Objective To study the influence of RNA interference Caspase-3 gene on apoptosis of transformed human embryonic kidney 293 cells induced by cadmium in vitro. Methods Transformed human embryonic kidney 293 cell were treated with siRNA for 36 h. The treated cells were incubated with 40 ?mol/L CdCl2 for 12-24 h and the cells viability were measured with methyl thiazolyl tetrazolium(MTT) assay. In addition,the expression of caspase-3 gene in 293 cells was detected by the methods of reverse transcription polymerase chain reaction (RT-PCR) and Western-blot analysis,and the occurrence of apoptosis was determined by flowcytometry with Annexin-V and propidium iodide (PI) double labeling method. Results The results of RT-PCR and Western-blot analysis revealed that in incubated cells with 40 ?mol/L cadmium 12 h,the expression of Caspase-3 mRNA and 20 kDa protein significantly increased when compared with controls(n=4,P

Objective To investigate the single nucleotide polymorphisms (SNPs) at codons 16 and 27 of β2-AR gene in pancreatic carcinoma and non-neoplastic pancreatic tissues,and the correlations between these SNPs and the expression of β2-AR protein in pancreatic carcinoma.Methods A total of 64 cases of pancreatic carcinoma (PC) and 20 non-neoplastic pancreatic tissues (NPC) were genotyped at codons 16 and 27 by PCR-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing.The correlations between the distribution of genotypes and clinicopathological characteristics of pancreatic carcinoma were analyzed.The expression of β2-AR protein was detected by immunohistochemistry in pancreatic carcinoma.Results The distributions of genotype frequency at codons 16 and 27 in PC and NPC were in accordance with the Hardy-Weinbery equeilibrium.The frequencies of their genotypes (AA,AG and GG) and frequencies of alleles A and G at codon 16 between PC and NPC showed no difference.The genotype frequencies were associated with TNM grade,lymph node metastasis,one-year survival rate (P=0.03,0.05,0.04),but they were not associated with patients' gender,age,histological differentiation and size of tumor.The allele G at codon 16 was frequently appeared in tumors with high TNM grade,lymph node metastasis,low one-year survival rate (P＝ 0.01,0.03,0.02),and high expressions of β2-AR protein (P =0.02).The frequencies of two genotypes (CC and CG) and frequencies of alleles C and G at codon 27 showed no difference between PC and NPC.The genotype frequencies and allele frequencies of codon 27 were not associated with patients' clinicopathological features,and expressions of β2-AR protein.Conclusions SNPs of β2-AR gene were associated with biological behaviors of pancreatic carcinoma.Allele G at codon 16 was associated with high risks of lymph node metastasis,high TNM grade,low one-year survival rate,and high expressions of β2-AR protein.Allele G at codon 16 might facilitate the progression and metastasis of pancreatic carcinoma through elevating the expression of β2-AR.SNPs at codon 16 of β2-AR are new useful biomarkers for predicting biological behaviors and survival of pancreatic carcinoma and might be used as a new gene therapeutic target.

Objective To construct markless gene deletion mutant at the clpP loci on the chromosome of Streptococcus mutans(S.mutans).Methods ASp resistance gene was amplified by PCR,to construct the Sp resistance cassette where the Sp resistance gene was flanked with two loxP site.After the clpP gene was cloned into the pGEM-T-Easy TA cloning vector,it was digested and linked with the Sp resistance cassette,yielding homologous recombination vector pIB △ clpP-Sp.The vector was linearized and used for the transformation of S.mutans UA159,with transformants selected on TPY plates containing Sp.The selected strain was transformed with the thermosensitive plasmid pCrePA to excise the Sp resistance gene.The pCre-PA was then easily eliminated at nonpermissive temperature,resulting in a markless mutant strain carrying a deletion at the clpP loci,which was verified by PCR and DNA sequencing.Results The result of the PCR analysis and DNA sequencing indicated that a part of the clpP gene was deleted.There was a loxP at this loci without the Sp resistance gene.Conclusion The markless clpP-deletion mutant of S.mutans was constructed successfully,which laid a foundation for further study of its biological function and its influence on the cariogenicity of S.mutans.

Objective To evaluate the usefulness of real-time three-dimensional transesophageal echocardiography(RT-3D-TEE)in cardiac valvular diseases.Methods Preoperative and postoperative RT-3D-TEE studies were performed in 32 patients who had undertaken cardiac valve repair or valvular replacement using Philips iE33 with X7-2 probe,and quantitative analysed the mitral structure in cases of mitral valve prolapse by using online QLAB7.0 quantitative analysis software.Results RT-3D-TEE can demonstrate the anatomicsl structure clearly,evaluate the function of cardiac valves precisely,and reveal details of type,position and extent of lesions vividly in surgical view.RT-3D-TEE helped correct misdiagnose in 2 cases,revised surgical plans in 3 cases,and guided implementation of remedial surgery in 1 case.Conclusions RT-3D-TEE can present images lively,and it can provide adequate information for preoperative diagnosis,aid the formulation of surgical plan and evaluate surgical effect.

ObjectiveTo optimize the condition of multiple fluorescence quantitative PCR and establish a new assay of four human herpes virus (HHV) detected by AllGlo quadruple fluorescence quantitative PCR.MethodsFour HHV including HSV-1,HSV-2,EBV and CMV were identified by sequence analysing the qualitative PCR production.Furthermore,they were quantitatively detected by AllGlo and TaqMan multiple fluorescence quantitative PCR respectively.ResultsBoth the positive rate and specificity of AllGlo and TaqMan in detecting single HHV achieved 100％.And AllGlo single fluorescence quantitative PCR prevailed over TaqMan's by Ct of 1-3.Four HHV can be simultaneously detected by AllGlo quadruple fluorescence quantitative PCR,comparing to the only two by TaqMan.ConclusionAllGlo fluorescence quantitative PCR assay allows a higher throughput,sensitivity and specificity than TaqMan in detection and thus provides a board prospect.

AIM: To investigate the expression of Smad2 signal protein in peritoneal mesothelial cells and how transforming growth factor ?1 (TGF-?1) affects its expression. METHODS: Rat peritoneal mesothelial cells were cultured in different levels of TGF-?1 (0,1.25,2.5,10 ?g/L) for different time (0,5,15,30,60,120 min). Endogenous Smad2 expression was evaluated by RT-PCR and immunohistochemical assay. The alteration of subcellular location of Smad2 was determined by immunohistochemical assay. RESULTS: TGF-?1 induced Smad2 mRNA expression, which increased at 5 min, peaked at 30 min, and declined to baseline levels by 120 min, in a time-dependent manner. Smad2 mRNA expression induced by TGF-?1 was also in a dose -dependent manner. TGF-?1 induced Smad2 phosphorlylation and nuclear localization in both time-dependent and dose-dependent manner, which was concordant with mRNA expression. Smad2 translocated from cytoplasm to nuclear accumulation in response to TGF-?1, and peaked at 30 min. CONCLUSIONS: Smad2 is present in peritoneal mesothelial cells. TGF-?1 may activate Smad2 expression and translocation to nuclear in a time-dependent and dose-dependent manner. [

Objective To study the pathological,ultrastructural and immunohistochemical features of kidney oncocytoma (RO). Methods The clinical and histological features of 13 patients (5 men and 8 women;mean age,58 years,age 44 -78 years) with RO were observed.Immunohistochemical staining was performed on paraffin-embedded tissues with a panel of antibodies including vimentin,EMA,ckpan,CK7,CK18,E-cadherin,CD10,RCC,CD117,34βE12,HMB45,s-100 and Ki-67.Of the 13 cases,10were found incidentally during a health examination while the other three cases presented with lumbago and discomfort of the lumber.Four cases were analyzed by electron microscopy.Eleven were treated with radical nephrectomy and 2 with partial nephrectomy. Results Histologically,the tumor cells were mainly arranged in closely packed nests,aciniform or tubule with an occasional microcystic pattern in loose edematous hypocellular fibrous stroma.Tumor cells had moderate to abundant granular eosinophilic cytoplasm with a small,round and uniform nucleus containing finely and evenly dispersed chromati.However,focal nuclear atypia were observed with no mitotic activity.Immunohistochemically,all cases were diffusely positive for CK18 and showed variable immunoreactivity for E-cadherin,CD117,CD10 and CK7.Twelve cases were positive for EMA. All cases were negative for vimentin,34βE12,HMB45 and s-100.The proliferative index (Ki-67) was very low in all cases,with less than 1％ of the nuclei labeled.Electronic microscopy showed the cytoplasm had abundant mitochondria with lamellar cristae.Follow-up on 10 patients ( range from 2 to 67months) showed no recurrence or metastasis. Conclusions Tumor cells arranged in nests with loose hypocellu]ar and edematous stroma is the most important histological feature of RO.The immunohistochemical features could be helpful for both diagnosis and differential diagnosis.

Objective To investigate the expression and significance of estrogen receptor alpha in renal tissue of immunoglobulin A (IgA) nephropathy.Methods Fifty renal tissue samples including forty five cases of IgA nephropathy and five cases of normal expression were collected.The expression of estrogen receptor αt (ERα) was detected by immunohistochemistry method,and its relationship with clinical parameters and the degree of glomerular damage were analyzed.Results ERα was located in the glomerular and renal tubules.With the severity of the lesion,the expression of ERα in renal tissue of IgA nephropathy decreased gradually (P =0.001) and its expression was associated with estimated glomerular filtration rate (eGFR),serum creatinine (Scr),and pathologic grade of IgA nephropathy (r =0.876,-0.818,and -0.736,P ＜ 0.05),The expression of ERα was significantly decreased in hypertensive patients with IgA nephropathy,and the difference was statistically significant compared with that in non-hypertensive group (P =0.011).Conclusions The expression of ERα in renal tissue of IgA nephropathy was significantly decreased and there was a correlation between the degree of renal tissue damage,suggesting that ERα might play a role in the development of renal disease.

AIM: To investigate the role of Smad7 in the Smad2 expression induced by transforming growth factor-?_1(TGF-?_1) in rat peritoneal mesothelial cells(PMCs).METHODS: Rat PMCs were cultured at different doses of TGF-?_1 (0,1.25,2.5,10 ?g/L) for different time(0,5,15,30,60,120 min).PCDNA3-Smad7 was then transfected into cultured rat PMCs by lipofectamine,and the cells were stimulated like the above.Endogenous Smad2 and Smad7 expression was evaluated by RT-PCR and Western blotting.RESULTS: TGF-?_1 induced increase in Smad2 mRNA and protein expression at 5 min,peaked at 30 min,and declined to baseline levels at 120 min, which was in a time-dependent manner.TGF-?_1 also induced Smad7 mRNA expression at 5 min,and then declined,down to the lowest at 30 min,but at 60 min it increased again.Smad2,Smad7 mRNA and protein expression induced by TGF-?_1 were also dose-dependent.After transfection,overexpressions of Smad7 mRNA and protein in rat PMCs were observed,which did not decline with time.The expression of Smad2 mRNA significantly decreased by 33%,56%,67%,71%,63% and 57%(P

AIM:To investigate the effects of brain-derived neurotrophic factor(BDNF)on extracellular proteolytic enzymes including matrix metalloproteinases(MMPs)and serine proteases,in particular,the urokinase-type plasminogen activator(uPA)-plasmin system in a human umbilical vein endothelial cell(HUVEC)model.METHODS:The HUVEC was activated with different doses of BDNF(25-200 ?g/L)for different time period(6-48 h).Reverse transcriptase-polymerase chain reaction(RT-PCR)was used to assay MMP-2,MMP-9,TIMP-1,and TIMP-2 mRNA in HUVEC.The cultured conditioned medium was analyzed for MMP and uPA activity by gelatin zymography and fibrin zymography,respectively.uPA,PAI-1,TIMP-1,and TIMP-2 were quantified by Western blotting analysis.RESULTS:The stimulation of serum-starved HUVECs with BDNF caused marked increase in MMP-2 and MMP-9 mRNA expression and induced the pro-MMP-2 and pro-MMP-9 activation without significant differences in proliferation.However,BDNF had no effect on TIMP-1 and TIMP-2 production.BDNF increased uPA and PAI-1 production in a dose dependent manner up to 100 ?g/L,while effects of 200 ?g/L were approximately equal to those of 100 ?g/L.BDNF stimulated uPA and PAI-1 production beyond that in control cultures from 12 h until 48 h after BDNF addition.Protease activity for uPA was also increased by BDNF in a dose dependent manner.CONCLUSION:BDNF activates MMP and uPA/PAI-1 proteolytic network in HUVEC.