白藜芦醇对人胰腺癌PANC-1细胞增殖与侵袭的影响

Objective To investigate the effect of resveratrol on the proliferation and invasion of human pancreatic cancer PANC-1 cells. Methods Five groups including blank control group, 0. 1% dimethylsulfoxide (DMSO) group and resveratrol groups (50, 100, 200 μmol/L) were established. The proliferation of PANC-1 cells was detected by MTT assay. The apoptosis and cell cycle change were analyzed by flow cytometry. The invasive ability of PANC-1 cells was observed with a Transwell cell culture chamber. The expressions of Bax, Bcl-2,matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) of the PANC-1 cells were assayed by real-time quantitative PCR and Western blot. All data were analyzed using the analysis of variance. Results ( 1 ) The inhibition rate of resveratrol on the proliferation of PANC-1 cells was 0 in the blank control group, 3.25% ±0.42% in the 0. 1% DMSO group, 13.23% ± 1.68% in the 50 μmol/L of resveratrol group, 42.25% ± 3.20% in the 100 μmol/L of resveratrol group, and 56.94% ±5.31% in the 200 μmol/L of resveratrol group. There was a significant difference in the inhibition rate among the five groups (F=460. 10, P<0.05). (2) The apoptosis rate was 0.05% ±0.03% in the blank control group, 3.39% ± 1.77% in the 0. 1% DMSO group, 6.92% ± 1.85% in the 50 μmol/L of resveratrol group, 19.05% ± 2.01% in the 100 μmol/L of resveratrol group, and 27. 17% ±6.43% in the 200 μmol/L of resveratrol group. There was a significant difference in the apoptosis rate among the five groups (F = 38.84, P < 0.05). (3) There was no significant effect of 0. 1% DMSO on the cell cycle of PANC-1 cells. The number of PANC-1 cells in the G0/G1 and S phase was increased. (4) The average number of invading PANC-1 cells was 61 ± 13 in the blank control group, 54 ± 13 in the 0. 1% DMSO group, 48 ± 15 in the 50 μmol/L of resveratrol group, 23 ±6 in the 100 μ mol/L of resveratrol group and 18 ±7 in the 200 μmol/L of resveratrol group. There was a significant difference in the number of invading PANC-1 cells among the five groups (F = 69.08, P < 0.05 ). (5) There were up-regulated mRNA and protein expressions of Bax and down-regulated mRNA and protein expressions of Bcl-2, and the expressions of MMP-2 and MMP-9 of the PANC-1 cells were inhibited in the resveratrol groups. The changes of the protein expressions of Bax, Bcl-2, MMP-2, MMP-9 were consistent with the changes of the mRNA expressions of the four indexes. Conclusion Resveratrol can significantly inhibit the proliferation and invasion, as well as induce apoptosis of PANC-1 cells in vitro.     

绿脓杆菌制剂对人肝癌MHCC97L细胞增殖和侵袭能力的影响

Objective To investigate the effects of Pseudomonas aeruginosa vaccine (PA) on proliferation and invasiveness of the hepatocellular carcinoma cell line MHCC97L with metastatic potential. Methods Proliferation, growth curve, plate efficiency, flow cytometry, transwell invasion assay, cell motility assay, scarification test, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2) protein activity were evaluated after cells were treated with PA at various concentrations. Results PA can inhibit the proliferation and plate efficiency of MHCC97L cell markedly in a dose-dependent manner. The IC50 of cells treated with PA for 48 h and 72 h was 3.1 ×108/ml and 1.9 × 108/ml, respectively. The doubling time increased and plate efficiency decreased gradually when cells treated with 0.5 × 108/ml, 1 × 108/ml and 2 × 108/ml PA (P＜0.01). PA could induce cell cycle arrest at the G1 phase in a dose-dependent manner by flow cytometric analysis. The average amount of invading cell per field in cell invasion assay and motility assay were 4. 8 ± 1.3 and 8. 8±2.2 when cells treated with 1× 108/ml PA, which was significantly lower than that of control group (8. 6±2. 1 and 15. 6±1.2 ) (P＜0.01) Scarification test showed that the metastatic ability of cells treated with 1 × 108/ml PA significantly lower than that in the control group. Comparison between cells treated with 1 × 108/ml PA and control group, no remarkable difference was found regarding expression of VEGF and MMP2 in supernatant of cell culture. Conclusion PA can inhibit proliferation and plate efficiency of HCC cell line MHCC97L, which is in part mediated by the cell cycle arrest at the G1 phase. PA could inhibit invasiveness of HCC cell line MHCC97L, which is unrelated to the VEGF and MMP2 protein activity.     

绿脓杆菌制剂对人肝癌MHCC97L细胞增殖和侵袭能力的影响

Objective To investigate the effects of Pseudomonas aeruginosa vaccine (PA) on proliferation and invasiveness of the hepatocellular carcinoma cell line MHCC97L with metastatic potential. Methods Proliferation, growth curve, plate efficiency, flow cytometry, transwell invasion assay, cell motility assay, scarification test, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2) protein activity were evaluated after cells were treated with PA at various concentrations. Results PA can inhibit the proliferation and plate efficiency of MHCC97L cell markedly in a dose-dependent manner. The IC50 of cells treated with PA for 48 h and 72 h was 3.1 ×108/ml and 1.9 × 108/ml, respectively. The doubling time increased and plate efficiency decreased gradually when cells treated with 0.5 × 108/ml, 1 × 108/ml and 2 × 108/ml PA (P＜0.01). PA could induce cell cycle arrest at the G1 phase in a dose-dependent manner by flow cytometric analysis. The average amount of invading cell per field in cell invasion assay and motility assay were 4. 8 ± 1.3 and 8. 8±2.2 when cells treated with 1× 108/ml PA, which was significantly lower than that of control group (8. 6±2. 1 and 15. 6±1.2 ) (P＜0.01) Scarification test showed that the metastatic ability of cells treated with 1 × 108/ml PA significantly lower than that in the control group. Comparison between cells treated with 1 × 108/ml PA and control group, no remarkable difference was found regarding expression of VEGF and MMP2 in supernatant of cell culture. Conclusion PA can inhibit proliferation and plate efficiency of HCC cell line MHCC97L, which is in part mediated by the cell cycle arrest at the G1 phase. PA could inhibit invasiveness of HCC cell line MHCC97L, which is unrelated to the VEGF and MMP2 protein activity.     

胰岛素对脂肪干细胞生长因子分泌功能的影响

Objective To study the effect of insulin in different concentrations on secretion function of growth factors of adipose-derived stem cells (ADSCs). Methods ADSCs were isolated from human ab-dominal adipose tissue and cultured. The immunophenotype and adipose induced-differenciation were identi-fied, and the third generation cells were collected. The collected cells were assigned to 1 × 10-8, 1 × 10-7, 1 × 10-6 mol/L insulin groups according to the concentration of added insulin. When cells grew into 70% confluence in conventional medium, ADSCs were cultured further in serum-free DMEM containing insulin in different concentrations for 3 days. ADSCs cultured in medium without insulin were used as control group. Secretion amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) of ADSCs were determined by enzyme-linked immunosorbent assay. The effects of the supernatant fluid of ADSCs' nutrient solution on the proliferation and collagen synthesis of the cultured fibroblast were detected by MTT chromatometry and hydroxyproline chromatometry. Results The secretion amounts of VEGF and HGF of ADSCs in 1 × 10-8 and 1 × 10-7 mol/L insulin groups [ (471±41, 762±66 ng/L), (643±64, 930±67 ng/L) , respectively ] were significantly higher as compared with those in control group (286±47, 577±84 ng/L) ( P <0.05 orP <0.01). No change occurred in the secretion amount of VEGF and HGF of ADSCs in 1±10-6 mol/L insulin group ( P >0.05 ). The supernatant fluid of ADSCs' nutrient medium of 1 ± 10-8 ,1 ± 10-7 mol/L insulin groups showed obvious stimulative effect on the proliferation and collagen synthesis of fibroblasts, and it was most obvious in the 1 ± 10-7 mol/L group ( P < 0.05 or P < 0. 01 ). Conclusions Insulin in the concentrations of 1 ± 10-8 and 1 ± 10-7 mol/L can notably promote ADSCs' function of secreting VEGF and HGF.     

顺铂联合加热对体外血液中肝肿瘤细胞的杀灭作用及对红细胞的影响

Objective To investigate the effectiveness of cis-diamminedichloroplatinum (DDP) combined with hyperthennia in killing liver tumor cells and its influence on erythrocytes in vitro. Methods Cultured liver tumor cells (2 ml) were mixed with erythrocyte suspension (10 ml) and then the mixture was separated into 6 centrifuge tubes with 2 ml in each one. The centrifuge tubes were randomly divided into A-F groups and the experiment was repeated for 30 times. Normal saline 2 ml was added in A and D groups. DDP 2 ml (200 μg/ml) was added in B and E groups. DDP 2 ml (400 μg/ml) was added in C and F groups. The cells were then incubated in warm bath of 37 ℃ for 30 min in A, B and C groups and in warm bath of 42 ℃ for 30 min in the other three groups.After hyperthermic treatment, tumor cells were isolated from erythrocytes using density gradient centrifugation, the inhibition rate of tumor cells was determined by MTT method and the clone formation of tumor cells was checked.The erythrocyte osmotic fragility and content of 2,3-diphosphoglyceric acid in erythrocytes were measured. Results The inhibition rate of tumor cells was gradually increased, while the rate of tumor cell clone formation decreased with the increase in the temperature and DDP concentrations ( P < 0.01) . The rate of tumor cell clone formation was more than 98% and no clone formation was tested in group F. There was no significant difference in the content of 2,3-diphosphoglyceric acid in erythrocytes between before and after hyperthermic treatment in group F ( P >0.05 ) . The rate of hemolysis of erythrocytes was less than 1 % in the 0.68 % sodium chloride solution in group F.Conclusion DDP 200 μg/ml combined with hyperthermic treatment with temperature of 42 ℃ for 30 min can make the liver tumor cells lose the capability of proliferation, however, it exerts slight effect on erythrocyte membrane and no influence on the oxygen-carrying capacity of erythrocytes.     

顺铂联合加热对体外血液中肝肿瘤细胞的杀灭作用及对红细胞的影响

Objective To investigate the effectiveness of cis-diamminedichloroplatinum (DDP) combined with hyperthennia in killing liver tumor cells and its influence on erythrocytes in vitro. Methods Cultured liver tumor cells (2 ml) were mixed with erythrocyte suspension (10 ml) and then the mixture was separated into 6 centrifuge tubes with 2 ml in each one. The centrifuge tubes were randomly divided into A-F groups and the experiment was repeated for 30 times. Normal saline 2 ml was added in A and D groups. DDP 2 ml (200 μg/ml) was added in B and E groups. DDP 2 ml (400 μg/ml) was added in C and F groups. The cells were then incubated in warm bath of 37 ℃ for 30 min in A, B and C groups and in warm bath of 42 ℃ for 30 min in the other three groups.After hyperthermic treatment, tumor cells were isolated from erythrocytes using density gradient centrifugation, the inhibition rate of tumor cells was determined by MTT method and the clone formation of tumor cells was checked.The erythrocyte osmotic fragility and content of 2,3-diphosphoglyceric acid in erythrocytes were measured. Results The inhibition rate of tumor cells was gradually increased, while the rate of tumor cell clone formation decreased with the increase in the temperature and DDP concentrations ( P < 0.01) . The rate of tumor cell clone formation was more than 98% and no clone formation was tested in group F. There was no significant difference in the content of 2,3-diphosphoglyceric acid in erythrocytes between before and after hyperthermic treatment in group F ( P >0.05 ) . The rate of hemolysis of erythrocytes was less than 1 % in the 0.68 % sodium chloride solution in group F.Conclusion DDP 200 μg/ml combined with hyperthermic treatment with temperature of 42 ℃ for 30 min can make the liver tumor cells lose the capability of proliferation, however, it exerts slight effect on erythrocyte membrane and no influence on the oxygen-carrying capacity of erythrocytes.     

PI3K-Akt-eNOS信号转导通路在吗啡促人脐静脉内皮细胞一氧化氮合成中的作用

Objective To evaluate the role of phosphatidyhnositol 3-kiuase-Akt-endothelial nitric oxide synthase (PI3K/Akt/eNOS) signal transduction pathway in nitric oxide (NO) synthesis increased by morphine in cultured human umbilical vein endothelial cells (HUVECs). Methods HUVECs were inoculated in 24 well palates in vitro. Three experiments were performed in this study. In experiment Ⅰ , HUVECs were randomly divided into 4 groups of 6 wells each: control group (group C) and morphine group (group MM1～3). In group M1～3,morphine with the final concentration of 1 μmol/L was added, and then the cells were incubated for 3, 6 and 12 h respectively. NO synthesis was viewed with fluorescence microscope and NO content was measured. In experiment Ⅱ , HUVECs were randomly divided into 4 groups of 6 wells each: group C and group M1～3. In group M1～3,morphine with the final concentrations of 0.01, 1 and 100 μmol/L was added respectively, and then the cells were incubated for 6 h. NO content was measured. In experiment Ⅲ, HUVECs were randomly divided into 5 groups of 6 wells each: group C, group M, group M + wortmannin (W) (group MW), group M + SH-5 (group MS) and group M + L-NAME (group ML). In group M, morphine with the final concentration of 1 μmol/L was added. In group MW, wortmannin (a PI3K specific inhibitor) was added and 15 min later, morphine with the final concentrations of 5 and 1 μmol/L was added. In group MS, SH-5 (an Akt specific inhibitor) was added and 15 min later, morphine with the final concentrations of 10 and 1 μmol/L was added. In group ML, L-NAME (an eNOS specific inhibitor) was added and 15 min later, morphine with the final concentrations of 0.5 and 1 μmol/L was added. The cells were then incubated for 6 h and NO content was measured in each group. Results In experiment Ⅰ , morphine treatment increased the endothelial NO synthesis in a time-dependent manner (P <0.05). In experiment Ⅱ , morphine treatment increased the endothelial NO synthesis in a concentration-dependent manner (P < 0.05). In experiment Ⅲ, increased NO synthesis by morphine treatment was abolished by wortmannin, SH-5, or L-NAME. Conclusion Morphine can increase NO synthesis in cultured HUVECs in concentration- and time-dependent manners, and the mechanism may be related to the activation of the PI3K-AKT-eNOS signal tranaduction pathway.     

Effect of Ureaplasma urealyticum infection on IL-laand IL-6 secretion by rat Sertoli cells

Objective: To observe the effect of Ureaplasma urealyticum (UU) infection on the IL-1α and IL-6 secretion by rat Sertoli cells. Methods: Eight 20-day-old UU-free male SD rats (average weight 40 g) were used. Under sterile condition, the testes were removed and separately digested with collagenase typeⅡand hyaluronidase. High purity Sertoli cells were then isolated and adjusted to a concentration of 8×105/mL with DMEM/Ham's F-12. In the infected group, 1 mL Sertoli cell suspension and 100 mL UU (serotype 8, T960) were introduced into one well of a 24 well culture plate. In the control group, 1 mL Sertoli cell suspension and 100 mL medium were introduced. IL-1αand IL-6 were determined in the culture supernatant with ELISA. Results: The production of IL-1αwas significantly lower and of IL-6 significantly higher in the infected than those in the control groups (P<0.01). Conclusion: UU infection reduces the IL-1αand increases the IL-6 secretion by rat Sertoli cells. UU infection is probably involved in     

Radioprotective effects of the expression of FLT3 ligand regulated by Egr-1 regulated element on radiation injury of SCID mice

Objective:In order to explore the radioprotective effects of the expression of hematopoietic growth factors regulated by radio-inducible promoter on radiation injury. Methods:The human FL (Flt3 ligand) cDNA and EGFP (enhanced green fluorescent protein) cDNA were linked together with IRES and then inserted into the eukaryotic expression vector pCI-Egr, which was constructed by substituting CMV promoter in pCIneo with the Egr-1 promoter (Egr-EF). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin. The transduced cell clones (HFCL/EF) had been selected by the addition of G418. The cells were exposed to γ-radiation by 60 Co source for 0.5-20Gy. The expressions of transduced cells were detected with FACS, Northern blot ELISA and CFU assay. The HFCL/EF and CD34+ cells from human umbilical cord blood were one after the other transplanted i.v. into sublethally irradiated severe combined immunodeficient (SCID) mice. The white blood cell amount in peripheral blood and human cell engrafted in recipent mice were detected by flow cytometry and CFU-GM etc. Results:The activity of EGFP in transduced cells increased by 3.1 fold as compared to non-transduced cells at 18h after exposure to 2.5Gy. The amounts of secreted FL in serum-free supernatants of Egr-EF increased by 605.46±107.21pg/ml, which were significantly higher than the control group (214.45±35.61pg/ml). The effects of FL in HFCL/EF cultural supernatants on expansion of CD34+ cells derived from cord blood in the presence of SCF, IL-6 and IL-3 were also studied. The results showed that at day 10 of culture the number of CD34+ cells increased by 173. 09±11.58×103/ml, which was significantly higher than that of non-radiation group(68. 04± 13. 73 × 103/ml). It showed that radiation can enhance the ability of the supernatants containing FL of HFCL/EF to expand early hematopoietic progenitor cells and protect hematopoietic cells from radiation-injury effects. The HFCL/EF and CD34+cells from human umbilical cord blood were one after the other transplanted i. v. into sublethally irradiated severe combined immunodeficient (SCID) mice. In contrast to two control groups (HFCL and HFCL/F), HFCL/EF (the Egr-1 regulatory element-drived expression of FL gene therapy) resulted in a proportionally obvious increase in the number of the white blood cell at early stage after radiation, while no significant differences were found for CD45+ 、CD34+ cells in bone marrow cells. In contrast to two control groups (HFCL and HFCL/F), HFCL/EF (the Egr1 regulatory element-drived expression of FL gene therapy) resulted in a proportionally obvious increase in the number of the white blood cell at early stage after radiation, without significant differences being found for CD45+、CD34+ 、CFU-GM and marrow nucleared cells in bone marrow cells. Conclusions:The results suggested both in vivo and in vitro use of the gene therapy of FL gene regulated by Egr-1 promoter could protect hematopoiesis from irradiation-induced damage.     

神经生长因子对胆管癌细胞67-kDa层黏连蛋白受体表达的影响

Objective To investigate the effects of nerve growth factor (NGF) on the expression of 67-kDa laminin receptor (67LR) in human bile duct carcinoma QBC939 cells, and study the possible mechanism of perineural invasion and metastasis of bile duct carcinoma. Methods ( 1 ) The expression of a high-affinity receptor for NGF, TrkA, was detected by immunofluorescence staining. ( 2 ) QBC393 cells were pretreated by β-NGF at different concentrations ( 1, 10, 100,200 μg/L), and then the mRNA and protein expressions of 67LR were examined by Real-Time PCR and Western blot assay. QBC939 cells were divided into control group and β-NGF (1, 10, 100,200 μg/L) groups. (3) The ideal concentration of β-NGF was selected according to the results of previous tests, and then the mRNA and protein expressions of 67LR were re-examined by adding specific TrkA inhibitor K252a at different concentrations ( 100,200,300 nmol/L). QBC939 cells were divided into control group, β-NGF 100 μg/L group and K252a ( 100,200,300 nmol/L) groups. All data were analyzed by one-way analysis of variance or LSD-test. Results (1) A strong expression of TrkA was detected in the membrane of QBC939 cells. (2) The mRNA and protein expressions of 67LR in QBC939 cells were 0.35 ± 0.06 and 0. 32 ± 0.05 in the control group, 0.38 ±0.14 and 0.50 ±0.09 in the β-NGF 1 μg/L group, 0.62 ±0.14 and 0. 69 ±0. 13 in β-NGF 10 μg/L group, 0.90 ± 0.08 and 0.93 ± 0.07 in the β-NGF 100 μg/L group, and 0. 70 ± 0. 10 and 0. 76 ±0.07 in the β-NGF 200 μg/L group, there were significant differences among the five groups (F = 22. 4, 14. 6,P ＜0.05). The mRNA and protein expressions of 67LR in the β-NGF 100 μg/L group were significantly higher than those in the control group ( t = 19. 0, 21.0, P ＜ 0. 05 ). (3) The mRNA and protein expressions of 67LR in the QBC939 cells were 0.35 ±0.10 and 0.41 ±0.10 in the control group, 0. 88 ±0. 14 and 0.84 ±0.10 in the β-NGF 100 μg/L group, 0.80±0.08 and 0.76 ±0.04 in the K252a 100 nmol/L group, 0.67 ±0.12 and 0.61 ± 0.09 in the K252a 200 nmol/L group, and 0. 43 ± 0.07 and 0. 50 ± 0. 12 in the K252a 300 nmol/L group, there were significant differences among the five groups ( F = 14. 1, 8. 9, P ＜ 0.05 ). There were no significant differences in the mRNA and protein expressions of 67LR between the K252a 300 nmol/L group and the control group (t =1.02, 0. 85, P＞0.05). Conclusion In bile duct carcinoma cells, NGF enhances the expression of 67LR by combining with TrkA, which might be the mechanism of NGF mediating perineural invasion of bile duct carcinoma.     

胸腰椎骨折椎弓根内固定断裂与植骨融合方式的相关性分析

[目的]探讨胸腰椎骨折椎弓根螺钉内固定系统内固定术后,椎弓根螺钉断裂与植骨融合方式之间的关系,以探讨胸腰椎骨折植骨融合的最佳方式。[方法]回顾性研究1995年5月~2005年12月本院脊柱外科收治的胸腰椎骨折病人197例,其中A组单纯内固定(不植骨)患者14例,B组“H”形椎板植骨21例,C组横突间植骨67例,D组椎间、椎内联合横突间植骨95例。[结果]术后随访6~32个月,内固定断裂12例,其中A组4例,B组3例,C组5例,D组0例,4组中D组内固定断裂率显著低于其他3组(P<0.05)。[结论]椎间、椎体内联合横突间植骨重建脊柱三柱的稳定性,符合人体生物力学原理,能有效降低内固定断裂的发生。     

Influence of the design of the prosthesis on the outcome after hemiarthroplasty of the shoulder in displaced fractures of the head of the humerus

We reviewed 39 patients with displaced three- and four-part fractures of the humerus. In 21 patients (group A) we had used an anatomical prosthesis for the humeral head and in 18 (group B) an implant designed for fractures. When followed up at a mean of 29.3 months after surgery the overall Constant score was 51.9 points; in group A it was 51.5 and in group B 52.4 points. The subjective satisfaction of the patients was assessed using a numerical rating scale and was similar in both groups. In group A complete healing of the tuberosities was found in 29% and 50% in group B. Partial integration was seen in 29% of group A and in only one patient in group B, while resorption was noted in 43% of group A and 44% of group B. The functional outcome was significantly better in patients with complete or partial healing of the tuberosities (p=0.022). The specific trauma prosthesis did not lead to better healing of the tuberosities. The difference in clinical outcome obtained by the two designs did not reach statistical significance.