[Preliminary study of the dual release baicalin and rhBMP-2 system to improve periodontal tissue regeneration in minipigs].

PURPOSE: To explore the feasibility of dual release baicalin and rhBMP-2 system for the treatment of periodontal diseases in minipigs. METHODS: Four miniature swines were selected to establish the model of periodontitis and randomly divided them into four groups: Dual release group, hydrogel with baicalin-GMS and rhBMP-2; Single Huang group, hydrogel with baicalin-GMS; BMP group, hydrogel with rhBMP-2; Negative control group, blank hydrogel. All parameters including clinical indicators of periodontitis, the level of IL-1 and TNF-α in the gingival crevicular fluid (GCF) were measured before and 6 weeks after modeling, and 4, 8 weeks after treatment. The data was analyzed using SPSS13.0 software package. The periodontitis model animals were sacrificed at the end of 8 weeks after treatment. The histological changes of periodontal tissues were observed under microscope with HE staining. RESULTS: (1)In all groups, there were significant differences of clinical indicators and levels of IL-1 and TNF-α in the GCF between periodontitis was established and 4, 8 weeks after treatment (P<0.05). Dual release group was significantly superior than that of other three groups (P<0.05). Moreover, all the parameters improved continuously. X-ray showed that bone mineral density and height in the remaining three groups increased compared with the control group, with highest in dual release group. (2)The results of histological observation showed that the inflammation reaction of periodontal tissues in negative control group was more serious than that in other groups 8 weeks after treatment. There was no obvious repair reaction in the control group, but different amount of new bone was seen in other three groups. Among them, dual release group had most obvious repair reaction. CONCLUSIONS: The dual-slow-release chitosan thermosensitive hydrogel system is employable to be used effectively in the regeneration of periodontal defects, which laid a foundation for further clinical use.

[Recombinant hFOXA2 and hPDX1 lentivirus induced dental pulp stem cells from deciduous teeth reprogramming for insulin-producing cells].

PURPOSE: The purpose of this study was to culture and identify dental pulp stem cells(DPSCs) from deciduous teeth in vitro and construct the recombinant hFOXA2 and hPDX1 lentivirus vectors and transfect the DPSCs to induce insulin-producing cells (IPCs). METHODS: DPSCs were separated and cultured by enzyme digest method, and purified by limited dilution method. Flow cytometry was used to determine the surface marker expression of the DPSCs, and the ability of multiple differentiations was determined by specific staining. hFOXA2 and hPDX1 genes were amplified by PCR, and the recombinant hFOXA2 and hPDX1 lentivirus vectors were reconstructed and transfected into 293T cells by lipofectamine2000 for virus packaging. The viral infection efficiency and titer were determined through fluorescence cell count. The recombinant virus was used to infect the DPSCs cells via multiplicity of infection (MOI) and induce the DPSCs reprogramming for IPCs. Immunofluorescence staining was used to measure the expression of proinsulin, FOXA2 and PDX1. ELISA method was used to detect the insulin secretion. The data was analyzed Using SPSS13.0 software package. RESULTS: DPSCs were isolated and cultured successfully. Cell surface highly expressed STRO-1 (98.01%), CDl46 (98.51%), CD34 (99.54%) and CD45 (24.08%). The multi-lineage differentiation capacity into osteoblasts, chondrocytes, and adipose was achieved. The recombinant hFOXA2 and hPDX1 lentivirus vectors were successfully constructed. Double enzyme digestion and sequencing appraisal showed that the sequence was fully consistent with GenBank retrieval. Virus packing efficiency was (96.15±0.17) % and (95.49±0.21) % respectively, and the infection titer was about 1.80±108 GTU/mL. The best MOI of the virus was 20. After inducing the cells to express proinsulin, FOXA2 and PDX1, insulin secretion volume was about 1.92 µmol/L. Compared with the uninduced group and control group, insulin secretion increased significantly (P<0.01). CONCLUSIONS: The recombinant transcription factor virus can activate cell reprogramming mechanism, form insulin-producing cells, and can be used for gene therapy of diabetes seed cells. Supported by Science and Technology Research Program of Shaanxi Province (2009K17-06) and Science and Technology Innovation as a Whole Plan Resources Leading Industry Key Technology (Chain) Project of Shaanxi Province (2011KTCL03-24).

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics.

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

[Construction and identification of BIRC5 shRNA lentiviral expression vector].

AIM: Design and synthesis complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence by RNA interference technique and construct and identify a lentiviral interference vector with human BIRC5 gene as target gene. METHODS: The designed and synthesised Single-Stranded primer were annealed to Double-Stranded oligo sequences and subcloned into linear pMAGic lentiviral plasmid vector digested by enzyme Age I and EcoR I. Screening positive clone after transformed into DH5α competent cells and identified by PCR amplification and DNA sequencing. RESULTS: 335 bp straps of positive clone and 298 bp straps of negative clone form PCR amplification production have been obtained after gel electrophoresis, the designed and synthesised sequences have been contained in these clone straps confirmed by the result of DNA sequencing. CONCLUSION: Four pairs of BIRC5 shRNA recombinant lentiviral expression vector were constructed successfully, which laid the foundation for researching the inhibition of BIRC5 siRNA target against tumor cells proliferation, induction apoptosis and gene therapy.

[Cloning of the glyceraldehydes 3-phosphate dehydrogenase gene of porphyromonas gingivalis and its expression in E. coli].

OBJECTIVE: To clone the glyceraldehydes 3-phosphate dehydrogenase (GAPDH) gene of Porphyromonas gingivalis (P. gingivalis) and to induce its fusion expression in E. coli. METHODS: GAPDH was obtained by PCR and was inserted into cloning vector pMD-18-T to construct clone recon. Double enzymes digest the recon pMD18-T-GAPDH and the prokaryotic expression vector pET-32a and then connect to get the expressing recon pET-32a-GAPDH. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent cells BL21 and induced the expression of GAPDH with isopropyl beta-D-1-thiogalactopyranoside (IPTG) of different density. RESULTS: DNA sequencing showed that the fragment was 99.802% the same to the sequence published in NCBI. Under the best density, IPTG could be highly expressed. CONCLUSION: The GAPDH had been successfully cloned and expressed in E. coli which gets ready for the following experiment to study the immunity of GAPDH and the homologues antibody preparation.

[Experimental study on the functional regulation of naringin in human periodontal ligament cells].

PURPOSE: To detect the effect of naringin on human periodontal ligament cells' (hPDLCs) proliferation,bone formation and OPG mRNA expression. METHODS: hPDLCs were primarily cultured and identified in vitro through enzyme digestion combined tissue culture method. With 3-(4, 5-Dimethylthiazol-2- yl)-2, 5-diphenyltetrazolium bromide (MTT) method, enzyme linked immunosorbent assay and RT-PCR, the hPDLCs' proliferation, alkaline phosphatase (ALP) activity, expression of collagen protein-I and OPG mRNA were observed after treatment with different concentrations (100,10,1.0,0.1,0.01mg/L) of naringin at different times. SPSS16.0 software package was used for statistical analysis. RESULTS: Primary cultured hPDLCs had good shape; Significant promotion of proliferation,ALP activity and collagen protein-I expression of hPDLCs with naringin was found at the dose of 1.0 mg/L; and 1.0mg/L naringin regulated the expression of OPG mRNA in time-dependent manner. CONCLUSION: Naringin might significantly promote the proliferation of hPDLCs and conversion into the osteoblast.

[Analysis of outer membrane proteins in various strains of Porphyromonas gingivalis by surface enhanced laser desorption/ionization time-of-flight mass spectrometry].

OBJECTIVE: To analyze the common outer membrane proteins (OMP) from Porphyromonas gingivalis (Pg) by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and to provide antigens for the subsequent experiments in screening vaccine for periodontitis therapy. METHODS: Four strains of Pg were cultured under anaerobic conditions. The common OMP was extracted through ultracentrifugation and SELDI-TOF-MS was employed to detect the expressions of proteomes by chip H(50). The data was analyzed by Biomarker Wizard. RESULTS: Four kinds of strains of OMP fingerprint spectrum were obtained. Seventy-one proteins of PgATCC33277, 74 proteins of PgW83, 76 proteins of PgW301 and 72 proteins of Pg381 were captured by chip H(50). Thirteen common proteins were identified according to fingerprint spectrum. There was only 1 of the 13 common proteins identified in NCBI protein bank. CONCLUSIONS: SELDI-TOF-MS has good reproducibility and high sensibility and can be used to identify the common OMP of Pg. The 13 proteins have a potential value in the screening vaccine candidate antigen sites for periodontitis.

[Construction of prokaryotic expression vector of FimA gene from Porphyromonas gingivalis, fusion expression and purification in E. coli BL21(DE3)pLyS].

OBJECTIVE: To clone the FimA gene of fimbriae from Porphyromonas gingivalis (P. gingivalis) and to construct prokaryotic expression vector which was induced in E.coli BL21(DE3)pLyS in the form of fusion protein expression and to identify, purify the product of its expression. METHODS: To clone the FimA gene of fimbriae from P. gingivalis and to construct prokaryotic expression vector pET15b-FimA vector which was transformed into the competent cells of BL21(DE3)pLyS. The expression of fusion protein was induced by isopropyl beta-D-1-thiogalactopyranoside (IPTG). With anti-6xHis Tag monoclonal antibody as the first antibody, the expressed fusion protein was characterized by Western blot and purified by Co(2+)-NTA affinity chromatography. RESULTS: Cloned FimA gene sequences and inserted into expression vector of the FimA sequences were related to the sequence in GenBank database showed 100% homology. IPTG induced and then identified by Western blot showed a fragment of 4.1 x 10(4) has been expressed. Co(2+)-NTA affinity chromatography column was used to obtain high concentrations of FimA purified protein. CONCLUSION: The recombinant prokaryotic expression vector of pET15b-FimA was constructed and was expressed and purified successfully in E. coli BL21 (DE3)pLyS. This study laid the experimental foundation to further prepare for monoclonal antibodies of fimbriae of P. gingivalis and to develop the subunit protein vaccine of prevention of periodontitis.

[Isolation of mesenchymal stem cells from human umbilical cord blood and its bone inducing activity in vitro].

PURPOSE: To investigate the biological characters of mesenchymal stem cells obtained from human umbilical cord blood(HUCB-MSCs), and detect the influence of different serum concentrations of culture medium to primary HUCB-MSCs' growth kinetics and the osteogenic differentiation capacity of the passage cells. METHODS: The mononuclear cells were isolated from umbilical cord blood(UCB), the growth characters and morphology of the primary MSCs were observed. Primary cells were cultured in alpha-MEM with different serum concentrations, and immunophenotype was investigated. P3 cells were induced to osteoblast-like cells and the differentiation results were analysed. SPSS 16.0 software package was used for statistical analysis through repeated measures analysis of variances. RESULTS: Four subpopulations with different morphology could be distinguished in primary HUCB-MSCs. All the subpopulations were positive for CD44 and CD105, and negative for CD34 and CD45. HUCB-MSCs displayed faster and stable growth when cultured in alpha-MEM containing 15% serum. Passage cells of HUCB-MSCs could be induced into osteoblast-like cells, the formation of mineralized extracellular matrix and the positive staining of expression of ALP could be observed. CONCLUSIONS: HUCB-MSCs are high in viability but low in proliferation. HUCB-MSCs contain 4 subpopulations with different morphology. The appropriate concentration of serum in culture medium should be 15%. HUCB-MSCs could be induced into osteoblast-like cells in vitro.

[A preliminary study on screening for Porphyromonas gingivalis outer membrane protein antigen with two-dimensional liquid phase fractionation and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry].

OBJECTIVE: To screen a variety of Porphyromonas gingivalis (Pg) common outer membrane proteins with two-dimensional liquid phase fractionation (PF2D) and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) and provide candidate target antigen for the design of vaccines with cross protection against a variety of Pg. METHODS: The outer membrane proteins of Pg301, PgATCC33277 and PgW83 were extracted through ultracentrifugation, and then they were separated by ProteomeLab PF2D protein fractionation system. After separation, the outer membrane proteins were obtained through comparison, and the primary structure of the proteins was identified by MALDI-TOF/TOF-MS and database. RESULTS: Ninety-nine protein samples out of 3 strains of Pg were obtained after the high performance chromato focusing (HPCF) separation process. B7 fractions of 3 strains of Pg were separated by the reversed-phase high performance liquid chromatography (RP-HPCL) separation process. After comparison of peak and retention time of chromatogram, the 8 common protein peaks of 3 strains of Pg were confirmed. The protein samples were identified by MALDI-TOF/TOF-MS, and one of them was known protein arg-gingipain A. CONCLUSIONS: PF2D protein fractionation system is of good reproducibility and high resolution. A combination of PF2D and MALDI-TOF/TOF-MS can be used to identify the common outer membrane proteins of Pg.

[Expression of CD1a and CD207 in condyloma acuminatum epidermis].

AIM: To investigate the expression of CD1a and CD207 in condyloma acuminatum (CA) epidermis and to understand its significance. METHODS: The mRNA expression of CD1a and CD207 in six CA epidermal lesions and in six normal controls were detected using oligonucleotide microarrys and confirmed by semi-quantitative RT-PCR, and the protein level of CD1a and CD207 in six CA epidermal lesions and in six normal controls were measured by Western blot. RESULTS: With microarrys, the mRNA expression of CD1a and CD207 were detected markedly down-regulated in six CA epidermal lesions as compared with that in six normal controls. Moreover, the down-regulation of CD1a and CD207 was verified by semi-quantitative RT-PCR. Western blot analysis showed that the protein expression of CD1a and CD207 in six CA epidermal lesions were significantly lower than that in normal controls. CONCLUSION: The expression of CD1a and CD207 is markedly down-regulated in CA epidermis compared with that in normal epidermis, and the results may suggest that the number of LC in CA epidermis is decreased and the function is impaired.

[Mass chromatographic analysis on different protein expression of human periodontal ligament cell under static pressure].

PURPOSE: To analyze the protein expression of human periodontal ligament cells (HPDLCs) under static pressure. METHODS: The proteins of two groups of human periodontal ligament cell (one group was interfered by static pressure and one group was not) were analyzed by two-dimensional electrophoresis and mass chromatographic analysis. The silver stained proteins spots were analyzed by Image Master 2D Platinum Software 5.0. There were about 720 and 730 detectable spots on the two 2D-gels separately and nearly 30 spots which were differentially expressed. RESULTS: With direct MALDI-TOF mass spectrometry and protein database searching, 2 protein spots out of 5 new appeared spots were identified. Presenilin 2 and catechol O-methyltransferase were expressed only in the group which was interfered by static pressure. CONCLUSIONS: The results of mass chromatographic analysis demonstrate that Notch pathway and calcitonin gene-related peptide(CGRP) may be the partners of the channel of biomechanical signal conduction of the periodontal ligament cell.

[The effect of continuously compressive press on the shape of cells and expressions of MMP-9,TRAP in human osteoclasts].

PURPOSE: To investigate the effect of continuously compressive pressure on the function of human osteoclasts. METHODS: Monocytes were collected from umbilical cord cells, and were cultured for 14 days. alpha-MEM which contains RANKL and M-CSF was used as the culture medium. 100kPa compressive pressure was applied to the mature osteoclasts for 1 hour,2 hours and 3 hours. The shape change was observed. TRAP,MMP-9 enzymes were stained, and measured randomly. The data were analyzed with SPSS12.0 software package for Student's t test. RESULTS: Compressive pressure made the shape of osteoclasts incline to round. After adding pressure for 1 hour, there was significant difference on the expression of TRAP between pressure group(59.61+/-14.95)and non-pressure group(80.01+/-9.69) (P<0.05). Significant increase expression of MMP-9 occured after 5 hours (P<0.05). CONCLUSION: The changes of shape and levels of functional enzymes show that compressive pressure enhances the bone-absorbing activity. Supported by National Natural Science Foundation of China (Grant No.30471911).

[Culture and purification of olfactory ensheathing cells from human fetal using different attachment rates combined with the technique of using NT3 intermittently].

AIM: To explore a simple and pragmatic method to obtain sufficient olfactory ensheathing cells from human fetal by using different attachment rates in harvested cells with the combinating of the technique of using NT3 intermittently. METHODS: DMEM/F12 culture solution including 100 mL/L of fetal bovine serum or including NT3 was used to culture olfactory ensheathing cells intermittently every 48 h. The conditions and growth degree of OECs were observed, and P75 immunocytochemistry was used to estimate the purity of the cells. RESULTS: Human fetal OECs were positive after P75 immunocytochemistry. They appeared to be dipolar or tripolar cells and their processes formed a network in vitro. The purity of OECs in good conditions reached about 95% on 9 d and 83% on 12 d. CONCLUSION: The method of using different attachment rates combined with the technique of using NT3 intermittently can culture and purify OECs simply and effectively.

[Expression of Langerhans cell-related chemokine genes in condyloma acuminatum epidermis].

AIM: To investigate differential gene expression of Langerhans cell-related chemokines in condyloma acuminatum (CA) epidermis and normal epidermis. METHODS: Gene expression of Langerhans cell-related chemokines in three CA epidermal lesions and in three normal controls was screened using Affymetrix oligonucleotide microarrays HG-U 133A 2.0, and part of the above differential gene expression was confirmed by semi-quantitative RT-PCR. RESULTS: With microarrays, seven down-regulated genes of Langerhans cell-related chemokine were detected in three CA epidermal lesions as compared with three normal controls, and the down-regulation of CXCR4 and CCL20 was verified by semi-quantitative RT-PCR. CONCLUSION: Several Langerhans cell-related chemokine genes are found down-regulated in CA epidermis as compared with normal epidermis, and the down-regulation of these genes may contribute to the decreased number and the homing disturbance of LC in CA epidermis.

[Effects of bcl-2 antisense oligodeoxynucleotide on proliferation and apoptosis of human melanoma A375 cell line].

AIM: To investigate effects of bcl-2 fully phosporothioated antisense oligodeoxynucleotide(bcl-2 ASODN) on proliferation and apoptosis of human melanoma A375 cell, and its possible mechanism. METHODS: Proliferation and apoptosis of A375 cell with bcl-2 ASODN treatment were evaluated by MTT colorimetric assay, laser scanning confocal microscope (LSCM), TUNEL and Annexin V/propidium iodide(PI), and the level of bcl-2 mRNA expression in A375 cell was detected by RT-PCR before and after being treated by bcl-2 ASODN. RESULTS: MTT assay demonstrated that bcl-2 ASODN could inhibit the proliferation of the cells in both time and concentration dependent manner. Characteristic morphologic apoptosis changes were observed by LSCM after incubated with bcl-2 ASODN for 48 h. Most nucleus were labeled in brown by TUNEL in ASODN group, but not markedly labeled both in SODN and control group. The apoptosis rate of A375 cells in 30 micromol/L bcl-2 ASODN group was significantly higher than that in bcl-2 SODN and in control group. The bcl-2 ASODN-induced apoptosis of A375 cells, which was accompanied by declined expression of bcl-2 mRNA was distinctly lower than that in SODN and control groups. CONCLUSION: These results suggest that bcl-1 ASODN can not only inhibit proliferation but also induce apoptosis of human melanoma A375 cells in vitro, and the apoptosis-induced mechanism is down-regulating expression of bcl-2 mRNA.

[Sexual hormone levels in semen and germ cell apoptosis].

OBJECTIVE: To explore the relationship between sexual hormones in semen and germ cell apoptosis in male population. METHODS: Sixty-six infertile patients and thirty fertile males were selected randomly. The levels of folicle stimulating hormone ( FSH), prolactin (PRL), luteinizing hormone (LH), and testosterone (T) in semen were measured by ELISA. Terminal deoxynucleotidyl transferase mediated UTP nick end labeling (TUNEL) was used for the detection of germ cell apoptosis. RESULTS: The levels of FSH, LH, PRL, T in thirty fertile men were (1.63 +/- 0.15) U/L, (2.18 +/- 0.21) U/L, (6.34 +/- 0.30) nmol/L, (1.85 +/- 0.11) nmol/L, respectively, and germ cell apoptosis rate was (4.61 +/- 1.23)%. FSH, LH, PRL, T levels in infertile group were (1.25 +/- 0.18) U/L, (1.76 +/- 0.32) U/L, (5.86 +/- 0.13) nmol/l, (1.45 +/- 0.13) nmol/, respectively, and germ cell apoptosis rate was (18.36 +/- 2.04)%. There were significant differences in all parameters between infertile group and fertile group. The levels of FSH, LH, PRL, T were negatively correlated with germ cell apoptosis rates( r = -0.88, -0.93, -0.90, -0.98). The volume of apoptotic germ cell decreased, and chromatin was compacted to form cell-membrane blebs and apoptotic bodies. CONCLUSION: Low concentration of sexual hormones may increase the apoptosis of germ cells, which can induce male infertility.

[Cloning of the RgpAcd gene of Porphyromonas gingivalis and its expression in E. coli].

OBJECTIVE: To clone the catalytic domain gene sequence of RgpAcd of Porphyromonas gingivalis (P. gingivalis) and to induce its fusion expression in E. coli. METHODS: The desired DNA fragment RgpAcd was obtained by PCR and was separately sequenced and identified by inserting into inter-vector pMD18-T vector. The correctly fragment was linked with and cloned into a prokaryotic expression vector pET-15b. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent cells BL21 (DE3) and expression of fusion protein was induced by IPTG. RESULTS: A 1 476 bp specific fragment was obtained and DNA sequencing showed that the fragment was consistent with those of the published. After induction with IPTG, a fusion protein of 5 x 10(4) was visualized on SDS-PAGE gel. CONCLUSION: The protein of RgpAcd will be obtained for further study and its protein was correctly expressed in E. coli BL21 cells.

[Apoptosis induced by curcumin and its effect on c-myc and caspase-3 expressions in human melanoma A375 cell line].

OBJECTIVE: To investigate the effect of curcumin on cell apoptosis and c-myc and caspase-3 expressions in human melanoma A375 cell line. METHODS: A375 cells were exposed to curcumin treatment and growth inhibition of the cells was examined by MTT assay. Annexin V/propidium iodide double staining and DNA fragmentation analysis were employed for assay of the cell apoptosis and morphological changes of the cells were observed with inverted microscopy and transmission electron microscopy, respectively. In situ hybridization and SABC immunohistochemistry were performed for detection of the expressions of c-Myc and caspase-3 in the A375 cells. RESULTS: Curcumin inhibited the growth of A375 cells in both time- and concentration-dependent manners. After treatment with 30 micromol/L curcumin for 48 h, apoptotic morphological changes were observed in the cells and an oligonucleosomal DNA ladder was clearly visualized in DNA fragmentation analysis. The apoptotic rates of the cells treated with curcumin at the concentration above 20 micromol/L were significantly higher than that of the control cells. c-myc expression level was decreased whereas caspase-3 expression increased with the increase in curcumin concentrations. CONCLUSION: Curcumin can inhibit the proliferation and induce apoptosis of A375 cells in vitro, and the genes encoding c-myc and caspase-3 may play a role in the process.