Abstract:Objective:To explore the migration,differentiation and the therapeutic effect of intracerebral transplantation of human mesenchymal stem cells (hMSCs) after hypoxic-ischemic brain damage in rats,and to test whether three anti-hMSCs monoclonal antibodies prepared by our group can detect hMSCs in rat brains.Methods: hMSCs were isolated and purified by density gradient centrifugation and adherence to culture flask.Cells of passage 3-5 were prelabeled with bromodeoxyuridine (BrdU) for 72 h before transplantation.Animal models of hypoxic-ischemic brain damage (HIBD) were built with 1 month old Wistar rats.Three days after hypoxia-ischemia,HIBD rats in hMSCs-treated group (n=18) received intracerebral transplantation of 5×105 hMSCs.In control group (n=18) HIBD rats received phosphate buffered saline of the same volume.In sham-operated group (n=6) and HIBD group (n=6),rats did not receive any transplantation.Rats in hMSCs-treated group and control group were allowed to survive for 0 day,3 days,1 weeks,2 weeks (all n=3) and 4 weeks (n=6,after transplantation).

Abstract:Objective:To investigate the effect of bone marrow mesenchymal stem cells (mMSC) infusion on the reconstruction of hematopoiesis function in mice with critical irradiation injury.Methods: Female C57BL/6 mice irradiated with 8 Gy 60Co γ-rays were randomly divided into control group and mMSCs-treated group.Mice in the mMSCs-treated group received mMSC of male C57BL/6 mice.The peripheral leukocyte,platelet,and erythrocyte counts,the pathological lesions,granulocyte-macrophage progenitors (CFU-GM),and sex chromosome ratio were all examined.Results: All the animals in the control group (n=6) died of hematopoiesis exhaustion 20 days after irradiation.The peripheral leukocyte of mMSC-treated group decreased greatly after bone marrow transplantation and quickly recovered 2 weeks later; till 28 days after transplantation,it recovered to 60% of the pre-irradiation level.mMSCs infusion promoted the quick recovery of the nucleated bone marrow cells and CFU-GM,improving the marrow morphology.The transplanted mMSC could be detected in the recipients 42 days after transplantation.Conclusion: Solo-injection of mMSCs can enhance hematopoietic reconstitution in mice with critical irradiation injury. The period of imbedding is related to the dose of infusion.

Abstract:Objective:To study the effects of mda-7/IL-24 on the growth,proliferation,apoptosis of different hepatic carcinoma cell lines and the related mechanisms.Methods: A recombinant adenovirus Ad-mda-7 was constructed and was used to transfect human hepatic carcinoma cell lines (HepG2,Hep3B and PLC/PRF/5) and normal liver cell line L02.MTT assay and FACS were employed to assess the growth and apoptosis of cells; the expression of related protein expression was examined by Western blotting.The cells were treated with calpastatin Ⅰ (ALLN,25 μmol/L) for 30 min to block the endoplasmic reticulum stress (ER-stress) and the above indices were examined again.Results: Treatment with Ad-mda-7 resulted in selective inhibition of cell proliferation and induced apoptosis,especially in HepG2 cells; Ad-mda-7 showed no influence on normal cells. Pretreatment with ALLN partially inhibited the above effects of Ad-mda-7.Western blotting revealed that Ad-mda-7 induced up-regulation of BiP/GRP78 and Bax protein,activation of caspase-12,caspase-3 and phosphorylation of p38 MAPK in HepG2 cells.Blocking ER-stress with ALLN down-regulated Bax,caspase-12 expression and inhibited activation of caspase-3 and caspase-12,but showed no effect on the expression of BiP/GRP78 or phosphorylation of p38 MAPK.Conclusion: mda-7/IL-24 can cause growth inhibition and promote apoptosis of hepatic carcinoma cells through the ER-stress pathway.

Abstract:Objective:To design and prepare siRNAs targeting ski gene and to observe its influence on the biological functions of HepG2 cells,such as proliferation,cell cycle,apoptosis,etc..Methods: Three specific siRNAs of ski were designed and synthesized,and were transiently transfected into HepG2 via cathodolyte liposome transfection method.Real-time PCR and Western blotting were used to measure ski expression at mRNA and protein levels.The cell proliferation was assessed by MTT assay and the changes in cell cycle and apoptosis were evaluated by flow cytometry.Results: All the 3 specific ski-siRNA(A,B,C) effectively inhibited the expression of ski gene,with ski-siRNA-B having the highest inhibition rate(70%).Furthermore,the ski expression had a decreasing tendency with the transfection time.The proliferation of HepG2 cells was markedly inhibited by ski-siRNAs(P<0.05); the number of cells at S stage was obviously decreased,being 2 folds that of the negative control group.Conclusion: The siRNA of ski gene can effectively induce growth inhibition of HepG2 cells and reduce cells of S stage, which is possibly through down-regulation of ski gene.

Abstract:Objective:To establish a mouse hepatic cancer cell line Hca-F transfected with shRNA (small hairpin RNA) targeting annexin A7,so as to provide a basis for future study. Methods: Three shRNAs(shRNA1,2 and 3) were designed and inserted into the pSilencer vector to silence annexin A7 gene. The three pSilencer-shRNA vectors were transfected into Hca-F cells separately,and the most effective pSilencer-shRNA vector was selected based on the results of RT-PCR and Western blotting. The Hca-F cells were transfected with the most effective pSilencer-shRNA vector and the transfectants were selected by 400 μg/ml G418. The cells with annexin A7 stably knockdown were passaged and the expression of annexin A7 was confirmed by Western blotting,and the result was compared with those transfected with empty vector and normal controls.Results: The sequencing results confirmed that the sequences of the 3 shRNAs were correct,and shRNA1 was found to have the best inhibitory effect against annexin A7. Compared with normal Hca-F cells and those transfected with empty vectors,the annexin A7 protein expression was significantly down-regulated in cells transfected with pSilencer-shRNA(0.318 6 vs 0.824 3,0.798 7,P<0.05),with no significant difference found between the former 2 groups. Conclusion: We have successfully established a Hca-F cell line with annexin A7 stably down-regulated using shRNA technique,paving a way for future study.

Abstract:Objective:To prepare rabbit polyclonal antibody against human P-element-induced wimpy testis like 2 (HIWI2) protein,identify its properties and investigate its distribution in normal and tumor tissues by means of tissue chip. Methods: PIWIL2 peptide was synthesized chemically and conjugated to Keyhole limpet hemocyanin (KLH) as an immunogen. Then the PIWIL2-KLH conjugation was injected into rabbits subcutaneously to produce polyclonal antibodies. The specificity and sensitivity of antibodies were identified by ELISA and Western blotting after purification by affinity chromatography. PIWIL2 was then immuno-stained on the tissue chip to study its distribution in the normal and tumor tissues. Results: Rabbit’s antibodies against human PIWIL2 were prepared after the injection of PIWIL2-KLH conjugation subcutaneously. These antibodies were identified as PIWIL2 peptides by ELISA and Western blotting assay. PIWIL2 protein expression was tissue-specific in tumor tissues,with PIWIL2 protein found in the cytoplasm of smooth muscle cells of most normal and tumor tissues. Conclusion: The polyclonal antibodies against human PIWIL2 protein have been successfully prepared,which provides a basis for further study on the role of PIWIL2 in the pathway of miRNA/RNA.

Abstract:Objective:To explore the relationship between CDR1 and CDR2 expression and fluconazole-resistance in clinical Candida albicans strains, so as to explore the mechanism of drug-resistance in Candida albicans.Methods: The minimal inhibitory concentrations (MICs) of clinical Candida albicans strains to fluconazole were determined by broth microdilution method. The total RNA of fluconazole-susceptible and fluconazole-resistant Candida albicans strains were extracted，and the expression of the CDR1 and CDR2 genes was examined by real-time RT-PCR. The efflux of Rhodamine 6G was determined in the strains highly expressing CDR1 and CDR2 genes.Results: The incidences of CDR1 and (or) CDR2 overexpression in fluconazole-resistant strains were significantly higher than those in the fluconazole-sensitive stains. The efflux of Rhodamine 6G was significantly increased in the fluconazole-resistant strains when glucose was added, and overexpression of CDR1 and CDR2 were observed in theses strains. Some resistant strains showed no overexpression of CDR1 and CDR2.Conclusion: Fluconazole-resistance in clinical Candida albicans strains is related to the overexpression of ABC transporter proteins．Overexpression of ABC transporters can increase the efflux of drugs and therefore lead to drug-resistance. Other mechanisms may also participate in the development of drug resistance in the clinical Candida albicans strains.

Abstract:Objective:To investigate the influencing factors of cell-free fetal DNA level in the maternal plasma during blood-processing.Methods: Aliquots of blood samples from pregnant women with male fetus were processed at 6 h and 36 h after sampling.The SRY and β-globin genes and the total DNA level were quantified by real-time quantitative PCR.Death of white blood cells was assayed by flow cytometry after stained with AnnexinⅤ/PI.The plasma DNase activity was assayed by radial enzyme-diffusion method and plasma lactate dehydrogenases(LDH) by rate method.Results: A 36 hour delay in blood-processing led to a significant increase in the total DNA and decrease in the fetal DNA (SRY gene) in the maternal plasma.The ratio of fetal DNA decreased from (10.3±5.6)% at 6 h after sampling to (3.0±2.1)% at 36 h after sampling under 4℃(P<0.05).No dead cells were identified in the blood sample 6 h after sampling; however,apoptosis and necrosis of white blood cells were identified 36 h after sampling.The activity of LDH at 36 h was significantly higher than that at 6 h (P<0.05).Radial enzyme-diffusion result showed that,though greatly decreased at 4℃,the DNase was still able to degrade DNA.Conclusion: Delay in blood-processing can lead to increase of the total free DNA in maternal plasma but decrease of fetal DNA,which might be related to the death of white blood cells and degradation of fetal DNA by plasma DNase,so the extraction of fetal DNA should be done as early as possible after sampling (within 6 h).

Abstract:Objective:To explore the effects of hydroxy-methyl-glutaryl coenzymeA reductase inhibitor (HMG-CoA RI) simvastatin on renal expression of apoptosis related gene Fas/Fas-L in aged diabetic rats after uninephrectomy.Methods: Elderly uninephrectomized SD rats were randomly divided into uninephrectomized control group (group C),streptozocin(STZ)-induced diabetic model group( group D),and diabetes + simvastatin group (group S).The apoptosis indices in renal tisses of three groups were detected by terminal deoxynucleotide mediated nick end labeling 12 weeks later.Immunofluorescence and real-time quantitative PCR were employed to observe the expression changes of Fas/Fas-L genes at the transcription and translation levels.Results: Compared with group C,group D had significantly higher apoptosis index and expression level of Fas/Fas-L; the above parameters were lower in group S compared with those in group D (P<0.05).The expression of Fas/Fas-L protein was mainly in the renal tubules.Conclusion:Both Fas and Fas-L genes are associated with the development and progression of diabetic nephropathy in aged rats.Simvastatin can down-regulate the translation and transcription of the genes,inhibit cell apoptosis and protect the kidney.

Abstract:Objective：To study the anti-proliferation effects of thiazolidinedione compounds-troglitazone, which is a high affinity ligand of PPAR-γ , on rat pituitary adenoma GH3 cell line and explore the related mechanisms.Methods: GH3 cells were separately treated with troglitazone (10-7,10-6 and 10-5 mol/L), dimethyl sulfoxide (DMSO) (DMSO control group) and phenol red- and serum-free F-12 medium (blank group). MTT was used to examine the cell growth in each group and FACS was used to detect the distribution of cell cycle. Semi-quantitative RT-PCR method was utilized to determine the expression of CyclinD1 mRNA. ANOVA was used for statistical analysis.Results:The 72 h treatment with troglitazone inhibited GH3 cell proliferation in a dose-dependent manner. The treatment also induced cell cycle arrest in G1/S phase and significantly decreased the expression of CyclinD1 mRNA as compared to the other 2 groups (P＜0.05).Conclusion:Troglitazone can obviously inhibit the proliferation of GH3 cells; the molecular mechanism may be the decrease of CyclinD1 mRNA due to binding to PPAR-γ.

Abstract:Objective:To study the therapeutic effect of tertiary butyl alcohol (TBA) eyedrops on selenite-induced rat cataract (simulating senile cataract) .Methods: Rat cataract model was induced by sodium selenite. The right eyes of the rats were treated with 25, 50, or 75 mmol/L TBA eyedrops (1 drop/time, 3-5 times/day); the left eyes were taken as control. The eyes were observed everyday and were examined using slit lamp at the fifth and tenth day after treatment for nuclear plaques of the lens. The maximal diameter changes of the cataract plaques were detected using vernier cursor (with the minimum division value being 0.02 mm). Meanwhile, the right eyes of normal SD rats were stimulated with 100 mmol/L TBA eyedrops (1 drop/time, 3-5times/day) to investigate the toxic effect of the eyedrops and other adverse reactions.Results: The diameter and degree of cataract plaques of the nuclear cataract in the right eyes were less than those in the left eyes, and the opacification degree in the right eyes was slighter than that in the left eyes. The TBA eyedrops had less toxicity and stimulation to the eye.Conclusion: TBA eyedrops have obvious therapeutic effect on selenite cataract.

Abstract:Objective:To observe the effect of bevacizumab on the proliferation of human heptoma cell line HepG2. Methods: The expression of vascular endothelial growth factor(VEGF) and its receptors (VEGFRs) in HepG2 cells were examined by immunocytochemical staining and RT-PCR；ELISA was used to determine the level of VEGF in culture supernatants of HepG2 cells. The proliferation of HepG2 cells was analyzed by MTT assay after treatment with rhVEGF and bevacizumab separately; the expression of VEGF was examined by RT-PCR and Western blotting. Results: VEGF and VEGFRs (Flt-1 and KDR) were expressed in human HepG2 cells. rhVEGF increased the proliferation of HepG2 cells in a dose-dependent manner within a concentration range of 0-100 ng/ml; bevacizumab inhibited the proliferation of HepG2 cells; the inhibition rates were (8.76%±1.15)%,(26.83±1.20)%,(31.87±1.30)% and (28.20±1.28)%, when the concentrations of bevacizumab were 0.1,1,10,and 20 μg/ml,respectively. Expression of VEGF in the HepG2 cells was increased by rhVEGF and inhibited by bevacizumab. Conclusion: Bevacizumab might inhibit the proliferation of HepG2 cells through blocking the effect of VEGF.

Abstract:Objective:To investigate the expression of CD40 and MMP9 in carotid atherosclerotic plaques, so as to assess the role of CD40 in the stability of the plaque and the possible mechanism. Methods: The expression of CD40 and MMP9 mRNA and protein in carotid atherosclerotic plaques obtained from carotid eversion endarterectomy (CEE) of 28 patients with high-grade stenosis (>70%) (stroke group, n=15; non-stroke group, n=13) and 8 normal postmortem arteries (control group) were detected by real-time quantification polymerase chain reaction(PCR) and Western blotting. The correlation between expression of CD40 and MMP9 was analyzed. Results: The expression of CD40 mRNA and MMP9 mRNA in the non-stroke group and stroke group was significantly higher than that in control group (P＜0.01); and that of the stroke group was significantly higher than that of the non-stroke group (P＜0.01). There was a linear correlation between expression of CD40 and MMP9 mRNA (r=0.964，P＜0.01). Extremely rare expression of CD40 and MMP9 protein was found in the normal carotid artery; the protein expression was evidently higher in the carotid artery atherosclerosis than in the normal carotid artery, and the expression in carotid artery atherosclerosis in the stroke group was higher than that in the non-stroke group. Conclusion: The increased expression of CD40 and MMP9 in the carotid atherosclerositic plaques may be closely related to the stability of plaques.

Abstract:Objective:To study the influence of glucosidorum tripterygii tororum (GTT) and Mesalazine on the expression of IL-23, IL-17 and IL-12 in the colonic tissues of mice with rinitrobenzene sulphonic acid (TNBS)-induced acute colitis, and to study the possible mechanism. Methods: The C57BL/6 mice were divided into 4 groups, namely, a control group, a model group, a Mesalazine group and a GTT group. Colitis was induced by TNBS in the last 3 groups. The mice in the model group received no additional treatment; those in the GTT group received GTT daily by oral gavage 4 days before exposure to TNBS till the end of the experiment, and those in the Mesalazine group received Mesalazine enema solution daily 4 days before exposure till the end of the experiment. All the mice were sacrificed at 48 h after enema with TNBS. The macroscopic and histological scores of colon damage and myeloperoxidase (MPO) activity in colonic tissue were evaluated in every group. IL-23p19 and IL-17 contents in colonic tissues were measured by ELISA; the expression of IL-23p19, IL-17 and IL-12p35 mRNA in colonic tissues were examined by real-time fluorescent quantitative reverse transcriptase polymerase chain reaction (RT-FQ-PCR) with SYBR Green Ⅰ.Results: Compared with the model group, GTT group and Mesalazine group had significantly lower macroscopic and histological scores and MPO activity (P<0.05). Expression of IL-23p19, IL-17 and IL-12p35 mRNA in the colonic tissues of the model group was significantly higher than that of the other 3 groups(P<0.05); the expression of IL-23p19, IL-17 protein was significant higher in the model group than that in the other 3 groups (P<0.05), with no significant difference found between the later 3 groups.Conclusion: GTT, like Mesalazine, can effectively inhibit inflammation in mice with TNBS-induced

Abstract:Objective:To summarize the major ultrasonic manifestations of various hepatic vascular anomaly(HVA), so as to improve the ultrasonic diagnosis rate of HVA.Methods: The chief complaints, timing and mode of ultrasonic diagnosis of 18 patients with congenital HVA or postliver transplantation HVA were retrospectively analyzed.Six of the 18 patients were subjected to detailed analysis.The major ultrasonic manifestations of various HVA were observed and experience of ultrasonic diagnosis was summarized.Results: The major vascular abnormalities in our group included hepatic vascular fistula (including fistulas between artery and vein, artery and portal vein, portal vein and hepatic vein, also a complex fistula among artery, portal vein and hepatic vein), portal aneurysm, congenital portal atresia and portal vein cavernous transformation; among which the portal atresia and the complex hepatic fistula involving hepatic artery, hepatic vein and portal vein were rarely described in the literature.Color Doppler ultrasound was the first choice for detection and diagnosis of HVA;contrastenhanced ultrasound was sensitive and specific in diagnosis of all vascular fistulas due to its ability to display homodynamic phase.Conclusion: The wide application of Doppler and contrastenhanced ultrasound examination improves the detection and diagnosis of HVA; diagnosis should be made based on scientific diagnosis mode and precise diagnosis planning.

Abstract:Objective:To observe the ultrasound characteristics of galactostasis at various physiological stages,so as to improve the diagnosis of galactostasis.Methods: The ultrasound data of 56 patients with galactostasis were retrospectively analyzed and the ultrasound characteristics were summarized.The ultrasonic manifestations of different physiological stages were compared.Results: During the lactation period,there were extensive mammary duct ectasias and floating weak signals were found in the mammary duct during pure galactostasis; large non-responsive fluid areas exhibited formations of honeycomb-like multilocular fibrous septa; and these cavities ruptured with abscess when secondary infection occurred.During the later lactation period,the dilated galactophores became hypoechoic or anechoic,which,together with the dot-like echoes of residual breast milk or macular strong echoes, formed mixed signals of retention cysts.After menopause,the galactophores atrophy occurred; the accumulation of galactostasis desiccated into patches or powder,which caused thick hyperechoic dots or multiple hyperechoic streaks in the galactophores or parenchyma.Conclusion: Ultrasonic images of galactostasis vary with the physiological and pathological changes of the mammary glands,and are related to the duration of galactostasis,absorption,concentration and desiccation of lactation.

Abstract:Objective:To evaluate the effect of histopathologic factors in patients with hepatocellular carcinoma after liver transplantation(LT) on the prognosis of liver transplantation. Methods: The clinical data of 272 HCC patients, who had received liver transplantation, were retrospectively analyzed. The survival rates were analyzed using the actuarial life-table method. Multivariate and univariate COX proportional hazards model were used to investigate the correlation between histopathologic factors and survival time. Kaplan-Meier method was used to plot the curves of accumulative survival rates and Log-rank tests were used to compare the curve of the survival rates. Results: Univariate analysis using a COX model revealed that scores of model of endstage liver disease(MELD), alphafetoprotein, size of tumor, capsule invasion, Eggel’s classification, Edmonson-Steiner grade, microvascular invasion, regional lymph node metastasis and TNM staging were significantly related to the prognosis of the patient after LT(P<0.05) . Multivariate COX model analysis showed that alphafetoprotein(RR:1.459, P=0.002) , Eggel’s classification(RR:1.617, P=0.004) , microvascular invasion(RR:2.631, P<0.001) and MELD(RR:2.194, P=0.011) are independent factors of patient prognosis.Conclusion: Alphafetoprotein, Eggel’s classification, microvascular invasion and MELD are the independent prognostic factors of HCC patients after LT. More attention should be paid to the influence of MELD on prognosis of HCC patients after LT.

Abstract:Objective:To establish a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for determination of the losartan concentrations in rat plasma,urine and bile．Methods: The method was based on a protein precipitation procedure for rat plasma and liquid-liquid extraction for rat urine and bile,then the diluted extract was analyzed by LC/MS/MS using turbo ion spray (TIS) positive ion mode.Chromatography was performed on an Agela Venusil MP-C18 column (5 μm particle,2.1 mm×50 mm) by gradient elution.Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile.The mobile phase B increased from 20% to 95% within 1.5 min,with the flow rate being 0.35 ml/min and the feeding rate being 10 μl.Electrospray ionization (ESI) ion source was used.Results: The linear range of losartan was 1-1 000 μg/L in the plasma,urine and bile of rats (r>0.99).The mean relative recovery rate was higher than 80.0%;RSD values of intra-day and inter-day were both less than 15%.Conclusion: The present method is easy to perform,accurate,and specific; it can be used for determination of losartan concentration in rat plasma,urine and bile．

Abstract:Objective:To study the pharmacokinetics of the transdermal absorption of Sinomenine in patch under electroporation condition by comparing with the transdermal absorption of passive diffusion. Methods: Sinomenine was taken as a model drug and was given to rabbits by passive diffusion (PD group) or by electroporation (EP group). The plasma drug concentration was determined by HPLC. The statistical moment theory was used for analyzing the values of plasma drug concentration.Results: The AUC0→∞ was 43.396 in the EP group, which was 1.32 folds that of the PD group. The mean residence time (MRT) was 20.025 in the EP group, which was 86% of the PD group. The cmax was 1.825 in the EP group, which was 1.31 folds that of the PD group. The decrease of tmax in the EP group was 4 hours earlier than that in the PD group. Their elimination rate constants λ were the same. The drug-time curve of EP group was more smooth and slow when declining compared with that of the PD group.Conclusion: The combination of electroporation and the patch can improve the speed and extent of transdermal absorption of sinomenine, and thus enhance the bioavailability.

Abstract:Objective:To evaluate the value of Cook MOB-15 system in guiding wire insertion during endoscopic retrograde cholangiopancreatography (ERCP). Methods: The clinical data of 51 patients who received Cook MOB-15 system-guided wire insertion during ERCP between Jan. 2005 to Dec. 2007 were retrospectively analyzed. Forty patients who received conventional ERCP catheter for malignant jaundice between Jan. 2002 and Dec. 2004 were taken as control. The successful insertion rates were compared between the 2 groups. Results: The successful insertion rate was 90.2% (46/51) in the Cook MOB-15 system group and 72.5% (29/40) in the conventional group; there was significant difference between the 2 groups (P<0.05). Conclusion: The Cook MOB-15 system can significantly raise the successful insertion rate in ERCP.

Abstract:Ovarian cancer is sex hormone-dependent.Gonadotropin releasing hormone (GnRH) analogues inhibit ovarian cancer not only through the hypothalamus-pituitary-gonadal axis, but also through directly inhibiting the proliferation and inducing the apoptosis via GnRH receptors on the ovarian carcinoma cells.In addition, GnRH analogues target GnRH receptors on the cancer cells and can serve as a carrier for cytotoxic agents, improving the efficiency of cytotoxic agents and lowering the side effect.In a word, treatment with GnRH analogues may be a valuable alternative for advanced and recurrent ovarian cancer.

Abstract:Regulation between genes is a dynamic event associated with changes of time and circumstances.Gene regulatory network is a complicated and dynamic system.Time series gene microarray provides a tool for creating dynamic gene regulatory network.In this paper,we review several models of dynamic gene regulatory network based on time series gene expression data,including temporal Boolean network,differential equation,dynamic Bayesian networks,etc..The advantages and disadvantages of the models were analyzed and the future of the research is predicted.

Abstract:目的:建立稳定表达人FcγRⅡ(human Fc gamma receptor Ⅱ ,huFcγRⅡ)的真核细胞系，为后续研究奠定基础。方法:采用RT-PCR方法从人外周血白细胞合成FcγRⅡcDNA，构建huFcγRⅡ表达质粒pcDNA3-huFcγRⅡ，经酶切和PCR鉴定后，将pcDNA3-huFcγRⅡ质粒通过脂质体转染法转染COS7细胞，并经G418筛选获得稳定表达huFcγRⅡ的细胞克隆。采用免疫荧光法和玫瑰花环试验检测huFcγRⅡ的表达。结果:构建的pcDNA3-huFcγRⅡ真核表达质粒经酶切鉴定与预期一致，稳定转入COS7细胞后，经免疫荧光染色，约90%的转染细胞可见荧光，而未转染的COS7对照细胞未见荧光。结论:成功构建pcDNA3-huFcγRⅡ真核表达质粒，建立了稳定表达huFcγRⅡ的细胞系，为进一步研究奠定了基础。

Abstract:目的:观察不同CO2分压对乳腺癌MCF-7细胞增殖和凋亡的影响，探讨临床腔镜技术的安全性。方法:体外建立人工气腔，MCF-7细胞在0、7、15 mmHg(1 mmHg=0.133 kPa)CO2分压下暴露l h，处理后0、24、48、72 h测定细胞增殖率，FCM(流式细胞术)测定MCF-7细胞凋亡率和Fas、Fas-L表达。采用0 mmHg的N2(1 h)作为缺氧对照组；正常培养细胞作为正常对照组。结果:与正常对照组相比，0 mmHg CO2可促进MCF-7细胞增殖(P＜0.05)，7、15 mmHg CO2抑制细胞增殖(P＜0.05)；7、15 mmHg CO2组细胞凋亡率明显增高(P＜0.05)；15 mmHg CO2组MCF-7细胞Fas表达明显增高(P＜0.05)；7 mmHg CO2组Fas表达在处理后0 h时增高(P＜0.05)；7、15 mmHg CO2组在处理后24、48 h Fas-L表达显著增高(P＜0.05)。结论:CO2分压对乳腺癌细胞增殖和凋亡的影响较复杂，临床常用的7~15 mmHg CO2分压能增加肿瘤细胞Fas/Fas-L表达，抑制增殖，促进凋亡。

Abstract:目的:观察Na+通道阻滞剂河豚毒素(tetrodotoxin,TTX)停搏液对离体大鼠缺血再灌注损伤心肌桥粒蛋白(desmoplakin)表达及心功能的影响，探讨其对缺血再灌注损伤心肌的可能保护机制。方法:20只Wistar大鼠随机分为St.Thomas-2停搏液灌注组(STH-2组，n=10)和TTX停搏液灌注组(TTX组，n=10)，分别应用相应的停搏液停搏，建立大鼠离体心脏Langendorff-Neely灌注模型；待搏动稳定，灌注30 min后停搏60 min，再灌注60 min，观察各组心脏停搏前后的心率(HR)、冠状动脉流量(CAF)、左心室收缩末期压力(LVESP)和压力变化速率(±dp/dt)的恢复率，采用免疫组化和Western印迹法检测心肌桥粒蛋白的分布及量的变化。结果:恢复灌注后，与TTX组比较，STH-2组心肌桥粒蛋白分布紊乱，表达明显减少(P<0.01)，心功能指标(HR、CAF、LVESP和±dp/dt)的恢复率也明显降低(P<0.01或P<0.05)。结论:河豚毒素停搏液有利于缺血再灌注损伤心肌功能的恢复，效果优于经典的STH-2停搏液，其保护作用可能与其保护心肌桥粒蛋白表达有关。

Abstract:目的:观察人胃癌组织中增殖诱导配体(APRIL)及其受体B细胞成熟抗原(BCMA)和穿膜蛋白活化物(TACI)蛋白的表达,探讨其临床意义。方法:采用H-E染色及间接免疫荧光法检测30例人胃癌组织及相应10例癌旁正常组织APRIL及其受体蛋白的表达,分析不同临床病理特征下相关蛋白的表达水平。结果:胃癌组织中APRIL阳性率为86.7%,癌旁正常组织未见表达(P<0.05)；而其受体BCMA和TACI蛋白表达水平在两者间无统计学差异。胃癌组织不同临床病理指标下(肿瘤大小、分化程度、浸润深度、有无淋巴结转移和TNM临床分期),APRIL及其受体蛋白表达无统计学差异。结论:胃癌组织中APRIL蛋白高表达,检测其表达水平可能有助于胃癌的早期诊断。

Abstract:目的:评价升膝汤联合α-受体阻滞剂/巴氯芬治疗尿潴留导尿管依赖状态的疗效。方法:115例尿潴留导尿管依赖状态患者，单纯应用升膝汤组65例，主药为中药煎剂，2次/d；升膝汤联合α-受体阻滞剂/巴氯芬治疗组50例，应用升膝汤治疗同时加用α-受体阻滞剂/巴氯芬，治疗2周后复诊，比较两组拔管成功率。结果:107例解除导尿管依赖状态，恢复自主排尿，9例失败，有效率为92.2%。单纯应用升膝汤治疗组拔管成功率为89.2%，低于升膝汤联合α-受体阻滞剂/巴氯芬治疗组(96.0%，P<0.05)。两组患者均无明显不良反应。结论:中西医结合治疗尿潴留导尿管依赖状态效果良好，值得推广使用。

Abstract:目的:观察乳果糖和前列腺素E1对恶性梗阻性黄疸患者术后肾功能保护的干预效应。 方法:将48例恶性梗阻性黄疸患者随机分为乳果糖治疗组、前列腺素E1治疗组、乳果糖和前列腺素E1合用组以及对照组,每组12例。分别于患者入院后1 d及术后1、2、3、7 d清晨6:00抽取外周静脉血,观察血中内毒素水平及肌酐(Cr)、尿素(urea)变化情况。 结果:恶性梗阻性黄疸患者血中内毒素水平明显升高(P<0.01)；两药合用组患者血中内毒素、Cr、BUN水平与对照组有统计学差异(P<0.01)；单独使用一种药物组患者血中内毒素水平与对照组有统计学差异,但Cr、BUN值与对照组无统计学差异。 结论:乳果糖、前列腺素E1均可以降低恶性梗阻性黄疸患者血中内毒素水平(P<0.01),对肾功能亦有保护作用;两种药物合用时其临床效果更加明显。

Abstract:目的:观察严重二尖瓣狭窄合并左室功能减退患者行二尖瓣置换的手术死亡率和远期效果。方法:回顾性分析自1995年4月至2006年10月间55例严重二尖瓣狭窄合并左室收缩功能减退行二尖瓣置换术患者的临床资料和长期随访结果,其中男性23例,女性32例,平均年龄(44.25±9.38)岁。均为风湿性心脏病,二尖瓣瓣口面积平均为(0.76±0.15) cm2,左室射血分数(EF)平均为(42.65±5.40)％。术前心功能Ⅳ级12例,Ⅲ级40 例。术前无合并冠心病患者。结果:本组术后30 d内死亡4例,手术死亡率为7.27%。死亡原因:左室破裂2例,严重呼吸功能衰竭1例,猝死1例。生存的51例患者早期均得到随访,随访至2007年8月,失访3例,随访率94.55％,随访时间平均为(5.07±3.17)年。5年、11年的生存率分别为(96.00±3.00)%、(74.00±14.00)%；没有出现栓塞相关并发症；出现1例脑出血；没有因细菌性心内膜炎或瓣膜功能障碍等再次手术的病例。44例存活患者的NYHA心功能分级Ⅰ级7例,Ⅱ级35例,Ⅲ级2例。心脏超声显示EF 41％～73％,平均(52.58±7.19)％(P<0.05)。结论:对严重二尖瓣狭窄合并左室收缩功能减退的患者如能加强围术期的处理,也有良好的手术及远期效果。

Abstract:目的：探讨长期肝内胆管结石伴胆管癌变的临床特征及有效治疗方法,提高肝内胆管结石并胆管癌变的诊治水平。方法：回顾性分析2000年1月至2006年12月间54例肝内胆管结石伴胆管癌变患者(占同期所有肝内胆管结石患者的2.8%)的临床资料,分析其临床表现、治疗结果及随访情况,总结临床诊治经验。结果：54例患者中,男18例,女36例,年龄29~72岁,平均（46.8±10.9）岁。发现结石病程均超过8年。临床体征常缺乏特异性。4例行内镜下逆行胆汁内引流及内支架植入术,50例行开腹手术,其中41例行肝切除术。结石部位与肿瘤部位均位于同一侧肝叶。术后病理均提示腺癌。随访51例(94.4%)。35例死亡,主要原因为胆管癌复发转移；16例至今存活,其中2例行肝切除患者存活超过6年。结论：临床医师应充分认识到长期肝内胆管结石有胆管癌变的可能,掌握合并癌变时的临床特征表现,重视对高危对象的定期检查。肝切除术是治疗肝内胆管结石合并癌变的有效手段,可提高部分患者的长期生存率。

Abstract:1临床资料71岁男性患者，因“突发左下肢不能活动4 h”就诊。既往有“高血压、心脏病”史，无“糖尿病、房颤、风湿性心脏病、脑梗死”史。体格检查:HR 80/min，BP 140/90 mmHg(1 mmHg=0.133 kPa)，肺部检查正常，心律齐。神志清楚，双瞳等大，直径2 mm，对光反应灵敏，伸舌居中。左下肢感觉减退，右下肢感觉正常。左下肢肌力0级，双上肢、右下肢肌力5级。拟诊“脑梗死”。骨科会诊发现患者胸闷，左下肢、左髋部疼痛。检查发现左髋部压痛(±)。检查:双下肢皮温对称，肤色无苍白，能自行活动，足背动脉搏动差，左股动脉搏动弱。拟诊“左下肢动脉栓塞”。予5％葡萄糖100 ml、链激酶(思凯通)25万U、普通胰岛素6 U静滴，要求1 h内滴完。辅助检查:Hb 110 g/L,RBC 3.6×1012/L，WBC 22.3×109/L，中性分叶核细胞0.82，淋巴细胞0.15，单核细胞0.03，PLT 175×109/L；血糖13 mmol/L。头颅CT:脑中线结构居中,双侧脑室前后角旁及额顶叶皮质下白质内见斑片状低密度影；脑室系统及脑池扩大，脑沟裂增宽；右侧颈内动脉海绵窦段见高密度影。诊断印象:脑萎缩，脑白质病；右侧颈内动脉海绵窦段硬化可能。腰椎正侧位片:腰椎生理曲度存在，L1-5椎体边缘可见不同程度骨质增生，L1椎体轻度楔形变，其内密度增高；余椎体密度普遍降低，椎间隙未见狭窄，椎间孔、骨突关节及余附件未见异常。诊断结论:腰椎退行性变，骨质疏松;L1椎体轻度楔形变;压缩性骨折？请结合临床。髋关节正位:左髋关节间隙、关节面未见异常，关节诸骨结构未见异常，软组织未见异常。诊断结论:左髋关节平片未见异常。超声描述:两侧下肢动脉外形正常，内膜欠光滑，回声增强，彩色血流充盈好，无狭窄。超声提示:(1)双侧下肢动脉轻度硬化；(2)左侧下肢动脉流速明显减低、血流平缓；下肢静脉未见异常。

Abstract:新生血管形成在肿瘤的形成与转移中起重要作用。通过干扰血管形成来抑制肿瘤已成为临床生物治疗策略\[1-2\]，其中血管内皮细胞生长因子（VEGF）单克隆抗体贝伐单抗（bevacizumab，商品名Avastin），已被美国食品药品监督管理局（FDA）批准为大肠癌肝转移的临床治疗一线用药\[3\]。在VEGF的胞内信号转导过程中，Ca2+作为第二信使分子，其浓度变化，可以通过下游的信号途径使多种相关酶的活性改变，促进内皮细胞的增殖与分化，调节新生血管形成\[4-5\]。为了明确一种新的钙通道蛋白瞬间受体电位通道C亚家族（TRP-canonical, TRPC）是否参与VEGF的调节过程，本研究选用TRPC3、6、7通道的阻滞剂SKF96365观察其对血管内皮细胞内游离钙离子浓度的影响。

Abstract:1临床资料患者女性，51岁。因“腰背部疼痛1个月余，伴皮肤瘀斑2周”于2007年9月6日入院。患者于2007年8月无明显诱因自觉腰背部疼痛，活动后加剧，2周前四肢皮肤出现数个大小不等的紫红色瘀斑，伴间断低热，无寒战、畏寒，大便每日1~2次，色黑，基本成形，在当地医院查血小板示38×109/L；骨髓细胞学检查示粒系、红系增生活跃，巨核数增多、成长障碍，血小板小簇散在分布；胸椎MRI检查示第8、11胸椎椎体变扁伴信号异常；予口服泼尼松50 mg/d治疗，皮肤瘀斑有所消退，但腰背痛进行性加重，复查血小板示27×109/L。既往无胃病史。入院查体：T 37.0℃，贫血貌，四肢皮肤散在大小不等深紫色瘀斑，全身浅表淋巴结未及肿大，心肺腹检查未见异常，第10~12胸椎、第5腰椎压痛，双下肢无水肿，病理征阴性。辅助检查：血常规示白细胞6.9×109/L，中性67%，血红蛋白63 g/L，血小板35×109/L，网织红细胞5.0%，幼红细胞4%；白蛋白31 g/L，乳酸脱氢酶1 560 U/L，碱性磷酸酶495 U/L；癌胚抗原19.42 μg/L，CA19-9 37.23 U/ml，甲胎蛋白、CA125、CA15-3正常，肝肾功能、凝血功能正常，M蛋白阴性，大便潜血阳性；全身骨扫描检查示肋骨、胸椎、腰椎、骨盆、股骨等多处骨骼可见异常放射性浓集区（图1A）；骨髓病理学检查示骨髓中可见大量腺癌细胞（图1B）；胃镜示胃小弯侧约一直径1.5 cm溃疡（图1C），病理提示低分化腺癌（图1D）。诊断：胃癌骨髓转移，继发性血小板减少性紫癜。

Abstract:1临床资料患者男性,16岁,因牙列不齐要求矫正。临床检查:恒牙,左上侧切牙先天缺失,左上尖牙为乳牙滞留且松动Ⅰ°,头颅正侧位片均显示左上尖牙埋伏阻生:牙冠向近中,牙根向远中横斜向位阻生。右上侧切牙唇倾,上中切牙内倾,咬及下前牙唇侧根部,使下前牙龈萎缩,牙根暴露且下颌牙列拥挤。诊断为:左上尖牙埋伏阻生+深覆+牙列拥挤(图1)。

Abstract:

Abstract:1临床资料患者，女，44岁，因“肝炎4年余，发现肝脏占位20 d”于2003年1月9日收住我院移植科。T 36.8℃，P 80次/min，R 18次/min，BP 120/75 mmHg(1 mmHg=0.133 kPa)。全腹软，无压痛及反跳痛，未触及包快。肝、脾肋下未触及。于2003年1月22日在全麻下行原位肝移植。全麻成功后取肝移植倒“T”形切口，进腹后可见肝脏硬化明显，呈结节状改变，腹腔中有少量腹水，术中行经典肝脏移植。手术顺利，供肝热缺血时间为0，冷缺血时间为6 h，无肝期65 min。病理示平滑肌肉瘤。术后给予普乐可复（FK506）、甲泼尼龙抗排异，给予谷胱甘肽(泰特)保肝，给予头孢哌酮-舒巴坦（舒普深）抗感染。化疗后34个月死亡。病理示肿物表面呈结节状，包膜完整，质软有弹性。切面呈灰白色、灰红色或深红色，鱼肉状，细嫩湿润，间有不规则片状出血坏死区。囊性感，切开见囊内有咖啡样液体，内壁不平、粗糙，淡褐红色。镜下肿瘤组织纵横交错或呈编织状排列，瘤细胞大、梭形，胞质较丰富、粉红，核圆形、卵圆形或椭圆形，两端钝或细杆样，核染色质粗颗粒状，核仁清楚，异型性明显，核大、深染，核分裂像易见，每高倍视野约1～2个（图1A）。肿瘤表面被有包膜，包膜外肝细胞受挤压萎缩。间质血管丰富，有出血、坏死。免疫组化染色结果：SMA（图1B）、Vim、Desmin+，AFP、CEA、α-AT、α-ACT、Lysozyme、CD68、CD34、CD117、CK、Hepatocyte、FⅧ、HMB45均阴性。