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.     

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

目的 研究外源性神经生长因子(NGF)对QBC939细胞67-kDa层黏连蛋白受体(67LR)表达的影响,探讨胆管癌神经浸润的机制.方法 (1)细胞免疫荧光染色法检测QBC939细胞表面NGF高亲和力酪氨酸激酶受体A(TrkA)表达情况.(2)用不同浓度的重组人神经生长因子(β-NGF)对QBC939细胞进行处理,采用Real-Time PCR和Western blot检测QBC393细胞67LR mRNA和蛋白表达情况.实验分为对照组、β-NGF(1、10、100、200μg/L)组.(3)根据上述实验结果选定最佳β-NGF浓度,再加入不同浓度的TrkA阻断剂K252a,再次检测QBC939细胞67LR mRNA和蛋白表达情况.实验分为对照组、100μg/Lβ-NGF组、K252a(100、200、300 nmol/L)阻断组.数据采用单因素方差分析,两两比较采用LSD法.结果 (1)QBC939细胞膜上TrkA表达明显.(2)对照组和β-NGF(1、10、100、200 μg/L)组QBC939细胞67LRmRNA和蛋白表达水平分别为0.35±0.06、0.38±0.14、0.62±0.14、0.90±0.08、0.70±0.10和0.32±0.05、0.50±0.09、0.69±0.13、0.93±0.07、0.76±0.07,两者比较,差异有统计学意义(F=22.4,14.6,P＜0.05),其中100ng/ml β-NGF作用最明显(t=19.0,21.0,P＜0.05).(3)对照组、100 μg/L β-NGF组、K252a(100、200、300 nmol/L)阻断组QBC939细胞67LR mRNA和蛋白表达水平分别为0.35±0.10、0.88±0.14、0.80±0.08、0.67±0.12、0.43±0.07和0.41±0.10、0.84±0.10、0.76±0.04、0.61±0.09、0.50±0.12,两者比较,差异有统计学意义(F=14.1,8.9,P＜0.05),其中经300 nmol/L K252a处理后,QBC939细胞67LR mRNA和蛋白表达水平与对照组比较,差异无统计学意义(t=1.02,0.85,P＞0.05).结论 外源性NGF通过与其高亲和力受体TrkA结合,可增强QBC939细胞67LR表达,这可能是促进胆管癌细胞延外周神经纤维间隙浸润的机制之一.

Abstract：

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.     

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.     

AG1478对U87细胞增殖、细胞周期和凋亡的影响

目的 观察表皮生长因子受体抑制剂Tyrphostin AG1478对人脑胶质瘤U87细胞增殖、细胞周期和细胞凋亡的影响.方法 噻唑蓝(MTT)比色法检测不同浓度Tyrphostin AG1478作用于体外培养的人脑胶质瘤U87细胞24 h后的细胞生存率,流式细胞仪检测不同浓度Tyrphostin AG1478作用于体外培养的人脑胶质瘤U87细胞24 h后细胞周期分布和细胞凋亡.结果 5、10、15、20 μmol/L的Tyrphostin AG1478作用人脑胶质瘤U87细胞24 h后细胞生存率分别为(7 8.93±11.95)%、(46.42±4.12)%、(42.13±7.54)%和(37.48±4.69)%;0、10、20 μmol/L的Tyrphostin AG1478作用人脑胶质瘤U87细胞24 h后细胞凋亡率分别为(10.19±3.15)%、(32.02±1.60)%和(54.35±2.80)%;0、10、20 μmol/L的Tyrphostin AG1478作用人脑胶质瘤U87细胞24 h后细胞主要分布在G0～G1期,分别为(51.20±1.21)%、(78.61±1.57)%和(82.73±0.77)%,实验组与对照组比较差异均有统计学意义(P＜0.05).结论 Tyrphostin AG1478抑制体外人脑胶质瘤细胞增殖,阻滞细胞周期在G0～G1期,诱导细胞凋亡,均呈浓度依赖性.

Abstract：

Objective To investigate the effects of Tyrphostin AG1478 on glioma U87 cells proliferation, cells cycle and apoptosis. Methods U87 cells were cultured for 24 h in the medium which contained AG1478 with different concentrations (0, 5, 10, 15, 20 μmol/L). The methyl thiazolyl tetrazolium(MTT) assay was used to detect the survival rate, and the cells cycle and apoptosis of the cells were examined by using flow cytometry. Results The cells proliferation was obviously inhibited by AG1478 in a dose-dependent manner. The survival rate of the cells in 5, 10, 15, 20 μmol/L AG1478 groups was (78.93 ±11.95)%, (46.42 ±4. 12)%, (42. 13 ±7.54)% and (37.48 ±4.69)% respectively, which was all significantly higher than that in 0 μ mol/L AG1478 group (P ＜0. 05 ). The apoptotic rate in 10, 25μmol/L AG1478 groups was (32.02 ± 1.60)% and (54. 35 ± 2. 80)% respectively, significantly higher than that in 0 μmol/L AG1478 group ( P ＜ 0. 05 ). The cells cycle was obviously inhibited by AG1478 in a dose-dependent manner. The percentage of cells treated with 10 and 20 μmol/L AG1478 in Go-G1 phase was ( 78. 61 ± 1.57 ) % and ( 82. 73 ± 0. 77 ) % respectively, significantly higher than that in 0 μmol/L AG1478 group (P ＜ 0. 05 ). Conclusion The growth inhibition of glioma U87 cells caused by AG1478 may be associated with apoptotsis induction and the G0-G1 arrest. AG1478 was expected to become a new anti-tumor drug in human glioma.     

The effects of β-NGF controlled delivery system on growth of primary cultured chick embryo dorsal root ganglion axons

Objective To construct β-NGF controlled delivery system and evaluate the biological effects of β-NGF on the growth of chick embryo dorsal root ganglion (DRG) axons in vitro. Methods Delivery systems releasing β-NGF at 50/μL, 100/μg/L and 250 μg/L concentration were constructed. To determine the optimal dose response effects of NGF in the controlled delivery system, DRG were co-cultured with of β-NGF at above concentrations while using DRG basic culture as control. Axonal growth was observed. DRG were also cocultured with the components in the controlled delivery system to detect the effects on growth of DRG axons. The experiment was divided into 5 experimental groups and 1 control group: control group, DRG+ fibrin; Group A,DRG+ fibrin+ peptide + heparin + 100 μg/L β-NGF; Group B, DRG + fibrin + heparin + 100/μg/L β-NGF;Group C, DRG + fibrin + peptide + 100 μg/L β-NGF; Group D, DRG + fibrin + 100 μg/L β-NGF; Group E,DRG + fibrin + peptide + heparin. Results The growth of DRG axons in 50 μg/L, 100μg/L and 250/μg/Lconcentration of β-NGF controlled delivery system was 1.31 ( P > 0. 05), 3.78 ( P < 0. 01 ) and 3.05 ( P <0.01) folds of the control respectively. The growth of DRG axons in 100 μg/L group was significantly better comparing to that in 250 μg/L group. The growth of DRG axons in Groups A, B, C, D and E was 3.75, 1.15,1.12, 1.10 and 1.09 folds of the control group, respectively. The difference was only statistically significant between Group A and the control group ( P < 0. 01 ). Conclusion β-NGF released from the β-NGF controlled delivery system was bioactive. It could promote the growth of DRG axons.     

The effects of β-NGF controlled delivery system on growth of primary cultured chick embryo dorsal root ganglion axons

Objective To construct β-NGF controlled delivery system and evaluate the biological effects of β-NGF on the growth of chick embryo dorsal root ganglion (DRG) axons in vitro. Methods Delivery systems releasing β-NGF at 50/μL, 100/μg/L and 250 μg/L concentration were constructed. To determine the optimal dose response effects of NGF in the controlled delivery system, DRG were co-cultured with of β-NGF at above concentrations while using DRG basic culture as control. Axonal growth was observed. DRG were also cocultured with the components in the controlled delivery system to detect the effects on growth of DRG axons. The experiment was divided into 5 experimental groups and 1 control group: control group, DRG+ fibrin; Group A,DRG+ fibrin+ peptide + heparin + 100 μg/L β-NGF; Group B, DRG + fibrin + heparin + 100/μg/L β-NGF;Group C, DRG + fibrin + peptide + 100 μg/L β-NGF; Group D, DRG + fibrin + 100 μg/L β-NGF; Group E,DRG + fibrin + peptide + heparin. Results The growth of DRG axons in 50 μg/L, 100μg/L and 250/μg/Lconcentration of β-NGF controlled delivery system was 1.31 ( P > 0. 05), 3.78 ( P < 0. 01 ) and 3.05 ( P <0.01) folds of the control respectively. The growth of DRG axons in 100 μg/L group was significantly better comparing to that in 250 μg/L group. The growth of DRG axons in Groups A, B, C, D and E was 3.75, 1.15,1.12, 1.10 and 1.09 folds of the control group, respectively. The difference was only statistically significant between Group A and the control group ( P < 0. 01 ). Conclusion β-NGF released from the β-NGF controlled delivery system was bioactive. It could promote the growth of DRG axons.     

The effects of β-NGF controlled delivery system on growth of primary cultured chick embryo dorsal root ganglion axons

Objective To construct β-NGF controlled delivery system and evaluate the biological effects of β-NGF on the growth of chick embryo dorsal root ganglion (DRG) axons in vitro. Methods Delivery systems releasing β-NGF at 50/μL, 100/μg/L and 250 μg/L concentration were constructed. To determine the optimal dose response effects of NGF in the controlled delivery system, DRG were co-cultured with of β-NGF at above concentrations while using DRG basic culture as control. Axonal growth was observed. DRG were also cocultured with the components in the controlled delivery system to detect the effects on growth of DRG axons. The experiment was divided into 5 experimental groups and 1 control group: control group, DRG+ fibrin; Group A,DRG+ fibrin+ peptide + heparin + 100 μg/L β-NGF; Group B, DRG + fibrin + heparin + 100/μg/L β-NGF;Group C, DRG + fibrin + peptide + 100 μg/L β-NGF; Group D, DRG + fibrin + 100 μg/L β-NGF; Group E,DRG + fibrin + peptide + heparin. Results The growth of DRG axons in 50 μg/L, 100μg/L and 250/μg/Lconcentration of β-NGF controlled delivery system was 1.31 ( P > 0. 05), 3.78 ( P < 0. 01 ) and 3.05 ( P <0.01) folds of the control respectively. The growth of DRG axons in 100 μg/L group was significantly better comparing to that in 250 μg/L group. The growth of DRG axons in Groups A, B, C, D and E was 3.75, 1.15,1.12, 1.10 and 1.09 folds of the control group, respectively. The difference was only statistically significant between Group A and the control group ( P < 0. 01 ). Conclusion β-NGF released from the β-NGF controlled delivery system was bioactive. It could promote the growth of DRG axons.     

The effects of β-NGF controlled delivery system on growth of primary cultured chick embryo dorsal root ganglion axons

Objective To construct β-NGF controlled delivery system and evaluate the biological effects of β-NGF on the growth of chick embryo dorsal root ganglion (DRG) axons in vitro. Methods Delivery systems releasing β-NGF at 50/μL, 100/μg/L and 250 μg/L concentration were constructed. To determine the optimal dose response effects of NGF in the controlled delivery system, DRG were co-cultured with of β-NGF at above concentrations while using DRG basic culture as control. Axonal growth was observed. DRG were also cocultured with the components in the controlled delivery system to detect the effects on growth of DRG axons. The experiment was divided into 5 experimental groups and 1 control group: control group, DRG+ fibrin; Group A,DRG+ fibrin+ peptide + heparin + 100 μg/L β-NGF; Group B, DRG + fibrin + heparin + 100/μg/L β-NGF;Group C, DRG + fibrin + peptide + 100 μg/L β-NGF; Group D, DRG + fibrin + 100 μg/L β-NGF; Group E,DRG + fibrin + peptide + heparin. Results The growth of DRG axons in 50 μg/L, 100μg/L and 250/μg/Lconcentration of β-NGF controlled delivery system was 1.31 ( P > 0. 05), 3.78 ( P < 0. 01 ) and 3.05 ( P <0.01) folds of the control respectively. The growth of DRG axons in 100 μg/L group was significantly better comparing to that in 250 μg/L group. The growth of DRG axons in Groups A, B, C, D and E was 3.75, 1.15,1.12, 1.10 and 1.09 folds of the control group, respectively. The difference was only statistically significant between Group A and the control group ( P < 0. 01 ). Conclusion β-NGF released from the β-NGF controlled delivery system was bioactive. It could promote the growth of DRG axons.     

The effects of β-NGF controlled delivery system on growth of primary cultured chick embryo dorsal root ganglion axons

Objective To construct β-NGF controlled delivery system and evaluate the biological effects of β-NGF on the growth of chick embryo dorsal root ganglion (DRG) axons in vitro. Methods Delivery systems releasing β-NGF at 50/μL, 100/μg/L and 250 μg/L concentration were constructed. To determine the optimal dose response effects of NGF in the controlled delivery system, DRG were co-cultured with of β-NGF at above concentrations while using DRG basic culture as control. Axonal growth was observed. DRG were also cocultured with the components in the controlled delivery system to detect the effects on growth of DRG axons. The experiment was divided into 5 experimental groups and 1 control group: control group, DRG+ fibrin; Group A,DRG+ fibrin+ peptide + heparin + 100 μg/L β-NGF; Group B, DRG + fibrin + heparin + 100/μg/L β-NGF;Group C, DRG + fibrin + peptide + 100 μg/L β-NGF; Group D, DRG + fibrin + 100 μg/L β-NGF; Group E,DRG + fibrin + peptide + heparin. Results The growth of DRG axons in 50 μg/L, 100μg/L and 250/μg/Lconcentration of β-NGF controlled delivery system was 1.31 ( P > 0. 05), 3.78 ( P < 0. 01 ) and 3.05 ( P <0.01) folds of the control respectively. The growth of DRG axons in 100 μg/L group was significantly better comparing to that in 250 μg/L group. The growth of DRG axons in Groups A, B, C, D and E was 3.75, 1.15,1.12, 1.10 and 1.09 folds of the control group, respectively. The difference was only statistically significant between Group A and the control group ( P < 0. 01 ). Conclusion β-NGF released from the β-NGF controlled delivery system was bioactive. It could promote the growth of DRG axons.     

The effects of β-NGF controlled delivery system on growth of primary cultured chick embryo dorsal root ganglion axons

Objective To construct β-NGF controlled delivery system and evaluate the biological effects of β-NGF on the growth of chick embryo dorsal root ganglion (DRG) axons in vitro. Methods Delivery systems releasing β-NGF at 50/μL, 100/μg/L and 250 μg/L concentration were constructed. To determine the optimal dose response effects of NGF in the controlled delivery system, DRG were co-cultured with of β-NGF at above concentrations while using DRG basic culture as control. Axonal growth was observed. DRG were also cocultured with the components in the controlled delivery system to detect the effects on growth of DRG axons. The experiment was divided into 5 experimental groups and 1 control group: control group, DRG+ fibrin; Group A,DRG+ fibrin+ peptide + heparin + 100 μg/L β-NGF; Group B, DRG + fibrin + heparin + 100/μg/L β-NGF;Group C, DRG + fibrin + peptide + 100 μg/L β-NGF; Group D, DRG + fibrin + 100 μg/L β-NGF; Group E,DRG + fibrin + peptide + heparin. Results The growth of DRG axons in 50 μg/L, 100μg/L and 250/μg/Lconcentration of β-NGF controlled delivery system was 1.31 ( P > 0. 05), 3.78 ( P < 0. 01 ) and 3.05 ( P <0.01) folds of the control respectively. The growth of DRG axons in 100 μg/L group was significantly better comparing to that in 250 μg/L group. The growth of DRG axons in Groups A, B, C, D and E was 3.75, 1.15,1.12, 1.10 and 1.09 folds of the control group, respectively. The difference was only statistically significant between Group A and the control group ( P < 0. 01 ). Conclusion β-NGF released from the β-NGF controlled delivery system was bioactive. It could promote the growth of DRG axons.     

复方麝香注射液在人脐带间充质干细胞神经分化中的作用

目的 观察复方麝香注射液在体外对人脐带间充质干细胞(hUMSCs)神经分化的影响.方法 取足月妊娠剖宫产健康新牛儿脐带,获取间充质干细胞,采用流式细胞术进行细胞表型鉴定.以碱性成纤维细胞生长因子(bFGF)预诱导24 h,再以含5、10、15、20、25g/L复方麝香注射液的无血清培养基进行神经分化诱导,采用免疫细胞化学检测神经元特异性烯醇化酶(NSE)、微观相关蛋白-2(MAP-2)和胶质纤维酸性蛋白(GFAP)的表达.结果 流式细胞检测显示hUMSCs表达CD13、CD29、CD44、CD73、CD90、CD105、CD106、CD166和HLA-ABC抗原,不表达CD31、CD34、CD38、CD45、CD133和HLA-DR抗原.经不同浓度复方麝香注射液诱导5 d后,各诱导组中均出现神经样细胞.5、10、15、20、25 g/L组显微镜每随机视野中NSE阳性细胞数分别为19.00±3.61、66.60±5.37、60.20±10.06、44.80±9.44、47.80±10.45,MAP-2阳性细胞数分别为9.20±3.27、53.40±10.92、59.40±10.41、46.60±8.88、46.80±8.93.以10 g/L和15 g/L组中的NSE、MAP-2阳性细胞数为最多.各诱导组中仅极少数细胞呈GAFP弱阳性.结论 人脐带富含问充质干细胞(MSCs),适量复方麝香注射液可有效诱导人脐带间充质干细胞分化为神经元细胞.     

白藜芦醇对人胰腺癌PANC-1细胞增殖与侵袭的影响

目的 探讨白藜芦醇对人胰腺癌PANC-1细胞增殖与侵袭能力的影响.方法 实验分为空白对照组,0.1%DMSO组,白藜芦醇组(50、100、200 μmol/L).MTT法检测白藜芦醇对细胞增殖的影响;流式细胞仪检测细胞凋亡及周期变化;Transwell侵袭小室检测白藜芦醇对细胞侵袭的影响;荧光实时定量PCR和Western blot检测白藜芦醇对细胞Bax、Bcl-2、MMP-2、MMP-9表达的影响.数据以-x±s表示,多组比较采用方差分析.结果 (1)空白对照组抑制率为0;0.1%DMSO组细胞抑制率为3.25%±0.42%;白藜芦醇50 μmol/L组细胞抑制率为13.23%±1.68%;白藜芦醇100μmol/L组细胞抑制率为42.25%±3.20%;白藜芦醇200μmol/L组细胞抑制率为56.94%±5.31%.各组比较,差异有统计学意义(F=460.10,P<0.05).(2)空白对照组细胞凋亡率为0.05%±0.03%;0.1%DMSO组细胞为凋亡率为3.39%±1.77%;白藜芦醇50 μmol/L组细胞凋亡率为6.92%±1.85%;白藜芦醇100 μmol/L组细胞凋亡率为19.05%±2.01%;白藜芦醇200 μmol/L组细胞凋亡率为27.17%±6.43%.各组比较,差异有统计学意义(F=38.84,P<0.05).(3)0.1%DMSO组对细胞周期无显著影响.白藜芦醇引起PANC-1细胞G0/G1期和S期阻滞,G2/M期细胞减少.(4)空白对照组平均穿膜细胞数为61±13;0.1%DMSO组为54±13;白藜芦醇50 μmol/L组为48±15;白藜芦醇100 μmol/L组为23±6;白藜芦醇200 μmol/L组为18±7.各组比较,差异有统计学意义(F=69.08,P<0.05).(5)白藜芦醇可使PANC-1细胞Bax表达升高,Bcl-2表达下调.MMP-2、MMP-9的表达明显受到抑制,mRNA和蛋白水平变化一致.结论 白藜芦醇可明显抑制胰腺癌PANC-1细胞增殖,诱导细胞凋亡并抑制其侵袭能力.     

雷帕霉素抑制结缔组织生长因子诱导的成肌纤维细胞增殖及细胞外基质蛋白的分泌

目的 探讨雷帕霉素（RAPA）对于结缔组织生长因子（CTGF）生物学作用的影响及可能机制。 方法 分别用RAPA 20 μg/L 和40 μg/L预处理成肌纤维细胞（MyoF）30 min后再加入CTGF 100 μg/L,与单独加入CTGF（CTGF组）和未加任何刺激的MyoF（对照组）进行比较。采用BrdU掺入法检测细胞的增殖反应;用Western印迹法检测细胞上清中纤连蛋白（FN）的水平及细胞ERK1/2信号的磷酸化。 结果 与对照组相比,CTGF（100 μg/L）显著促进MyoF增殖（P < 0.01）,增加上清中FN的蛋白水平（P < 0.05）。与CTGF组相比,RAPA 20 μg/L及 40 μg/L 预处理可使细胞增殖率显著下降,分别下降了62% 和70%（均P < 0.05）,但两个剂量之间差异无统计学意义;使细胞上清中FN的蛋白水平分别下降了15%和44%,后者与CTGF组的差异有统计学意义（P < 0.05）。CTGF（100 μg/L ）刺激10 min可致MyoF的ERK1/2发生磷酸化,RAPA 40 μg/L预处理细胞30 min可显著降低CTGF诱导的ERK1/2磷酸化。以ERK1/2活化特异抑制剂PD98059（50 μmol/L）预处理30 min后,可以抑制CTGF诱导的细胞增殖效应（7%±5%比85%±7%,P < 0.01）和FN的分泌效应（1.0±0.1比1.6±0.3,P < 0.05）。 结论 RAPA具有部分抑制CTGF的促增殖和促细胞外基质分泌的作用,其作用可能通过抑制ERK1/2信号通路实现。     

丹参酮ⅡA对兔纤维环细胞能量代谢的保护作用

[目的]考察丹参酮ⅡA(TSⅡA)对白细胞介素-1β(IL-1β)诱导的兔纤维环细胞能量代谢障碍的保护作用.[方法]藻酸盐串珠立体培养兔纤维环细胞,将细胞分为7组,在培养过程中加入不同浓度的药物:A组为空白对照不加入药物,B组加入4 μg/ml TSⅡA,C组加入10ng/ml IL-1β,D～G组在给予10 ng/mlIL-1β同时分别加入0.5、1、2和4 μg/ml TSⅡA.于培养3 d后行Na+-K+-ATP酶活性检测、,琥珀酸脱氢酶活性检测、MTT法细胞增殖情况检测以及细胞凋亡的流式细胞仪检测.[结果]G组Na+-K+-ATP酶活性(3.23±0.28U/mgprot)较C组(1.118±0.15U/mgprot)明显增高(P<0.01),与A组接近(3.57±0.15 U/mgprot)(P>0.05).G组琥珀酸脱氢酶活性(12.48±0.97U/mgprot)较c组(3.03±0.60 U/mgprot)明显增高(P<0.01),与A组接近(14.24±1.56 U/mgprot)(P>0.05).G组MTT试验吸光度(0.77±0.06)较C组(0.31±0.07)明显增高(P＜0.01),随着TSⅡA浓度的升高,D～G组吸光度随着TsIIA上升而上升.G组细胞死亡细胞比例和凋亡细胞比例分别为21.08±1.46%和8.99±0.33%,均显著低c组(43.11±2.7,P<0.01和11.71±0.32,P<0.01).[结论]TSIIA能够减轻IL-1β对纤维环细胞能量代谢的抑制作用,从而改善纤维环细胞的增殖、死亡及凋亡.     

胃泌素促进大肠癌细胞增生与STAT3信号转导通路的关系

目的 探讨胃泌素(GAS)对体外培养的人大肠癌SW480细胞株增生的影响,以及与STAT3信号转导通路的关系.方法 通过体外对大肠癌SW480细胞株的培养,分别运用MTT法观察细胞增生活力改变情况,确立胃泌素和丙谷胺处理SW480细胞的最佳浓度;流式细胞仪检测各组最佳药物浓度作用下细胞增生指数的变化;RT-PCR检测各组最佳药物浓度作用下STAT3 mRNA表达水平的变化以及应用SP法检测各组最佳药物浓度作用下SW480细胞PCNA、STAT3蛋白表达水平的变化.结果 5-肽胃泌素在一定范围内(6.25-100μg/mL)能促进大肠癌SW480细胞的增生,且当浓度达25 μg/mL时为最佳浓度(P<0.01);胃泌素受体拮抗剂丙谷胺对大肠癌SW480细胞增生无明显影响,但丙谷胺在一定范围内(8～128 μg/mL)能明显拮抗5-肽胃泌素促进SW480细胞的增生作用,其最佳浓度为32 μg/mL(P<0.01).5-肽胃泌素组细胞增生指数(37.54±5.17)%显著高于对照组(27.72±5.00)%和联合用药组(27.31±5.76)%(P<0.01).STAT3 mRNA及蛋白表达在5-肽胃泌素组(0.65±0.03,0.19±0.03)明显高于对照组(0.46±0.05,0.12±0.02)及联合用药组(0.48±0.09,0.13±0.03)(P<0.01).结论 5-肽胃泌素对体外大肠癌SW480细胞有促增生作用,但能够被其受体拮抗剂丙谷胺抑制.STAT3信号转导通路参与了胃泌素对大肠癌细胞增生的调控.     

吲哚胺2,3双加氧酶对小鼠CD4+T细胞增殖的抑制作用

目的 探讨吲哚胺2,3双加氧酶(IDO)抑制T细胞增殖的机制。方法 通过低色氨酸(10μmol/L)培养基(LTM)和/或添加不同浓度(50、100、200和400μmol/L)色氨酸代谢产物(TC),观察小鼠树突细胞(DC)与异系CD4+T细胞培养体系中后者的增殖。Annexin-V及碘化丙锭(PI)双染法测定CD4+T细胞凋亡。结果 LTM中CD4+T细胞增殖指数(2.718 ±0.010)及抑制CD4+T细胞增殖的TC浓度(200μmol/L KYN或50 μmol/L 3-HAA)较正常色氨酸培养基(NTM)(3.385 ±0.013,400 μmol/L KYN或100 μmol/L 3-HAA)显著降低,差异有统计学意义(P＜0.01)。LTM中CD4+T细胞早期凋亡率(33.163±0.556)％显著高于NTM(8.867 ±0.565)％ (P ＜0.01)。NTM中CD4+T细胞凋亡率随3-HAA浓度升高而增加[3-HAA浓度50、100、200和400 μmol/L组中分别为(14.433 ±0.640)％、(22.273±0.629)％、(37.363±0.953)％和(46.643±0.633)％]。结论 LTM及TC均可通过诱导凋亡来抑制CD4+T细胞增殖,两者具有叠加效应。     

黄连素体外诱导人脐带间充质干细胞向神经样细胞定向分化的实验研究

目的:探讨黄连素体外诱导人脐带间充质干细胞(hUMSCs)向神经细胞分化的作用。方法:体外对hUMSCs进行培养,以不同浓度黄连素(分4组：对照组即0 mg/L组、50 mg/L组、100 mg/L组和200 mg/L组)诱导其向神经样细胞分化,显微镜下观察细胞形态改变并记录,并通过细胞免疫化学及免疫荧光技术检测细胞表...     

小檗碱对结肠癌细胞HT-29增殖、凋亡及细胞膜钾通道的作用

目的 观察小檗碱(Berberine,以下简称"Ber")对结肠癌细胞株HT-29增殖、凋亡的作用并探讨其离子通道机制.方法 Ber 0.1～30.0μmol/L处理HT-29细胞.氮蓝四唑盐法检测细胞增殖,流式细胞术检测凋亡率,膜片钳检测延迟整流钾通道[IK(V)].结果 0.1～30.0μmoL/L Ber均可抑制HT-29细胞增殖(P<0.05),其抑癌率与作用时间及浓度相关;HT-29细胞经Ber处理24 h后,凋亡率明显增高(P<0.05),0.1、0.3、3.0、30.0μmoL/L Ber组凋亡率分别为(7.6±1.8)%、(9.8±2.1)%、(22.3±4.2)%、(40.6±4.5)%,对照组凋亡率为(2.4±1.1)%;0.3、3.0、30.0μmoL/L的Ber能浓度依赖性抑制结肠癌细胞IK(V)(P<0.01),阶跃刺激为+80 mV时,对照组、0.3、3.0、30.0μmol/L Bet组IK(V)分别为(488±42)、(376±22)、(296±25)、(225±34)pA,0.3、3.0、30.0μmol/L Ber处理组IK(V)分别为对照组的(77.16±5.41)%、(61.35±6.09)%、(45.87±7.62)%.结论 Ber具有抑制结肠癌细胞HT-29增殖并诱导其凋亡的作用,此作用可能与Ber抑制肿瘤细胞细胞膜钾离子通道开放有关.