PPAR-γ对放射诱导成纤维细胞分泌PAI-1和TGF-β的影响

目的 分析过氧化物酶增殖体活化受体-γ(PPAR-γ)配体(匹格列酮)对X射线诱导纤溶酶原抑制物-1(PAI-1)、转化生长因子-β(TGF-β)在小鼠肺成纤维细胞(L929细胞)中的mRNA表达和L929细胞增殖活性的影响.方法 用RT-PCR证实L929细胞表达PPAR-γmRNA.分别采用10、4、2 GyX射线照射L929细胞,观察照射前后PAI-1、TGF-βmRNA的表达情况.照射+匹格列酮处理L929细胞,用RT-PCR方法观察对PAI-1、TGF-βmRNA的表达影响,用MTT法观察对细胞增殖活性影响.结果 RT-PCR检测结果显示10 Gy照射48 h后L929细胞中PAI-1、TGF-βmRNA表达明显升高[0.18∶0.78、0.22∶0.76(F=3.70,P=0.010)],2 Gy和4 Gy有类似趋势[0.18∶0.43、0.18∶0.44和0.22∶0.39、0.22∶0.40(F=3.40,P=0.090)];10 Gy照射+匹格列酮48 h后明显降低[0.78∶0.45、0.76∶0.54(F=3.90,P=0.010)],2 Gy和4 Gy的也有类似趋势[0.43∶0.37、0.44∶0.35和0.39∶0.32、0.40∶0.32(F=2.40,P=0.210)].MTT结果显示与空白对照比较2、4、10 Gy照射和单用匹格列酮均会抑制L929细胞增殖[0.44∶0.39、0.36、0.34和0.32(F=3.90,P=0.040)],2、4、10 Gy照射+匹格列酮更能抑制L929细胞增殖[0.44∶0.27、0.26、0.25(F=2.50,P=0.005)].结论 X射线照射L929细胞后出现促纤维化因子PM-1、TGF-β表达升高.匹格列酮使PAI-1、TGF-β高表达降低,也会抑制成纤维细胞的增殖.

Abstract：

Objective To observe the influence of peroxisome proliferator activated receptor-γligand (PPAR-γ, pioglatazone) on expression of PAI-1 and TGF-β mRNA and proliferation in fibroblast cells before and after X-ray radiation, and to study the effect of PPAR-γon normal cells during radiation induced fibrosis process. Methods RT-PCR method was used to measure PPAR-γgene expression in L929 cells.After X-ray irradiation of 10 Gy,4 Gy or 2 Gy, the expressions of PAI-1 and TGF-β mRNA in mouse lung fibroblast cells (L929) were measured using RT-PCR. After X-ray irradiation and pioglatazone treatment,the influence of pioglatazone on PAI-1 and TGF-β was measured using RT-PCR method. MTT method was used to test cell proliferation after the treatment of irradiation and pioglatazone. Results PPAR-γ mRNA expression was observed in L929 cells. Expression of PAI-1 and TGF-β mRNA reached the highest level 483.40,P =0. 090) ). At 48 h after the treatment of pioglatazone and 10 Gy radiation, pioglatazone decreased 0. 36, 0. 34 and 0. 32( F = 3.90, P = 0. 040) ). The inhibitory effect was significantly increased when L9292. 50,P =0. 005)). Conclusions X-ray irradiation can increase the expression of PAI-1 and TGF-β in L929 cells. Pioglatazone can decrease the expression of radiation-induced PAI-1 and TGF-β, and restrain the fibroblast proliferation.     

Changes of hMSH2 and hMLH1 Expression in Nasopharyngeal Carcinoma Cells after X-Radiation

OBJECTIVE To study the effect of X-radiation on expression of hMSH2 and hMLH1 in nasopharyngeal carcinoma （NPC） CNE-1 cells, and explore the mechanism of DNA mismatch repair after radiation. METHODS The cells were divided into experimental and control groups. Experimental cells were exposed to 10 Gy radiation administered as 2 Gy per fraction. Control cells were not radiated. The distribution of cells in the cell cycle was analyzed using flow cytometry. The expression of hMSH2 and hMLH1 was examined by RT-PCR and Western blots. RESULTS The expression of hMSH2 mRNA in experimental cells was significantly greater compared to control cells at 0-3rd weeks and decreased at the 4th week following radiation （P〈0.01）. The expression of hMSH2 protein in experimental cells was up-regulated and significantly greater compared to control cells at the 2nd-4th weeks after radiation （P〈0.01）. There were no significant differences in hMLH1 mRNA and protein expression between experimental and control cells （P 〉0.05）. CONCLUSION Radiation induces hMSH2 expression; hMSH2 has a role in the process of DNA repair, which maybe responsible for reduction of radiosensitivity after radiation.     

The Effect of 17 β-Estradiol on Invasion by the Ovarian Clear Cell Adenocarcinoma Cell Line ES-2 and the Molecular Mechanism Involved

OBJECTIVE To assess the effect of 17 β-estradiol（E2） on cell proliferation, cell invasiveness and its regulation of MTA3, Snail and matrix metalloproteinase 2 （MMP-2） expression in the ovarian clear cell adenocarcinoma cell line ES-2, and to further investigate the mechanism involved. METHODS We first investigated expression of ERα, ERβ, PR and E-cadherin of ES-2 cells by RT-PCR and Western blots. Before all experiments, the ES-2 cells were grown in medium depleted of steroid for more than 7 days. Following treatment with 10^-7,10^-8 and 10^-9 M E2, cell viability of the ES-2 cells was determined by the MTT method, and the cell cycle distribution and apoptosis were examined by flow cytometry （FCM）. Invasion and mobility assays were performed using modified Boyden chambers. MTA3, Snail and MMP-2 mRNA expression was measured by RT-PCR, and Snail, MMP-2 protein levels were determined by IHC. MMP-2 activity was assayed by zymography. RESULTS RT-PCR and Western Blots showed that theexpression of ERα and E-cadherin mRNA and protein in the ES-2 cells was negative, while ERβ and PR expression was positive. E2 at 10^-7,10^-8 or 10^-9M stimulated cell proliferation. A level of 10^-8M E2 reduced the proportion of G0-G1 phase cells and increased the proportion of cells in the S phase, but it had no effect on apoptosis. Invasiveness and mobility of the ES-2 cells was significantly increased by 10^-8M E2. Treatment with 10^-8M E2 led to reduced MTA3 mRNA expression, and elevated Snail and MMP-2 mRNA and protein levels. CONCLUSION E2 enhanced invasion by the ES-2 cells. The effects observed maybe mediated by down-regulation of MTA3 and up-reguation of Snail and MMP-2.     

Effects of Bradykinin on the Expression of Interleukin-6 in C6 Glioma Cells

OBJECTIVE There is a close correlation between interleukin-6 （IL-6） and malignant proliferation of gliomas. We investigated the effect of bradykinin on the expression of IL-6 in C6 glioma cells. METHODS Semi-quantitive RT-PCR and radioimmunoassay were used to examine the effect of bradykinin on the expression of IL-6 in C6 glioma cells and on the level of IL-6 in the culture medium. RESULTS Using semi-quantitive RT-PCR, the expression of IL-6 mRNA was examined in C6 glioma cells at 0, 5, 10,15, 30, 60 rain following addition of 1μmol/L bradykinin. There was no statistical difference in expression of IL-6 mRNA between the treatment and control groups （P〉0.05） and IL-6 was not detected in the cell culture medium. CONCLUSION Within an hour, IL-6 expression in C6 glioma cells is not induced by bradykinin, suggesting that its clinical application may be useful as a potential therapeutic agent for tumors of the central nervous system .     

TIME- AND DOSE-DEPENDENT UP-REGULATION OF TNF-α mRNA AFTER IRRADIATION OF HUMAN NSCLC CELL LINES IN VITRO

Objective： Even though radiotherapy plays a major role in the local treatment of non-small cell lung cancer （NSCLC）, little is known about the molecular effects of irradiation in this tumor. In the present study, we examined two NSCLC cell lines for their endogenous production of TNF-α after irradiation. To investigate the radiation-induced TNF-α production in NSCLC cell lines. Methods： Two human NSCLC cell lines （A549： squamous; NCI-H596： adenosquamous） were investigated for their TNF-α mRNA （real-time RT-PCR） after exposure to different irradiation doses （2, 5, 10, 20, 30, 40 Gy） and time intervals （1, 3, 6, 12, 24, 48 or 72 h）. The TNF-α mRNA expression was quantified by real-time RT-PCR. The clonogenic survival was evaluated after irradiation with 2, 4, 6 and 8 Gy. Results： Non-irradiated NSCLC cells exhibited no or very low TNF-α expression. For the NCI-H596 cell line, TNF-α expression was significantly elevated 1～12 h （maximum 6h： 568fold increase relative to unirradiated cells） in a time-dependent manner. The radiation-induced increase could be observed after irradiation with 2 Gy reaching maximal at 40 Gy, with 83 times higher than normal controls. The clonogenic survival of these cell lines was nearly identical. Conclusion： NCI-H596 cells produce significant quantities of TNF-α following irradiation in a time- and dose-dependent manner. The pro-inflammatory cytokine TNF-α is a key mediator for the pathogenesis of radiation pneumonitis. Radiation-induced endogenous TNF-α expression in NSCLC cells may affect the normal lung adjacent to the tumor and may be associated with an adverse clinical outcome of the patient.     

Experimental study of the differentiation HPV16 subgenes-immortalized human endocervical cells induced by all-trans-retinoic acid

Objective： To investigate the differentiation-inducing effects of all-trans-retinoic （ATRA） to HPV16 subgenesimmortalized human l：ndocervical cells （H8 cell） in vitro. Methods： HPV16 subgenes-immortalized human endocervical cells （H8 cells） were cultured in vitro. After treated with ATRA, the proliferation of immortalized human endocervical cells was measured by MTT assay; morphological changes were observed using M and TEM; cell cycle was analyzed by FCM; expression of Ki67 was tested using immunocytochemistry and the activity of telomerase was tested using PCR-ELISA. Results： ATRA could inhibit proliferation of H8 cells significantly and induce their morphodifferentiation. According to FCM, H8 cells accumulated in G1 phase and expression of Ki67 and activity of telomerase reduced significantly after treatment with ATRA. Conclusion： ATRA could induce the differentiation of H8 cell line obviously, which might be achieved by inhibiting proliferation, blocking cell cycle, and reducing activity of telomerase.     

Effect of Celecoxib on Apoptosis of Endometrial Carcinoma Cell

Objective:To investigate the effect of Celecoxib on proliferation and apoptosis of the endometrial carcinoma cell HEC-1B and the effect on the expression of Fas and Survivin mRNA.Methods:The inhibition on the growth of human endometrial carcinoma cell HEC-1B was investigated by cell culture and MTT experiment when treated with different concentrations of Celecoxib.The cell apoptosis was detected by flow cytometry and DNA Ladder Electrophoresis.The change of the expression of Fas and Survivin mRNA after the treatment of Celecoxib was detected With RT-PCR.Results:Celecoxib could effectively inhibit the growth of HEC-1B cells and induce apoptosis.Survivin mRNA expression was decreased and Fas mRNA expression was increased after treating with Celecoxib.Conclusion:Celecoxib could inhibit HEC-1B cell proliferation and induce its apoptosis.     

X射线照射对鼻咽癌细胞多药耐药基因表达影响初步研究

Objective To invstigate the effect of high dose X-ray irradiation on the expression of multidrug resistance-1 (MDR1), Bcl-2, MMP7 genes. Methods A nasopharyngeal carcinoma cell line,CNE1, were irradiated with a total dose of 50 Gy. The resistance to the cisplatin of CNE1 cells and the irradiated CNE1 (CNE1 R) cells was detected by MTT. mRNAs expression of MDR1 , Bcl-2 and MMP7 was measured by quantitative RT-PCR. Results The expression of MDR1 increased in CNE1 R cells. The semiquantitative A value of MDR1 mRNA was 0.47 ±0.04, and the value of CNE1R cells (1st, 7th, 21st,28th, 35th, 42nd and 49th days after irradiation) were 0.67 ± 0. 06 (t = -5.44, P = 0. 003) ,0.70 ± 0. 01(t=-5.90,P=0. 002),0.73±0. 01(t= -6. 45,P=0. 001) ,0. 67 ± 0. 03 (t= -3.97,P=0.011),0.65 ±0.01(t = -4.43,P=0. 007),0. 62±0. 05(t= -2. 64,P=0.046) and 0.62 ±0.02(t = -3.34,P=0.021), respectively. Bcl-2 mRNA expression were 0.55 ±0.02 and 1.05 ±0.04(t = -9.93,P=0. 000) and MMP7 mRNA expression were 0.51 ±0.01 and 0.82 ±0.02(t = -8.51,P=0.000) in CNE1and CNE1 R cells. Conclusions The MDR1 expression was increased after a total dose of50 Gy irradiation,which may be related to the synchronous change of Bcl-2 and MMP7 genes.