Antisense oligodeoxynucleotide inhibits expression of recombinant porcine follicle-stimulating hormone receptor

Ｓｙｎｔｈｅｔｉｃｏｌｉｇｏｄｅｏｘｙｎｕｃｌｅｏｔｉｄｅｓ（ＯＤＮｓ）ｒｅｐｒｅｓｅｎｔａｎｅｗｔｏｏｌｆｏｒｔｈｅｄｉｓｃｏｖｅｒｙｏｆｐｈｙｓｉｏｌｏｇｉｃａｌｍｅｃｈａｎｉｓｍｓｉｎｃｅｌｌｃｕｌｔｕｒｅｓ,ｉｎｔｉｓｓｕｅｓａｎｄｉｎｖｉｖｏ．Ｉｄｅａｌｌｙ,ａｎａｎｔｉｓｅｎｓｅＯＤＮｉｓｔａｒｇｅｔｅｄｉｎａｓｅｑｕｅｎｃｅ－ｓｐｅｃｉｆｉｃｍａｎ－ｎｅｒｔｏｎｕｃｌｅｉｃａｃｉｄｓ（ＲＮＡｏｒＤＮＡ）ｔｏｏｆｆｅｒｔｈｅｅｘｃｉｔｉｎｇｐｏｓｓｉｂｉｌｉｔｙｏｆｓｅｌｅｃｔｉｖｅ…     

Role of Connective Tissue Growth Factor in Extracellular Matrix Degradation in Renal Tubular Epithelial Cells

In order to investigate the effects of connective tissue growth factor (CTGF) antisense oligodeoxynucleotide (ODN) on plasminogen activator inhibitor-1 (PAI-1) expression in renal tubular cells induced by transforming growth factor β1 (TGF-β1) and to explore the role of CTGF in the degradation of renal extracellular matrix (ECM), a human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipid-mediated CTGF antisense ODN was transfected into HKC. After HKC were stimulated with TGF-β1 (5 μg/L), the mRNA level of PAI-1 was detected by RT-PCR. In-tracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 in the media was determined by Western blot. The results showed that TGF-β1 could induce tubular CTGF and PAI-1 mRNA expression. The PAI-1 mRNA expression induced by TGF-β1 was significantly inhib-ited by CTGF antisense ODN. CTGF antisense ODN also inhibited intracellular PAI-1 protein syn-thesis and lowered the levels of PAI-1 protein secreted into the media. It was concluded that CTGF might play a crucial role in the degradation of excessive ECM during tubulointerstitial fibrosis, and blocking the biological effect of CTGF may be a novel way in preventing renal fibrosis.     

Reversal of multidrug resistance and inhibition of phosphorylation of AKT in human ovarian cancer cell line by wild-type PTEN gene

Summary The reversing effect of wild-type PTEN gene on resistance of C13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B (AKT) were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were analyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C13K cells were 2.04 ± 0.10, 0.94 ± 0.04 respectively and the expression of p-Akt protein (0.94 ± 0.07) was lower than those in control groups (1.68 ± 0.14, 1.66 ± 0.10) (P < 0.05). The IC₅₀ of DDP to C13K cells transfected with PTEN (7.2 ± 0.3 μmol/L) was obviously lower than those of empty-vector transfected cells and non-transfected cells (12.7 ± 0.4 μmol/l, 13.0 ± 0.3 μmol/L) (P<0.05). The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were (41.65 ± 0.87)%, (18.61 ± 0.70)% and (15.28 ± 0.80)% respectively, and the difference was statistically significant (P<0.05). PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C13K with multidrug-resistance by decreasing the expression of p-Akt. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30571950) and National Key Basic Research Program Foundation (NO.2002CB513107).     

Effects of betulinic acid on proliferation and apoptosis in Jurkat cells and its in vitro mechanism

Summary  The anti-cancer effects of betulinic acid (BA) on Jurkat cells and its in vitro mechanism were examined by using MTT assay. Apoptosis was detected by using Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effects of betulinic acid on the cell cycle of Jurkat cells were studied by propidium iodide method. RT-PCR and Western blotting were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid. Our results showed the proliferation of Jurkat cells was decreased in betulinic acid-treated group with a 24-h IC50 value being 70.00 μmol/L. Betulinic acid induced apoptosis of Jurkat cells in a time- and dose-dependent manner. The number of Jurkat cells treated with betulinic acid showed an increase in G₀/G₁ phase and decrease in S phase. After treatment with 0, 20, 60, 100 μmol/L betulinic acid for 24 h, the number of Jurkat cells was increased from (31.00±1.25)% to (58.84±0.32)% in G₀/G₁ phase, whereas it was decreased from (61.45±1.04)% to (35.82±1.95)% in S phase. PBMCs were less sensitive to the cytotoxicity of betulinic acid than Jurkat cells. The expressions of cyclin D3, bcl-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid. It is concluded that betulinic acid is able to inhibit the proliferation of Jurkat cells by regulating the cell cycle, arrest cells at G₀/G₁ phase and induce the cell apoptosis. The anti-tumor effects of betulinic acid are related to the down-regulated expression of cyclin D3 and bcl-xl. Zi CHEN, Female, born in 1980, Resident This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30500686).     

Effects of Bushen Tiaochong Recipe (补肾调冲方) containing serum on ovarian granulosa cell proliferation, steroidogenesis and associated gene expression in rats

Objective:To observe the effect of Bushen Tiaochong Recipe (补肾调冲方,BSTCR) on rats' ovarian granulosa cell (GC) proliferation,steroidogenesis and follicle-stimulating hormone receptor (FSHR),and insulin-like growth factor-1 (IGF-1) mRNA expression using serum pharmacological method.Methods:Rats' GCs were incubated with 10% blank serum (as negative control group),follicle- stimulating hormone (FSH)-containing serum (S-FSH,as positive control group),or BSTCR (in different dosages) containing serum (S-BSTCR,as the BSTCR groups) for 48 h.~3H-TdR incorporation was then performed;DNA was measured to analyze the distribution of GCs in the cell cycle and their proliferation index (PI) using a flow cytometer;estradiol (E_2) and progesterone (P) content in the culture fluid were examined by radioimmunoassay;and levels of FSHR and IGF-1 mRNA expression in GCs were measured by real-time RT-PCR.Results:A dose-dependent increase of ~3H-TdR incorporation in GC was shown in the BSTCR groups.Cells in G_0/G_1 phase had markedly less,while those in S phase had a significantly higher increase in the BSTCR groups compared with the negative control group.A high value of PI was also shown in the BSTCR groups,especially in the high dose group where the influence of cell proliferation was stronger than that in the positive control group.The levels of E_2 and P in the BSTCR groups of all dosages were significantly higher than those in the negative control group,and did not show any significant difference compared with those in the positive control group.Levels of FSHR and IGF-1 mRNA expression in the BSTCR groups increased in a dose-dependent manner at levels higher than those in the negative control group.Conclusion:S-BSTCR can obviously stimulate the proliferation and steroidogenesis of ovarian GCs.It is speculated that BSTCR could play a regulatory action on ovarian function through two different pathways of endocrine and autocrine by promoting FSHR and IGF-1 mRNA expression.     

The Effects of PDTC on Interleukin-1β-induced Nitric Oxide Production in Chondrocytes

In order to find new drugs to inhibit nitric oxide （NO） production, the effects of pyrrolidine dithiocarbamate （PDTC）, a nuclear factor-kappa B （NF-κB） inhibitor, on recombinant human interleukin-1β （rhIL-1β）-induced NO production in chondrocytes were investigated. Rat chondrocytes were isolated and cultured, divided into control, P0, P1, P2, P3 and P4 groups. The chondrocytes in the P0, P1, P2, P3 and P4 groups were treated with different concentrations of PDTC （0, 3, 10, 30, and 50 p.mol/L respectively） for 1 h and then incubated with 5 U/mL rhIL-1β for 24 h. NO assay kit and RT-PCR were used to detect the NO content and the iNOS mRNA expression in the chondrocytes The expression level of iNOS mRNA in control, P0, P1, P2, P3 and P4 groups was 0.02±0.01, 1.24±0.13, 1.21±0.14, 0.61±0.11, 0.40±0.09, 0.21±0.06, and the relative content of NO was 15.8±2.7, 100±14.8, 92.6±9.3, 68.3±14.2, 27.5±9.8, 19.8±3.6, respectively. In the P0, P1, P2, P3 and P4 groups, the expression of iNOS mRNA and NO production were significantly increased as compared with those in the control group. As compared with the P0 group, the expression of iNOS mRNA and NO content in control group were lower. In the P2, P3 and P4 groups, PDTC could significantly inhibit the expression of iNOS and NO production induced by rhIL-1β in a concentration-dependent manner. It is suggested that PDTC can inhibit NO production and iNOS mRNA expression induced by IL-1β, which may provide an alternative method for the treatment of osteoarthritis.     

Effect of Dexamethasone and Aquaporin-1 Antisense Oligonucleotides on the Aquaporin-1 Expression in Cultured Human Trabecular Meshwork Cells

The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with antisense oligonucleotides （AS-ODN） were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group （0 μg/mL）. In the 4 μg/ mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.     

Wild-type smad3 gene enhances the osteoblastic differentiation of rat bone marrow-derived mesenchymal stem cells in vitro

Summary This study examined the effect of wild-type Smad3 gene on the osteoblastic differentiation of rat bone marrow-derived mesenchymal stem cellsin vitro. Bone marrow-derived mesenchymal stem cells (MSCs) were stably transfected with the complexes of pcDNA3. 0-Myc-Smad3 or pcDNA3. 0-Myc-Smad3ΔC and Lipofectamine reagent. Immunofluorescence staining was performed to evaluate the c-Myc signal in MSCs. The cell proliferation was detected by MTT method. To clarify the osteoblastic characteristics in stably transfected MSCs, alkaline phosphatase (ALP) mRNA and core binding factor α1 (Cbfa1) mRNA were investigated by RT-PCR, and ALP activity and mineralization were examined by p-nitrophenolphosphate method and alizarin red staining respectively. PD98059, a specific inhibitor of the ERK signaling pathway, was used to determine the role of ERK in Smad3-MSCs osteoblastic differentiation. c-Myc signal was detected in Smad3-MSCs and Smad3 ΔC-MSCs. The proliferation of Smad3-MSCs was slower than that of Smad3 ΔC-MSCs or V-MSCs. The relative levels of ALP mRNA and Cbfal mRNA in Smad3-MSCs, as well as ALP activity and mineralization, were markedly higher than those in Smad3 ΔC-MSCs or V-MSCs. Although ALP activity and mineralization were slightly lower in Smad3-MSCs treated with PD98059 than in those without PD98059 treatment, no significant difference was found between them (P>0.05). It is concluded that the wild-type Smad3 gene, which is a crucial component promoting bone formation, can inhibit the proliferation of MSCs and enhance the osteoblastic differentiation of uncommitted MSCs and the maturation of committed MSCs independent of the ERK signaling pathway. ZHENG Qixin, male, born in 1952, Professor This study was supported by a grant from the National Natural Science Foundation of China (No. 30170270).     

Growth inhibition and apoptosis induction in human hepatoma cells by tanshinone II<Subscript>A</Subscript>

Summary In order to study the effect of tanshinone II_A on growth and apoptosis in human hepatoma cell line BEL-7402in vitro, the human hepatoma cell line BEL-7402 was treated with tanshione I_A at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-related alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone II_A could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC₅₀ value being 6.28 μg/ml. After treatment with 1–10 μg/ml tanshione II_A for 72 h, BEL-7402 cells apoptosis with nuclear chromatin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at 5 μg/ml concentration for 12 h, 24 h, 36 h, 48 h and 72 h were (2.32±0.16)%, (3.01±0.35)%, (3.87±0.43)%, (6.73±0.58)% and (20.85±1.74)% respectively, which were all significantly higher than those in the control group (1.07±0.13)%. It is concluded that Tanshione II_A could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition. TANG Zhongzhi, male, born in 1966, Doctor in Charge This project was supported by a grant from Natural Sciences Foundation of Hubei Province (No. 2000J064).     

Expression of macrophage inflammatory protein 1α in the endothelial cells exposed to diamide

Summary In order to study whether the endothelial cells (ECs) with lipid peroxidation induced by diamide can express and secrete macrophage inflammatory protein 1α (MIP-1α), the expression of MIP-1α protein in the cells was detected by cell enzyme-linked immunosorbent assay (ELISA) and that of MIP-1α mRNA was determined by cell in situ hybridization and nuclease S1 protection assay after the ECs were exposed to different concentrations of diamide for 4 h. The chemotactic activity of MIP-1α was tested by micropore filter method using modified Boyden chambers. Cell ELISA showed that the expression of MIP-1α protein in endothelial cells exposed to 1 μmol/L, 5 μmol/L and 10 μmol/L diamide was 1.9-fold, 2.3-fold and 1.7-fold respectively as much as that in the control cells, which was statistically significant by analysis of variance. In situ hybridization revealed that the mRNA expression of ECs treated with 1 μmol/L, 5 μmol/L and 10 μmol/L diamide was 1.3-fold, 3.0-fold and 1.7-fold as much as that in the control group, which had statistical significance (F=188. 93,P<0.01). The mRNA expression in 5 μmol/L dimide treated ECs, measured by nuclease S1 protection assay, was 3.4-fold as much as that in the control group (t=8.70,P<0.05). Chemotactic response (99.50±4.31 μm) to the culture medium conditioned by 5 μmol/L diamide treated ECs, which was stronger than that (66.47±3.25 μm) conditioned by the ECs (F=404.31,P<0.05), was significantly decreased (F=192.25,P<0.05) after adding MIP-1α antibody. It suggests that diamide, a lipid peroxidation inducer, could stimulate ECs to produce high level of MIP-1α, and might play an important role in atherogenesis by promoting the migration of peripheral blood monocytes into arterial intima. YANG Limin, female, born in 1973, M. D., Ph.D. This project was supported by a grant from National Natural Sciences Foundation of China (No. 39730220).     

Influence of osteopontin short hairpin RNA on the proliferation and activity of rat vascular smooth muscle cells

To investigate the influence of osteopontin （OPN） short hairpin RNA （shRNA） on the proliferation and activity of rat vascular smooth muscle cells （VSMCs）, the expressing vector of shRNA targeting OPN was constructed and transferred into the rat VSMCs. After amplification and purification, pGenesil-1/OPNshRNA1 （PG1）, pGenesil-1/OPNshRNA2 （PG2） and pGenesil-1/OPNshRNAHK （PGH） were transfected into the cultured rat VSMC by LipofectamineTM 2000. Transfected cells were visualized by using an inverted fluorescent microscope. VSMCs transfected by optimal recombined plasmid was selected by culturing in G418 48 h later. Nude cells and cells transfected by PGH were used as control. The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. The OPN of VSMCs was suppressed by transfection of optimal recombined plasmid, and the changes in cell proliferation, adhesion and motility were evaluated by MTT, adhesion test and transwell chamber test. Levels of type I and Ⅲ collagen were measured with ELISA kit. Our results showed that VSMCs stably transfected by OPN shRNA accounted for over 50% of total cells. OPN mRNA and protein were reduced by 81% and 67% （P〈0.01） by PG1, 73% and 52% （P〈0.01） by PG2, respectively while no change was found in PGH and non-treated VSMCs. PG1 significantly suppressed the proliferation, adhesion, mobility of VSMCs and reduced the amount of type Ⅰ and Ⅲ collagen. It is concluded that recombinant plasmid can be success-fully transfected into VSMCs by LipofectamineTM 2000 and inhibit the expression of OPN. The proliferation, adhesion and mobility of VSMCs can be inhibited by knocking down OPN expression. Moreover, the transferring capability of cells is attenuated, and the secretion of type Ⅰ and Ⅲ collagen is inhibited aftter knocking-down of OPN expression. The study provides experimental evidence for clinical prevention of restenosis after percutaneous coronary intervention （PCI） by RNA interference （RNAi） technology.     

Antagonistic effects of trasilast on proliferation and collagen synthesis induced by TGF-\sB<Subscript>2</Subscript> in cultured human trabecular meshwork cellsin cultured human trabecular meshwork cells

Summary Whether tranilast had antagonistic effect on proliferation inhibition and collagen synthesis promotion induced by TGF-\sB₂ in cultured human trabecular meshwork cells was investigated. Suspension of 1×10⁴ cultured human trabecular meshwork cells of 3–5 passage was distributed in each well of a 96-well disk and divided into control group and experimental group. After 24 h, 0 μg/ml (control), 12.5 μg/ml, 25 μg/ml, 50μg/ml tranilast with 3.2 ng/ml TGF-\sB₂ were added into the incubation medium. Another 24 h later, proliferation and collagen synthesis in cultured human trabecular meshwork cells were examined respectively by using tetrazolium-based semiautomated colormetric (MTT) assay and³H-proline incorporation with liquid scintillation technique. The results showed absorbance (A) values of the experimental groups were 0.9036±0.3017, 1.1361±0.1352, 1.2457±0.1524 according to the different concentrations of tranilast, and 0.8956±0.1903 of the control group. In comparison with the control group, 25 μg/ml (q=3.23,P<0.05), 50 μg/ml (q′=4.70,P<0.01) tranilast significantly antagonized the decrease of theA values induced by TGF-\sB₂ in the cultured human trabecular meshowrk cells. In comparison with the control group [817.37±124.21 cpm/10⁴ cells], 12.5 μg/ml (620.33±80.46 cpm/10⁴ cells,q′=4.26,P<0.05), 25 μg/ml (59.4.58±88.13 cpm/10⁴ cells,q′=4.81,P<0.01), 50 μg/ml (418.64±67.90 cpm/10⁴ cells,q′=8.62,P<0.01) tranilast significantly inhibited the incorporation of³H-proline into the cultured human trabecular meshwork cells promoted by TGF-\sB₂ in a dose-dependent manner. It was concluded that tranilast had the antagonistic effect on the proliferation inhibition and collagen synthesis promotion induced by TGF-\sB₂ in the cultured human trabecular meshwork cells. Da Banghong, female, born in 1973, Resident. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 38970758).     

Trichostatin A Regulates hGCN5 Expression and Cell Cycle on Daudi Cells in vitr

Summary The expression of human general control of amino acid synthesis protein 5 (hGCN5) in human Burkitt’s lymphoma Daudi cells in vitro, effects of Trichostatin A (TSA) on cell proliferation and apoptosis and the molecular mechanism of TSA inhibiting proliferation of Daudi cells were investigated. The effects of TSA on the growth of Daudi cells were studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. The effect of TSA on the cell cycle of Daudi cells was assayed by a propidium iodide method. Immunochemistry and Western blot were used to detect the expression of hGCN5. The proliferation of Daudi cells was decreased in TSA-treated group with a 24 h IC₅₀ value of 415.3979 μg/L. TSA induced apoptosis of Daudi cells in a time-and dose-dependent manner. Treatment with TSA (200 and 400 μg/L) for 24 h, the apoptosis rates of Daudi cells were (14.74±2.04) % and (17.63±1.25) %, respectively. The cell cycle was arrested in G₀/G₁ phase (50, 100 μg/L) and in G₂/M phase (200 μg/L) by treatment with TSA for 24 h. The expression of hGCN5 protein in Daudi cells was increased in 24 h TSA-treated group by immunochemistry and Westem blot (P<0.05). It was suggested that TSA as HDACIs could increase the expression of hGCN5 in Daudi cells, and might play an important role in regulating the proliferation and apoptosis of B-NHL cell line Daudi cells. This project was supported by grant from the National Natural Sciences Foundation of China (No. 30271672).     

Ruxolitinib对人红白血病HEL细胞增殖、凋亡作用的研究

目的 探讨JAK2抑制剂Ruxolitinib对人红白血病HEL细胞增殖、凋亡作用的机制.方法 用不同浓度(0、1、5、10、50、100、500 nmol/L)的Ruxolitinib处理HEL细胞,其中0nmol/L为对照组.CCK-8法检测细胞活力;Hoechst33342荧光染色检测细胞凋亡;流式细胞术检测细胞周期;罗丹明123检测线粒体膜电位变化;试剂盒检测Caspase-3/7活性;RT-PCR检测JAK2 mRNA水平;蛋白质印迹法检测p-JAK2、p-ERK、Bcl-2、Bim蛋白表达.结果 不同浓度Ruxolitinib作用HEL细胞48h后,细胞活力分别为(97.0±4.4)％、(92.0±3.9)％、(88.0±3.7)％、(81.0±3.1)％、(64.0±2.9)％、(38.0±2.2)％;Hoechst33342凋亡细胞染色显示100 nmol/L Ruxolitinib处理细胞48 h后,亮蓝色凋亡细胞[(49.21±1.80)％]较对照组[(10.02±1.40)％]增多(P＜o.05);流式细胞术结果显示100 nmol/L Ruxolitinib作用细胞48 h后G0/G1期细胞比率[(73.1±3.6)％]高于对照组[(45.2±3.0)％];1～500 nmol/L Ruxolitinib作用12、24 h后,HEL细胞线粒体膜电位降低,Caspase-3/7活性增强;RT-PCR结果显示,不同浓度Ruxolitinib处理HEL细胞48 h后JAK2 mRNA表达呈剂量依赖性减低;蛋白质印迹检测结果显示,实验组细胞p-JAK2、p-ERK、Bcl-2蛋白表达较对照组降低(均P＜0.01),Bim蛋白表达增加(P＜0.01).结论 Ruxolitinib可能通过抑制JAK2及ERK激酶途径诱导HEL细胞凋亡.     

The Effect of Connective Tissue Growth Factor on Human Renal Tubular Epithelial Cell Transdifferentiation

Summary To investigate the role of connective tissue growth factor (CTGF) in transdifferentiation of human renal tubular epithelial cell (HKC),in vitro cultured HKC cells were divided into 3 groups: negtive control, low dose CTGF-treated group (rh CTGF, 2.5 ng/ml) and high dose CTGF-treated (rhCTGF, 5. 0 ng/ml). Then the expression of α-smooth muscle actin (α-SMA) were assessed by indirect immuno-fluorescence, and the percentage of α-SMA positive cells were assessed by flow cytometry. RT-PCR were also performed to examine the mRNA level of α-SMA. Upon the stimulation of different concentrations of rhCTGF, the expression of α-SMA were markedly stronger than that in negative controls. The percentages of α-SMA positive cells were significantly higher in the stimulated groups than that of negative controls (38.9%, 65.5% vs 2.4%,P<0.01). α-SMA mRNA levels were also up-regulated by the stimulation of rhCTGF (P<0.01). These results suggest that CTGF can promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblast (Myo-F). ZHANG Chun, male, born in 1977, M. D., Ph. D. This work was supported by a grant from the Science & Technology Foundation of Hubei Province (No. 2003AA301C14).     

Effect of IL-Iβ and TNF-α on the expression of monocyte chemotactic protein-1 in endometriotic cells

Summary To investigate the clinical significance of monocyte chemotactic protein-1 (MCP-1) produced by endometriotic tissues, the endometriotic tissues were taken from 15 patients with endometriosis. MCP-1 mRNA and MCP-1 protein were determined by dot blot analysis and enzyme linked immunosorbent assay (ELISA) in endometriotic cells cultured with or without interleukin-1β (IL-lβ, 2 μg/L), tumor necrosis factor-α (TNF-α, 20 g/L). After exposure to IL-1β or TNF-α, the expression of MCP-1 mRNA in the endometriotic cells (8.635 ±0.826, 7.031 ±0.970, respectively) were significantly higher than that in the control group (4.482±0.435, P<0.05); The expression of MCP-1 protein in IL-lβ and TNF-α group was 4.52±0.09 μg/L,2.87±0.27 μg/L, respectively, which were significantly higher than 1.74±0.16 μg/L in control (P<0.01). The results suggested that IL-l\ and TNF-α could up-regulate the expression of MCP-1 in endometriotic cells, which might be related to the development of endometriosis.     

Modulatory Effects of EPA and DHA on Proliferation and Apoptosis of Pancreatic Cancer Cells

In order to investigate the effects of eicosapentaenoic acid （EPA） and docosahexaenoic acid （DHA） on the proliferation, apoptosis of pancreatic cancer cell line SW1990 cells and the expression of cyclin E mRNA, the SW1990 cells were treated with different concentrations of EPA or DHA （20, 40, 60 μg/mL） for 0, 12, 24, 36 and 48 h respectively. By using MTF method, the inhibitory effects of EPA or DHA on the cell growth were assayed. Real time PCR was used to detect the expression changes of cyclin E mRNA after the SW1990 cells were treated with 40μg/mL EPA or DHA for different time. Flow cytometry was used to test the changes of apoptostic rate in the SW1990 cells treated with different concentrations of EPA or DHA for 24 h. The results showed that EPA and DHA could inhibit the growth of SW1990 cells in a time- and concentration-dependent manner （P〈0.01）. EPA or DHA could also significantly inhibit the expression of cyclin E mRNA in a time-dependent manner （P〈0.05）. EPA or DHA could induce the apoptosis of SW1990 cells in a concentration-dependent manner （P〈0.01）. It was concluded that ω-3 fatty acid could inhibit the proliferation of pancreatic cancer cell line SW1990 cells and promote their apoptosis. The down-regulation of the cyclin E expression by ω-3 fatty acid might be one of the mechanisms for its anti-tumor effect on pancreatic cancer.