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ABSTRACT: To investigate whether nerve growth factor (NGF) induced angiogenesis of bone marrow mesenchymal stem cells (MSCs) and the underlying mechanisms.
Bone marrow MSCs were isolated from femors or tibias of Sprague-Dawley rat, and cultured. The cells were purified after 3 to 5 passages, seeded on Matrigel-coated 24-well plates and treated with NGF. Tube formation was observed 24 h later. Tropomyosin-related kinase A (TrkA) and p75NTR gene expression was examined using PCR analysis and flow cytometry. Growth curves were determined via cell counting. Expression of VEGF and pAkt/Akt were analyzed with Western blot.
NGF (25, 50, 100 and 200 μg/L) promoted tube formation of MSCs. The tubular length reached the maximum of a 2.24-fold increase, when the cells were treated with NGF (50 μg/L). NGF (50 μg/L) significantly enhanced Akt phosphorylation. Pretreatment with the specific PI3K inhibitor LY294002 (10 μmol/L) blocked NGF-stimulated Akt phosphorylation, tube formation and angiogenesis. NGF (25-200 μg/L) did not affect the expression of TrkA and vascular endothelial growth factor (VEGF), but significantly suppressed the expression of p75NTR. NGF (50 μg/L) markedly increased the proliferation of MSCs.
NGF promoted proliferation of MSCs and activated the PI3K/Akt signaling pathway, which may be responsible for NGF induction of MSC angiogenesis.
Acta Pharmacologica Sinica 12/2011; 32(12):1483-90. · 1.95 Impact Factor
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ABSTRACT: To investigate whether the proteasomes inhibitor MG262 exerts its anti-cancer function by inducing apoptosis in human ovarian cancer cells, and whether the extracellular signal regulated kinase (ERK) signaling pathway is involved in the regulation of apoptosis induction.
Human ovarian cancer cell line SKOV3 was incubated with different concentrations of MG262 for 24 and 48 hours. Cell viability was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay at different time points of culturing. Flow cytometry was used to detect cell apoptosis rate. The expression of vascular endothelial growth factor (VEGF) was evaluated with western blot and enzyme-linked immunosorbent assay (ELISA). Western blot was used to detect the expression of phosphorylated ERK (p-ERK).
The viability of SKOV3 cells was decreased by MG262 in a concentration-dependent fashion (P < 0.05). After 24 h incubation with MG262 at 1, 10, 20, 40, 60 and 80 nmol/L, the viability rates of SKOV3 were (94.6 +/- 3.1)%, (92.7 +/- 3.7)%, (89.5 +/- 7.7)%, (84.2 +/- 5.1)%, (82.0 +/- 7.4)% and (76.8 +/- 11.0)% respectively, and after 48 h incubation, those figures were further decreased to (91.3 +/- 10.1)%, (86.8 +/- 4.5)%, (74.6 +/- 4.2)%, (56.8 +/- 2.1)%, (49.3 +/- 4.5)% and (37.4 +/- 5.4)%, respectively (P < 0.05). Apoptosis rate of SKOV3 cells induced by MG262, PD98059 or their combination was (30.7 +/- 4.3)%, (26.8 +/- 8.6)% and (50.3 +/- 10.6)%, respectively, which were significantly different compared with controls (P < 0.05). In contrast to SKOV3 cells, apoptosis rate of 293T cells induced by MG262, PD98059 or their combination was (14.5 +/- 5.3)%, (16.2 +/- 7.5)% and (10.8 +/- 7.3)%, respectively, which were not significantly different compared with controls (P > 0.05). p-ERK expression decreased gradually in a time-dependent manner. And wild-type p53 expression was not significantly different. There was no significant difference between experimental and control 293T cells (P < 0.05). In addition, MG262 down-regulated VEGF secretion and expression in SKOV3 cells (P < 0.05).
Proteasome inhibitors can induce apoptosis and inhibit cell proliferation and angiogenesis through ERK signal pathway in SKOV3 cells.
Zhonghua fu chan ke za zhi 09/2008; 43(9):690-4.
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ABSTRACT: To construct a short hairpin RNA(shRNA) interference expression plasmid vector of survivin gene, transfect tongue squamous cell carcinoma line Tca8113 which expressed survivin gene in a high level, and choose the cells whose survivin gene were suppressed significantly.
Two pairs of oligonucleotide sequences specific for survivin gene were designed and synthesized, and cloned into pSilencer-2.1U6-neo plasmid. The recombinant plasmids (named PS1 and PS2) were amplified in Ecoli. DH5alpha was identified by restriction digestion, PCR and sequencing. The vectors were transfected into Tca8113 cells with lipofectamine 2000. After selection with G418, the stable cell clones were attained. Survivn expression was assayed with real-time quantitative PCR and Western blotting. SAS8.0 software package was used for Student t test.
Two vectors were constructed successfully and stable cell clones with PS1 or PS2 plasmid were obtained. As compared with those of control, survivin expression of transfected cell with PS1 or PS2 in mRNA level was significantly suppressed (P<0.05). In protein level, only those of transfected cell with PS2 was significantly suppressed (P<0.01).
The shRNA interference expression plasmid vectors of survivin gene are successfully constructed, and Tca8113 cells which express survivin gene in a stable lower level are attained, which enable us to carry out further research on gene therapy of oral squamous cell carcinoma. Supported by National Natural Science Foundation of China (Grant No.30572056).
Shanghai kou qiang yi xue = Shanghai journal of stomatology 06/2008; 17(3):275-9.
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ABSTRACT: To study the effect of curcumin on invasion and migration of the tongue squamous cell line Tca8113.
Tca8113 cells were treated with curcumin (0 - 100 micromol/L) for 24 h and the conditional medium was collected. The gelatinases - matrix metalloproteinase -2 and -9 (MMP-2, -9) in the conditional medium were detected by gelatin zymography. The cell invasion and migration model in vitro was conducted using transwell chamber with or without matrigel. Using this model, the effects of 50 micromol/L curcumin on invasion and migration of Tca8113 were detected.
Curcumin reduced the activities of MMP-2 and MMP-9 on a dose-dependent manner. Curcumin can suppress cell invasion and migration significantly (P <0.01).
Curcumin can suppress Tca8113 invasion and migration by reducing the activities of MMP-2 and MMP-9.
Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology 03/2008; 43(2):101-4.
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ABSTRACT: The aim of the present study was to assess the prevalence and risk factors of chronic renal disease in hyperglycemic population of Shanghai Caoyang Community.
Microalbuminuria was determined by measuring urinary albumin-to-creatinine ratio (ACR) and glomerular filtration rate (GFR) was estimated from fasting serum creatinine.
A total of 406 Shanghai Chinese, with the average age of 67.5 +/- 13.8 years (244 with diabetes mellitus and 162 with impaired glucose regulation) from the established hyperglycemic cohort were included. (1) The prevalence of microalbuminuria was 20.9% and 10.5% in the subjects with diabetes and impaired glucose regulation (IGR); (2) The prevalence of a cGFR >or= 60 and < 90, >or= 30 and < 60, < 30 mlxmin(-1)x(1.73 m(2))(-1) were 41.6%, 37.0%, 1.2% respectively in the patients with diabetes, and 34.2%, 47.2%, 1.9% in the patients with IGR. Impaired renal function was 38.2% and 49.1% respectively in the subjects with diabetes and IGR; (3) The prevalence of microalbuminuria was significantly higher in the diabetic patients with hypertension, central obesity, dyslipidemia, history of cardiovascular disease or selinity. Systolic blood pressure, waist circumstance, fasting plasma glucose and history of cardiovascular disease were all independently associated with hyperglycemic microalbuminuria; (4) cGFR was diminished with increased age and the impaired renal function was more frequent in the patients with hypertension (48.5%). There was a significant positive correlation between a diminished cGFR and increasing levels of ACR after the patients with macroalbuminuria were deleted and adjusted age. Serum creatinine, age and systolic blood pressure were independently associated with diabetic cGFR < 60 ml/min/1.73 m(2).
The high prevalence of microalbuminuria and impaired renal function in the hyperglycemic population of Caoyang Community underlines the need for cost-effective programs for the detection of chronic renal disease, and approaches to screen it in the hyperglycemic patients should incorporate assessment of GFR in addition to monitoring urine albumin excretion.
Zhonghua yi xue za zhi 10/2006; 86(36):2527-32.
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ABSTRACT: To investigate the apoptosis of implanted primary gastric cancer cells in nude mice induced by resveratrol and the relation between this apoptosis and expression of bcl-2 and bax.
A transplanted tumor model was established by injecting human primary gastric cancer cells into subcutaneous tissue of nude mice. Resveratrol (500 mg/kg, 1,000 mg/kg and 1,500 mg/kg) was directly injected beside tumor body 6 times at an interval of 2 d. Then changes of tumor volume were measured continuously and tumor inhibition rate of each group was calculated. We observed the morphologic alterations by electron microscope, measured the apoptotic rate by TUNEL staining method, detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and PT-PCR.
Resveratrol could significantly inhibit carcinoma growth when it was injected near the carcinoma. An inhibitory effect was observed in all therapeutic groups and the inhibition rate of resveratrol at the dose of 500 mg/kg, 1,000 mg/kg and 1,500 mg/kg was 10.58%, 29.68% and 39.14%, respectively. Resveratrol induced implanted tumor cells to undergo apoptosis with apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation. The inhibition rate of 0.2 mL of normal saline solution, 1,500 mg/kg DMSO, 500 mg/kg resveratrol, 1 000 mg/kg resveratrol, and 1 500 mg/kg resveratrol was 13.68+/-0.37%, 13.8+/-0.43%, 48.7+/-1.07%, 56.44+/-1.39% and 67+/-0.96%, respectively. The positive rate of bcl-2 protein of each group was 29.48+/-0.51%, 27.56+/-1.40%, 11.86+/-0.97%, 5.7+/-0.84% and 3.92+/-0.85%, respectively by immunohistochemical staining. The positive rate of bax protein of each group was 19.34+/-0.35%, 20.88+/-0.91%, 40.02+/-1.20%, 45.72+/-0.88% and 52.3+/-1.54%, respectively by immunohistochemical staining. The density of bcl-2 mRNA in 0.2 mL normal saline solution, 1,500 mg/kg DMSO, 500 mg/kg resveratrol, 1,000 mg/kg resveratrol, and 1,500 mg/kg resveratrol decreased progressively and the density of bax mRNA in 0.2 mL normal saline solution, 1,500 mg/kg DMSO, 500 mg/kg resveratrol, 1,000 mg/kg resveratrol, and 1,500 mg/kg increased progressively with elongation of time by RT-PCR.
Resveratrol is able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated by down-regulating apoptosis-regulated gene bcl-2 and up-regulating the expression of apoptosis-regulated gene bax.
World Journal of Gastroenterology 02/2005; 11(2):280-4. · 2.47 Impact Factor
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ABSTRACT: To investigate the apoptosis in primary gastric cancer cells induced by genistein, and the relationship between this apoptosis and expression of bcl-2 and bax.
MTT assay was used to determine the cell growth inhibitory rate in vitro. Transmission electron microscope and TUNEL staining were used to quantitatively and qualitatively detect the apoptosis of primary gastric cancer cells before and after genistein treatment. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosis-associated genes bcl-2 and bax.
Genistein inhibited the growth of primary gastric cancer cells in dose-and time-dependent manner. Genistein induced primary gastric cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that after the treatment of primary gastric cancer cells with genistein for 24 to 96 h, the apoptotic rates of primary gastric cancer cells increased time-dependently. Immunohistochemical staining showed that after the treatment of primary gastric cancer cells with genistein for 24 to 96 h, the positivity rates of Bcl-2 proteins were apparently reduced with time and the positivity rates of Bax proteins were apparently increased with time. After exposed to genistein at 20 micromol/L for 24, 48, 72 and 96 respectively, the density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time.
Genistein is able to induce the apoptosis in primary gastric cancer cells. This apoptosis may be mediated by down-regulating the apoptosis- associated bcl-2 gene and up-regulating the expression of apoptosis-associated bax gene.
World Journal of Gastroenterology 07/2004; 10(12):1822-5. · 2.47 Impact Factor
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ABSTRACT: To study the distribution of Candida spp. mainly Candida albicans in the oral cavities of health children.
Four groups children of different ages, A1: newborn babies, A2: 3.2 years old (average) children,A3, 7.2 years old,A4:12.7 years old and B control group 20.4 years old,mucosal swab sampling with centrification,CHROMagar Candida identified culture medium for culture and identification, and different methods for isolating Candida albicans for A3 group.
The isolation rates of Candida spp were A1 7.5%,A2 70%,A3 56.36%,A4 49.12%,B 27.5%,the proportion of Candida albicans also diversely,and the method of PCR was more sensitive than the one of culture.
Candida spp can be isolated from the normal oral cavities in different aged children, most of them were Candida albicans,both the isolation rates of Candida spp and the proportion of Candida albicans were different.
Shanghai kou qiang yi xue = Shanghai journal of stomatology 09/2003; 12(4):288-91.
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ABSTRACT: To investigate the expression and alteration (including homozygous deletion and mutation) of MTS1 gene in precancerous lesions and squamous cell carcinomas (SCC) of oral mucosa, and to analyse the function of MTS1 gene alteration in oral mucosal carcinogenesis.
The expression of p16 protein produced by MTS1 gene was examined with immunohistochemical SP method in 10 normal oral mucosas, 30 precancerous lesions (10 mild, 10 moderate and 10 severe dysplasia respectively) and 45 squamous cell carcinomas (SCCI18, SCCII 19, SCCIII 8). The deletion and mutation of exon1 and exon2 of MTS1 gene were examined with methods of PCR and SSCP in these same samples.
All the precancerous lesions had p16 protein expression and no alteration of MTS1 gene. In SCC, the positive rate of p16 protein was 60.0% with 72.2% in SCCI, 57.9% in SCCII, 37.5% in SCC III, and there were no significant difference among the three groups by chi2 test (P>0.05). Gene homozygous deletion of exon1 and/or exon2 was detected in 10 cases, and gene mutation in 4 cases. The whole rate of gene alteration was 31.1% (14/45). The MTS1 gene alteration rate was 27.8% in SCCI, 31.6% in SCCII, 37.5% in SCC III and there was also no significant difference among the three groups by chi2 test (P>0.05). In SCC with local lymph nodes metastasis, MTS1 alteration rate was 57.1%, while in SCC with no lymph nodes metastasis was 8.3%, and there was significant difference by chi2 test (P<0.05).
MTS1 gene alteration is not an early event in the carcinogenesis of oral mucosa and can not be used as a biology mark to examine oral precancerous lesions. MTS1 gene may play a certain role in the progression of oral squamous cell carcinomas.
Zhonghua kou qiang yi xue za zhi = Zhonghua kouqiang yixue zazhi = Chinese journal of stomatology 09/2003; 38(5):361-3.
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ABSTRACT: To explore the enhanced cell-killing effect in vivo of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapeutic system using tegument viral protein (VP22) intercellular trafficking.
Human ovarian epithelial cancer cell line 3AO was infected by lentivirus containing HSV-TK and HSV-VP22-TK respectively. Tumors were induced in nude mice by subcutaneous injection of the mixture of 90% 3AO cells and 10% 3AO cells carried with HSV-TK (3AO/TK) or HSV-VP22-TK (3AO/VP22-TK). Nude mice injected with 3AO cells were used as blank control. When the volume of tumor was 150 mm(3), GCV was administered at 10 mg/kg or 50 mg/kg intraperitoneally.
There were significant differences, in the tumor volume and weight between 3AO/TK group and 3AO/VP22-TK group after administration of 10 mg/kg GCV (P < 0.01), and the later was more efficient than the former. But there was no significant difference after administration of 50 mg/kg GCV (P > 0.05). The tumor inhibition rates in 3AO/VP22 group and 3AO/VP22-TK group were 37.7% and 91.5% respectively after administration of 10 mg/kg GCV (P < 0.01), and were 81.8% and 96.7% respectively after administration of 50 mg/kg GCV (P > 0.05).
These results clearly indicate that VP22 enhances the efficiency of the suicide gene transfer, thereby increases the cell-killing effect on tumor in vivo.
Zhonghua fu chan ke za zhi 05/2003; 38(4):195-8.
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ABSTRACT: To study whether Tongxinluo (TXL) can induce angiogenesis in bone marrow mesenchymal stem cells (MSCs), and to investigate the underlying mechanism.
Bone marrow MSCs were obtained from male Sprague-Dawley rats. We established an angiogenesis model in vitro via matrigel experiment. MSCs were seeded on matrigel coated 24-well plates, and treated by TXL 50 and 100 mg/L. After 24 h, we observed the tube formations of MSCs in the matrigel. Cell migration ability was examined by wound scratch test and transwell assay. Expressions of vascular endothelial growth factor (VEGF), fetal liver kinase-1 (Flk-1), matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinase-2 (TIMP-2) were analyzed at the protein level by Western blot. Gelatin zymography assay was applied to investigating the MSC paracrine abilities of pro-MMP-2 and activated MMP-2.
TXL promoted MSC tube formation in matrigel. The ratio of TXL 100 mg/L treated-MSC tubular length was increased 3.04-fold compared to the control group (P<0.05). Scratch test and transwell assay showed that TXL could improve the cell migration ability of MSCs. Western blot experiments showed that TXL promoted MSC synthesis of MMP-2, but it had no influence on the expressions of MMP-9 and TIMP-2. This effect was confirmed by gelatin zymography assay, which showed that TXL increased MSC secretion of pro-MMP-2 and activated MMP-2. VEGF expression of TXL treated-MSCs was increased compared to the control group. The expression of Flk-1 was not different among the groups.
This study demonstrates that TXL can promote the tube formation of MSCs, and the underlying mechanisms are associated with increased migration ability of MSCs and the up-regulation of MMP-2 and VEGF expressions.
Journal of Zhejiang University SCIENCE B 12(8):644-51. · 1.10 Impact Factor
