BACKGROUND: Previous studies have demonstrated that berberine represses multiple tumors and tumor stem cells, but the effect of berberine on lung cancer stem cells (LCSCs) remains unclear. OBJECTIVE: To explore the effect of berberine on the proliferation and apoptosis of LCSCs and the possible mechanism. METHODS: CD133+ LCSCs were separated from A549 cells by immunomagnetic beads. The effects of different concentrations (0, 2.5, 5, 10, 20, 40 mg/L) of berberine on the proliferation and apoptosis of LCSCs were determined by MTT and flow cytometry analysis, respectively. In order to further affirm the effect of berberine on the proliferation and apoptosis of LCSCs, the expression levels of Ki67, Bax and Bcl-2 protein were detected by western blot. In addition, to investigate the potential mechanism by which berberine exerts regulatory effects on LCSCs, the expression levels of Hedgehog signaling pathway-associated proteins (PTCH1, SHH, Gli-1 and SMO) were determined. RESULTS AND CONCLUSION: After magnetic cell sorting, the content of the CD133+ fraction was enriched up to 84.13%. MTT and flow cytometry assays showed that berberine inhibited proliferation and promoted apoptosis of LCSCs in a concentration-dependent manner. Western blot analysis showed that the expression levels of Ki67, Bcl-2, PTCH1, SHH, Gli-1 and SMO proteins of LCSCs cultured in the medium with 20 mg/L berberine were dramatically decreased compared to the control, while the expression level of Bax protein was markedly increased compared to the control. These findings suggest that berberine may inhibit proliferation and promote apoptosis for LCSCs through the Hedgehog signaling pathway.BACKGROUND: Previous studies have demonstrated that berberine represses multiple tumors and tumor stem cells, but the effect of berberine on lung cancer stem cells (LCSCs) remains unclear. OBJECTIVE: To explore the effect of berberine on the proliferation and apoptosis of LCSCs and the possible mechanism. METHODS: CD133+ LCSCs were separated from A549 cells by immunomagnetic beads. The effects of different concentrations (0, 2.5, 5, 10, 20, 40 mg/L) of berberine on the proliferation and apoptosis of LCSCs were determined by MTT and flow cytometry analysis, respectively. In order to further affirm the effect of berberine on the proliferation and apoptosis of LCSCs, the expression levels of Ki67, Bax and Bcl-2 protein were detected by western blot. In addition, to investigate the potential mechanism by which berberine exerts regulatory effects on LCSCs, the expression levels of Hedgehog signaling pathway-associated proteins (PTCH1, SHH, Gli-1 and SMO) were determined. RESULTS AND CONCLUSION: After magnetic cell sorting, the content of the CD133+ fraction was enriched up to 84.13%. MTT and flow cytometry assays showed that berberine inhibited proliferation and promoted apoptosis of LCSCs in a concentration-dependent manner. Western blot analysis showed that the expression levels of Ki67, Bcl-2, PTCH1, SHH, Gli-1 and SMO proteins of LCSCs cultured in the medium with 20 mg/L berberine were dramatically decreased compared to the control, while the expression level of Bax protein was markedly increased compared to the control. These findings suggest that berberine may inhibit proliferation and promote apoptosis for LCSCs through the Hedgehog signaling pathway.

Objective: To express glycosyl-phosphatidylinositol (GPI) modified Met- RANTES fusion protein on Chinese hamster ovary (CHO) cells and to develop a novel immunosuppressant GPI anchored form of Met-RANTES. Methods: The eukaryotic expression vector PEF/GPI-Met-RANTES were constructed and transfected into CHO cells by electroporation. The transfectants were selected with methotrexate (MTX). Expression of the recombinant protein was assessed by flow cytometric analysis, cell immunofluorescence staining and immunogold electron microscopy. Results: The chimeric molecules of GPI anchored form of Met-RANTES including the whole reading frame were constructed, and the sequence was identical to the designed sequence. GPI anchored form of Met-RANTES was stably expressed on CHO- DHFR- cells. Conclusion: A large amount of GPI modified Met-RANTES fusion protein was expressed on CHO cells. GPI anchored form of Met-RANTES may be used as novel immunosuppressant for suppressing reaction in graft rejection.

Objective To study the expression of telomerase and its possible clinical significance in patients with condyloma acuminatum,bowenoid papulosis,Bowen's disease and vulvar squamous cell car- cinoma.Methods The human telomerase reverse transcriptase (hTERT) was detected by immunohisto- chemistry technique in all patients.Histopathological analysis was done in patients with bowenoid papulosis or Bowen's disease.Results There was a significant difference in the expression of hTERT among normal tissue,condyloma acuminatum and squamous carcinoma,and the expression rate increased successively from normal control to squamous carcinoma.In bowenoid papulosis,the expression of bTERT was significantly higher as compared to that in condyloma acuminatum,significantly lower as compared to that in squamous carcinoma,and was as the same as that in Bowen's disease.Histopathologically,the degree of cell atypia was significantly higher in Bowen's disease than that in bowenoid papulosis.Conclusions The expression of telomerase increases successively from normal skin,benign tumors to malignant carcinomas,which sug- gests that the expression of telomerase may play an important role in both cell proliferation and immortaliza- tion.

Background The rat model of N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell apoptosis is often used to study retinal degeneration.But the changes in the gene expression patterm in retinal degeneration in rats have not been reported.Objective This study was undertaken to investigate regulation of gene expression in the retina of MNU-induced retinal degeneration in rats by performing microarray analysis of retinal RNA.Methods Fifty 6-week-old SD rats were numbered and randomized into the normal group and the model group.The retinal degeneration model was established by a single hypodermic injection of 40 mg/kg of MNU,and the rats in the normal group received equivalent volume of physiological saline in the same way.The rats were sacrificed 12 hours or 24 hours after injection.Retinal sections from the right eyes were prepared for the measurement of the retinal thickness by histopathological examination,and retinas from the left eyes were used to confirm the differential gene expression as detected by microarray (normal group and 12 hours model group).Genes exhibiting changes in expression by ≥2.0 folds were further confirmed using real-time PCR.Results The whole thickness of the retina declined in the rats from the 24 hours model group compared to the normal group and 12 hours model group (t =9.926,P=0.002;t=2.736,P=0.028).The thickness of the outer nuclear layer was (26.58±2.90) μm in the 24 hours model group,showing a significant decrease in comparison with (38.11 ± 1.01) μm in the normal group and (35.07t3.03) μm in the 12 hours model group (t=6.028,P=0.009;t=6.839,P=0.006).However,there was no significant difference in retinal thickness between the normal group and the 12 hours model group (whole thickness:t=1.541,P=0.324;outer nuclear layer thickness:t=2.040,P=0.134).Microarray analysis of the rat genes showed that out of 17 000 genes,142 genes involved in biological process and 94 genes involved in molecular functions were differentially expressed,where most of them participate in the mitogen activated protein kinase signaling pathway,Tolllike receptor signaling pathway and apoptosis pathway.Real-time PCR analysis demonstrated that the expression of CCL2,IL-1b,CCL3,c-fos,c-myc,p53 and MMP3 were consistently up-regulated,conforming with the results from microarray analysis.Conclusions The changes in gene expression pattern appear in the early stage of MNUinduced retinal degeneration.These microarray results provided clues to understanding the molecular pathways underlying photoreceptor degeneration and indicating the directions for future studies.

ObjectiveTo explore the influences of age and duration of status convulsivus （SC） on mitochondrial membrane potential (△Ψm） in hippocampus. MethodsConvulsive seizures for 30 min or 3 h (30 min SC or 3 h SC) were induced in 80 infant (20 d after birth) and 80 adult Wistar rats (IRs ＆ ARs respectively) with lithium-pilocarpine ip. The rats were sacrificed at 6 different time points from the 3rd hour to 7th day after SC termination. The mitochondrial △Ψm in hippocampal cells was determined with flow cytometry. ResultsThe mitochondrial △Ψm in hippocampal cells started to decrease at the 3th hour after SC in both IRs and ARs. The bottom level was reached at the 6th hour after SC ［(6.08±0.43) in IRs and (5.70±0.63) in ARs ) ］. Both of them were significantly lower than that of control group (P<0.01） and began to increase at 12th hour after SC. On the 7th day after 30 minutes SC, the level of mitochondrial △Ψm in IRs increased to the level of control, while the level in ARs was still lower than that of control (P<0.05）. At the 3rd hour, the 3rd and the 7th day after SC, the levels of mitochondrial △Ψm in IRs were obviously higher than those in ARs. Compared with the same time point after 30 min SC, the levels of mitochondrial △Ψm at the 3rd and the 6th hour after 3 h SC were much lower in different age groups (P<0.05）. Except the effect of the age-related difference, there was a positive correlation between the duration of SC and the changes of mitochondrial △Ψm in partial correlation analysis （r=0.71，P<0.05）. ConclusionSevere seizure could induce the mitochondrial △Ψm decreased in hippocampus. Age and duration of SC were important factors associated with the mitochondrial △Ψm decrease. There may be an internal protective response against brain damage in premature brain.

Acoustic Stimulation ; Animals ; Corpus Striatum ; physiology ; Estrogens ; metabolism ; Estrus ; physiology ; Evoked Potentials, Auditory ; physiology ; Female ; Rats ; Rats, Sprague-Dawley

Anemia, Refractory ; Humans ; Male ; Middle Aged ; Thrombocytosis

10.3969/j.issn.1000-8179.2013.12.012

Objective To investigate the effects of allogenic intra-bone marrow bone marrow transplantation (IBM-BMT) on re-establishing hematopoiesis in mice. Methods Bone marrow mononuclear cells (BMNCs) from BALB/ c mice were transplanted into the C57BL/6 mice treated with a lethal dose of ~(60)Coγ-ray radiation through intra-bone marrow injection or intravenous injection. Sixty of the C57BL/6 mice were randomly divided into three groups as higher dose intra-bone marrow injection group (IBM1 group), lower dose intra-bone marrow injection group (IBM2 group) and intravenous injection group (IV group). The nucleated cell numbers of whole bone marrow from the tibia of each recipient mouse were counted respectively at the day 1, day 3, day 6 and day 9 after the transplantation. The donor-derived total nucleated cells and myeloid cells were quantified by flow cytometry. Results At 6th day after transplantation, more total bone marrow nucleated cells, total donor-derived nucleated cells and donor-derived myeloid cells in the tibia of injected side in both IBM1 group and IBM2 group were found than that in IV group (P<0.05 or P<0.01). Conclusion Compared with traditional bone marrow transplantation (IV-BMT),IBM-BMT improves the bone marrow hematopoiesis in the early hematopoietic re-establishing stage in allogenic bone marrow transplantation.

Objective To investigate the relationship between carotid intima-media thickness (IMT) and coronary atherosclerosis plaque through coronary CT angiography (CTA) in asymptomatic individuals.Methods Two hundred and fifty cases with CTA screened were selected in asymptomatic individuals,and according to the inspection result they were divided into no plaque group (detection of no coronary atherosclerosis plaque,180 cases) and plaque group (detection of coronary atherosclerosis plaque,70 cases).All cases were checked carotid IMT with B-ultrasonic.Results ANOVAs analysis showed that age and body mass index (BMI) had significant correlation with coronary atherosclerosis plaque (P ＜ 0.05).x2 test was used to show the smoking and family history of coronary heart disease had significant correlation with coronary atherosclerosis plaque (P ＜ 0.05).Univariate Logistic regression analysis showed that carotid IMT,systolic pressure,diastolic pressure,low density lipoprotein cholesterol,fasting serum glucose and creatinine had significant correlation with coronary atherosclerosis plaque (P ＜ 0.05).Multifactor Logistic regression analysis showed that carotid IMT had correlation with coronary atherosclerosis plaque (P ＜ 0.05).Conclusion There is significant relationship between carotid IMT and coronary atherosclerosis plaque in asymptomatic individuals.