眼结膜印迹细胞法及其影响因素

本文摸索眼结膜印迹细胞法及其影响因素，证明该方法是一项简便，快速，有效地评价机体维生素Ａ营养状况的方法；取样材料，染色时间，温度，透明方法均可影响制片结果。该方法适合基层推广应用。

结膜印迹细胞法检查维生素A缺乏能力的确证研究

１９９５年８月至１９９６年１月，作者先后三次（基线时、补充后三个月及补充后五个月）用结膜印迹细胞法（ＣＩＣ）及血清学方法调查了河北省易县山区两所中学２８３名学生的维生素Ａ营养状况及补充后的变化情况，以验证ＣＩＣ用于现场检查维生素Ａ缺乏的能力。结果显示：ＣＩＣ的灵敏度、特异度和阳性预测值分别为：５１．４％、７２．２％及２３．５％。因此，它不宜用于检查个体维生素Ａ缺乏。但是，当用它来评价群体维生素Ａ的营养状况时，发现它能够将维生素Ａ营养状况良好的群体和维生素Ａ缺乏的群体区分开，而且，它的这一能力相对稳定。补充２０×１０４ＩＵ的维生素Ａ后，补充组的ＣＩＣ异常率在第二次调查时显著降低且低于对照组（Ｐ＜０．０１）。然而，此时补充组仍有１７．４％的受试者ＣＩＣ异常，第三次调查时仍维持于这一水平。试验结果提示结膜炎可能是影响ＣＩＣ结果的混杂因素。但是，没有发现砂眼和ＣＩＣ结果间存在相关关系。结膜印迹细胞转移法（ＩＣＴ）和ＣＩＣ结果的一致率高达９４．４％。单眼的ＣＩＣ异常率显著高于双眼的结果。但是，单眼的ＣＩＣ结果也能将相对剂量反应（ＲＤＲ）异常率相差较大的群体分开。

维生素A缺乏的检测方法及其进展

本文对当前用于检测维生素Ａ缺乏的方法进行了系统的综述，着重介绍了相对剂量反应试验（ＲＤＲ）及结膜印迹细胞法（ＣＩＣ／ＩＣＴ）的研究及应用情况。

凝血酶对血管内皮细胞核因子κB活化及基质金属蛋白酶2表达的影响

研究凝血酶是否通过激活核因子κB信号传导途径,诱导体外培养的人脐静脉内皮细胞上调基质金属蛋白酶2表达,以及重组水蛭素对该途径是否有拮抗作用。体外培养人脐静脉内皮细胞,用免疫细胞化学和免疫印迹法分析核因子κB活化时由细胞浆至细胞核的动态改变,用逆转录聚合酶链反应法评价基质金属蛋白酶2mRNA的表达。结果发现,凝血酶诱导脐静脉内皮细胞后15min即有核因子,κB的活化,30min核内大量活化:凝血酶诱导脐静脉内皮细胞表达基质金属蛋白酶2mRNA,较高表达在6-24h。重组水蛭素可以抑制该过程。结果提示,通过参与核因子κB激活传导信号途径,凝血酶诱导脐静脉内皮细胞上调基质金属蛋白酶2mRNA表达。水蛭素可以有效地抑制凝血酶诱导脐静脉内皮细胞上调基质金属蛋白酶2表达,但不能完全抑制核因子κB的活化。

人巨细胞病毒感染对宿主细胞周期蛋白表达影响的实验研究

目的观察人巨细胞病毒(HCMV)感染对宿主细胞周期蛋白(Cyclins)表达的影响。方法用HCMV感染同步化于G0/G1期的人胚肺成纤维细胞(HEL),分别于感染后0 h、3 h、6 h、12 h、24 h、48 h、72 h、96 h终止培养。用免疫蛋白印迹法(Western Blot)检测CyclinE、CyclinA、CyclinD1蛋白的表达。结果HCMV感染接触抑制细胞12 h时CyclinE蛋白被诱导,感染后24 h出现CyclinE峰值;HCMV不能诱导CyclinA蛋白表达;CyclinD1没有被诱导,且在感染后24 h开始下降。结论HCMV感染同步化于G0/G1期的细胞后,诱导CyclinE蛋白明显升高,激活CyclinE/Cdk2激酶,使细胞周期越过G1/S限制点,将细胞周期阻止于晚G1期。

印迹细胞检查法在眼表疾病诊断中的应用

印迹细胞法是近年来在临床上应用的一种简单、微创的眼表取材技术,该技术通过特殊的方式获取眼球表面的细胞样本,进行各种病理染色,帮助诊断眼表疾病。该技术不但可以对结膜鳞状上皮化生进行分期,而且可以对眼表复层鳞状上皮新生物、干眼症、角巩膜缘干细胞缺乏、眼表病毒感染、维生素A缺乏症、眼表过敏性炎症、结膜色素痣和结膜恶性黑色素瘤等眼表疾病进行辅助诊断。本文对印迹细胞法的技术特点及其在眼科中的应用进行综述。

RNA干扰对SKOV3卵巢癌细胞株Skp2表达的抑制作用

目的:探讨RNA干扰(RNAi)技术对SKOV3卵巢癌细胞株Skp2表达的抑制作用。方法:设计合成针对Skp2的小干涉RNA(siRNA)并进行转染。实验分为空白对照组、转染组1、转染组2、转染对照组及阴性对照组。采用Real time PCR和Western blotting技术检测各组Skp2mRNA和蛋白水平的变化,通过细胞计数试剂盒-8(CCK-8)法检测各组卵巢癌细胞增殖情况。结果:应用Skp2-siRNA干扰SKOV3卵巢癌细胞株后,转染组1、转染组2的Skp2mRNA和蛋白表达水平明显降低,细胞增殖明显受限,与空白对照组相比较,差异有统计学意义(P<0.05),转染组1与转染组2之间差异无统计学意义(P>0.05)。转染组1与转染组2基因沉默效率分别为75.31%和76.86%,蛋白抑制率分别为62.10%和63.11%,细胞增殖抑制率分别为52.75%和53.06%。结论:RNAi技术能够抑制SKOV3卵巢癌细胞株Skp2的表达,从而抑制卵巢癌细胞的增殖。

AS患者PBMCMCP-1蛋白表达及其临床意义

目的：研究单核细胞趋化因子蛋白-1（MonocyteChemoattractantProtein-1，MCP-1）在动脉粥样硬化（Atherosclerosis，AS）患者外周血单个核细胞（peripheralbloodmononuclearcells，PBMC）蛋白表达及其与疾病发生、发展的关系。方法：用蛋白质印迹法（Westernblotting）检测20例As患者和20例健康者PBMCMCP-1的蛋白表达。结果：AS患者PBMCMCP-1蛋白表达明显高于健康者，差异有统计学意义（P〈O．05o结论：MCP-1表达与As存在一定相关性，在As发病过程中起重要的作用。

Pro-apoptotic effects of tectorigenin on human hepatocellular carcinoma HepG2 cells

AIM:To investigate the effects of tectorigenin on human hepatocellular carcinoma(HCC)HepG2 cells.METHODS:Tectorigenin,one of the main components of rhizome of Iris tectorum,was prepared by simple methods,such as extraction,filtration,concentration,precipitation and recrystallization.HepG2 cells were incubated with tectorigenin at different concentrations,and their viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Apoptosis was detected by morphological observation of nuclear change,agarose gel electrophoresis of DNA ladder,and flow cytometry with Hoechst 33342,Annexin V-EGFP and propidium iodide staining.Generation of reactive oxygen species was quantified using DCFH-DA.Intracellular Ca2+was monitored by Fura 2-AM.Mitochondrial membrane potential was monitored using Rhodamine 123.Release of cytochrome c from mitochondria to cytosol was detected by Western blotting.Activities of caspase-3,-8 and-9 were investigated by Caspase Activity Assay Kit.RESULTS:The viability of HepG2 cells treated by tectorigenin decreased in a concentration-and timedependent manner.The concentration that reduced the number of viable HepG2 cells by 50%(IC50)after 12,24 and 48 h of incubation was 35.72 mg/L,21.19 mg/L and 11.06 mg/L,respectively.However,treatment with tectorigenin at 20 mg/L resulted in a very slight cytotoxicity to L02 cells after incubation for 12,24 or 48 h.Tectorigenin at a concentration of 20 mg/L greatly inhibited the viability of HepG2 cells and induced the condensation of chromatin and fragmentation of nuclei.Tectorigenin induced apoptosis of HepG2 cells in a time-and dose-dependent manner.Compared with the viability rate,induction of apoptosis was the main mechanism of the anti-proliferation effect of tectorigenin in HepG2 cells.Furthermore,tectorigenininduced apoptosis of HepG2 cells was associated with the generation of reactive oxygen species,increased intracellular[Ca2+]i,loss of mitochondrial membrane potential,translocation of cytochrome c,and activation of caspase-9 and-3.CONCLUSION:Tectorigenin induces apoptosis of HepG2 cells mainly via mitochondrial-mediated pathway,and produces a slight cytotoxicity to L02 cells.

Influence of chitosan nanofiber scaffold on porcine endogenous retroviral expression and infectivity in pig hepatocytes

AIM: To investigate the influence of chitosan nanofiber scaffold on the production and infectivity of porcine endogenous retrovirus (PERV) expressed by porcine hepatocytes. METHODS: Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Nano group and Hep group) for 7 d. The daily collection of culture medium was used to detect reverse transcriptase (RT) activity with RT activity assaykits and PERV RNA by reverse transcription-polymerase chain reaction (PCR) and real time PCR with the PERV specific primers. And Western blotting was performed with the lysates of daily retrieved cells to determine the PERV protein gag p30. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryo kidney 293 (HEK293) cells. RESULTS: The similar changing trends between two groups were observed in real time PCR, RT activity assay and Western blotting. Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline. No significant difference was found between two groups except the significantly high level of PERV RNA at Day 6 and PERV protein at Day 5 in Nano group than that in Hep group. And in the in-vitro infection experiment, no HEK293 cell was infected by the supernatant. CONCLUSION: Chitosan nanofiber scaffold might prolong the PERV secreting time in pig hepatocytes but would not obviously influence its productive amount and infectivity, so it could be applied in the bioartificial liver without the increased risk of the virus transmission.

Uncoupling protein 2 regulates glucagon-like peptide-1 secretion in L-cells

AIM:To investigate whether uncoupling protein 2(UCP2) affects oleic acid-induced secretion of glucagonlike peptide-1(GLP-1) in L-cells.METHODS:mRNA and protein expression of UCP2 were analyzed in human NCI-H716 cells,which serve as a model for enteroendocrine L-cells,by quantitative reverse transcription-polymerase chain reaction and Western blotting before and after treatment with oleic acid.Localization of UCP2 and GLP-1 in NCI-H716 cells was assessed by immunofluorescence labeling.NCI-H716 cells were transiently transfected with a small interfering RNA(siRNA) that targets UCP2(siUCP2) or with a nonspecific siRNA using Lipofectamine 2000.The concentrations of bioactive GLP-1 in the medium were measured by enzyme linked immunosorbent assay.RESULTS:Both GLP-1 and UCP2 granules were expressed mainly in the cytoplasm of NCI-H716 cells.NCI-H716 cells that secreted GLP-1 also expressed UCP2.Time-course experiments revealed that release of GLP-1 from NCI-H716 cells into the medium reached a maximum at 120 min and remained stable until at least 180 min after treatment with oleic acid(the level of GLP-1 increased about 2.3-fold as compared with the level of GLP-1 in the control cells,P < 0.05).In an experiment to determine dose dependence,stimulation of NCI-H716 cells with ≤ 8 mmol oleic acid led to a concentration-dependent release of GLP-1 into the medium;10 mmol oleic acid diminished the release of GLP-1.Furthermore,GLP-1 secretion induced by oleic acid from NCI-H716 cells that were transfected with siUCP2 decreased to 41.8%,as compared with NCI-H716 cells that were transfected with a non-specific siRNA(P < 0.01).CONCLUSION:UCP2 affected GLP-1 secretion induced by oleic acid.UCP2 plays an important role in L-cell secretion that is induced by free fatty acids.

Identification of deregulated miRNAs and their targets in hepatitis B virus-associated hepatocellular carcinoma

AIM： TO identify the differentially expressed miRNAs and their targets in hepatitis B virus （HBV）-associated hepatocellular carcinoma （HCC）. METHODS： Six hundred and sixty seven human miRNAs were quantitatively analyzed by Taqman lowdensity miRNA array （TLDA） in HBV-HCC tissues. Gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway analyses were used to analyze the significant function and pathway of the differentially expressed miRNAs in HBV-HCC. TargetScan software was used to predict the targets of deregulated miRNAs. Western blotting and luciferase assay were performed to verify the targets of these miRNAs.RESULTS： Ten up-regulated miRNAs （miR-217, miR- 518b, miR-517c, miR-520g, miR-519a, miR-522, miR- 518e, miR-525-3p, miR-512-3p, and miR-518a-3p） and 11 down-regulated miRNAs （miR-138, miR-214, miR-214#, miR-199a-5p, miR-433, miR-511, miR-592, miR-483-3p, miR-483-5p, miR-708 and miR-1275） were identified by Taqman miRNAs array and confirmed quantitatively by reverse transcription polymerase chain reaction in HCC and adjacent non-tumor tissues. GO and KEGG pathway analysis revealed that ＂regulation of actin cytoskeleton＂ and ＂pathway in cancer＂ are most likely to play critical roles in HCC tumorigenesis. MiR- 519a and ribosomal protein S6 kinase polypeptide 3 （RPS6KA3） were predicted as the most significant can-didates by miRNA-mRNA network. In addition, cyclin D3 （CCND3） and clathrin heavy chain （CHC）, usually up-regulated in HCC tissues, were validated as the di- rect target of miR-138 and miR-199a-5p, respectively.

Antisense expression of PKCα improved sensitivity of SGC7901/VCR cells to doxorubicin

AIM:To explore whether antisense blocking of protein kinase C alpha(PKCα)would reverse multi-drug resistance(MDR)in the vincristine(VCR)-resistant human gastric cancer cell line SGC7901/VCR. METHODS:SGC7901/VCR cells expressing antisense PKCα,SGC7901/VCR/aPKC,were established by transfection with a recombinant plasmid reversely inserted with PKCαcDNA.Empty vector(PCI-neo)transfected cell clones,SGC7901/VCR/neo,served as the control.Western blot method was used to detect PKCαcontent in SGC7901,SGC7901/VCR,SGC7901/ VCR/neo and SGC7901/VCR/aPKC cells,using PKCα-specific antibody.The sensitivity of SGC7901,SGC7901/ VCR,SGC7901/VCR/neo and SGC7901/VCR/aPKC cells to doxorubicin(DOX)in vitro was determined by MTT assay.The uptake of DOX in these cells was detected with fluorescence spectrophotometer.RESULTS:Western blot analysis showed that the PKCαprotein level was about 8.7-fold higher in SGC7901/ VCR cells than that in SGC7901 cells,whereas the protein expression of PKCαwas reduced by 78%in SGC7901/VCR/aPKC cells when compared with the SGC7901/VCR cells.SGC7901/VCR/aPKC cells had a 4.2-fold increase in DOX cytotoxicity,accompanied by a 1.7-fold increase of DOX accumulation in comparison with SGC7901/VCR cells. CONCLUSION:PKCαpositively regulates MDR in SGC7901 cells,and inhibition of PKCαcan partially attenuate MDR in human gastric cancer cells.

siRNA-mediated downregulation of TC21 sensitizes esophageal cancer cells to cisplatin

AIM： To determine the functional significance of TC21 in esophageal squamous cell carcinoma （ESCC）. METHODS： TC21 siRNA transfection was carried out using Hyperfectamine to knock down TC21, and tran- scripts were analyzed by reverse transcription-poly- merase chain reaction and protein by Western blotting.We demonstrated the effect of TC21 downregulation of cell signaling in esophageal cancer cells by assess- ing the phosphorylation status of its downstream tar- gets, phosphoinositide 3-kinase （PI3K）, phosphatase and tensin homolog （PTEN）, protein kinase B （pAl〈t）, nuclear factor-KB （NF-~B） and cyclinD1 using specific antibodies. Cell survival analysis after cisplatin treat- ment was carried out by cell viability assay and cell cycle analysis using flow cytometry. RESULTS： TC21 knockdown in human ESCC cell line TEl3 cells, showed only a marginal increase （14.2%） in cell death compared with control cells. The expres- sions of the signaling proteins PI3K and pAkt, transcrip- tion factor NF-KB, and cell cycle protein cyclin D1 were markedly decreased in response to TC21 downregula- tion, whereas the level of pPTEN, an antagonist of PI3K, was increased. In addition, we evaluated the potential of TC21 as a putative target for sensitizing ESCC cells to the chemotherapeutic agent cisplatin. Increased cell death （38.4%） was observed in cells treated with cis- platin after TC21 knockdown compared with cells which were treated with cisplatin alone （20% cell death）. CONCLUSION： Results suggest that TC21 mediates its effects via the PI3K-Akt pathway, NF-KB and cyclin D1, and enhances chemoresistance in esophageal cancer cells.

Notch2 regulates matrix metallopeptidase 9 via PI3K/AKT signaling in human gastric carcinoma cell MKN-45

AIM：To clarify the role of activated Notch2 in the invasiveness of gastric cancer.METHODS：To investigate the invasiveness of silencing Notch2 gene expression,we established a Notch2small interfering RNA（siRNA） transfected cell line using the MKN-45 gastric cancer cell line.After the successful transfection confirmed by real-time reverse transcription-polymerase chain reaction（RT-PCR） and Western blotting,migration and invasion assays were employed to evaluate the aggressiveness of the gastric cancer.RT-PCR and Western blottings were employed to confirm the down-regulation of Notch2 and to evaluate the expression of epithelial mesenchymal transition-related gene matrix metallopeptidase 9（MMP9）,Akt,p-Akt.To confirm the relationship between PI3KAkt and MMP9,the PI3K inhibitor LY294002 was used to treat MKN-45 cells.RESULTS：Notch2 expression was dramatically decreased after Notch2 siRNA transfection（100.00% ± 9.74% vs 11.61% ± 3.85%,P 〈 0.01 by qRT-PCR）.There was also a marked reduction of Notch target gene Hes1（100.00% ± 4.74% vs 61.61% ± 3.58%,P 〈 0.05） at the mRNA,indicating an inhibition of Notch signaling.Inhibition of Notch signaling was also confirmed by the marked reduction of Notch2 intracellular domain at the protein levels（100.00% ± 9.74% vs 65.61% ± 7.58%,P 〈 0.05）.Down-regulation of Notch2 by siRNA enhanced tumor cell invasion（100.00% ± 21.64% vs 162.22% ± 16.84%,P 〈 0.05） and expression of MMP9（1.56 fold,P 〈 0.05）,and activated the pro-MMP9 protein to its active form（1.48 fold,P 〈 0.05）.There was no significant difference in the protein levels of Akt between the two groups（100.00% ± 10.87% vs 96.61% ± 7.33%,P 〉 0.05）,while down-regulation of Notch2 elevated p-Akt expression（100.00% ± 9.87% vs 154.61% ± 13.10%,P 〈 0.05）.Furthermore,p-Akt and MMP9 was down-regulated in response to the inhibitor LY294002（p-Akt 100.00% ± 8.87% vs 58.27% ± 5.01%,P 〈 0.05;MMP9 100.00% ± 9.17% vs 50.03% ± 4.88%,P 〈 0.05）.CONCLUSION：Notch2 may negatively regulate cell invasion by inhibiting the PI3K-Akt signaling pathway

Up-regulation of Fas Ligand Expression by Sirtuin 1 in both Flow-restricted Vessels and Serum-stimulated Vascular Smooth Muscle Cells

Objective To study the role of sirtuin 1 （SIRT1） in Fas ligand （FasL） expression regulation during vascular lesion formation and to elucidate the potential mechanisms. Methods SIRT1 and FasL protein levels were detected by Western blotting in either mouse arteries extract or the whole rat aortic vascular smooth muscle cell （VSMC） lysate. Smooth muscle cell （SMC）-specific human SIRT1 transgenic （Tg） C57BL/6 mice and their littermate wild-type （WT） controls underwent complete carotid artery ligation （ligation groups） or the ligation-excluded operation （sham groups）. The carotid arteries were collected 1 day after operation. Reverse transcription-polymerase chain reaction was performed to detect the mRNA levels of SIRT1 and FasL. Luciferase reporter assays were performed to detect the effect of WT-SIRT1, a dominant-negative form of SIRT1 （SIRT1H363Y）, and GATA-6 on the promoter activity of FasL. Flow cytometry assay was applied to measure the hypodiploid DNA content of VSMC so as to monitor cellular apoptosis. Results SIRTI was expressed in both rat aortic VSMCs and mouse arteries. Forced SIRT1 expression increased FasL expression both in injured mouse carotid arteries 1 day after ligation （P〈0.001） and VSMCs treated with serum （P〈0.05 at the transcriptional level, P〈0.001 at the protein level）. No notable apoptosis was observed. Furthermore, transcription factor GATA-6 increased the promoter activity of FasL （P〈0.001）. The induction of FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 （P〈0.001）, while SIRT1H363Y significantly relieved the enhancing effect of WT-SIRT1 on GATA-6 （P〈0.001）. Conclusions Overexpression of SIRT1 up-regulates FasL expression in both flow-restricted mouse carotid arteries and serum-stimulated VSMCs. The transcription factor GATA-6 participates in the transcriptional regulation of FasL expression by SIRT 1.

MiR-210 regulates cell cycle in nasopharyngealcarcinoma cell line (CNE-1) under hypoxic condition by reducing the expression of cyclin D1

Objective： The aim of our study was to determine the underlying mechanism of miR-210 on regulation of the cell cycle in nasopharyngeal carcinoma cell line CNE-1, particularly through regulation of cyclin D1, under hypoxic conditions. Methods： The CNE-1 cell line was induced with hypoxia, and the expression levels of endogenic miR-210 and cyclin D1 were detected by real-time PCR and Western blotting. Next, the luciferase assay was used to confirm that cyclin D1 is a target gene for miR-210. Cell cycle and cell proliferation were detected in CNE-1 cells that were cultured under hypoxic conditions with either overexpression or knockout of miR-210 using flow cytometry and MTT assay, respectively. Results： Hypoxia induced the expression of miR-210, resulting in reduced mRNA and protein levels of cyclin D1 and repression of cyclin D1 in CNE-1 cells. Further analysis indicated that miR-210 directly binded to the 3＇UTR of the cyclin D1 gene, thus regulated the expression of cyclin DI. The flow cytometry assay showed that, under hypoxic conditions, miR-210 blocked CNE-1 cells in the G1 phase, and miR-210 also inhibited the proliferation of CNE-1 cells. Conclusion： Under hypoxic conditions, miR-210 directly reduced the expression of cyclin D1, leading to CNE-1 cells blocked in G1 phase.

Study on effect of BSO on esophageal cancer cell line TE-1

Objective: The aim of the study was to investigate the sensitizing effect of buthionine sulfoximine (BSO) and radiation on esophageal cancer cell line TE-1. Methods: Methyl thiazolyl tetrazolium (MTT) assay was used to observe the inhibition of BSO and radiation on cell proliferation, and to investigate the sensitizing effect of BSO on esophageal cancer cell line TE-1. Flow cytometry (FCM) was used to observe the effect of BSO and radiation on cell apoptosis and cycle. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot were used to observe the effect of BSO on manganese superoxide dismutase (MnSOD) mRNA and protein expression. Results: BSO could inhibit the proliferation of TE-1 esophageal cancer cells, and had significant dose- and time-dependent radiosensitizing effects on TE-1 esophageal cancer cells. After the combined effects of BSO and radiation on TE-1 cells, the rate of apoptosis and G2/M phase proportion increased significantly, and MnSOD mRNA and protein expression decreased. Conclusion: BSO may reduce MnSOD mRNA and protein expression by affecting TE-1 cell cycle, thus inhibiting and inducing the apoptosis of esophageal cancer cells and enhancing the killing effect of the radiation on esophageal cancer cells.

c-Jun N-terminal kinase is required for thermotherapyinduced apoptosis in human gastric cancer cells

AIM:To investigate the role of c-Jun N-terminal kinase(JNK) in thermotherapy-induced apoptosis in human gastric cancer SGC-7901 cells.METHODS:Human gastric cancer SGC-7901 cells were cultured in vitro.Following thermotherapy at 43 ℃ for 0,0.5,1,2 or 3 h,the cells were cultured for a further 24 h with or without the JNK specific inhibitor,SP600125 for 2 h.Apoptosis was evaluated by immunohistochemistry [terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)] and flow cytometry(Annexin vs propidium iodide).Cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.The production of p-JNK,Bcl-2,Bax and caspase-3 proteins was evaluated by Western blotting.The expression of JNK at mRNA level was determined by reverse transcription polymerase chain reaction.RESULTS:The proliferation of gastric carcinoma SGC-7901 cells was significantly inhibited following thermotherapy,and was 32.7%,30.6%,43.8% and 52.9% at 0.5,1,2 and 3 h post-thermotherapy,respectively.Flow cytometry analysis revealed an increased population of SGC790l cells in G0/G1 phase,but a reduced population in S phase following thermotherapy for 1 or 2 h,compared to untreated cells(P < 0.05).The increased number of SGC-790l cells in G0/G1 phase was consistent with induced apoptosis(flow cytometry) following thermotherapy for 0.5,1,2 or 3 h,compared to the untreated group(46.5% ± 0.23%,39.9% ± 0.53%,56.6% ± 0.35% and 50.4% ± 0.29% vs 7.3% ± 0.10%,P < 0.01),respectively.This was supported by the TUNEL assay(48.2% ± 0.4%,40.1% ± 0.2%,61.2% ± 0.29% and 52.0% ± 0.42% vs 12.2% ± 0.22%,P < 0.01) respectively.More importantly,the expression of p-JNK protein and JNK mRNA levels were significantly higher at 0.5 h than at 0 h post-treatment(P < 0.01),and peaked at 2 h.A similar pattern was detected for Bax and caspase-3 proteins.Bcl-2 increased at 0.5 h,peaked at 1 h,and then decreased.Furthermore,the JNK specific inhibitor,SP600125,suppressed p-JNK,Bax and caspase-3 at the protein level in SGC790l cells following thermotherapy,compared to mock-inhibitor treatment,which was in line with the decreased rate of apoptosis.The expression of Bcl-2 was consistent with thermotherapy alone.CONCLUSION:Thermotherapy induced apoptosis in gastric cancer cells by promoting p-JNK at the mRNA and protein levels,and up-regulated the expression of Bax and caspase-3 proteins.Bcl-2 may play a protective role during thermotherapy.Activation of JNK via the Bax-caspase-3 pathway may be important in thermotherapy-induced apoptosis in gastric cancer cells.

Study of sensitivity of different chemotherapeutic agents on colon cancer cell line SW480

Objective:The aim of this study was to investigate the sensitivity of chemotherapeutic agents 5-FU,cisplatin(DDP) or TAXOL on colon cancer cell line SW480 with different methods,to find out the best examine time period for further study of chemotherapeutic sensitivities.Methods:The SW480 cell was treated with 5-FU(200μg/mL),DDP(150μg/mL) or TAXOL(50μg/mL) respectively for 4h,8h or 12h.MTT assay was used to examine the cell survival rate,Annexin V/PI assay was used to analysis the apoptosis rate,Western Blot assay was applied to examine the expression of apoptotic protein.Results:(1) Results of MTT assay showed that the survival rate of SW480 cells at 4h,8h or 12h was:5-FU(200μg/mL)96.0%±8.2%,85.4%±7.8%,74.4% ±10.2%(P<0.05);DDP(150μg/mL) 99.0%±6.4%,88.7%±4.7%(P<0.05),46.9%±2.6%(P<0.01);TAXOL(50μg/mL) 51.5%±4.2%(P<0.01),31.9%±3.1%,17.6%±2.3%,or blank group 97.2%±5.8%,98.7%±7.2%,97.5%±7.5% respectively.(2) The apoptosis rate of cancer cells at 4h,8h or 12h was:control group:3.4%±0.2%,6.2%±0.4%,7.0%±0.5%;5-FU(200μg/mL) 4.0%±0.3%,4.8%±0.4%,7.7%±0.7%;DDP(150μg/mL) 8.5%±0.9%,18.6%±1.6%(P<0.05),67.0%±6.2%(P<0.01);or TAXOL(50μg/mL) 32.0%±5.2%(P<0.01),76.6%±8.5%,94.0% ±8.2%,respectively.(3) Western Blot assay showed that the expression of apoptosis associated protein.PARP,X-linked inhibitor of apoptasis(XIAP),Caspase-9 and Bcl-xL were changed.Conclusion:The sensitivity of chemotherapy could be assessed by MTT assay,Annexin V/PI assay and Western Blot.The best examine time of the three chemotherapuc agents was 5-FU(200μg/mL):>12h,DDP(150μg/mL):8-12h,or TAXOL(50μg/mL):<4h.