclear cells to CD3AK, LAK and CIK cells as well as changes of killing activity to gastric cancer cell strain in vitro. Methods： CD3mAb and IL-2 were used to induce CD3AK cells, and IL-2 was used to induce LAK cells; IFN-γ was used in the beginning, then IL-1, CD3mAb and IL-2 were used to induce CIK cells after 24 h for observing amplification and analyzing their relationship. The phenotypes of the cultured CIK cells were analyzed by flow cytometry. Subsequently, by using MGC-803 gastric cancer cell strain as target cells, the killing activity of CD3AK, LAK and CIK cells was evaluated by using MTT method. Results： The amplification activity of CD3AK and CIK cells was all far higher than LAK cells （P〈0.05）. The amplification activity had no obvious difference between CIK cells and CD3AK cells at prophase, but that was far higher in CIK cells than CD3AK cells at about 20^th day （P〈0.05）. The flow cytometry revealed that the amount of CD3^＋ CD56^＋ cells, major effector cells after CIK cells being cultured was significantly increased （P〈0.05）, moreover, the amount of CD8^＋ cells was significantly increased as well （P〈0.05）. The killing activities of CD3AK and CIK cells to the MGC-803 gastric cancer cell strain were all significantly higher than LAK cells, while the killing activity of CIK cells was stronger than CD3AK cells （P〈0.05）. Conclusion： CIK cells have stronger amplification activity and killing activity, and can be taken as more effective killing cells applied to the tumor adoptive immunotherapy.

3D-QSAR Studies on the Anti-tumor Activity of N-Aryl-salicylamide Derivatives

In the present work,comparative molecular field analysis(CoMFA)techniques were used to perform three-dimensional quantitative structure-activity relationship(3D-QSAR)studies on the anti-tumor activity(pHi,i=1,2,3,4)of N-aryl-salicylamide derivatives against four cancer cell lines,including A549,MCF-7,SGC-7901,and Bel-7402.12 compounds were randomly selected as the training set to establish the prediction models,which were verified by the test set of 5 compounds containing template molecule.The contributions of steric and electrostatic fields to pH1,pH2,pH3,and pH4 were 23.8% and 76.2%,20.1% and 79.9%,18.7% and 81.3%,and 14.3%and 85.7%,respectively.The cross-validation(Rcv 2)and non-cross-validation coefficients(R2)were 0.826 and 0.963 for pH1,0.867 and 0.974 for pH2,0.941 and 0.989 for pH3,and 0.797 and 0.961 for pH4,respectively.The CoMFA models were then used to predict the activities of the compounds,and it was found that the models had strong stability and good predictability.Based on the CoMFA contour maps,some key structural factors responsible for the anticancer activity of the series of compounds were revealed.The results provide some useful theoretical references for understanding the mechanism of action,designing new N-aryl-salicylamide derivatives with high anti-tumor activity,and predicting their activities.

Free radical scavenging property and antiproliferative activity of Rhodiola imbricata Edgew extracts in HT-29 human colon cancer cells

Objective:To investigate the in vitro antioxidant and antiproliferative activity of rhizome extracts of Rhodiola imbricata(R.imbricata) in HT-29 human colon cancer cell line.Methods:The successively extracted rhizome of R.imbricata using various solvents was analyzed for their total phenolics,tannins and flavonoid contents.In vitro antioxidant activity was evaluated by employing different assays,including DPPH,ABTS radical scavenging assays,FRAP, phosphomolybdenum reduction assay,superoxide anion,hydroxyl radical scavenging activities and metal chelating ability.Results:Acetone and methanol extracts recorded higher phenolic content and showed comparable antioxidant activity with standard reference.Additionally,they also inhibited the proliferation of HT-29 cells upon treatment at higher concentration(200μg/mL) (acetone and methanol,84%and 84%.respectively).On examination acetone extract exhibited antiproliferative activity in a concentration dependent manner whereas,methanol extract showed both dose dependent and lime dependent inhibitory activity.Conclusions:The results obtained justify the traditional usage of R.imbricata from their promising antioxidant activity.

T cell immune response is correlated with fibrosis and inflammatory activity in hepatitis B cirrhotics

AIM： TO explore the relationship among interferon-γ （IFN-γ） activity, fibrogenesis, T cell immune responses and hepatic inflammatory activity. METHODS： Peripheral blood samples from a total of 43 hepatitis B cirrhotic patients （LC） and 19 healthy controls （NC） were collected to measure their serum levels of IFN-γ, interleukin-2 （IL-2）, soluble interleukin-2 receptor （sIL-2R）, interleukin-10 （IL-10） and three serological markers of fibrosis including hyaluronic acid （HA）, procollagen type III peptide （PIIIP）, and type iV collagen were measured using a double antibody sandwich ELISA. Also, serum total bilirubin （TB） and alanine aminotransferase （ALT） were measured by routine measures. RESULTS： The concentrations of serological markers of fibrosis in patients with active cirrhosis （ALC） were significantly higher than those in stationary liver cirrhosis （SLC） or NC groups. The levels of serological markers in HBeAg-positive patients were significantly higher than those in HBeAg-negative patients. In SLC and ALC patients, a negative linear correlation was found between IFN-γ levels and the serological markers of fibrosis. IFN-γ and IL-2 levels in the ALC group were significantly higher than those in the SLC and NC groups, but the statistical difference was not significant between the latter two. In contrast, IL-10 levels in the SLC group were significantly higher than that in the NC group, but no significant difference was found between SLC and ALC groups. The sIL-2R level was elevated gradually in all these groups, and the differences were significant. Positive linear correlations were seen between IFN-γ activity and ALT levels （r = 0.339, P 〈 0.05）, and IL-2 activity and TB levels （r = 0.517, P 〈 0.05）. sIL-2R expression was positively correlated with both ALT and TB levels （r = 0.324, 0.455, P 〈 0.05）, whereas there was no statistically significant correlation between IL-10 expression and serum ALT and TB levels （r = -0.102, -0.093, P 〉 0.05）. Finally, there was a positive correlation between IFN-γ and IL-2 levels. CONCLUSION： T cell immune responses are correlated with fibrosis and hepatic inflammatory activity and may play an important role in liver cirrhosis.

Synthesis and Anti-tumor Activity of Novel Amide Derivatives of Ursolic Acid

Ursolic acid was modified at C3 and C28 position to obtain fourteen derivatives including twelve novel compounds, and their chemical structures were characterized by IR, ^1H NMR and MS. Cell growth inhibitory effects of the derivatives against Hela cell were evaluated by MTT assay. All these derivatives were found to have stronger cell growth inhibitory than their parent compound, ursolic acid. The derivatives with a substituted acetyl group at C3 hydroxyl group show better activities than those with an unsubstituted hydroxyl group.

Berberine displays antitumor activity in esophageal cancer cells in vitro

To investigate the effects of berberine on esophageal cancer (EC) cells and its molecular mechanisms. METHODS Human esophageal squamous cell carcinoma cell line KYSE-70 and esophageal adenocarcinoma cell line SKGT4 were used. The effects of berberine on cell proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. For cell cycle progression, KYSE-70 cells were stained with propidium iodide (PI) staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min and cell cycle was analyzed using a BD FACSCalibur flow cytometer. For apoptosis assay, cells were stained with an Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using BD ModFit software. Levels of proteins related to cell cycle and apoptosis were examined by western blotting. RESULTS Berberine treatment resulted in growth inhibition of KYSE-70 and SKGT4 cells in a dose-dependent and time-dependent manner. KYSE-70 cells were more susceptible to the inhibitory activities of berberine than SKGT4 cells were. In KYSE-70 cells treated with 50 mu mol/L berberine for 48 h, the number of cells in G2/M phase (25.94% +/- 5.01%) was significantly higher than that in the control group (9.77% +/- 1.28%, P < 0.01), and berberine treatment resulted in p21 upregulation in KYSE-70 cells. Flow cytometric analyses showed that berberine significantly augmented the KYSE-70 apoptotic population at 12 and 24 h post-treatment, when compared with control cells (0.83% vs 43.78% at 12 h, P < 0.05; 0.15% vs 81.86% at 24 h, P < 0.01), and berberine-induced apoptotic effect was stronger at 24 h compared with 12 h. Western blotting showed that berberine inhibited the phosphorylation of Akt, mammalian target of rapamycin and p70S6K, and enhanced AMP-activated protein kinase phosphorylation in a sustained manner. CONCLUSION Berberine is an inhibitor of human EC cell growth and could be considered as a potential drug for the treatment of EC patients.

Research on stress-induced apoptosis of natural killer cells and the alteration of their killing activity in mouse liver

AIM:To investigate the stress-induced apoptosis of natural killer(NK)cells and the changes in their killing activity in mouse livers.METHODS:A restraint stress model was established in mice.Flow cytometry was employed to measure the percentage of NK cells and the changes in their absolute number in mouse liver.The cytotoxicity of hepatic and splenic NK cells was assessed against YAC-1 target cells via a 4 h 51Cr-release assay.RESULTS:The restraint stress stimulation induced the apoptosis of NK cells in the liver and the spleen,which decreased the cell number.The number and percentage of NK cells in the spleen decreased.However,the number of NK cells in the liver decreased,whereas the percentage of NK cells was significantly increased.The apoptosis of NK cells increased gradually with prolonged stress time,and the macrophage-1(Mac-1)+NK cells were more susceptible to apoptosis than Mac-1-NK cells.Large numbers of Mac-1-NK cells in the liver,which are more resistant to stress-induced apoptosis,were observed than the Mac-1-NK cells in the spleen.The stress stimulation diminished the killing activity of NK cells in the spleen was significantly decreased,but the retention of numerous Mac-1-NK cells in the liver maintained the killing ability.CONCLUSION:Significant stress-induced apoptosis was observed among Mac-1+NK cells,but not Mac-1-NK cells in the mouse liver.Stress stimulation markedly decreased the killing activity of NK cells in the spleen but remained unchanged in the liver.

Effect of Lidamycin on Telomerase Activity in Human Hepatoma BEL-7402 Cells

Objective To investigate the effect of lidamycin （LDM） on telomerase activity in human hepatoma BEL-7402 cells under the condition of LDM inducing mitotic cell death and senescence. Methods Chromatin condensation was detected by co-staining with Hoechst 33342 and PI. Cell multinucleation was observed by Giemsa staining and genomic DNA was separated by agarose gel electrophoresis. Fluorescent intensity of Rho123 was determined for mitochondrial membrane potential. MTT assay and SA-13-gal staining were employed to analyze the senescence-like phenotype. The expression of proteins was analyzed by Western blot. Telomerase activity was assayed by telomerase PCR-ELISA. Results Mitotic cell death occurred in LDM-treated cells characterized by unique and atypical chromatin condensation, multinucleation and increased mitochondrial membrane potential. However, no apoptotic bodies or DNA ladders were found. In addition, apoptosis-related proteins remained nearly unaltered. Senescence-like phenotype was identified by increased and elongated size of cells, growth retardation, enhanced SA-13-gal activity and the changes of senescence-related protein expression. Telomerase activity markedly decreased （P〈0.01） in LDM-treated hepatoma BEL-7402 cells. Conclusion Mitotic cell death and senescence could be triggered simultaneously or sequentially after exposure of hepatoma BEL-7402 cells to LDM. The decrease in telomerase activity may play a key role in the defective mitosis and aging morphology. Further investigation of detailed mechanism is needed.

Decreased phagocytic activity of Kupffer cells in a rat nonalcoholic steatohepatitis model

AIM: To investigate Kupffer cell dynamics and phagocytic activity,using a rat nonalcoholic steatohepatitis (NASH) model. METHODS: Male F344 rats were fed either a control diet or a choline-deficient L-amino acid-defined (CDAA) diet,followed by contrast enhanced ultrasonography (CEUS) using Levovist. The uptake of latex beads by the Kupffer cells was determined by fluorescent microscopy. The status of the Kupffer cells was compared between the two groups,using the immunohistochemical staining technique. RESULTS: After 4 or more wk of the CDAA diet,CEUS examination revealed a decrease in the signal intensity,20 min after intravenous Levovist. Fluorescent microscopic examination showed that the uptake of latex beads by the Kupffer cells was reduced at week 1 and 2 in the study group,compared with the controls,with no further reduction after 3 wk. Immunohistochemical staining revealed no significant difference in the Kupffer cell counts between the control group and the CDAA group. CONCLUSION: CEUS examination using Levovist demonstrated reduced contrast effect and phagocytic activity in the liver parenchymal phase,although the Kupffer cell numbers were unchanged,indicating reduced phagocytic function of the Kupffer cells in the rat NASH model. We believe that CEUS examination using Levovist is a useful screening modality,which can detect NASH in fatty liver patients.

Clinical significance of telomerase activity in peritoneal lavage fluid from patients with gastric cancer and its relationship with cellular proliferation

AIM： To evaluate the efficacy of telomerase activity assay and peritoneal lavage cytology （PLC） examination in peritoneal lavage fluid for the prediction of peritoneal metastasis in gastric cancer patients, and to explore the relationship between telomerase activity and proliferating cell nuclear antigen expression.METHODS： Telomeric repeated amplification protocol （TRAP）-enzyme-linked immunosorbent assay （ELISA） was performed to measure the telomerase activity in 60 patients with gastric cancer and 50 with peptic ulcer. PLC analysis of the 60 patients with gastric cancer was used for comparison. The proliferating cell nuclear antigen （PCNA） in gastric carcinoma was immunohistochemically examined.RESULTS： The telomerase activity and PLC positive rate in peritoneal lavage fluid from patients with gastric cancer was 41.7% （25/60）, and 25.0% （15/60）, respectively. The positive rate of telomerase activity was significantly higher than that Qf PLC in the group of pT, （15/16 vs 9/16, P 〈 0.05）, P1-3 （13/13 vs 9/13, P 〈 0.05） and diffuse type （22/42 vs 13/42, P 〈 0.05）. The patients with positive telomerase activity, peritoneal metastasis, and serosal invasion had significantly higher levels of average PCNA proliferation index （PI）, （55.00 ± 6.59 vs 27.43 ± 7.72, 57.26 ±10.18 vs 29.15 ±8.31, and 49.82 ± 6.74 vs 24.65 ±7.33, respectively, P 〈 0.05）.CONCLUSION： The TRAP assay for telomerase activity is a useful adjunct for cytologic method in the diagnosis of peritoneal micrometastasis and well related to higher proliferating activity of gastric cancer. The results of this study also suggest a promising future therapeutic strategy for treating peritoneal dissemination based on telomerase inhibition.

Activity of T Cells Stimulated by Hemagglutinin-neuraminidase of Newcastle Disease Virus in vivo

To investigate the stimulated activity of T cells and the anti-tumor properties of hemagglutinin-neuraminidase（HN） of Newcastle disease virus（NDV） strain Changchun（NDVcc）, the expression of HN gene in hepatoma cells（human HepG-2 and mouse H22 cells） infected with the recombinant adenovirus（Ad-HN） was identified by Western blot analysis and flow cytometry. Sialidase activity of NDVcc HN expressed by Ad-HN was assayed by the periodate-resorcinol method. The in vivo anti-tumor effects of NDVcc HN were evaluated in the H22 solid tumor model. Regional lymph nodes of the mouse model treated with Ad-HN were removed to harvest T lymphocytes and evaluating the specific cytotoxicity of cytotoxic T lymphocyte（CTL） and natural killer（NK） cells by an L-lactate dehydrogenase（LDH） assay, in the mean time, the secretion of cytokines was analyzed by enzyme linked immunosorbent assays（ELISA）. The results show that NDVcc HN was effectively expressed by Ad-HN in HepG-2 and H22 cells. The sialidase activity assay showed that Ad-HN significantly reduced sialic acid level of the hepatoma cells compared with the cells infected the empty adenovirus vector（Ad-mock）. When treated with Ad-HN, the growth of subcutaneous H22 primary tumors in C57BL/6 mice was suppressed, and the mean mice survival increased. In addition, the treatment of Ad-HN elicited strong NK and CTL responses, and high levels of Th1 cytokines, such as IL-2 and IFN-γ. In conclusion, NDVcc HN effectively elicits T cell-mediate anti-tumor cytotoxicity via sialidase activity and may be a novel strategy for cancer immunotherapy.

Effect of Nano Red Elemental Selenium on GPx Activity of Broiler Chick Kidney Cells in vitro

A new selenium source, Nano red elemental selenium (Nano-Se) was used to study the effect on the GPx activity of broiler chick kidney cells (BCKC) in vitro, Sodium selenite (Na_ 2 SeO_ 3 ) and seleno-1-methionine (Se-Met) were used as the controls. The results showed that the effects of three kinds of Se forms on the GPx activity of BCKC were accordant(p>0.05) compared with each other at 0.01,0.05 and 0.10 μmol/L Se concentrations treatments. In the range of 0.00-0.10 μmol/L Se concentrations, the GPx activity increased with elevation of Se concentrations in medium. For the three kinds of Se forms, the GPx activity reached the climax at 0.10 μmol/L Se concentration. At 0.20 and 0.30 μmol/L Se concentrations, the influnces of three kinds of Se forms were not accordant with one another. For Nano-Se, the GPx activity at 0.20 and 0.30 μmol/L Se concentrations remained the same as that at 0.10 μmol/L Se concentration treatment. For Se-Met, the GPx activity at 0.20 μmol/L Se concentration treatment remained the same with 0.10 μmol/L treatment; the GPx activity at 0.30 μmol/L Se concentration treatment was declined significantly(p<0.05) compared with 0.10 or 0.20 μmol/L treatment. For Na_ 2 SeO_ 3 , the GPx activity falled gradually with Se concentration increasing from 0.10 μmol/L to 0.30 μmol/L, and at 0.30 μmol/L Se concentration treatment, the GPx activity was less than the original of BCKC. The results implicated, on the GPx activity of BCKC in vitro, the ranking of width range of the most suitable Se concentration for nutrition curve of the three Se formes is Nano-Se>Se-Met>Na_ 2 SeO_ 3 .

Characterization and Anti-tumor Activity of Glycopeptides from Ganoderma sinensis

The water-soluble part（GS） of Ganoderma sinense Zhao, Xu et Zhang was divided into high molecular（GS-H） and low molecular（GS-L） parts by Cellulose Super Filtration, and GS was also fractionated into four fractions, GS-1, 2, 3, and 4 by ethanol precipitation according to their molecular weights. Chemical analysis shows that GS and GS-1, 2, 3, 4 were complexes of polysaccharide and peptide. The fractions with molecular weights over 4000, GS-1, 2, 3, and GS-H show anti-tumor activities, however, the fractions with molecular weights lower than 4000, GS-4, and GS-L have no anti-tumor activity, indicating that the anti-tumor activity of Ganoderma Sinensis was caused by glucopeptides with molecular weight ranging from 4000 to 20000. Two purified glucopeptides, GS-6b and GS-7b were obtained from GS-H by ion-exchange and gel-permeation chromatography. Their molecular weights, glycosidic linkages, and configurations were detected by means of IR spectrum, sugar composition analysis, and methylation analysis. The polysaccharide parts of GS-6b and GS-7b had glucan backbone consisting of β-1→3 Glc, and side chain containing glucosyl, mannosyl, fucosyl, xylosyl, galactosyl, and glucuronic acid residues attached on 1-2, 1-4, 1-6 positions of the backbone of GS-6b, or 1-6, 1-4 positions of the backbone of GS-7b. The peptide parts in GS-6b and GS-7b were composed of 10 kinds of amino acids, including Asp, Ser, Arg, Gly, Thr, Pro, Ala, Val, Met, and Lys.

Immune Killing Activity of Lymphocytes on Hela Cells Expressing Interleukin-12 In Vitro

The killing effects of lymphocytes on Hela cells expressing interleukin-12 （IL-12） in vitro were explored. By using gene transfection technique, full length IL-12 gene was transfected into Hela cells. The expression of IL-12 in Hela cells was detected quantitatively by ELISA; Changes in killing effects of lymphocytes on Hela cells expressing IL-12 were observed by MTT. It was found that Hela cells could express IL- 12 between 24 h and 72 h after transfection. Killing activity of lymphocytes on Hela cells expressing IL-12 was significantly enhanced. It was concluded by cell transfection technique, Hela cells could express 1L-12 and were more easily killed by lymphocytes.

SHP2 regulates skeletal cell fate by modifying SOX9 expression and transcriptional activity

Chondrocytes and osteoblasts differentiate from a common mesenchymal precursor, the osteochondroprogenitor（OCP）, and help build the vertebrate skeleton. The signaling pathways that control lineage commitment for OCPs are incompletely understood. We asked whether the ubiquitously expressed protein-tyrosine phosphatase SHP2（encoded by Ptpn11） affects skeletal lineage commitment by conditionally deleting Ptpn11 in mouse limb and head mesenchyme using ＂Cre-lox P＂-mediated gene excision.SHP2-deficient mice have increased cartilage mass and deficient ossification, suggesting that SHP2-deficient OCPs become chondrocytes and not osteoblasts. Consistent with these observations, the expression of the master chondrogenic transcription factor SOX9 and its target genes Acan, Col2a1, and Col10a1 were increased in SHP2-deficient chondrocytes, as revealed by gene expression arrays, q RT-PCR, in situ hybridization, and immunostaining. Mechanistic studies demonstrate that SHP2 regulates OCP fate determination via the phosphorylation and SUMOylation of SOX9, mediated at least in part via the PKA signaling pathway. Our data indicate that SHP2 is critical for skeletal cell lineage differentiation and could thus be a pharmacologic target for bone and cartilage regeneration.