[ ABSTRACT] AIM:To study the influence of insulin resistance on fatty liver in the mice fed with high-fat diet (HFD).METHODS:Male 8-week-old C57BL/6J mice were randomly divided into HFD group (with 60% calories by high saturated fatty acid) and control group (with chow diet).The mice in both groups were fed for 12 weeks.The body weight, liver weight, serum triglyceride (TG) and total cholesterol (TC), and blood glucose and insulin levels were meas-ured.Hyperinsulinemic euglycemic clamp experiment was applied to reflect insulin sensitivity .The lipid deposition in the liver was analyzed by HE staining , Sudan IV staining and measurement of liver fat content .The phosphorylation levels of IRS1 and Akt, and the protein levels of SREBP-1 and FAS were determined by Western blot to reflect the activities of insu-lin signaling and lipid synthesis .RESULTS:Compared with control group , the body weight and liver weight were signifi-cantly increased in HFD group .TG and TC contents in serum and liver tissues were remarkably increased in HFD group . High-fat diet induced insulin resistance , as evidenced by increased serum insulin levels , reduced glucose infusion rate and decreases in IRS1 and Akt phosphorylation levels .In livers of HFD group, HE staining showed that the cytoplasm of hepa-tocytes was filled with vacuoles .Sudan IV staining also displayed that many different sizes of red lipid drops existed in the hepatocytes , and the protein levels of SREBP-1 and FAS were significantly increased .In primary normal hepatocytes with exogenous oleic acid intervention for 48 h, the phosphorylation levels of IRS 1 and Akt were reduced , and the protein ex-pression of SREBP-1 and FAS was significantly increased in a dose-dependent manner .CONCLUSION: Feeding with HFD leads to insulin resistance , resulting in activation of lipid synthesis and accumulation of lipid deposition in the liver , thus inducing fatty liver .

Objective To explore the Cep70 by adjusting the stability of acetylated alpha tubulin,participate in breast cancer drug resistance mechanisms.Methods (1) In order to induce taxol drug resistance cell line Michigan cancer foundation-7 (MCF-7)/pac,high-dose shock treatments taxol MCF-7 was used for 6 months,until the cells can grow in 3.5 μmol/L of paclitaxel.(2) The 3-(4,5-dimenthylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method was used to detect inhibition rate by taxol to MCF-7 and MCF-7/pac cell.(3) Immunofluorescence and Western blot were used to test acetylated alpha-tubulin and Cep70 expression levels in MCF-7 and MCF-7/pac cells.(4) Chemical intervention was used to acetylate apha-tubulin expression,Western blot and polymerase chain reaction (PCR) were used to detect the change of acetylated alpha-tubulin and Cep70 in MCF-7 and MCF-7/pac groups.Flow cytometry and Western blot were used to detect the change of cell cycle.Results (1) IC50 of MCF-7 and MCF-7/pac was 22.47 μ mol/L and 31.38 μmol/L,respectively.(2) Immunofluorescence and Western blot results showed that the expression of acetylation of alpha-tubulin in resistant MCF-7 cell/pac was obviously decreased.(3) Real time polymerase chain reaction (RT-PCR) and Western blot showed Cep70 expression was consistent of acetylation of alpha-tubulin.(4) After incubation with paclitaxel for 24 hours,the expressions of acetylation of alpha-tubulin and Cep70 in MCF-7 and MCF-7/pac were increased,but the extent of MCF-7 cell was much higher.Instead,incubation with nocodazole after 24 hours,the acetylation of alpha-tubulin and Cep70 in MCF-7 and MCF-7/pac cells were obviously lowered.(5) After paclitaxel intervention,compared to the same group MCF-7 cells,the G2 phase ratio in MCF-7/pac cells was lower.In addition,given nocodazole after the intervention,compared to the same group MCF-7 cells,the ratio of G2 phase in MCF-7 cell/pac was significantly decreased.Conclusions Cep70 decreased the expression of the acetylated alpha-tubulin,reduced the stability of microtubules,which could be an important mechanism of taxol drug resistance.

Objective To reveal acetylated a-tubulin acts as pressure sensors,sensing the changes in extracellular matrix to impact on the invasion and metastasis of breast cancer.Methods The acetylated alpha microtubule protein expressions were detected,and its relationship with invasion and metastasis in breast cancer clinical specimens and different molecular subtypes of breast cancer cell lines were analyzed.The expression of acetylated a-tubulin was interfered through chemical methods,and cell morphology and the change of the invasion and metastasis ability were detected under the condition of different matrix hardness.Results The expression of acetylated a-tubulin was highest in basal-like breast cancer tissue and cell lines,and was lowest expressed in the luminal B breast cancer tissue and the breast cancer cell lines.The expression of acetylated a-microtubule was positively correlated with the occurrence of breast cancer.Under the condition of soft substrate cultivation,cell polarization was declined,becoming the circular or oval shape.The acetylated a-tubulin caused reduction in cell polarity,accompanied with less invasion and metastasis ability.Conclusions The acetylated a-tubulin acts as pressure sensors,sensing the sclerosis of extracellular matrix in the process of tumorigenesis,promoting invasion and metastasis of breast cancer.

Objective To evaluate the feasibility and the safety of the direct coronary stent implantation Methods Retrospectively reviewed the clinical and angiographic data of 139 patients received direct coronary stent implantation, follow up was performed in 95 of these patients Results The direct coronary stenting procedure was successful in 133 (95 7%) of 139 cases In residual 6 (4 3%) failure cases, the stent could not cross the lesion and was successful retrieved in 2 cases, balloon predilation was added In 4 cases the stent did not completely cover the lesion or dissection appeared after direct stenting, the second stent was deployed Angiographic success was achieved in all the 139 cases without major adverse coronary events during in hospital In 7 (7 4%) of 95 cases the target lesion needed repeat PTCA because of significant restenosis during 1～23 months (median 4 7 months) of clinical follow up Conclusion Direct coronary stent implantation is feasible and safe in selective patients It can save the cost of one balloon catheter

Objective To explore the characters of the aged patients with coronary disease undergoing percutaneous transluminal coronary angioplasty in clinical presentation, procedural success, and in-hospital outcomes. Methods Data were collected as a part of a prospective registry of all percutaneous coronary interventions performed by authors between January 2001 and October 2002. Comparisons between 2 age groups (≥70 years and

Objective To investigate the mechanism of glycoprotein serglycin (SRGN) promoting metastasis of breast cancer cells and the possible mechanism of SRGN expression.Methods Real time quantitative polymerase chain reaction (PCR) and bioinformation retrieval were used to detect the expression of SRGN in lymph node metastasis and non-metastasis breast cancer.MDA-MB-231 shRNA and MCF-7-SRGN of breast cancer stable cell line were established by lentivirus shRNA interferencc and overexpression.Transwell assay was used to test the effect of SRGN on invasion and metastasis of breast cancer cell line in vitro.Western blot assay was used to detect the changes of epithelial-mesenchymal (EMT) related markers.The possible regulatory mechanism of SRGN expression was detected by Western blot assay.Results SRGN expression was significantly increased in lymph node metastasis of breast cancer in clinical specimens.SRGN interference inhibited the invasion and metastasis of tumor cells.SRGN promoted breast cancer cells EMT.Transforming growth factor β1 (TGFβ1) promoted the expression of beta SRGN transcription.Conclusions SRGN can induce the change of EMT in breast cancer cells and promote the invasion and metastasis of breast cancer cells.

Objective To study the induction of amelogenin in formation of hard tissues in the pulp tissues,to provide experimental foundation for research in formation of hard tissues induced with amelogenin.Methods Sixteen male Wistar rats were divided into control group and experimental group.Cells and matrix structures and amelogenin expression in the growing ends of cyclophosphamide(CP)-affected incisors were examined by histological and immunohistochemical methods.Results Experiment group:extensive edematous changes were noted in the apical pulp tissue of the incisor associated with disappearance of odontoblasts facing to presecretory and secretory ameloblasts. The cell layer of young ameloblasts was therefore lined directly with pulp tissue in which some osteodentin-like tissue was present near the ameloblasts. Immunoreactions for amelogenins were observed in the labial side of the pulp,particularly around the newly induced hard tissues in the affected region. Conclusion Amelogenins has induction in formation of hard tissues in dental pulp following the inhibition of formation of odontoblasts by CP.

Objective To establish the method to express leukemia inhibitory factor(LIF) in fetal fibroblasts in order to provide theoretical foundation for establishment of transgenic animal model.Methods Using the fetal of 13.5 d ICR mouse,the primary fetal fibroblasts were cultivated by trypsin enzyme digestion.The lineared eukaryotic expression vector pcDNA3.1-LIF was transferred to the fetal fibroblasts of mouse with liposome induction.The positive cells were selected by G418,genome DNA of the positive cells was extracted and sequenced. Results The primary fetal fibroblasts of mouse were successfully obtained by isolating and culturing,and fetal fibroblasts expressing LIF were established by transferring.The sequencing result demonstrated that the homology of clone plasmid of positive cells was about 99%.Conclusion Eukaryotic expression vector pcDNA3.1-LIF is successfully transferred to the fetal fibroblasts of mouse.

[ ABSTRACT] AIM:To observe the effect of endogenous nitric oxide synthase ( NOS) inhibitor asymmetric dimeth-ylarginine ( ADMA ) and its signaling pathways on NO levels and skeletal muscle contractility in 4-week running rats. METHODS:The 4 weeks running rat model was established.The twitch tension, tetanic tension and the fatigue test of sole-us muscle induced by electrical stimulation ex vivo were detected.The ATP content, mitochondrial DNA levels and the mR-NA expression of peroxisome proliferator-activated receptor γcoactivator-1α(PGC-1α), nuclear respiratory factor (NRF) were measured to reflect the mitochondrial function and biosynthesis in the skeletal muscle.Serum ADMA concentration was detected by high performance liquid chromatography.The endogenous ADMA enzymes PRMT1 and 2 subtypes of ADMA me-tabolism enzyme DDAH, 3 subtypes of NOS protein expression in the skeletal muscle were determined by Western blot.NOS activity and nitric oxide ( NO) content were analyzed by colorimetric method.RESULTS: Compared with normal control group, the twitch tension, tetanic tension and the anti-fatigue capability of soleus muscle in running group were significantly enhanced, ATP content, mitochondrial DNA content and the mRNA expression of PGC-1αand NRF were significantly in-creased (P<0.01).In addition, the protein expression of constitute type NOS (cNOS) and NOS activity were significantly increased (P<0.01), but the increase in NO content was relatively smaller in soleus muscle in exercise group (P<0.05). Moreover, serum ADMA concentration in running group was increased, while the DDAH2 expression in skeletal muscle was decreased.CONCLUSION:Short-term endurance exercise enhances the twitch tension, tetanic tension and fatigue resist-
ance of soleus muscle.The mechanism may be that increased cNOS expression feedbacks to increase ADMA concentration, thus maintaining the increase in NO synthesis at a relatively low level, and resulting in promoting skeletal muscle mitochon-dria biosynthesis and mitochondrial function.

Objective:To evaluate the toxic effect of 3 different gingival retraction cords.Methods:DMEMextraction of DL-adren-aline HCl,aluminium sulphate and non-drug retraction cords with the extraction time of 5,10,15 and 30 min were respectively pre-pared and were used to culture human gingival fibroblasts(HGFs)in vitro respectively.Cell proliferation was tested by MTT assay. Cell apoptosis was examined by Annexin/PI method.Results:The 3 gingival retraction cord extractions inhibited the roliferation,pro-moted the apoptosis of HGFs(P <0.05),the effects were related to the extraction time.Conclusion:The 3 retraction cords have time-dependant cytotoxity.