2003年 · 第116卷第05期

Objective To investigate the biological characteristics of cell lines of healthy and diseased human dental alveoli. Methods Primary cell lines from either healthy or diseased human dental alveoli were obtained. Two cell lines, H-258 and H-171 derived from healthy and diseased human tissues respectively, were selected for morphological study and research on their growth and aging, using cell counting, and histochemical and immunohistochemical staining. Results Primary cell lines were successfully established from innormal dental alveoli. After freezing and thawing for three times, cell growth was continued and no morphological alterations were observed. The doubling time was 53. 4 hours and mean division index(MDI)was 4‰. Cells were kept normal after twenty generations with no obvious reduction of doubling time and MDI. Of twenty-six primary cell lines derived from healthy human dental alveoli, only three cell lines achieved generation. After freezing and thawing for twice, cultured cells were still alive at a decreased growth speed, with doubling time of 85. 9 hours and MDI of 3‰. Both cell lines, H-171 and H-258, shared the characteristics of osteoblast. Conclusions Primary cell lines of diseased human dental alveoli show greater growth potential. All cell lines of dental alveoli share characteristics of osteoblast. The technique we developed may be put into practice for the treatment of abnormal dental alveoli. Chin Med J 2003; 116(5): 781-784

Objective To investigate the correlation between glutathione S-transferase(GST)M1 and T1 genotypes and endometriosis risk(EM). Methods Polymerase chain reaction(PCR)technique was used to detect the presence or absence of the GSTM1 and GSTT1 genes in genomic DNA isolated from the blood samples of 68 Han Chinese women with endometriosis and 28 without endometriosis. Results The frequencies of GSTM1 and GSTT1 null genotypes in women with endometriosis were 0. 721(49/68)and 0. 779(53/68), respectively, and in women without endometriosis were 0. 429(12/28)and 0. 321(9/28), respectively. There was a significant difference with regard to the frequencies of GSTM1 and GSTT1 null genotypes between the women with and without endometriosis(P<0. 01). Furthermore, the frequencies of GSTM1 and GSTT1 null genotypes were significantly higher in the patients with stage Ⅲ and IV endometriosis [0.731(38/52)and 0. 788(41/52), respectively] than in women without endometriosis(P<0.01), and the frequency of GSTT1 null genotype was statistically higher in patients with stage Ⅰ and Ⅱ endometriosis [0. 75(12/16)] than in the women without endometriosis(P < 0. 01). No correlation between GSTM1 and GSTT1 null genotypes and age, induced abortion or dysmenorrhea was detected in this study(P>0. 05). Conclusion GSTM1 and GSTT1 null genotypes may be risk factors for the development of endometriosis. Chin Med J 2003; 116(5): 777-780

Objectives To determine the pre-therapeutic serum level of vascular endothelial growth factor(VEGF)in patients with hepatocellular carcinoma(HCC)and to elucidate the relation between the serum level and clinical characteristics and metastasis of HCC. Methods One-hundred and fifteen HCC patients, 40 patients with benign liver lesions, and 30 healthy control subjects were included in this study. The serum VEGF level was measured with the quantitative sandwich enzyme linked immunosorbent assay(ELISA, R&D systems). Results The serum VEGF levels in the HCC group(465. 62 ±336.24 pg/ml)was significantly elevated as compared with those in patients with benign liver lesions(159. 54 ±120. 58 pg/ml)and those in normal controls(123. 53 ±51.84 pg/ml). The VEGF levels were not significantly different between the patients with benign liver lesions and the normal controls. The serum VEGF levels showed a positive rate of 77. 4%, 25%, and 3. 3% in the HCC patients, benign liver lesion patients and normal controls, respectively. In the 115 HCC patients, the serum VEGF levels in patients with portal vein(PV)emboli(n =26, 582. 76 ±441. 89 pg/ml), with metastasis(n =43, 548. 29 ±438. 57 pg/ml)or with large HCC lesions(≥ 5 cm in diameter)(n = 69, 554. 43 ±369. 99 pg/ml)were significantly higher than those without PV-emboli(n =89, 431.39 ± 292. 84 pg/ml), without metastasis(n = 72, 416. 24 ±247. 27 pg/ml)or with small HCC lesions(n = 42, 328. 67 ±227. 47 pg/ml). The serum VEGF levels in stage Ⅰ, Ⅱ,Ⅲ, IVa and IVb HCC patients were 340. 6 pg/ml, 451.55 ± 307. 84 pg/ml, 397. 44 ± 257. 18 pg/ml, 486. 10 ± 397. 73 pg/ml and 647. 93 ± 344. 56 pg/ml, respectively. Conclusion The pre-therapeutic serum VEGF levels in HCC patients appear to reflect the disease's potential activity of vascular invasion and metastasis. Chin Med J 2003; 116(5): 772-776

Objective To investigate the effects of lactulose on intestinal bacterial overgrowth(IB0), bacterial translocation(BT), intestinal transit and permeability in cirrhotic rats. Methods BT in all animals was assessed by bacterial culture of mesenteric lymph node(MLN), liver and spleen, and IBO was assessed by a jejunal bacterial count of the specific organism. Intestinal permeability was determined by the 24-hour urinary99mTc-diethylenetriamine pentaacetatic acid(99mTc-DTPA)excretion, and intestinal transit was determined by measuring the distribution of51 Cr in the intestine. Results BT and IBO were found in 48% and 80% of the cirrhotic rats, respectively, while not in the control rats. Cirrhotic rats with IBO had significantly higher levels of intestinal endotoxin higher rates of bacterial translocation, shorter intestinal transit time and higher intestinal permeability than those without IBO. It was also found that BT were closely associated with IBO and injury of the intestinal barrier. Compared with the placebo group, lactulose-treated rats had lower rates of BT and IBO, which were closely associated with increased intestinal transit and improved intestinal permeability by lactulose. Conclusions Our study indicate that endotoxin and bacterial translocation in cirrhotic rats may attribute to IBO and increased intestinal permeability. Lactulose that accelerates intestinal transit and improves intestinal permeability might be helpful in preventing intestinal bacterial and endotoxin translocation. Chin Med J 2003; 116(5): 767-771

Objective To evaluate the effects of Carvedilol on cardiopulmonary bypass(CPB)-induced myocardiocyte apoptosis and its effects on regulation of Fas, FasL expression, caspase-3 activity and oxidative stress in the left ventricle(LV)in this setting. Methods Ten adult dogs undergoing conventional hypothermic CPB were randomly divided into control and Carvedilol treated groups(n =5, respectively). Dogs in Carvedilol treated group received a bolus of Carvedilol(1 mg/kg)intravenously and a maintenance dosage of Carvedilol(3 μg · min-1 ·kg-1)for 3 hours after the reperfusion of the heart. Dogs in control group received no Carvediolol. LV samples were obtained before, during and 3 hours after CPB. In situ nick end-labeling(TUNEL)technique was used to detect the apoptotic cells. The expressions of Fas and FasL were detected immunohistochemically and quantified by fluorescence activated cell sorting(FACS). The activity of caspase-3 enzyme and malondialdehyde(MDA)level were measured by cleavage of Z-DEVD-AMC substrate and thiobarbituric acid reactive substance(TBARS)method, respectively. Results Before and during CPB, all the parameters were not significantly different intra-or between groups(P>0. 05). After CPB, these parameters in both groups were significantly elevated compared with those of before and during CPB(P < 0. 028, respectively). However, the number of apoptotic cells in Carvedilol treated group was significantly decreased compared with that of the control group(P<0. 021). The expressions of Fas and FasL were significantly downregulated by Carvedilol(P<0. 001 and 0. 003, respectively). The caspase-3 activity and the content of MDA in the Carvedilol treated group was also significantly reduced(P<0. 026 and 0. 005, respectively). Conclusions Carvedilol significantly reduces CPB-induced cardiomyocyte apoptosis in dog hearts and the reduction of cardiomyocyte apoptosis is associated with downregulation of Fas and FasL expression, inhibition of caspase-3 activity and oxidative stress in LV. Chin Med J 2003; 116(5): 761-766

Objective To investigate the effects of Salvia miltiorrhiza(SM)extracts on the expression of heme oxygenase-1(HO-1)and nitric oxide synthase(NOS)in small pulmonary arteries(SPAs)of rats with chronic hypoxia. Methods After two weeks of hypoxia, rats were treated with diltiazem, and small, median, and large doses of SM extracts. The lungs were tested for the expression and distribution of HO-1, endothelial NOS(eNOS)and inducible NOS(iNOS)by using immunohistochemistry and Western blot method. Results Median and large doses of SM extracts significantly reduced hypoxia-induced media thickening in the SPAs(similar with diltiazem), recovered repaired ultrastructure injury, decreased HO-1 and iNOS levels, and increased eNOS expression in the SPAs. Conclusions Median and large doses of SM extracts play significant roles in inhibiting structural remodeling in rats with hypoxic pulmonary hypertension. These effects might attribute to the suppression of HO-1 and iNOS, and the promotion of eNOS expression under the conditions of hypoxia. Chin Med J 2003; 116(5): 757-760

Objective To investigate the signaling pathway through testing the effects of dexamethasone(Dex)on the activation of the extracellular signal-regulated protein kinase 1/2(ERK1/2)and p38 kinase(p38)in H0-8910 cells. Methods Activation of the ERK1/2 and p38 was detected by Western blotting using the antibodies against the total ERK1/2 and p38 mitogen-activated protein kinases(MAPKs)protein and the phosphorylated forms of them. Results Dex could suppress the activation of ERK1/2, while enhance the activation of p38 rapidly and strongly in a dose-and time-dependent manner. Neither effect could be blocked by RU486, the antagonist of glucocorticoid receptor(GR). Conclusion Dex has rapid effects on the activation of ERK1/2 and p38, and these effects are not mediated by GR. Chin Med J 2003; 116(5): 753-756

Objective To evaluate the role of nuclear factor-kappa B(NF-κB) and inhibitory kB alpha(IκBα)in hepatocellular cacinoma(HCC)SMMC7721 cells, the consequence of NF-κB inhibition in SMMC7721 cells transfected with mutated IkBoc(mIkBa)plasmid and the effect of stable inhibition of NF-κB activity in combination with Doxorubicin. Methods Western blot was used to determine the expression of NF-κB and IkBoc in SMMC7721 cells and normal liver cells. Nuclear protein was used to evaluate the binding of the 32P-labeled tandem kB sequence using electrophoretic mobility shift assay and the expression of NF-κB using Western blot between SMMC7721 cells transfected with mlKBa plasmid(SMMC7721-MT)and control cells. Furthermore, cell viability was plotted between SMMC7721-MT and control cells. The binding of kB sequence and cell viability between SMMC7721-MT and control cells at different concentrations of Doxorubicin were also investigated. Results Western blot analysis for nuclear extract showed more P50(NF-κB1)and P65(RelA)expression in SMMC7721 cells compared with normal liver cells. The expression of cytosolic IkBα protein in SMMC7721 cells was less than that in normal cells. SMMC7721-MT cells inhibited NF-κB nuclear translocation at 0, 24, 48 and 96 hours. Furthermore, NF-κB cannot be detected in the nuclear protein of SMMC7721-MT cells by Western blot. By calculating cell viability, the proliferation of SMMC7721-MT cells was shown to be suppressed more significantly than that of control cells. NF-κB in untransfected cells was activated by Doxorubicin in a dose-dependent manner, but that in SMMC7721-MT cells was not induced at low concentrations of Doxorubicin. Compared with untransfected cells, the viability of SMMC7721-MT cells was significantly suppressed at the same concentration of Doxorubicin(P<0. 01). Conclusions The present study demonstrates that upregulation of NF-κB and downregulation of inhibitory kappa B(IkBα) in SMMC7721 cells are related with the growth of hepatocellular cacinoma cells. Stable expression of mIkBα in SMMC7721-MT cells can inhibit NF-κB nuclear translocation and suppress cell growth. Furthermore, stable inhibition of NF-κB activity in combination with Doxorubicin can significantly inhibit cell proliferation in SMMC7721-MT cells. Thus, modulation of NF-κB may represent an improvement in the efficacy of HCC therapies and be worthy of further research and investigation. Chin Med J 2003; 116(5): 747-752

Objective To assess the effects of nuclear translocation of tissue transglutaminase(TTG)and the release of cytochrome C on hepatocyte apoptosis and to reveal the mechanism of signal transduction of early apoptosis in injured hepatocytes. Methods Hepatocytes isolated from tissue transglutaminase gene knock-out mice and rats were stimulated with ethanol. Proteins from whole cell, cytoplasm and nuclei were extracted for determination of TTG activity by 14C-putrescine incorporation. Distribution of TTG throughout the entire cell, as well as just nucleus was observed under a confocal scanning microscope. The amount of cytochrome C released from mitochondria was determined by ELISA. Cell apoptosis was observed by fluorescent cytochemistry. Results TTG activity in whole cells and nuclei was significantly increased after the hepatocytes were treated with ethanol. Cytochrome C release was remarkably increased in the cells isolated from rat and wild-type mouse after treatment with ethanol but not in TTG gene knock-out mice. Cellular apoptosis appeared in hepatocytes isolated from rats and wild-type mice but not in the hepatocytes from TTG gene knock-out mice after stimulation with ethanol. Conclusions Increased TTG in hepatocytes can be translocated into the nucleus and promote release of mitochondrial cytochrome C into the cytoplasm. Passing through a series of signal pathways, hepatocyte apoptosis is induced eventually. Chin. Med J 2003; 116(5): 742-746

Objective To observe the antileukemic effect in relapse patients by infusion of donor immunocompetent cells with or without granulocyte colony-stimulating factor(G-CSF)mobilization. Methods Twenty patients with leukemia in relapse after allogeneic bone marrow transplantation(allo-BMT)were treated with chemotherapy followed by donor-derived lymphocytes(DDL)without G-CSF mobilization(Group A, n = 11), or donor peripheral blood progenitor cells(PBPCs)with G-CSF mobilization(Group B, n=9). Results Five patients in Group A were in hematologic relapse. After DDL infusion, 3 of 5 patients had a temporary complete remission(CR)and relapsed after 3, 7 and 10 months, respectively. One achieved partial remission and died of interstitial pneumonia; and the other one showed no response. Another 6 patients in Group A were in cytogenetic relapse or central nerve system(CNS)leukemia, and all achieved CR and remained in disease free survival(DFS)for 10 to 98 months after DDL infusion. All 9 patients in group B were in hematologic relapse. Three patients with chronic myeloid leukemia(CML)had cytogenetic and molecular remission for 16, 35 and 51 months, respectively after PBPC infusion; and 5 patients with acute lymphoid leukemia(ALL)had CR and were still in CR for 10 to 18 months except 1 patient relapsed soon. And the other one with AML showed no response to the therapy. Conclusion Donor immunocompetent cells infusion is an effective therapy for relapsed leukemia after allo-BMT, especially for the patients with early(molecular and cytogenetic)or CNS relapse. Infusion of donor PBPC mobilized by G-CSF seems to have more potentiated graft-versus-leukemia(GVL)effect than DDL infusion. Chin Med J 2003; 116(5): 736-741

Objective To explore low density lipoprotein(LDL)oxidation by macrophage myeloperoxidase(MPO)at molecular level. Methods Using a mouse macrophage model, we examined the relationship between LDL oxidation and macrophage MPO by measuring macrophage MPO activity, LDL oxidation products, MPO gene expression and cellular orientation of LDL oxidation. Results MPO gene expression increased to its maximum gradually when the concentration of LDL was increased, and then maintained at that level. NaN3 inhibied the elevation of MPO activity and LDL oxidation, which was LDL concentration-dependent. After the composition of macrophage membrane was roughly analyzed, it was determined that the contents of MPO and LDL in 5% sucrose were 7. 667 and 21 times higher than those in 10% sucrose, respectively. Conclusion LDL is attached to the "microdomain"of the macrophage membrane in which LDL is oxidized by MPO. Chin Med J 2003; 116(5): 791-793

Objective To investigate the effects of fresh and preserved amniotic membrane on polymorphonuclear neutrophils(PMNs)so as to understand the anti-inflammatory mechanism of amniotic membrane transplantation. Methods Conditioned medium was collected 48 hours after fresh or preserved amnions were cultured in DMEM and 5% C02 at 37℃. Then, polymorphonuclear cells were cultured in conditioned culture or DMEM. Fluorescent microscopy with 4', 6-diamidino-2-phenylindole(DAPI)staining and cytometry were performed 6, 9, 12, and 15 hours later. Results Apoptotic neutrophils were found in each group at different time points. The percentage of apoptotic cells at 6, 9, 12, and 15 hours after culture in the fresh and preserved amnion groups and the control group was 17. 3%, 24. 4%, 29.8%, 37.1 %, and 16. 2%, 20.1 %, 23. 7%, 27.7%, and 10. 2%, 13. 7%, 21.1%, 26. 4%, respectively(t test, P1 <0. 01, P2 <0. 01 and P3 <0. 01). Conclusion Amniotic membrane can accelerate apoptosis of polymorphonuclear neutrophils, reduce inflammation, and prevent ocular surface collagen from resolution, indicating that fresh amnion might have a stronger effect than preserved amnion. Chin Med J 2003; 116(5): 788-790

Objective We designed a multi-center, double-blind, randomized, parallel, with metformin controlled clinical trial to evaluate the efficacy and safety of low dose rosiglitazone combined with sulphonylurea therapy in type 2 diabetic patients who were inadequately controlled with sulphonylurea alone. Methods Patients were treated with 4 mg rosiglitazone once daily plus sulphonylurea(test group)or 0. 5 g metformin twice daily plus sulphonylurea(control group)for 12 weeks. The mean levels of HbA1c, fasting and postprandial plasma glucose were recorded and compared between the two groups. Results The mean levels of HbA1c decreased by 1. 09% and 0. 95% in the test group(n = 102)and control group(n =96)respectively. Fasting and postprandial plasma glucose levels in the test group decreased by 25. 0% and 35. 6%, and in the control group, decreased by 17. 7% and 23.8% as compared with the baseline(both P<0. 01). No liver damage was found. Conclusion Combination treatment of rosiglitazone and sulphonylurea can effectively improve glycemic control in type 2 diabetic patients inadequately controlled with sulphonylurea alone. Chin Med J 2003; 116(5): 785-787