1.Discrimination of anticancer agent action loci at G(2) and M phases by flow cytometry and confocal microscopic imaging.
Yi-Sheng ZHONG ; Chang-Chuan PAN ; Chang-Nan JIN ; Jian-Jun LIN ; Gong-Peng XIONG ; Jian-Xi ZHANG ; Jian-Pin GONG
Journal of Experimental Hematology 2009;17(4):965-968
This study was purposed to evaluate a method to discriminate the action loci of anticancer agents in G(2) and M phases of cell cycle. The meta-amsacrine (m-AMSA) and vinblastine (VBL), already known as G(2) and M phase arrest agent respectively, were used to induce the arrest of MOLT-4 cells at G(2) and M phases, the change of DNA content was detected by flow cytometry, the morphology of arrested cells was observed by confocal microscopy so as to find the arrest efficacy difference of 2 anticancer agents. As a result, the flow cytometric detection showed that the arrested MOLT-4 cells displayed the raise of peaks in G(2) and M phases, but flow cytometric detection alone can not discriminate the difference between them. The observation with confocal microscopy showed that the MOLT-4 cells arrested by m-AMSA displayed the morphologic features in G(2) phase, while the MOLT-4 cells arrested by VBL displayed the morphologic features in M phase. This observation with confocal microscopy is helpful to discriminate the difference between them. In conclusion, the combination of flow cytometry with confocal microscopy is one of the effective methods to discriminate the kind of G(2) or M phase arresting agent of anticancer drugs.
Antineoplastic Agents
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pharmacology
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Cell Cycle
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drug effects
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Cell Division
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drug effects
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Flow Cytometry
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G2 Phase
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drug effects
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Humans
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Microscopy, Confocal
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Tumor Cells, Cultured
2.Effects of hydrogen sulfide on pulmonary surfactant in rats with acute lung injury induced by lipopolysccharide.
Ping WANG ; Jian-Xin ZHANG ; Jian-Pin GONG ; Lan-Fang LI ; Pu-Le JIN ; Cui-Min DING
Chinese Journal of Applied Physiology 2011;27(4):485-489
OBJECTIVETo observe the changes of pulmonary surfactant (PS) in rats with acute lung injury(ALI) induced by lipopolysaccharide (LPS) and to explore the effects of hydrogen sulfide (H2S) on PS.
METHODSFourty- eight male rats were randomly divided into six groups (n = 8). They were control group, LPS group, LPS+ NaHS low, middle, high dose groups and LPS+ PPG group. Saline was administrated in Control group. LPS was administrated in LPS group. In LPS + NaHS low, middle, high dose groups or LPS + PPG group, sodium hydrosulfide (NaHS) of different doses or DL-propargylglycine (PPG) were respectively administrated when the rats were administrated of LPS after 3 hours. All the rats were killed at 6 hours after administration of Saline or LPS. The morphological changes of alveolar epithelial type II cells (AEC-II) were respectively observed by transmission electron microscopes. The content of H2S in plasma and activity of cystathionine-gamma-lyase (CSE) in lung tissues were respectively detected. The contents of total protein (TP) and total phospholipids (TPL) in bronchoalveolar lavage fluid (BLAF) were respectively measured. The pulmonary surfactant protein A (SP-A), surfactant protein B (SP-B) and surfactant protein-C (SP-C) mRNA expressions in lung tissues were analysed.
RESULTS(1) Compared with control group, the content of H2S in plasma, activity of CSE, content of TPL, and SP-A, SP-B and SP-C mRNA expressions were respectively decreased in LPS group (P < 0.05 or P < 0.01). But the content of TP was increased in LPS group (P < 0.01); (2) Compared with LPS group, the content of H2S, activity of CSE and SP-A mRNA expression were significantly increased in LPS + NaHS low, middle and high dose groups (P < 0.05). The SP-B mRNA expression and content of TPL were significantly increased in LPS + NaHS Middle and High dose groups (P < 0.05). The content of TP was decreased in LPS + NaHS High dose group (P < 0.05). The SP-C mRNA expression was not altered in LPS+ NaHS low, middle and high dose groups (P > 0.05); (3) Compared with LPS group, the content of H2S, activity of CSE, content of TPL, and SP-A, SP-B and SP-C mRNA expressions were respectively decreased, but content of TP was increased in LPS + PPG group (P < 0.05).
CONCUSIONThe decrease of PS is the important physiopathologic process of ALI induced by LPS. Exogenously applied H2S could attenuate the process of ALI that possibly because H2S could adjust the compose and secretion of PS.
Acute Lung Injury ; chemically induced ; metabolism ; Animals ; Hydrogen Sulfide ; metabolism ; pharmacology ; Lipopolysaccharides ; Male ; Pulmonary Surfactants ; metabolism ; Rats ; Rats, Sprague-Dawley
3.Establishment of hepatocellular carcinoma multidrug resistant monoclone cell line HepG2/mdr1.
Yong-Bing CHEN ; Mao-Lin YAN ; Jian-Ping GONG ; Ren-Pin XIA ; Li-Xin LIU ; Ning LI ; Shi-Chun LU ; Jing-Guang ZHANG ; Dao-Bing ZENG ; Jian-Guo XIE ; Jia-Yin YANG ; Lü-Nan YAN
Chinese Medical Journal 2007;120(8):703-707
BACKGROUNDThe multidrug resistance (MDR) associated with the expression of the mdr1 gene and its product P-glycoprotein is a major factor in the prognosis of hepatocellular carcinoma cell (HCC) patients treated with chemotherapy. Our study was to establish a stable HCC MDR cell line where a de novo acquisition of multidrug resistance specifically related to overexpression of a transgenic mdr1.
METHODSThe 4.5-kb mdr1 cDNA obtained from the plasmid pHaMDR1-1 was cloned into the PCI-neo mammalian expression vector, later was transferred by liposome to human hepatocarcinoma cell line HepG2. Then the transfected HepG2 cells resisting G418 were clustered and cultured and the specific fragment of mdr1 cDNA, mRNA and the P-glycoprotein (Pgp) in these HepG2 cells were detected by PCR, RT-PCR and flow cytometry, respectively. The accumulation of the daunorubicin was determinated by flow cytometry simultaneously. The nude mice model of grafting tumour was established by injecting subcutaneously HepG2/mdr1 cells in the right axilla. When the tumour diameter reached 5 mm, adriamycin was injected into peritoneal cavity. The size and growth inhibition of tumour were evaluated.
RESULTSThe mdr1 expression vector was constructed successfully and the MDR HCC line HepG2/mdr1 developed. The PCR analysis showed that the specific fragment of mdr1 cDNA in HepG2/mdr1 cells, but not in the control group HepG2 cells. Furthermore, the content of the specific fragment of mdr1 mRNA and Pgp expression in HepG2/mdr1 cells were (59.7 +/- 7.9)% and (12.28 +/- 2.09)%, respectively, compared with (16.9 +/- 3.2)% and (3.07 +/- 1.06)% in HepG2 cells. In the nude mice HCC model, the tumour genes of both groups were identified. After ADM therapy, the mean size of HepG2 cell tumours was significantly smaller than HepG2/mdr1 cell tumours.
CONCLUSIONThe approach using the transfer of mdr1 cDNA may be applicable to the development of MDR hepatocarcinoma cell line, whose MDR mechanism is known. This would provide the experimental basis of MDR research.
ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Animals ; Carcinoma, Hepatocellular ; drug therapy ; genetics ; pathology ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; therapeutic use ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Female ; Flow Cytometry ; Genetic Vectors ; genetics ; Humans ; Liver Neoplasms, Experimental ; drug therapy ; genetics ; pathology ; Mice ; Mice, Nude ; Mitomycin ; pharmacology ; therapeutic use ; Reverse Transcriptase Polymerase Chain Reaction ; Xenograft Model Antitumor Assays ; methods