Tumor-infiltrating regulatory T cells are positively correlated with angiogenic status in renal cell carcinoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Immune cells within a tumor microenvironment have shown modulatory effects on tumor angiogenic activity. Renal cell carcinoma (RCC) is a hypervascular tumor that reportedly increases the frequency of regulatory T cells (Tregs) in tumor tissues. This study investigated the correlation between Tregs infiltration and angiogenic status in RCC.</p><p><b>METHODS</b>Thirty-six patients with RCC were enrolled in the present study, and twenty age-matched healthy donors were included as the control. Tregs were defined as CD4(+)CD25(high)CD127(low/-) T cells. The frequency of Tregs in peripheral blood and tumor infiltrating lymphocytes (TILs) were determined by flow cytometry. The expression of vascular endothelial growth factor (VEGF) in surgical resection specimens were measured with a commercial enzyme-linked immunosorbent assay (ELISA) kit. Microvessel density (MVD) was calculated on slides stained with CD34 antibody. Spearman's rank correlation was performed to evaluate the correlation between the frequencies of Tregs in TILs and VEGF values, as well as between frequencies of Tregs and MVD determinations.</p><p><b>RESULTS</b>Compared to healthy controls, the frequency of peripheral blood Tregs was significantly increased in patients with RCC (P < 0.05). The percentage of tumor-infiltrating Tregs was higher than that of peripheral blood Tregs in patients with RCC (P < 0.01). In addition, the frequency of tumor-infiltrating Tregs was shown to significantly correlate with the pathological stage (P < 0.05) and nuclear grade (P < 0.01). Importantly, a significant positive correlation was observed between the frequency of tumor-infiltrating Tregs and VEGF protein expression (r = 0.51, P < 0.05), as well as between frequencies of Tregs and MVD score (r = 0.39, P < 0.05).</p><p><b>CONCLUSIONS</b>These observations suggest that the high pro-angiogenic status of RCC may be associated with the accumulation of Tregs in the local microenvironment. Angiogenesis networks may be connected with immune tolerance units and cooperate with each other to facilitate tumor growth and progression.</p>

Hypoxia regulates osteopontin expression of mature dendritic cells via adenosine 2 receptor / 中华微生物学和免疫学杂志

Objective To investigate the mechanism of hypoxia regulate osteopontin (OPN) secreting by mature dendritic cells (mDCs). Methods CD14 + cells were enriched using anti-CD14 immunomagnetic beads, for inducing to mDCs, CD14 + cells were cultured with GM-CSF and IL-4 in hypoxia or normoxiain vitro. Concentration of OPN and TGF-β1 in supernatant were detected by sandwich ELISA, OPN mRNA detected by RT-PCR. Approach regulating function of A2 R in expressing of OPN by mDCs by using NECA (surrogate of adenosine), A2R agonist (CGS21680), A2R antagonist (SCH58261) and investigate role of TGF-β1 in this process by using rhTGF-β1 and anti-TGF-β1 Ab. Results Hypoxia inreased the level of OPN and OPN mRNA in mDCs, and this effect could be reversed by A2 R antagonist. Under normoxia,both NECA and A2R agonist (CGS21680) could upregulate the level of OPN and OPN mRNA in mDCs significantly, but this positive effect could be reversed by A2 R antagonist. A2 R played a role in regulating TGF-β1, and confirmed TGF-β1 involved in regulation of OPN by using rhTGF-β1 and anti-TGF-β1 Ab. Conclusion High adenosine induce the generation of TGF-β1 through the A2R on mDCs, and then TGF-β1 raise the OPN secreting by mDCs.

Effect of hypoxia on adhesion and invasion of human tongue squamous cell carcinoma cell line Tca8113 / 实用口腔医学杂志

Objective: To evaluate the effect of synthesized small interfering RNA targeting to HIF-lα on the adhesion and invasion of human tongue squamous cell carcinoma cell line (Tca8113). Methods; A double strand small interference RNA (siRNA) targeting HIF-1α (siRNAH1Fla) was transfected into cultured Tca8113 cells by lipofectamine2000. The expression of HIF-1α was investigated on mRNA level by real time-PCR and protein level by Western blot. The adhesion and invasion of Tca8113 cells to extracellular matrix (ECM) was also analyzed. Results: Exposure to hypoxia induced a prolonged elevation of HIF-lα protein and siRNAHIF.la reduced HIF-la synthesis as measured on mRNA level and protein level compared with the controls. No matter under normoxic or hy-poxic conditions, the adhesion potency of siRNAHIF-1α treated Tca8113 cells was markedly inhibited compared with controls(P<0.05 or P <0.01). So did the invasion potency (P<0.01). The adhesion and invasion potency of siRNAHIF.,a treated Tca8113 cells were inhibited more greatly under hypoxic condition than under normoxic condition ((36.4±2.7)% vs(26±2.35);(44.2±2.2)% vs (35±1.75), P<0.01)). Conclusion; siRNAH1F.lo can knockdown the expression of HIF-la and inhibit the cell adhesion and invasion to ECM in Tca8113 cells. HIF-la may play an established role in the regulation of Tca8113 cells invasion and metastasis. Interfering with HIF-1α pathways by siRNA strategy may provide a therapeutic target for human tongue squamous cell carcinomas.

Expression and significance of chemokine CXC receptor 3, 4 and their ligands at the early pregnancy decidua and villi / 中华妇产科杂志

Objective To explore the expression and significance of chemokine CXC reeeptor (CXCR)3 and CXCR4 and their ligands(CXCL)at the early pregnancy decidua and villi.Methods Decidual mononuclear cells were isolated from the normal decidua of 5-8 weeks pregnant women by lymphocyte separation medium in vitro.CD56+natural killer(NK)cells were purified by dynabeads cell sorter kiL Purity and phenotype of CD56+decidua NK cells were analyzed by fluorescence-activated eell sorter (FACS).Gene expression of CXCR3 and CXCR4 in decidua NK cells and CXCL9,CXCL10 and CXCL12 in early pregnancy decidua and villi was assessed bv RT.PCIZ Protein expression of CXCL9,CXCL10 in normal endometrium and early pregnancy decidua was characterized and quantified by streptavidin-biotin pemxidase chain reaction(SP)immunohistochemistry and computered image analysis system.Correlations between the gray degree of CXCL9 and CXCL10 and the number of CD56+NK cells in upper tissue were analyzed by Spearman's correlation ceefficient rank tesL Results The phenotype of 98.7%decidua NK cells was CD56bright.The genes of CXCR3 and CXCR4 were expressed in decidua NK cells and that of CXCL9 and CXCL1O were expressed in early pregnancy decidua and CXCLI2 in early pregnancy villi.CXCL9 and CXCL10 were expressed in the cytoplasm of surface epithelia,glandular epithelia and stromal cells of early pregnancy deeidua and were not expressed in villi by immunohistochemistry.The gray degree of CXCL9 and CXCL10 in the secretory phase endometrium(56±43,59±47)was stronger than that in the proliferative phase(16±18,8±14,P<0.05)and reached the highest(143±35,158±29,P<0.05)in the early pregnancy decidua.The number of cD+56 NK cell in the secretory phase endometrium(60±20)was more than that in the proliferative phase endometrium(23±4,P<0.05)and was the most in the early pregnancy decidua(114±15,P<0.05).The gray degree of CXCL9 in upper tissue had a positive correlation with the number of CD+56 cells(r=0.88,P<0.05)and that of CXCL10 had a similar pattern to CXCL9(r=0.86,P<0.05).Condusion The interactions between CXCL9,CXCL10 and CXCL12 expressed in decidua and villi and CXCR3,CXCR4 expressed in CD+56 decidua NK cells may influence the CD+56 NK cell recruitment at the maternal-fetal interface.

Effect of extracelinlar signal regulated kinase signal pathway on apoptosis induced by MG262 in ovarian cancer cells / 中华妇产科杂志

Objective To investigate whether the proteasomes inhibitor MG262 exerts its anticancer function by inducing apoptosis in human ovarian cancer cells,and whether the extracellular signal regulated kinase (ERK) signaling pathway is involved in the regulation of apoptosis induction.Method Human ovarian cancer cell line SKOV3 was incubated with different concentrations of MG262 for 24 and 48 hours.Cell viability was evaluated with 3-(4,5-dimethyhhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at different time points of culturing.Flow cytometry was used to detect cell apoptosis rate.The expression of vascular endothelial growth factor (VEGF) was evaluated with western blot and enzyme-linked immtmosorbent assay (ELISA).Western blot was used to detect the expression of phosphorylated ERK(pERK) .Results The viability of SKOV3 cells was decreased by MG262 in a concentration-dependent fashion(P＜0.05).After 24 h incubation with MG262 at 1,10,20,40,60 and 80 nmol/L,the viability rates of SKOV3 were (94.6±3.1)%,(92.7±3.7)%,(89.5±7.7)%,(84.2±5.1)%,(82.0±7.4)%and(76.8±11.0) % respectively,and after 48 h incubation,those figures were further decreased to (91.3±10.1)%,(86.8±4.5)%,(74.6±4.2)%,(56.8±2.1)%,(49.3±4.5)% and (37.4±5.4) %,respectively(P＜0.05).Apoptosis rate of SKOV3 cells induced by MG262,PD98059 or their combination was (30.7±4.3)%,(26.8±8.6)% and (50.3±10.6)%,respectively,which were significantly different compared with controls (P＜0.05).In contrast to SKOV3 cells,apoptosis rate of 293T ceils induced by MG262,PD98059 or their combination was (14.5±5.3) %,(16.2±7.5) % and (10.8±7.3)%,respectively,which were not significantly different compared with controls (P＞0.05).pERK expression decreased gradually in a time-dependent manner. And wild-type p53 expression was not significantly different.There was no significant difference between experimental and control 293T cells(P＜0.05).In addition,MG262 down-regulated VEGF secretion and expression in SKOV3 ceils (P＜0.05).Conclusions Proteasome inhibitors can induce apoptosis and inhibit cell proliferation and angiogenesis through ERK signal pathway in SKOV3 cells.

Expression of BAFF in the trophoblast and decidua of normal early pregnant women and patients with recurrent spontaneous miscarriage / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>BAFF, the B cell activation factor, is a member of the tumor necrosis factor (TNF) ligand family that binds to BCMA, TACI, and BAFF-R. Previous studies have shown that members of the TNF family are detected in human placental trophoblast cells, but the expression patterns of BAFF involved in human decidua and the differential expression of BAFF between normal pregnancy and miscarriage are still incompletely documented or unknown. This study was designed to investigate the expression of BAFF and BAFF-R in the trophoblast and decidua of normal early pregnant women and recurrent spontaneous abortion (RSA) patients.</p><p><b>METHODS</b>Forty-five patients with RSA and 45 normal pregnant women were included in this study. By reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemical experiments, we explored the expression of BAFF and BAFF-R in the maternal-fetal interface of normal early pregnant women and RSA patients.</p><p><b>RESULTS</b>Analysis by RT-PCR and Western blotting revealed that BAFF was detected in both trophoblast and decidua of all the samples, and the expression level was higher in the tissues of normal early pregnant women (P<0.05) than that of recurrent spontaneous abortion patients under the same gestational weeks. Messages for BAFF-R were absent. Immunohistochemical experiments showed that expression of BAFF was cell-specific which was localized to villous cytotrophoblast and syncytiotrophoblast cells in trophoblast and to stromal cells in decidua. Whereas BAFF was prominent on the trophoblast and decidua of normal early pregnant women, it was decreased in the tissues of RSA patients.</p><p><b>CONCLUSIONS</b>BAFF might steer maternal leukocytes away from a harmful immune response and toward a favorable one and play a potentially vital role for successful pregnancy.</p>

Effect of hypoxia inducible factor-1 alpha on vascular endothelial growth factor expression in human tongue squamous carcinoma cells (Tca8113) under hypoxia / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of hypoxia inducible factor-1 alpha (HIF-1 alpha) on vascular endothelial growth factor (VEGF) expression in Tca8113 cells under hypoxia.</p><p><b>METHODS</b>The expression of the mRNA of HIF-1 alpha and VEGF in Tca8113 cells was examined by RT-PCR technique at different culture times (1/2 h, 1 h, 3 h, 6 h, 12 h, 24 h) under normoxic and hypoxic conditions.</p><p><b>RESULTS</b>The expression of HIF-1 alpha under hypoxia showed the trend of increasing first and then decreasing, and was higher than that of the control (normoxic group) at 6h and 12 h (P < 0.05). The expression of VEGF under hypoxia was higher than that of the control group at 1/2 h, 1 h, 3 h, 12 h, 24 h (P < 0.05). The expression of hypoxia-induced VEGF mRNA increased with the increased expression of HIF-1 alpha mRNA in the cell lines tested at the initial stage of hypoxia. But no statistical significant association was observed between HIF-1 alpha and VEGF expression within 24 h under hypoxia (rs = 0.5750, P > .005).</p><p><b>CONCLUSIONS</b>The increased expression of VEGF in Tca8113 cells might be mediated by multiple factors, including HIF-1 alpha.</p>

Experimental study on the phenotype of human embryonic lung fibroblasts transfected with Ad-BMP-2 / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the effects on the phenotype, especially the mineralization ability of human embryonic lung fibroblasts(HELFs) by transfection with adenovirus vector ecoding human bone morphogenetic protein-2 gene (Ad-BMP-2).</p><p><b>METHODS</b>The HELFs were primarily cultured, then transfected with Ad-BMP-2. The morphologic characteristics of the cells were observed. The cell proliferation, alkaline phosphatase activities, BMP-2 protein expression, and the mineralization ability were detected with the methods of MTT, ALP staining, Western blot, and alizarin red S staining, respectively.</p><p><b>RESULTS</b>After transfection, the shape of HELFs changed from silm spindle to multifigure, the cells became bigger than before. The colonies changed from unilaminar into multilaminar. The proliferation of HELFs was severely inhibited after transfection. An obvious BMP-2 lane was shown in Western blotting. Most cells presented positive in ALP staining, and large number of nacarat mineralized nodes were observed after alizarin red S staining.</p><p><b>CONCLUSION</b>HELFs were capable of transforming into osteoblast-like phenotype, and were endowed with the ability of mineralization while being transfected with Ad-BMP-2.</p>

Effect of some active Chinese herbal fraction on brain tissue proteomic profile of ischemic mouse / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of some active Chinese herbal fraction on protein expression of brain tissue in ischemic mouse with proteomic technique.</p><p><b>METHODS</b>Ischemia-reperfusion mice were treated with baicalin, geniposide, cholic acid and concha margaritifera respectively for 3 hrs, and then their brain tissue were taken to extract the total protein. Protein expression in ischemic mouse brain was analyzed with surface-enhanced laser desorption/inionation-time of flight-mass spectra (SELDI-TOF-MS) protein-chip.</p><p><b>RESULTS</b>The four components tested had effect on 3 target proteins at 5373Da, 5707Da and 15103Da, showing the nature of multi-target and with different action on protein expression.</p><p><b>CONCLUSION</b>Protein-chip is an effective approach for exploring the pharmacological mechanism of Chinese herbal fraction.</p>

Endoplasmic reticulum stress within hypoxia microenvironment and its related dieases / 基础医学与临床

Endoplasmic reticulum stress,an critical component of cell stress,is a protective response for eukaryotic cells. The change of partial pressure of oxygen (for instance hypoxia) is a primary condition for cell stress,significantly impacting the biological phenotype and behavior of cells (including survival,migration and invasion et al). Through endoplasmic reticulum stress,intracellular concentration of unfolded protein is reduced,and agglutination inhibited. Recently,endoplasmic reticulum stress has been demonstrated to play an important role in pathogenesis of some diseases such as cancer,diabetes and inflammation,which are characterized by the micro-environment hypoxia in local tissue. Therefore,investigations on the mechanism of endoplasmic reticulum stress caused by hypoxia may promote the novel therapeutic strategy for tumors,cardiovascular diseases and diabetes.

Anti-tumor effects of 10-hydroxycamptothecinc-treated DC-Hepa1-6 fusion vaccines / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the induction of antitumor immune responses and therapeutic effects of 10-hydroxycamptothecinc-treated (HCPT) DC-Hepa fusion vaccines by DC fused with hepal-6 cell from hepatoma.</p><p><b>METHODS</b>The fused cells were isolated by magnetic cell sorting and adherent culture. Cell apoptosis was detected by Rhodamine123/PI double-labeled assay, CTL activity by 4 h (51)Cr releasing assay. Protective and therapeutic effects of the fusion vaccine to the tumor-bearing mice was also observed.</p><p><b>RESULTS</b>The apoptosis rate was 29.7%+/-4.1% when DC-Hepa fusion vaccine was treated with 50 microg/ml HCPT for 24 h. After treatment with the HCPT-DC-Hepa fusion vaccine, the tumor grew obviously slowly, survival period of the mice was prolonged, induced more potent CTL cytotoxicity, and resisted against the rechallenge of Hepal-6 cells.</p><p><b>CONCLUSION</b>Vaccination with HCPT-DC-Hepa fusion vaccine could elicit potent antitumor responses, which will provide a new approach to the DC-mediated therapeutic antitumor immunity.</p>

Treatment of ovarian cancer cell line Skov3 with HSV-tk/GCV under the control of human telomerase reverse transcriptase gene promoter / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the in vitro effect of HSV-tk/GCV using a hTERT promoter-driven vector system on Skov3 ovarian cancer cells.</p><p><b>METHODS</b>An expression vector (pBTdel-279-tk) containing tk gene under the hTERT promoter was constructed by molecular biological methods, and then was transfected into Skov3 ovarian cancer cells, normal ovarian epithelial cells (NOEC) and human embryonic lung fibroblast by cationic liposome. Following the transfection with tk, GCV was added, and MTT and flow cytometry methods were applied to investigate its antitumor effect. RT-PCR was used to detect the tk gene in ovarian cancer cells and normal cells after the transfection of pcDNA3-tk or pBTdel-279-tk.</p><p><b>RESULTS</b>pBTdel-279-tk/GCV system induced apoptosis in hTERT-positive ovarian cancer cells, but not in hTERT-negative normal ovarian epithelial cells and fibroblasts. The hTERT promoter system was almost as efficient in inducing cancer cell death as the CMV promoter. tk gene was expressed in Skov3 cells and NOEC after pcDNA3-tk transfection, while positive was only in ovarian cancer cells after pBTdel-279-tk transfection.</p><p><b>CONCLUSION</b>The telomerasespecific transfer of the tk gene under the hTERT promoter is a novel targeting approach for the treatment of ovarian cancer and may lead to an effective and specific gene therapy.</p>

Study on the Relationship Between MDR1 Gene Expression in Liver Cell and Cholesterol Calculus in Gallbladder / 中国普外基础与临床杂志

Objective To explore the relationship between the expression of MDR1 gene in liver cell and the formation of cholesterol calculus in gallbladder.Methods The mRNA expression level of MDR1 gene in liver cell of the cholesterol calculus group and the normal control group were measured through reverse transcriptionpolymerase chain reaction (RT-PCR), and microglobulin ?_2 was used as internal contrast.Results The MDR1 mRNA expression level of the cholesterol calculus group was lower than that of the normal control group(1.30?0.19 vs 2.25?(0.28), P

Determination of CD34(+) cells in mobilized peripheral blood by flow cytometry / 中国实验血液学杂志

To explore a simple and effective method to determinate the volume of CD34(+) cells in the peripheral blood of donors received drug mobilization for stem cell transplantation by using flow cytometry, the mobilized peripheral blood from donors and 100 micro l fresh whole blood were labeled with monoclonal antibodies Anti-CD34-PE and Anti-CD45-FITC, after lying the red blood cells, and assessed with flow cytometer FL2 (log) vs SSC (log) and FL1 (log) vs SSC (log) were mainly used for analysis windows. The results showed that a level of CD34(+) cells in whole nucleated cells as low as 0.05% - 0.1% can be detected effectively using this method when 10(5) nucleated cells were counted. At day 5 or day 6, the level of CD34(+) cells in most samples of patients reached a peak volume, some of samples and the levels were more than one percent in. It was concluded that CD34(+) cells can be effectively determined by using this method. According to the relative rate of CD34(+) cells, the time to harvest the stem cells in blood can be determined.

DETECTION OF GENE MUTATIONS IN HBV PRECORE AND BASIC CORE PROMOTER REGION USING MICROARRAY TECHNIQUE / 解放军医学杂志

Objective To investigate the clinical value of micro-array technique in the detection of HBV pre-core and basic core promoter region variants. Methods Four spot mutations of A1896(nt1896G→A) and A1899( nt1899G→A) in precore region and T1762A1764(nt1762A→T,nt1764G→A) in basic core promoter region in 46 patients of acute and chronic liver diseases were detected by gene chips to investigate the clinical value of micro-array technique. Results Micro-array technique had a high specificity in the detection of specific mutation, and the positive rate was 87.0%. A1896 mutation was found in 18 cases (45.0%), A1899 mutation in 10 cases (25.0%), T1762 and A1764 double variants in 30 cases (75.0%), and poly-sites variants in 14 cases (35.0%). Significant difference in liver functions was found between each mutation group and non-mutation group (P

Experiment research of anti-PG cell metastasis and mechanism of a new Chinese herb Qcimum basilicum polysaccharide / 中国病理生理杂志

AIM: In this paper, we studied the efficiencies and the mechanisms of a new Chinese herb Qcimum basilicum polysaccharide (BP) on PG cell metastasis in vitro. METHODS: The number of tumor cells going through matrigel was assayed and used to represent the ability of the invasion and migration of PG cells. Using Scrape-loading and dye transfer (SLDT) technique, the efficiencies of BP on recovering PG cell gap junction -mediated intercellular communication (GJIC) was measured. The expressions of c-myc, nm23-H1 and Tiam-1 genes mRNA in PG cells treated with BP were determined by RT-PCR. RESULTS: Compared with the control, the action of invasion and migration of PG cells were decreased after treated with BP (P

Effect of pregnancy uterine microenvironment on the expression of NKG2A,NKG2D and their ligands in decidual NK cells / 中国病理生理杂志

AIM: To investigate the expression of NKG2A,NKG2D and their ligands in pregnancy uterine micro-environment and to probe the function of NKG2A and NKG2D imbalance expression during the immunotolerance at the fetal-maternal boundary.METHODS: Decidual lymphocytes and peripheral lymphocytes were obtained from 30 women during 6-9 weeks of pregnancy who were undergoing selective termination.FACS technology was used to detect NK cells number and NKG2A,NKG2D expression.RT-PCR was used to investigate HLA-E and MICA mRNA in trophoblast tissue.RESULTS: Natural killer cells predominate,accounting for 70% of pregnancy endometrial lymphocytes.FACS results indicated that NKG2A was significantly increased in decidual NK cells as compared with that in peripheral NK cells,accounting for 97.86%?1.75% and 33.35%?10.92%.The difference between them in NKG2A expression was significant(P

Research on the method of culture and identification of embryonic stem cells from human primordial germ cells / 中国免疫学杂志

Objective:To study the cooperated effect of Human Embryonic Lung Fibroblast(HELF) feeder layer and LIF in the culture of human Embryonic Stem(hES) cells and establish the method of identifying hES cells from Primordial Germ Cells(PGCs).Methods:Embryonic lungs were mechanically disaggregated,then cultured to establish HELF feeder layer.Gonadal ridges and mesenteries of embryos were mechanically disaggeregated,then cultured and passaged in vitro.Comparing the growth characters of hES cells in different conditions.To identify hES cells through their biological characteristics.Results:The coactions of HELF feeder layer and LIF play an important role in proliferation and undifferentiation of hES cells in vitro.High levels of AKP and telomerase activity are associated with hES cells. The cultrued cells have been continuously passaged for more than two months and found to be karyotypically normal and stable.When differentiating,they form embryoid bodies(EBs).Conclusion:To avoid indection of the heterogeneous protein,HELF feeder layer, in the presence of LIF,can be used in culture of hES cells;hES cells from PGCs can be identified through their biological characteristic. [

Construction of Recombinant Adenoviral Vector Carrying the Human BMP-2 Gene with the Technology of in vitro Ligation / 中国肿瘤生物治疗杂志

Objective: To provide an efficeut protocol for constructing recombinant adenovirus, an in vitro ligation was used instead of homologous recombination. Methods: Gene BMP-2 was ligated into pShuttle 2 vector ( pShuttle 2-BMP-2 ) and then fragment containing BMP-2 gene and promoter pcmvie excised by PI-SCe Ⅰ and Ⅰ-Ceu Ⅰ endonuclease. The fragment was further combined with adenovirus vector (Adeno-X-BMP-2) , which was finally linearized with Pac Ⅰ and trans-fered to HEK293 to package adenovirus particles. Results: Both PCR assay and restiction analysis showed that the recombined rectors pShuttle2-BMP-2 and Adeno-X-BMP-2 contains the target BMP-2 gene. THe packaged adenovirus was also i-dentified by PCR assay with specific primers for BMP-2. Conclusions: The BMP-2 incorporated recombinant adenovirus was obtained and this laid a foundation for further study on BMP-2 mediated gene therapy. The in vitro ligation method de-scinbed here for constructing recombined adenovirus was more efficient than traditional homologous recombination.

THE REGULATION EFFECTS OF INSULIN ON THE LYMPHOCYTES IN MICE / 中国药理学通报

The in vitro modulation effect of insulin on DNA and IL-2 production of spleen lymphocytes in diabetic mice were studied. The results suggest that the DNA and IL-2 production of the lymphoc-ytes are significantly inhibited in the mice. When suspend the lymphocytes to the culture medium containing insulin, the DNA and IL -2 production of the lymphocytes are remarkably increased. Therefore, insulin is an important immunoregulation hormone.