Objective:To explore the anti-tumor effect of Tanshinone Ⅱ A microemulsion and to research its reversal effect of multidrug resistance on SMMC-7721/VCR tumor.Methods The human hepatocellular carcinoma cell line SMMC-7721 and human hepatocellar carcinoma drug-resisitant cell line SMMC-7721/VCR were cultured in vitro.Each cell was divided three groups:experimental group (cell suspension + 3 kinds of anti-tumor drugs),negative control group (cell suspension + culture) and blank control group (culture).Tanshinone Ⅱ A microemulsion and Tanshinone Ⅱ A in five concentrations (0.5,1.0,2.0,4.0,8.0 μg · mL-1) intervention SMMC-7721 cells.The inhibition effect of Tanshinone Ⅱ A microemulsion of 0.5,1,2,4,8 μg · mL-1 in 24 h and 48 h on SMMC-7721 cell proliferation was tested by MTT assay.The IC50 of VCR,CDDP,5-fluorouracil(5-Fu) for the cell of SMMC-7721,SMMC-7721/VCR were determined by MTT assay.The IC50 of VCR,CDDP,5-Fu for the SMMC-7721/VCR after intervention with Tanshinone Ⅱ A microemulsion were determined also by MTT assay.The sensitivity change and drug reversal effect of Tanshinone Ⅱ A microemulsion before and after intervention on the SMMC-7721/VCR cell line was investigated by MTT assay.Results The inhibition rate after intervention with Tanshinone Ⅱ A microemulsion on SMMC-7721 cells for 48 h were 47.93％,55.92％,66.34％,95.61％.Accordingly,the inhibition rate of Tanshinone Ⅱ A were 38.83％,49.31％,58.24％,82.46％.The half inhibitory concentration were (0.67 ±0.32),(1.91 ±0.53) μg · mL-1,respectively.The difference had statistical significance (all P ＜ 0.01).With the increase of concentration,the inhibition rate increased of Tanshinone Ⅱ A microemulsion on SMMC-7721 cell with dose dependence and time dependence.When the concentration of Tanshinone ⅡA and its microemulsion for 0.5 ug · mL-1,on SMMC-7721 cells and SMMC-7721/VCR cell growth inhibition rate were 9.53％,7.74％,both less than 10％ and no obvious toxic effect,so concentration of 0.5 μg · mL-1 was choosed as liver cancer drug resistant safety experiment dose.When 0.5 ug · mL-1 Tanshinone Ⅱ A microemulsion was intervention on SMMC-7721/VCR cell,VCR to SMMC-7721/VCR half inhibitory concentration was from (12.27 ±0.84) ug · mL-1 to (3.25 ±0.14) ug · mL-1.Reverse ratio is 3.78.Half inhibitory concentration of CDDP on SMMC-7721/VCR cell is from (20.42 ± 0.18) μg · mL-1 to (6.98 ±0.99) μg · mL-1,reverse ratio is 2.93.Half inhibitory concentration of 5-FU on SMMC-7721/ VCR cell is from (35.21 ± 0.68) μg · mL-1 to (18.27 ± 2.18) ug · mL-1;reverse ratio is 1.93.Compared with the effect on SMMC-7721 ceils,VCR,CDDP,5-FU effect on SMMC-7721/VCR cells had significant difference (P ＜0.01).Compared with the effect on SMMC-7721/VCR cell which was dealed by Tanshinone Ⅱ A microemulsion,VCR,CDDP,5-FU effect on SMMC-7721/VCR cells had significant difference (P ＜ 0.01).Conclusion The Tanshinone Ⅱ A submicroemulsion can inhibit the growth of SMMC-7721 cells and SMMC-7721/VCR cells.The Tanshinone Ⅱ A submicroemulsion can partially reverse the multidrug resistance of SMMC-7721/VCR in vitro.

This study was aimed to explore the expression of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) in 3 different lymphoblastic cell lines with relation to their glucocorticoid (GC) sensitivity. The 11β-HSD2 expressions in acute lymphoblastic leukemia Jurkat cells, lymphoma Daudi and Raji cells, and peripheral blood T cells of a healthy volunteer were analyzed by real time PCR and Western blot. Glucocorticoid (GC)-induced apoptosis in 3 different cell lines was detected by flow cytometry. Cell growth in Jurkat cells treated with cortisol was analyzed by trypan blue dye exclusion. Flow cytometry was performed to observe GC-induced apoptosis in Jurkat cells treated by combination of GC with 11β-HSD2 inhibition 18β-glycyrrhetinic acid (18β-GA). The results demonstrated that 11β-HSD2 highly expressed in Jurkat cells, but not in Daudi, Raji cells and normal blood T cells. Compared to Daudi and Raji cells, Jurkat cells were more resistant to GC-induced apoptosis. Furthermore, the inhibition of 11β-HSD2 by 18β-GA resulted in increased cellular sensitivity to GC as shown by elevated induction of apoptosis. it is concluded that 11β-HSD2 is at least partly responsible for GC resistance in Jurkat cells. 11β-HSD2 may be a potential target for reduction of GC-resistance in therapeutic applications.
11-beta-Hydroxysteroid Dehydrogenase Type 2 ; metabolism ; Cell Line, Tumor ; Glucocorticoids ; pharmacology ; Glycyrrhetinic Acid ; analogs & derivatives ; pharmacology ; Humans ; Jurkat Cells ; Lymphocytes ; drug effects ; metabolism

BACKGROUND: Yaotu Granules have been proved to protect human nucleus pulposus cells and delay their degeneration. Notably, Yaotu Granules for lumbar disc herniation has achieved good clinical results.OBJECTIVE: To investigate the effect of the herbal compound formula Yaotu Granules on the Fas/FasL expression in a rabbit model of lumbar disc degeneration, and further elucidate the underling mechanism of preventing and treating lumbar disc degeneration.METHODS: Twenty New Zealand white rabbits were enrolled and the models of lumbar disc degeneration were established by minimally invasive puncture and rotation cutting, followed by randomized into normal saline, low-, middle-,and high-dose groups (n=5 per group). 10 mL of normal saline, 10, 20, and 40 mL of water decoction of Yaotu Granules were administered intragastrically into the normal saline, low-, middle-, and high-dose drug groups for 21 days, twice daily, respectively. Subsequently, the expression level of Fas/FasL in the rabbit nucleus pulposus cells in each group was detected.RESULTS AND CONCLUSION: The signal intensity of the rabbit lumbar disc on MRI was decreased, and ruptured annulus and posterior herniated disc were visible at 12 weeks after modeling. Masson staining showed that the nucleus pulposus cells arranged in disorder, and even ruptured. Additionally, safranin O staining found that the number of nucleus pulposus cells was decreased obviously. The order of the relative expression levels of Fas and FasL mRNA in the nucleus pulposus cells was as follows: normal saline group > low-dose drug group > middle-dose drug group > high-dose drug group (P < 0.05). These results suggest that Yaotu Granules delay the rabbit lumbar disc degeneration by downregulating the expression level of Fas/FasL.

Objective To study the effect of Sinisan on the ultra structure of hippo-campus to the intervention in rats with post-traumatic stress disorder(PTSD).Methods SD rats were divided equally into 5 groups,each group had ten rats:blank control group,model group,negative control group,positive control group and experimental group.The blank control group did not copy the model,the normal feeding.Model group,repetitive post-traumatic stress disorder model was induced by current stimulation,but not treated.Negative control group,equal volume of 0.9％ NaC1.Positive control group (paroxetine hydrochloride 0.42 mg · mL-1) and experimentalgroup (Sinisan,containing crude drug 0.24 g · mL-1).It given to drugs 1 h before the model establishment.The rats were administered with 10 mL · kg-1,once a day,for a total of 7 d.In each group,rats was cardiac perfusion and the hippo-campus tissue was collected.The changes of ultra structure of CA1 and CA3 area in hippo-campus with rats were observed by transmission electron microscope.Results In the blank control group,the CA1 and CA3 neurons in the hippocampus were rich in cytoplasmic organelles,and the mitochondria were round or long,the structure of the mitochondrial cristae was clear,the rough endoplasmic reticulum was like a cord like distribution,and the ribosome was abundant.Compared with the blank control group,the organelle of CA1 and CA3 area in hippo-campus of the model group were significantly damaged,the cytoplasm was open,the mitochondria were swollen,the mitochondrial membrane and cristae disappeared,and the rough endoplasmic reticulum was dilated.The results showed that the damage of the organelles in the hippocampal neurons of rats was induced by the electric shock.The model group was similar to the negative control group,which indicated that there was no significant effect on the stress of rats after intragastric administration.The intracellular organelles in the CA1 and CA3 neurons in the hippocampus of the positive control group and the experimental group were significantly recovered,and the changes in the two groups were similar.These results suggest that both paroxetine hydrochloride and Sinisan can significantly improve the structure of CA1 and CA3 neurons in the hippocampus of PTSD rats.Conclusion Snisan as traditional Chinese medicine compound can significantly improve the hippocampus of rats with PTSD CA1 and CA3 neurons.

OBJECTIVEA comparative proteomic approach was used to identify and analyze proteins relevant to metastasis of hepatocellular carcinoma (HCC).

METHODSProteins extracted from 12 liver tumor tissue specimens (6 with metastases and 6 without) were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns between the two groups were done using computerized image analysis. Selected proteins exhibiting statistically significant alternations were identified by mass spectrometry. Immunohistochemistry, Western blotting and RT-PCR were performed to examine the expressions of the candidate proteins.

RESULTS16 proteins including HSP27, S100A11, CK18 were identified using mass spectrometry, which were related to cell mobility, signal transduction, and energy metabolism respectively. Of these, HSP27 was found to be uniquely over-expressed in 2-DE maps of all metastatic HCCs when compared to the non-metastatic HCC tissues. Immunohistochemistry and Western blotting of HCC tissues confirmed this difference while RT-PCR did not.

CONCLUSIONThere are different proteins working together that affect the metastasis of HCCs. The overexpression of HSP27 may serve as a biomarker for early detection and therapeutic targets to the metastatic phenotype of HCC. The role of HSP27 in HCC metastasis warrants further investigation.

Carcinoma, Hepatocellular ; chemistry ; pathology ; Electrophoresis, Gel, Two-Dimensional ; Gene Expression Regulation, Neoplastic ; HSP27 Heat-Shock Proteins ; Heat-Shock Proteins ; analysis ; Humans ; Liver Neoplasms ; chemistry ; pathology ; Mass Spectrometry ; Neoplasm Proteins ; analysis ; Proteome ; analysis ; S100 Proteins ; analysis

OBJECTIVESTo compare expressions of tyrosine-phosphorylated proteins in different hepatocellular carcinoma cell lines with different metastasis potential and to screen key molecules associated with HCC metastasis and recurrence.

METHODSUsing two-dimensional electrophoresis, Western blotting and MALDI-TOF-MS/MS, we analyzed tyrosine-phosphorylated protein profiles of Hep3B, MHCC97L and MHCC97H, HCC cell lines with different metastasis potentials.

RESULTS10 spots were detected in Hep3B, 19 in MHCC97L and 17 in MHCC97H. Seventeen significantly different phosphotyrosine proteins in gel were identified by MALDI-TOF-MS/MS, including Annexin I.

CONCLUSIONThe changed expression of tyrosine-phosphorylated proteins is associated with HCC metastasis and recurrence.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Neoplasm Metastasis ; Neoplasm Proteins ; analysis ; Phosphotyrosine ; analysis

OBJECTIVETo study the role of cerebrovascular hemodynamic indexes (CVHI) changing in stroke and to provide reference for stroke prevention and risk factor study.

METHODSFrom 2003 to 2004, participants aged 40 years and above in two communities in Fengxian district were recruited by cluster sampling. Risk factors of stroke and CVHI were investigated and checked during baseline investigation. A total of 10 565 individuals completed the survey and met the inclusion criterion. After baseline investigation, the cohort was followed up for stroke occurrence. Relative risk (RR) of CVHI and common risk factors were estimated by cohort study design.

RESULTSAge of the cohort was (56.2 ± 11.4) years. 4444 (42.1%) were males and 6121 (57.9%) were females. Total follow-up duration was 67 885.7 person-years. A total of 195 stroke cases occurred and incidence density of stroke was 287.2 per 100 000 person-years. Stroke incidence in exposure groups of hypertension, heart disease and alcohol drinking was 3.47% (108/3118), 2.96% (21/710) and 2.50% (47/1882), respectively. The incidence in corresponding non-exposure group was 1.17% (87/7448), 1.77% (174/9855) and 1.70% (148/8683) respectively. There was significant difference between 2 groups (χ(2) value was 62.72, 4.56 and 4.94, respectively, P < 0.05). Stroke incidence in CVHI score < 25, 25 - 49, 50 - 74 and ≥ 75 groups was 9.12% (59/647), 5.68% (44/775), 2.52% (39/1545) and 0.72% (53/7403)(χ(2)trend = 273.57, P < 0.05), respectively. Incidence of stroke in 40 - 49, 50 - 59, 60 - 69, ≥ 70 years age group was 0.22% (8/3565), 1.28% (43/3357), 2.71% (50/1848) and 5.88% (94/1600) (χ(2)trend = 181.48, P < 0.05), respectively. Multiple Cox regression analysis indicated that RR (95%CI) value of hypertension and cigarette smoking was 1.40(1.02 - 1.92) and 1.59(1.19 - 2.12), respectively when comparing with non-exposure group. RR (95%CI) value in CVHI score < 25, 25 - 49 and 50 - 74 points group were 6.15 (4.08 - 9.26), 4.55 (2.98 - 6.96) and 2.68 (1.75 - 4.09), respectively when comparing with the score ≥ 75 points group. RR (95%CI) value in age 50 - 59, 60 - 69 and ≥ 70 years group was 4.61 (2.16 - 9.82), 7.81 (3.67 - 16.60) and 13.49(6.44 - 28.24), respectively when comparing with below 40 years group.

CONCLUSIONCVHI score is the strong independent predictive factor and hypertension, cigarette smoking and age are the independent risk factors of stroke.

Aged ; Brain ; physiopathology ; Cohort Studies ; Female ; Hemodynamics ; Humans ; Male ; Middle Aged ; Risk Factors ; Stroke ; epidemiology ; etiology ; physiopathology

OBJECTIVETo screen hepatocellular carcinoma (HCC) autoantibodies as diagnostic biomarkers or therapy targets by serologic proteome analysis (SERPA).

METHODSTotal proteins extracted from human HCC cell line HCCLM3 were separated by two-dimensional electrophoresis (2-DE) and then transferred onto PVDF membranes, which were subsequently incubated with sera from HCC, hepatitis B virus (HBV) infected patients or healthy volunteers. All immuno-reactive protein spots on blot films were matched to those on 2-DE gel maps by image analysis and identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS/MS).

RESULTS2-DE gel maps of HCCLM3 and corresponding blot films of good quality and reproducibility were established. The number of spots on HCCLM3 2-DE reference gel totaled 603 and those on HCC, HBV and healthy sera blotted films were 70.75+/-24.25, 68.5+/-23.44 and 41.38+/-15.05, respectively. Blot films of HCC and HBV groups had more spots than those of the healthy group (P < 0.05) while no significance was found between films of HCC and HBV groups. By identification, those HCC autoantibodies could be classified as nuclear proteins, cytoskeleton proteins, heat shock proteins and metabolic enzymes.

CONCLUSIONSerological proteome analysis is a high throughput technique for screening tumor autoantibodies. Those newly identified HCC associated tumor antigens and corresponding autoantibodies can be used in the early diagnosis or immuno-therapy of HCC.

Antibodies, Neoplasm ; analysis ; Autoantibodies ; analysis ; Carcinoma, Hepatocellular ; immunology ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Liver Neoplasms ; immunology ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Tumor Cells, Cultured

BACKGROUND:The vascular endothelial growth factor (VEGF) plays an important role in the development and formation of blood vessels.Up to now,there are few reports about the treatment of postoperative complications of vascular anastomosis surgery by mcrosutures with VEGF in China.OBJECTIVE:To synthesiize microsutures with VEGF and to evaluate its effect in revascularization following small vessel anastomosis.METHODS:The method of emulsification-diffusion was use to produce biodegradable polymer polylactic acid/glycolic acid (PLGA) copolymer microparticles containing VEGF,and then,the microparticles were added into microsutures to prepare microsutures with VEGF.Ninety Sprague-Dawley rats were enrolled to make animal models of caudal artery anastomosis using microsutures with VEGF in experimental group and microsutures alone in control group.Complications and VEGF level in the peripheral blood were detected and hematoxylin-eosin staining at the anastomotic site was performed at 2,12 hours,1,3,7 days after anastomosis.RESULTS AND CONCLUSION:(1) Postoperative complications:The postoperative incidence of skin necrosis was significantly lower in the experimental group than the control group (P ＜ 0.05).(2) VEGF level:Compared with the control group,the peripheral blood VEGF level was significantly higher in the experimental group at each time point after operation (P ＜ 0.05).(3) Hematoxylin-eosin staining:In the experimental group,proliferated endothelial cells were seen near the anastomotic site at 1 day after anastomosis;there were a large number of proliferated endothelial calls and subcutaneous tissues covering the sutures completely at 3 days after anastomosis;and endothelial cells and internal elastic lamina were completely repaired,smooth muscle cells proliferated further,and the outer membrane returned to normal at 1 week after anastomosis.In the control group,cell degeneration and necrosis were seen near the anastomotic suture,and only adventitial cells infiltrated and exhibited a traumatic proliferative response at 1 day after anastomosis;neonatal endothelial cells appeared in the exfoliated area of the endothelial cells,grew and migrated,and there was a few endothelial cells covering the anastomotic site at 3 days after anastomosis;and newborn endothelial cells got over the anastomotic crack and covered the suture.To conclude,microsutures with sustained-release VEGF microparticles can promote endothelial cell regeneration in rats at the anastomotic site.

BACKGROUND: Methylene blue is used as a developer to identify intervertebral disc degeneration in the transforaminal endoscopic surgery. However, many scholars have indicated that methylene blue can accelerate the degeneration process, whilst foreign researches have reported that it may play therapeutic effect on degenerative intervertebral discs under acidic conditions due to its acidophily. Therefore, whether methylene blue holds toxic effect on the disc remains controversial. OBJECTIVE: To determine whether methylene blue exerts toxic effect on nucleus pulposus cells by cell counting-kit 8 (CCK-8) assay. METHODS: The discarded nucleus pulposus from two patients with intervertebral disc herniation were selected. After digestion, nucleus pulposus cells were extracted and cultured until proliferated to 80% of the medium. Then, the cells were digested to make cell suspensions, divided into six groups and inoculated into the 96-well plates: blank control (only the medium, CCK-8 solution), control group (only medium, cells and CCK-8 solution), and the other groups were cultured with 1%, 2%, 3%, and 4% methylene blue, respectively. The absorbance values were measured by CCK-8 assay at 24, 48, 72 and 96 hours after incubation to calculate the cell viability, and the color change was observed. RESULTS AND CONCLUSION: The color in the control group was the deepest, and the color became lighter with the concentration of methylene blue increasing. The cell viability was the highest in the control group, and it was decreased with the concentration of methylene blue increasing. Thus, methylene blue may exert toxic effect on human nucleus pulposus cells.