Basic & Clinical Medicine

Identification of tumor markers with SILAC proteomics approaches

2007, 27(2): 113-121.

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Progress in glycoproteome research

2007, 27(2): 122-128.

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Proteomics study of SH3 and WW domains

2007, 27(2): 129-133.

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Research over the past decade have revealed that a fairly large set of protein are accomplished by peptide-binding domains that recognize short peptide motifs in their binding partners, such as SH2，WW，SH3，etc. Protein interaction domains play important roles in critical cellular functions, such as subcellular localization, signal transduction, and multiprotein complexes assembly. With the development of proteomics, high-throughput technology has been applied to explore binding properties and interaction networks of domains. In this review, several proteomic strategies for domain research were summarized with SH3 and WW domains as examples.

The of phosphorylated fdcal adhesion kinase in rat hepatic fibogenesis and the study on mechanism

2007, 27(2): 143-147.

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Objective To observe the relationship between phosphorylation of focal adhesion kinase(FAK) and hepatic stellate cell(HSC) proliferation in fibrotic rat liver, and to study the mechanism of phosphorylated FAK regulating HSC proliferation from the cell cycle point of view. Methods The rat hepatic fibrosis was induced by bile duct ligation (BDL). Histopathological changes were evaluated by hematoxylin and eosin staining, and by Masson's trichrome method. Numbers of activated HSCs were quantified after alpha smooth muscle actin(α-SMA) staining. The proteins of p-FAK(Tyr397), Cyclin D1 and cyclin dependent kinase4 (CDK4) in the liver tissue were assayed by Western blot. Results ①With the development of hepatic fibrosis, the positive cells of α-SMA increased obviously. The positive areas of the rat livers in model groups at week 1 to 4 (12.88%±2.63%, 22.65%±2.16%, 27.45%±1.86%, 35.25%±2.34%) were larger than that in control group (5.88%±1.46%), P＜0.01. ②Western blot analysis showed that in the sham-operated group, the p-FAK(Tyr397), Cyclin D1 and CDK4 was expressed a little. But with the development of liver fibrosis, the expression of the three kinds of proteins increased gradually in model groups at week 1 to 4. ③α-SMA correlated with p-FAK (Tyr397) positively, r value was 0.964, P＜0.01; α-SMA correlated with Cyclin D1 and CDK4 positively, r value were 0.953 and 0.906 respectively, P＜0.01; P-FAK(Tyr397) correlated with Cyclin D1 and CDK4 positively, r value were 0.969 and 0.893 respectively，P＜0.01. Conclusions In hepatic fibrogenesis process, HSC became active and proliferated, while the expression of p-FAK (Tyr397), Cyclin D1 and CDK4 increased. P-FAK (Tyr397) correlated positively with Cyclin D1 and CDK4. The mechanism of phosphorylated FAK promoting hepatic fibrosis probably concerned with regulating cell cycle related protein.

Effect of VEGF-C-ASODN on VEGF-C expression in human gastric cancer cell OCUM-2MD3 induced by liposome

2007, 27(2): 148-151.

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Objective To investigate the influence of VEGF-C antisense oligodeoxynuclecotide (ASODN) on the VEGF-C expression and transfection effects in human gastric cancer cell line OCUM-2MD3 induced by liposome. Methods The ASODN sequence, which was synthesized according to the coding region of VEGF-C gene, was transformed into human gastric cancer cell line OCUM-2MD3 induced by liposome. The transfection effects were observed by fluorescence microscopy. The changes in the expression of VEGF-C mRNA and protein were determined by RT-PCR and flow cytometry(FCM)，respectively. Results VEGF-C-ASODN could be successfully transformed into gastric cancer cell induced by liposome, and its optimal ratio was 87.93% after 12 hours. Compared with the control and oligodeoxynuclecotide (SODN) group, not only was the VEGF-C mRNA level (Ar=0.38±0.11) significantly decresaed(P<0.01), but also the protein of VEGF-C (31.26±5.17)% was dramatically lowed(P<0.01). Conclusion VEGF-C-ASODN that was transformed into human gastric cancer cell induced by liposome may inhibit the expression of VEGF-C mRNA and protein.

Analyze the indices of the screeing test of hyperlipidemia by logistie regression analysis and ROC study in chilren

2007, 27(2): 152-156.

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Objective The present study was designed to examine the serum lipid levels, investigate the risk factors of dyslipidemia in children and study the indices of screening test for dyslipidemia of children in Beijing. Methods Serum total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-c) and low density lipoprotein-cholesterol (LDL-c) were detected by HITACHI 7060 automatic biochemical analyzer in Beijing students. Body height, weight, upper body height, waist circumference, upper arm circumference and blood pressure were measured. Body mass index (BMI) was calculated according to the measurement of body height and weight. Waist-to-height ratio (WHR) was also calculated. Then a Logistic regression analysis and ROC study were conducted to examine the relationship between the above indices and dyslipidemia. Results Of 962 students, 107 were found dyslipidemia. The prevalence rate was 11.12%. The upper body height, upper arm circumference, BMI, WHR, systolic pressure and diastolic pressure in children with dyslipidemia were higher than those of normal subjects (P<0.05). BMI, WHR, diastolic pressure and positive familial history were risk factors of children dyslipidemia. Conclusion High risk group of dyslipidemia in children should be screened by BMI, WHR, pressure and familial history. And further fasting plasma lipid concentrations should be detected.

Differentiation of human mesenchymal stem cells into cardiomyocyte-like cells

2007, 27(2): 157-160.

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Objectives To prove the effects of porcine CMs extract on the differentiation of human MSCs. Methods The MSCs isolated from human bone marrow were induced by two different culture systems: 5-aza and CMs extract medium. Immunostaining against α-actin，cTnT，Cx43 and CD31 was performed. The effects of 5-aza and CMs extract on the proliferation of MSCs were analyzed by MTT method. Results MSCs cultured in CMs extract medium expressed cTnT,Cx43 and CD31; MSCs induced by 5-aza expressedcTnT and Cx43, but did not express CD31; 5-aza inhibited MSCs proliferation, whereas CMs extract stimulated MSCs proliferation. Conclusions The method of adding porcine CMs extract into the culture system can induce human MSCs to differentiate into CLCs and endothelial-like cells. CMs extract has significant effects on cell proliferation. This method with few side effects on MSCs is better than that of the well-established inducer 5-aza.

Modulation effect of aresunate on the expression of CDC25A and TGF-beta in human esophageal carcnoma

2007, 27(2): 161-165.

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OBJECTIVE To observe the effects of Art on the proliferation of human esophageal carcinoma Eca-109 cell line and explore relationship between cell cycle arrest effect of Art and the expression of CDC25A and TGF-β. METHODS Human esophageal carcinoma Eca-109 cell and healthy human peripheral blood mononuclear cell (hPBMC) were cultured. The inhibitory effects of Art on cell proliferation were determined by MTT method. The changes of cell cycle of tumor cells were assayed by flow cytometry (FCM). The expression of CDC25A mRNA was examined by RT-PCR and the protein expression of CDC25A as well as TGF-β was determined by Western blot. RESULTS Art could significantly inhibit the proliferation of Eca-109. Its IC50 was (68.80±0.76 )μmol/L. It had weaker effect on the proliferation of hPBMC. The cell cycle of Eca-109 was changed greatly after the administration of Art. The number of Eca-109 cells during G0/G1 was increased and that of cells among S phase was reduced under lower doses of Art. However, most of cells were arrested at G2/M phase under the concentration of 100μmol/L. Art could dramatically inhibit the expression of CDC25A in both mRNA and protein levels. The expression of TGF-β was increased at the same concentration of art. CONCLUSION Art inhibited the proliferation of tumor cell, modulated cell cycle by down regulation CDC25A and increased TGF-β.

Increased protrin level of PTEN in live and muscle of insulin resistance of diabetic KKAy mice

2007, 27(2): 166-168.

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Objective To detect protein PTEN in live and muscle tissue in KKAy diabetic mice and investigate whether PTEN is associated with the insulin resistance in diabetic KKAy mice. Methods: Study group was divided into normal diet C57BL group (n=7,sixteen-week-old C57BL mice) , high fat diet C57BL group (n=7), diabetic KKAy group (n=7), the latter two group were fed with normal diet until week 12, thereafter fed with high fat diet for 4 weeks. Blood glucose were measured from tail vein every week, it would be diagnosed of diabetes if blood glucose more than 300mg/dl (16.7mmol/L) in two consecutive weeks. Dispatched the mice ,took quadriceps of femoris muscle and liver tissue ,added tissue lytic solution, measured the protein concentration by Bradford method and detect the PTEN protein expression in muscle and liver tissue by Western bolts methods. Results: The PTEN protein level was significantly elevated in quadriceps muscle and live tissue in KKAy mice compared to the both age-matched C57BL control groups.(P <0.01). Conclusion: PTEN may be a potent negative modulator of the PI3K-Akt pathway and then play an important role in the development of insulin resistance.

Effects of High Glucose Concentration on P38MAPK and TGFβ2 Expression

2007, 27(2): 169-173.

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Abstract：Objective To examine the effects of glucose on the expression of P38MAPK and TGFβ2 in cultured human umbilical vein endothelial cells．Methods Human umbilical vein endothelial cells (HUVEC)were incubated in a culture medium with 11．2 mmol／l, 16．8 mmol／l and 33．6 mmol／l glucose concentrations for 24h，48 h，72 h respectively． The expression P38MAPK and TGFβ2 were measured by RT—PCR and Western-blot．Results W hen D—glucose concentrations rose, HUVEC exposed to high glucose concentration(11．2 mmol／l, 16．8 mmol／l and 33．6 mmol／L)showd increase in cell expression of P38MAPK and TGFβ2 after 48 h and 72 h exposure compared with those cultured in medium with a low glucose concentration(5．6 mmol／l)．Conclusion High concentration of glucose can arrest the proliferative response and show that even a short—term exposure of endothelial cells (ECs)to high glucose concentration leads to their activation associated with increased expression of P38MAPK and that this effect may appears more significantly along with time of exposuring high concentration of glucose．

efficacy and tolerability of alendronate daily in Chinese postmenopausal osteoporotic women

2007, 27(2): 174-177.

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Objective: To observe the efficacy and tolerability of a daily alendronate in improving bone mineral density (BMD) and bone turnover markers in Chinese osteoporotic women. Subjects and methods:274 Chinese osteoporotic women, 49-82 years old with more than 3 years of menopause period were included. 10mg Alendronate and 500mg calcium were prescribed daily. The bone mineral density (BMD, by Lunar DPX-L，Norland XR-36 or Hologic QDR-2000), urine NTX/Cr(by Elisa, kit of Osteomark, USA), serum calcium, phosphate and alkaline phosphatase levels(by autonomic analysis) were measured to evaluate the efficacy. The side effects, liver and kidney function (by autonomic analysis) were observed to assess the tolerability. Results: BMD at Lumbar 2-4, Neck, Wards and Trochanter sites were increased significantly by 4.1%, 2.5%, 3.3% and 2.4% respectively（P<0.001）. Urine NTX/Cr and alkaline phosphatase levels decreased by 31.6% and 26.7%（P<0.001 vs baseline）. It indicated that Alendronate effectively inhibited bone turnover. Body height of patients had no change. Except one case with foot phalange fracture caused by a trauma, no new fracture occurred during treatment. Blood and urine routine test, liver and kidney function were all in normal ranges. Mild upper gastrointestinal side effects were found in 4.4% cases possibly related to Alendronate. Conclusion: Alendronate was effective and well tolerated in Chinese postmenopausal osteoporotic women.

Effect of atorvastatin on MMP-9 and tissue factor induced by oxLDL in human monocytes-derived macrophages

2007, 27(2): 178-182.

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Objective To investigate the effect of atorvastatin-a HMG-CoA reductase inhibitor on the expression and activity of matrix metalloproteinase-9 (MMP-9) and tissue factor(TF) on human monocytes induced by Oxidized LDL (oxLDL).Methods Human monocytes-derived macrophages were incubated with oxLDL in presence or absence of different concentration atorvastatin.At the end of incubation,MMP-9 expression and its activity was assessed by ELISA and zymography analysis respectively,and TF expression and its procoagulant activity was assessed by ELISA and a one-stage clotting assay respectively. Results The results showed that atorvastatin inhibited human monocytes-derived macrophages proliferation in a methods of the concentration-dependment,which can be eliminated by adding 100μmol/L mevalonate. 0.1μmol/L and 0.5μmol/L atorvastatin can inhibit MMP-9 expression (1.32±0.06 VS 2.57±0.06μg/L，P＜0.05；1.25±0.04 VS 2.57±0.06μg/L，P＜0.05，respectively)and its activity of human monocytes-derived macrophages significantly,and inhibit TF expression(3.34±0.66 VS 5.78±0.86μg/L，P＜0.05 ；2.20±0.60 VS 5.78±0.86μg/L，P＜0.05，respectively) and its procoagulant activity in a methods of the concentration-dependment.Conclusion Atorvastatin inhibit the proliferation of human monocytes-derived macrophages,and also inhibit MMP-9 and TF expressions and their activities induced by oxLDL,which can lead to collagen degradation and blood clotting.

Anti-Tumor Effect and Immune Regulation of several Human Lactobacillus

2007, 27(2): 183-186.

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Objective To investigate three plant of vaginal Lactobacillus the regulative effects on macrophage and antitumor effects on murine uterocervical carcinoma number 14(U14) .Methods Established model mice transplanted with U14, then divided them into 5 groups at random.Model group and DDP group respectively were regarded as nagative and positive comparison ; Lactobacillus groups were injected 1010 each with Lactobacillus into peritoneum for 10 days successively. The TNF-α and NO content which the peritoneal macrophages secreted by KM mice secreted and the inhibitory rate on U14 cells were detected. Results The production of TNF-α and NO secreted by the peritoneal macrophages activated by Lactobacillus had greatly increased than Model control .The three strains of Lactobacillus all had the antitumor effects on KM mice bearing U14 significantly. Conclusion Lactobacillus showed a marked inhibition effects on the U14 cells and can activate macrophages to produce TNF-αand NO which may be related with the antitumor mechanism.

Characteristics of human insulinoma cells in vitro

2007, 27(2): 187-192.

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Objective To isolate human insulinoma cells, and investigate its functional characteristics. Methods Primary human insulinoma cells were isolated by using collagenase type P, and then purified by repeatedly selective cell culture. The cellular autofluorescence was checked by fluorescent microscope. Expression of insulin and islet cell autoantigens such as IA-2, IAR and ICA were checked by RT-PCR and Western blot. The cytotoxicity induced by anti-human IA-2 antibody was analyzed by MTT assay. Results The human insulinoma cells could form islet-like structure under the condition of suspension culture and presented autofluorescence under fluorescent microscope. The human insulinoma cells expressed lower level of insulin mRNA and protein as compared with a rat insulinoma cell line, INS-1. The insulinoma cells were also expressed autoantigens of islet cells such as IA-2, IAR and ICAp69. A dose dependent cytotoxicity was found when the insulinoma cells incubated with anti IA-2 antibody. Conclusion Human beta cells derived from insulinoma could be purified by selective cell culture. Since the insulinoma cells expressed insulin and islet cell autoantigens, it could be used as cell model for investigating the related mechanisms of type I diabetes.

Preliminary studies on blood sample processing and mass spectrometry standards

2007, 27(2): 193-197.

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Objective To study the influence of sampling, handling, shipping and storage on low-molecular-weight serum proteome profiling，using magnetic beads and mass spectrometry. Methods Serum samples instantly separated and aliquoted, some stored at -80℃ for up to 6 months, others stored at 4 ℃ or room temperature (25℃) from 2h to 72h. The variations of protein profiling among these conditions on WCX magnetic beads were profiled. Profiling influenced by hemolysis，multi freeze-thaw cycles，storage conditions. Results Different handling procedures and storage conditions can cause different effects on serum profiling. Serum stored at -80℃ up to 6 months, stored at 4℃ or 25℃ for 2h or one freeze-thaw cycle, had little effects on serum proteomic analysis. If serum diluted into 9 M urea buffer at room temperature, the result is stable within 24 hours. Hemolysis can cause great effect on serum profiling. Statistics analysis shows that 35.9% peaks have significant changes (p < 0.01) in hemolytic samples. Conclusion The sampling procedure and storage will affect the result of protein profiling. Serum should be prepared in 2 hours at 4℃. Serum could be shipped or experimentally processed at 25℃ after dilution with 9 M urea buffer for 24 hours. Serum may be stored at －80℃ for long-term purpose with one freeze-thaw cycle. Hemolytic sample should be avoided. Our data suggested that a standard procedure for proteomic research and biomarker discovery is necessary.

Seminal characteristics and enzymological biochmistry analyses of azoospermic males

2007, 27(2): 198-199.

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Objection To determine the predictive value of wheat germ agglutinin receptor (WGAR) on human spermatozoa surface, the frequency of abnormitic sperm morphology and human spermatozoa acrosin activities to fertilization rate of IVF-ET.Methods Spermatozoa and oocytes were obtained from both normospermic men and women patients with oviduct obstruction,They came to our center for IVF procedure. Statistics of the quantitation of WGAR on spermatozoa, the frequency of abnormitic sperm morphology, human spermatozoa acrosin activities and the fertilization rate of IVF-ET were made. Result Two groups based on the fertilization rate less than 65% (n=64)and over 65%(n=194) were divided, there was a significant difference about their amount of the WGAR(P<0.01);there was not significantly different about the frequency of abnormitic sperm morphology and human spermatozoa acrosin activities(P>0.01;P<0.05). Conclution The fertilization rate of IVF-ET was affected by the amount of WGAR on spermatozoa, the frequency of abnormitic sperm morphology and human spermatozoa acrosin activities,while the amunt of WGAR on spermatozoa might be more useful to predict fertilization rates in vitro

Cathepsin S expression in the aorta of goto-kakis rats and its relationship with atherosclerosis

2007, 27(2): 200-201.

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The empirical study on the expression of MMP13 in the xhondrocyte of rabbit's traumatic OA

2007, 27(2): 202-203.

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Genetic Polymorphism of β1 - AR Arg389Gly in the Man of beifang

2007, 27(2): 204-205.

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Abstract：　Objective 　To identify the distribut ion feature ofβ1 - ARArg389Gly gene in the Hans in Guangdong Province. Methods :224 health individuals were selected to genotyped atβ1 - ARArg389Gly receptor genes by the technique of polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Results: It was shown that the gene frequency ofβ1 -AR Arg389Gly was as followed ;genotype frequency : Gly/ Gly 9. 28 % ,Gly/ Arg 45. 46 % , Arg / Arg 45. 26 %. Conclusions :β1– AR Arg389Gly genes distribution in the Mans of beifang is different from those in other areas.

The diagnosis and management of adrenal myelolipoma

2007, 27(2): 210-212.

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Objective To review the diagnosis and therapy of adrenal myelolipoma (ML). Methods The medical records of 21 patients with adrenal myelolipoma were reviewed. All patients were examined by B-ultrasound, CT and endocrine tests while some were examined by MRI and 131I-MIBG in addition. Results All patients had undergone resection of adrenal ML. The mean diameter of tumors was 6.7cm (4.5cm~12cm). Pathological examination of all tumors confirmed the diagnosis of adrenal ML. No recurrence was observed during the follow-up period (1~15years).Conclusion Adrenal ML is a rare, benign tumor and is often discovered incidentally. Its prognosis is generally good.

Discovery brings the wish--Advances on tumor stem cells

2007, 27(2): 213-216.

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Nowadays, the study of tumor stem cells has become the heatpoint in cancer research. Many experiments have successfully demonstrated the existence of tumor stem cells, which have been isolated from some solid tumors successfully. As the research on the origin of tumor stem cell is developing, the knowledge of the occurrence and development of tumor has become more clear, which will influence the diagnosis and treatment for tumor significantly. Moreover it will bring benefit to following-up after surgical operation and giving hope to cancer sufferers.

Research and practice on new teaching model for bringing up high quality medical talent

2007, 27(2): 224-227.

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In order to meet the modern needs in medical talent education and bring up high quality medical talent, Medical College of Shantou University has aggressively and effectively conducted exploration and practice on high medical education, and developed a new teaching model for bringing up high quality medical talent under the strategies of "Reform of Course System" and "Innovation of Teaching Model". The new teaching model is designed to bring up excellent clinicians with innovative spirit and proficient basic skills. It keeps up with the global ideal of high medical education, and it is an originated teaching model in China with Chinese characteristics and a unique style of Medical College of Shantou University, which will make a positive and significant impact on Chinese high medical education.

Clinical scientific research workers calling for correct mind on statistics

2007, 27(2): 228-232.

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