Volume 88  ·   Issue 08  ·  2008

Surgical treatment is an important treatment for breast cancer. The rational application of adjuvant chemotherapy, endocrine therapy, molecular targeted therapy and preoperative neoadjuvant therapy to prevent recurrence and metastasis after operation has significantly improved the therapeutic effect of operable breast cancer patients and won more opportunities for cure.

The microenvironment of malignant tumor (tumor) is composed of tumor cells and their surrounding fibroblasts, epithelial cells, innate and specific immune cells, constituent cells of tumor blood vessels and lymphatic vessels, tissue-specific mesenchymal cells and their expression products, metabolites and other components. It is the local environment for tumor development and growth. All components except tumor cells are collectively referred to as tumor stroma.

Objective To evaluate the accuracy of proton magnetic resonance spectroscopy (1H MRS)in quantitative measurement of intra-hepatic triglyceride (TG) stores in fatty liver at the levels of animal model and human body.Methods 40 male Wistar rats were randomly divided into 2 equal groups:fatty liver model group,fed with high fat diet for 16 weeks,and control group,fed with standard diet.Every 4 weeks 5 rats from each group underwent 1H MRS to examine the intra-hepatic triglyceride contents and then were sacrificed.The liver tissues were stained with Sudan Ⅲ to detect the fat content.Eleven hospitalized patients with falty liver suspected or confirmed by CT and with indications of liver puncture underwent 1H MRS and then liver puncture within 7 days.Linear regression was conducted to analyze the correlation between the results of 1H MRS and liver pathology.Resuits Intrahepatic lipid was detected in all the rat models.some rats of the control group,and the patients with fatty live.The lipid peak area and the ratio of the CH2 lipid peak area relative to the water peak area increased along with the duration of high fat diet feeding.In the rat models there was a close positive correlation between the hepatic TG content measured in vivo by 1H MRS and pathological measurement of liver fat content [r=0.899;P＜0001;slope 14.45;y-intercept-0.51].The similar correlation existed in the patients too [r=0.814;P＜0001;slope 33.72;y-intercept 27.04].When the data of the rats and patients were analyzed together,the percentage of liver fat was highly positively correlated with the ratio of area under fat peak to the area under water peak (r=0.878:P＜0001;slope 56.33;y-intercept-0.68).Conclusion A relative quantitative method to measure intrahepatic lipid content by 1H MRS,a safe,non-invasive method,is primarily established at the animal level and has been verified in humans with a good correlation with the pathological examination.

Objective To investigate the superiority of curative resection with hemihepatectomy in treatment of hilar cholangiocarcinoma and the strategy to lower the incidence of complications and mortality.Methods The clinical data of 50 patients with hilar cholangiocarcinoma who underwent surgical procedures in Peking University Schod of Oncology from January 1998 to May 2006 were retrospectively analyzed.Results Forty-one patients (56.9%) underwent surgical resection.Twenty-two (53.6%) received a radical operation (RO).Thirty patients underwent preoperative percutaneous transhepatic cholangio-drainage (PTCD) to reduce the jaundice.Curative resection with hemihepatectomy was performed on 19 cases including 14 cases undergoing RO.Two patients who had undergone hemihepatectomy died of liver failure.The postoperative morbidity rate was 62% in all 50 cases,78% in the hemihepatectomy group.The 1-and 2-year survival rates of the hemihepatectomy group were 57.1% and 27.3% respectively.Conclusion Curative resection with hemihepatectomy is a safe and effective option for hilar cholangiocarcinoma,especially for Bismuth Ⅲ or Ⅳ type.Preoperative biliary drainage is necessary before a major hepatic resection.

Objective To investigate the effects of alprostadil (Lipo PGE1) in prevention of portal vein thrombogenesis (PVT) after splenectomy for portal hypertension.Methods Seventy-six patients with portal hypertension undergoing splenectomy and pericardial devascularization were randomly divided into 2 groups:treatment group (n=40),receiving intravenous drip of injection of radix Salviae miliorrhazae (RSM)40 ml and alprostadil 20μg,both once a day since the third day after operation for 2 weeks and then oral administration of dropping pill of SM,and control group (n=36),receiving intravenous drip of injection of RSM and taking enteric coated aspirin 3 times a day for 2 weeks and then taking dropping pill of SM.Platelets (PLT),prothrombin time (PT),and liver function were detected periodically.Color Doppler uhrasonography was conducted every week to observe the blood flow velocity and diameter of the portal and splenic veins.and if PVT event and ascites occurred.All patients were followed up for 8-20 months.Results No prolongation of coagulation time and bleeding tendency was found in both groups.The PLT number increased remarkably in the 7th to 14th days after operation without significant difference between the 2 groups(P＞0.05).The PVT rate of the treatment group was 5.0％,significantly lower than that of the control group(25.0％,χ2=6.12,P＜0.05).The ascites rate of the treatment group was 10.0％,significantly lower than that of the control group (33.3％,χ2=7.44,P＜0.01).The levels of ALT and total bilirubin 7 and 16 days after operation of the treatment group were all significantly lower than those of the contml group (all P＜0.05).Conclusion Use of alprostadil early after devascularization is an effective and safe measure to prevent PVT.improve liver function.and decrease ascites rate.

Objective To investigate the expression of macrophage migration inhibitory factor (MIF) in breast cancer and to analyze the relationship between MIF expression and clinicopathological features,tumor microvessel density(MVD),and prognosis.Method The expression of MIF in 121 samples of breast cancer,20 samples of breast benign tumors,and 20 samples of normal breast tissues were examined by tissue microarray using immunohistoehemistry.The values of MVD in the breast cancer samples were examined by immunohistochemistry using anti-CD34.The association of MIF expression with the clinicopathological characteristics and prognosis was further analyzed using the computer software Statistical Package for the Social Sciences 13.0.Results The MIF positive expression rate of the breast cancer tissues Was 29.8％,significantly higher than those of the benign tumors and normal breast tissue (P=0.031).MIF expression rate was positively correlated with the expression of interleukin-8(IL-8)(P=0.014),MVD (P=0.043),and HER-2 (P=0.030).The survival rate of the patients with positive MIF expression in breast tissues was 80.6％.not significantly different from that of the negative MIF expression group (87.1％,P=0.171),however,the tumor-free survival rate of the MIF positive expression group was 72.2％,significantly lower than that of the MIF negative group (87.1％,P=0.029).Conclusion MIF may predict a poor prognostic factor for human breast cancer,and may be used as a novel molecular marker to predicate the progression of breast cancer.Through promoting the secretion of IL-8,MIF participates in the angiogenesis in breast cancer.

Objectlve To study the clinical features and prognosis of hepatitis C virus (HCV)-related cirrhosis after the first occurrence of complications.Methods The clinical data of 89 decompensated HCV-related cirrhosis patients were analyzed.Univariate and multivariate analyses of the factors influencing the clinical decompensation were conducted.Results Ascites was the most frequent first decompensation (44.9％),followed by upper gastrointestinal bleeding (23.6％),and serf-originated peritonitis (20.2％),and hepatic encephalopathy (11.2％).During the follow-up of 62 months (60-66 months) ascites was the most frequent first decompensation (47.2％),followed by self-originated peritonitis (18.0％),upper gastrointestinal bleeding(15.7％),and hepatic encephalopathy(7.9％).The 5-year survival rates after of the patients with hepatic encephalopathy,ascites,upper gastrointestinal bleeding and self-originated peritonitis as the first decompensated complications were 64.5％,85.0％,75.0％,and 83.3％ respectively.Multivariate regression analysis showed that esophageal and gastro varices and billirubin were independently correlated with survival.Conclusion Hepatitis C iS a slowly progressing disease.Decompensation occurring in hepatitis C is significantly correlated with survival.

Objective To evaluate the inhibition of CD4+ CD25+ regulatory T cells (Treg) in the chronic hepatitis B patients.Methods Peripheral blood samples were collected from 22 patients with chronic hepatitis B (CHB) and 18 healyhy blood donors to isolate the peripheral blood mononuclear cells (PBMCs).Flow cytometry was used to analyze the proportion of CD4+ CDl27lo CD25hi-int Tregs in the CD4+ T cells so as to calculate the proportion of CD4+ CD25+ Tregs in the CD4+ T cells.BrdU incorporation method was used to evaluate the immune inhibition of the CD4+ CD25+ Tregs.CD4+ CD25- cells were isolated by magnetic bead sorting technique.The CD4- T cells and CD4+ CD25- T cells ere mixed and stimulated by HBVcore 18-27 peptide.The PBMCs of the CHB patients with the Treg depleted and Treg not depleted underwent detection of HBVcorel8-27 specific cytotoxic T lymphocytes(CTLs).The IFN-γ secretion of the CTLs in the PBMCs of CHB patients with Treg depleted and Treg not depleted was detected by HLA-pentamer and enzyme-linked immunospot assay (Elispot).Results The proportion of CD4+ CDl27lo CD25hi-int Treg in the CD4+ T cells used to reflect the percentage of CD4+ CD25+ Tregs in the CD4+ T cells of the CHB patients was 4.3±2.4％.significantly higher than that of the healthy controls (2.1％±1.3％,t=3.74,P＜0.01).There was no significant difierence in the inhibition of CD4+ CD25- T cells by autogeneous CD4+ CD25+ T cells between the CHB patients and healthy controls.The frequency of CTLs induced by HBV core 18-27 of the CHB patients with their CD4+ CD25+ cells in circulation depleted was 0.74％±0.31％,significantly higher than that of the patients whose CD4+ CD25+ cells in circulation were not depleted (0.17％±0.08％,t=4.75,P＜0.01).The frequency of IFN-γ secreting spots of HBVcorel8-27-specific CD8+ T cells of the CHB patients with their CD4+ CD25+ cells depleted was (112±33),significantly higher than that of the CHB patients whose CD4+ CD25+ cells in circulation were not depleted [(23±14),t=7.828,P＜0.01)].Conclusion The proportion of CD4+ CD25+ Treg in CHB patients iS increased compared to the healthy blood donor.The proliferative capacity of CD4+ CD25- T cells iS inhibited by the presence of CD4+ CD25+ Treg dose-dependently,and the inhibition of CD4+ CD25+ Tregs in the CHB patients iS similar to the inhibition of CD4+ CD25+ Tregs in healthy donors.The elimination of Treg cells followed by stimulation with HBVcore18-27 peptide significantly improves the antivirus CTL responses in CHB patients.

Objective To explore the effects of pravastatin in prevention of diabetes and mechanisms thereof.Methods 1183 to 4-week-old female non-obese diabetic (NOD) mice were randomly divided into 4 groups:control group (n=30),fed with regular diet,low-dose pravastatin group (n=29),glucose was measured every week.Eight 12-week old mice without onset of DM from each group were killed with the pancreas taken out to undergo insulitis scoring via microscopy.Another 8 12-week old mice without onset of DM from each group were killed with their spleens taken out.Suspension of splenoeytes was made,put into a 96-well plate,and stimulated by rGAD65;and[3H]-thymidine was incorporated.The stimulation index (SI) of the splenocytes was calculated.Flow cytometry was used to observe the population of CD4+ CD25+ regulatory T cells.ELISA was used to detect the interferon (IFN)-γ and interleukin (IL)-4 levels in the supernatants of splenoeytes.RT-PCR was used to detect the mRNA expression of IFN-γ and IL-4 in the spleen.Resuits At 30 weeks of age.the incidence rate of DM onset of the high-dose pravastatin group was significantly lower than that of tlle control group (P=0.003),and the incidence rates of DM onset of the medium and lOW dose groups were not significantly different from that of the control group (both P＞0.05).The severity of insulitis at 12 weeks of age of the high-dose pravastatin group was significantly lower than that of the controls group (P＜0.001).There were no significant difference in the SI level and in the percentage of CD4+ CD25+ regulatory T cells among the four groups (all P＞0.05).The IFN-γ level in the supernatant of splenocytes of the high-dose pravastatin group was (42±20) pg/ml,significantly lower than that of the control group[(157±32)pg/ml,P=0.000].The IFN-γ mRNA expression level in the spleen of the high-dose pravastatin group was 0.24±0.10,significantly lower than that of the control group (0.81±0.18.P=0.000).The IL-4 level in the supematants of splenocytes of the high-dose pravastatin group was (91±22)pg/m,significantly higher than that of the control group [(44±20)pg/ml,P=0.000)].The IL-4 mRNA expression level in spleen of the high-dose pravastatin group was 0.39±0.1 8,significantly higher than that of the control group (0.20±0.08,P=0.002).However,there were not significant differences in the IFN-γ and IL-4 levels in the supematant and the IFN-γ and IL-4 mRNA expression in spleen among the medium dose,low dose,and control groups (all P＞0.05).Conclusion High-dose pravastatin therapy at early time decreases the IFN-γ level and increases the IL-4 level,shins the immune response to the direction of Th2,and subsequently lessens insulitis and prevents DM.

：Objective To investigatethe influence of heat shock (HS) preconditioning on the ultra-structure of gastric mucosalcells and the activity of eytochrome oxidase (CCO) and superoxide dismutase (SOD) inmitochondria isolated from gastric mucosa of burned rats in order to investigate itsprotective mechanism on burn-induced acute gastric mucosal lesion.Methods Ninety-sixWistar rats were divided randomly into two groups:(1)burn model group (n=40) undergoingburning and made into model of acute gastric mucosal lesion;and then subdivided into 5equal subgroups:3,6,12,24,and 48 h after burning;(2)normal control group (n=8);(3) HSpreconditioning+burn group (n=40),undergoing burning 24 h after HS preconditioning;and (4)HS preconditioning group (n=8),without burn as experimental control A number of rats weresacrificed and laparotomized before and 3,6,12,24,and 48 h after the buruings.Specimenswere obtained from the gastric antrum,bodies if stomach and gastric lesion to undergomicroscopy and determination of the ulcer index (UI). Streptavidin-peroxidaseimmunohistochemistry was usod to detect the protein expression of heat shock protein(HSP)-60 and HSP-70.Mitochondria were isolated and the activities of CC0 and SOD inmitochondria were measured.The dynamic postburu changes in micro and ultra structure ofgastric mucosal cells of scalded rats were obserred.Results The rats of the burn groupshowed conspicuous gastric mucosal lesions.The UI levels at every time point of the HSgroup were all the lowest in comparison with other groups.The expression levels of HSP70and HSP60 of the HS group,especially those at the time points 3,6,12,and 24 h weresignificantly higher than those of the burn group (P＜0.05 or P＜0.01).Theactivities of mCCO and mSOD of the HS group did not decrease remarkably at any time pointin comparison with the control groups,and the activities of mCCO and mSOD at the timepoints 6,12,and 24 h ofthe HS group were all significandy higherthan those of the bumgroup (P＜0.05 of P＜0.01).Electron microscopyshowed that the ultrastructural lesion was mild and alteration of gastric mucosalmitochondria was not significant in the HS group compared with those ofthe burn group.Apositive correlation was shown between SOD and HSP70 (r=0.436,P＜0.05)and also between CCO and HSP60 (r=0.679.P＜0.05).Conclusion HS preconditioning ameliorates theburn-induced acute gastric mucosal lesion and has a protective role to gastric mucosalmitochondria.The role of HS preconditioning on mitochondria iS associated with theprotection of the activity of antioxidase and CCO bv HSP60 and 70.

Objeetive To investigate the effects of mifepristone (MIF) on the growth of breast cancer.Methods Forty female athymic BALB/c-nude mice underwent subcutaneous injection of breast cancer cells of the line MCF-7,ER+/PR+.Ten days later when tumor nodules were formed,the mice were randomly divided into 4 equal groups to be administered with MIF of the concentrations of 25 mg,50 till 3 weeks later.Ten mice were used as normal control group,undergoing gastric perfusion of vegetable oil.Parts of the animals were killed 2 weeks later,and tlle remaining mice were all killed 3 weeks later.The tumors were taken out and underwent immunoehemistry to measure the protein expression of CD34,estrogen receptor(ER),progesterone receptor(PR),vascular endothelial growth factor(VEGF),bcl-2,Ki67,p53,and CerbB-2.Microscopy was used to measure the microvessel density (MVD).Results The growth velocity of tumor of the mice of MIF groups were all slower than that of the control group (all P＜0.01).The MVD levels of the MIF groups all decreased time-and dose-dependently.Microscopy showed that in the tumor tissues heteromorphism was significant,pathological earyomitosis was more remarkable,karyoplasmic ratio was greater,endochylema was deep blue,mesenchyma was sparse,and zone of neoplasm necrosis became lager in comparison with the control group.The expression levels of VEGF,bcl-2,Ki67,p53,and CerbB-2 of the MIF groups were all significantly lower,time and dose-dependenfly,than those of the control group(all P＜0.05).Conclusion MIF inhibits the growth of breast cancer by the mechanisms related to apoptosis promotion and inhibition of angiogenesis,so it can be used in breast cancer endocrine therapy.

Objective To study the protective mechanism of ischemic preconditioning (IPC) and diazoxide preconditioning (DPC) against myocardium ischemia-reperfusion (I/R) injury.Methods The hearts were taken out from 30 male Wistar rats and were divided randomly into 3equal groups:I/R group undergoing 30-min equilibration perfusion and 30-min ischemia and then 60-min reperfusion,IPC group undergoing 10-min equilibration perfusion and then two cycles of 5 min ischemia interspersed with 5 min reperfusion prior to 30 min ischemia and a 60-min reperfusion,and DPC group undergoing 10-min equilibration perfusion and 2 cycles of 5 min of 100 μM diazoxide perfusion followed by 5-min drug-free period before the 30 min ischemia and 60-min reperfusion.Frozen sections of myocardium at the cardiac apex were made and immunohistochemical staining was used to detect the expression of peroxisome proliferator-activated receptor-γ coactivator 1α(PGC-1α).Uhrathin sections 70 nm thick were made and transmission electron microscopy was used to detect the structure of the mitochondria with the Flameng scoring system.Results The PGC-1α expression of the IPC and DPC groups were significantly higher than that of the I/R group(P＜0.01 and P＜0.05),however,there was no significant difference in PGC-1α is expression between the IPC and DPC groups (P＞0.05).The Flameng scores of the IPC and DPC groups were 0.44±0.13 and 0.47±0.10 respectively,both significantly higher than that of the I/R group (1.78±0.14,both P＜0.01),however,there was no significant difference between IPC and DPC groups (P＞0.05).Conclusion IPC and DPC can protect myocyte mitochondria from the injury of ischemia/reperfusion.The cardioprotective effects of IPC and DPC may be concerned with the activation and high expression of PGC-1α.

objective To investigate the effects of isocitrate lyase (ICL) from Mycobacterium tubereulosis (MTB-icl) on the survival of Mycobacterium smegmatis (MS) in macrophage and illuminate the possible mechanisms.Methods MTB-icl gene was amplified by PCR and cloned into Ecoli-Mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmid pUV15-icl expressing ICL-GFP.The recombinant shuttle plasmid pUV15-icl and blank plasmid pUV15 were induced into MS of the line 1-2c so as to obtain rMS-pUV15-icl and rMS-pUV15.Shuttle plasmid rMS-pUV15-IG expressing ICL-green fluorescent protein (GFP) was constructed.rMS-pUV15-IG and MS 1-2c were used to infect the murine macrophages of the line RAW264.7,fluorescence microscopy was used to observe the expression of ICL-GFP.The expression of ICL in the MS swallowed by the macrophages was verified by RT-PCR and Western blotting.Another macrophages RAW264.7 were cultured and infected with rMS-pUV15-icl and rMS-pUV15 respectively.0,24,and 48 hours later macrophages were collected and the number of MS colonies was calculated.The interferon (IFN)-γ and nitrogen oxide (NO) concentrations in the culture supernatants of macrophages infected by rMS-pUV15-icl and rMS-pUV15 were measured by ELISA and Griess assay respectively.The apoptotic rate of the macrophages was assayed by in situ TUNEL technique.Results Western blotting showed that the MTB ICL protein expression of the rMS-pUV15-icl was significantly higher than that of rMS-pUV15.Fluorescence microscopy showed green fluorescence in the RAW264.7 cells infected with rMS-pUV15-IG.but not ion the RAW264.7 cells infected with MS 1-2c.0 h after the infection of the macrophages there was not significant difference in the MS amount in the macrophages between the rMS-pUV15-isl and rMS-pUV15 groups.and 24 h and 48 h later the MS amounts of the rMS-pUV15-icl group were (32.78±2.90)×103 and(23.33±2.34)×103 respectively,both significantly higher than those of the rMS-pUV15 group[(14.67±2.45)×103 and (2.28±0.25)×103 respectively,both P＜0.01].There were not significant differences in the concentrations of IFN-γ and NO in the culture supematants between the 2 groups(both P＞0.05).48 h after the infection the apoptotic rate of the rMS-pUV15-icl group was significantly lower than that of the rMS-pUV15 group(P＜0.01).Conclusion MTB-ICL promotes the intracellular survival of MS.Suppressing the apoptosis of the host macrophage may be one of the important mechanisms involved in this increased intracellular survival.

Objective To observe the morphological changes in vascular ageing-related remodeling natural aging,screen out genes related to remodeling,analyze the relation between gene difierential expression and morphological changes in remodeling,and illustrate possible gene regulation mechanism of vascular ageing-related remodeling.Methods Sixty male Wistar rats were divided into 4 equal groups according to their ages:4-month,10-month,16-month,and 22-month groups.Their aortas were taken out to undergo HE staining to observe the aortic macrostructure,and undergo Masson staining to observe the relative contents of smooth muscle cells (SMCs) and collagen fibrils (CFs).Gene fishing difierential display,RT-PCR,and Western blotting were used to analyze and confirm the genes related to remodeling in ageing vessels and their dynamically differential expression in different months.Results The amounts of SMCs and CFs increased along with ageing.5 differentially expressed gene fragments were selected by gene fishing PCR,which were homologous to the known genes as L-type Ca2+ channel α1c subunit (Cacna1c),matrix metalloproteinase 9 (MMP-9),NAD (P) H oxidase α subunit (p22-phox),stromal cell-derived factor 1α(SDF-1α),and caspase 9 (Casp9).The expression levels of Canalc and p22-phox increased with ageing.The expression levels of Canalc and p22-phox of the 16 month group were the highest (0.74±0.13and 0.73±0.12 respectively),both significantly higher than those of the 10 month group(0.12±0.05 and 0.20±0.03 respectively,both P＜0.01).The expression level of SDF-1α decreased with aging.The expression levels of MMP-9 and Casp9 were two-phase,peaking in the 16-month group(0.56±0.15 and 0.47 ±0.16 respectively).Muhiple stepwise regression analysis showed that SMC(%)=10.81Cacna1c-32.34SDF-1α+48.11(r=0.92,P＜0.01) and that CF(%)=23.71p22-phox-24.36SDF-1α+26.85(r=0.93,P＜0.01).Conclusion With a slow startup,an obvious speedup and a following speed-down,the aortic ageing-related remodeling is not a constant velocity process.The phasic differentiation expression of genes is highly correlated with the vascular ageing-related remodeling.The phasic differentiation expression of Cacna1c,p22-phox,and SDF-1α seem to be the key genes for startup,speedup and keeping up of vascular ageing-related remodeling and will be the promising molecular targets for vascular anti-aging.

Objective To evaluate the expression of TNF-related apoptosis inducing ligand (TRAIL) and nuclear factor (NF)-κB in the kidney tissues of diabetic rats and the effects of valsartan,mycophenolate mofetil (MMF),and their combined application on the renal TRAIL and NF-κB expression.Methods Eighty uninephrectomized male Wistar rats were randomly divided into 2 groups:normal control (NC) group (n=28),undergoing intraperitoneal injection of citric acid buffer,and diabetes mellitus (DM) group,undergoing intraperitoneal injection of streptozotocin (STZ) to establish DM models.The 52 DM rats were randomly divided into 7 equal subgroups:DM without treatment for 4 weeks (DM4),DM without treatment for 8 weeks (DM8) DM without treatment for 12 weeks (DM12),and DM without treatment for 16 weeks (DM16),valsartan treatment (DM+V),MMF treatment (DM+M),and combined treatment (DM+V+M).The treatment subgroups were treated for 8 weeks immediately after the diabetic models ere established.Twenty-four hour urine was collected to measure the amount of protein 4,8,12,and 16 weeks after the induction of DM respectively.The rats were sacrificed.Blood samples were collected from the abdominal aorta to detect the blood urea nitrogen (BUN),serum creatinine (sCr),albumin,and glucose.The kidneys were taken out.Hypertrophy index (left kidney weight/body weight) was determined.Quantitative real time RT-PCR was performed to detect the expression of TRAIL and NF-κB The 24 h urine protein levels and hypertrophy indexes of all DM subgroups were significantly higher than those of the NC groups (all P＜0.05).24 h urine protein and hypertrophy index increased gradually and peaked in the12th week;blood albumin gradually decreased since the 8th week (P＜0.01),and BUN and sCr began to decrease only since the 16th week (both P＜0.01).Compared with the DM 8 subgroup,the hypertrophy index and 24 h urine protein of the different treatment subgroups,especially the DM+V+M with the NC group,the TRAIL expression levels of the DM subgroups were significantly lower before the 12th week after induction of DM model (all P＜0.01),and then significantly highet in the 16th week (all P＜0.01).The TRAIL expression of the treatment groups,especially that of the DM+M subgroup,were significantly higher than that of the DM8 group (all P＜0.05).Compared with the NC group,the NF-κB expression levels of the DM subgroups were significantly higher time-dependently (all P＜0.01).Compared with the DM8 group,the NF-κB expression levels of the treatment subgroups,especially that of the DM+Vthe convoluted tubtde of kidney,and no TRAIL protein expression was detected in the glomeruli or renal vasculature.The levels of NF-κB protein expression,shown in glomeruli and convoluted tubules,of all DM subgroups were all higher than that of the NC group.The NF-κB protein expression level of the DM+V+M subgroup was significantly lower.The number of NF-κB positive cells was significantly related to the mononuclear macrophage infiltration,kidney function,and structural lesion.Conclusion An important monitoring factor in the autoimmune system,TRAIL closely participates in the pathogenesis of diabetic nephropathy,possibly controlled by NF-κB.In the early stage combination of valsartan and MMF may up-regulate the expression of TRAIL,thus protecting the kidney function.

Objective To study the protective effects of metanephric mesenchymal cells (MMCs) on acute renal tubular damage and explore its possible mechanism.Methods MMCs were isolated and cultured from 13-day-old embryonic rats and labeled with 5-bromodeoxyuridine.Seventy-two male SD rats were randomly divided into 3 equal groups:MMC group,receiving MMC injection instantaneously when ischemia/reperfusion (I/R) renal injury was induced,L/R group,undergoing I/R to establish acute renal tubular damage models,and sham operation group.Six rats from each group were killed at different time points:24 h,48 h,72 h,and 96 h later.Blood sample was collected from the vena cava inferior,to examine the serum creatinine (SCr) and blood urea nitrogen (BUN).Specimens of kidney underwent microscopy.Apoptosis was conformed by TUNEL assay.Immunohistochemistry was used to detect the protein expression of Bcl-2 and Bax.The distribution of MMCs labeled with 5-bromodeoxyuridine in kidney was observed by immunofluorescence technique.Results The SCr and BUN levels in difierent time points of the MMC group were both significantly lower than those of the I/R group (both P＜0.05),HE staining showed that pathological damage of the MMC group was less than that of the I/R group (P＜0.05).TUNEL results investigated that the number of apoptosis renal tubular epithelial cells of the MMC group was (13.4±3.2/HPF),significantly less than that of the I/R group [(25.4±5.2/HPF)].In comparison with the I/R group,there were more Bcl-2 positive cells and fewer Bax positive cells in the MMC group.BrdU-labeled MMCs began to occur in the renal tissue (60±6/HP) In the 72 h subgroup of MMC group,and number of BrdU-labeled MMCs,the 96 h subgroup was (143±8/HP),significantly higher than that of the 72 h subgroup (P＜0.05).Conclusion MMCs have the ability to protect renal function in acute renal tubular damage in rats,migrate and repopulate in the I/R injured renal tubules,and inhibits renal tubular epithelial cell apoptosis.The mechanism may be involved in regulating the expression of Bcl-2 and Bax.

：Objective To explore thegene diagnostic method for autosomal recessive Alport syndrome (AR-AS).Methotis GenomicDNA was extracted from the peripheral leukocytes of the proband of an AR-AS family.All theexons of COL4A3 and COL4A4 introns were amplified by PCR,and then the PCR products weresequenced by direct sequencing.Meanwhile,the mRNA of the coding region of type IV collagenα3 and α4 chain was extracted from the PBL and EB virustransfected cell and analyzed by using RT-PCR and sequencing to confoYm the genomic DNAanalysis results.Results PCR-sequencing analysis identified two novel COL4A3 mutations.OBewas a 5' donor splice site mutation (C.3418+1 G to A) in exon 39,leading to the deletionof exon 39 in mRNA level by RT-PCR analysis.The other was a deletion mutation of 9 bp atexon 25 (C.1729-1737 del9).Conclusion Both genomic-DNA-PCR-sequencing andmRNA-RT-PCR-sequencing methods can be carried out to detect tlle pathogenic mutations.Inparticulr,mRNA-based approach Can identify the changes in transcript level,therefore it iSbetter than the genomic DNA-based method.

A 72-year-old male was admitted to the Second Affiliated Hospital of Zhejiang University School of Medicine with headache for 5 days. After admission, the physical examination and neurological signs were negative. All indexes of routine laboratory examination were within the normal range. He had a long history of head trauma and coma 19 years ago. Imaging examination: CT of the head in the outside hospital showed low-density space-occupying lesions in the left frontal lobe.