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 comprae the difference of Sex hormone,serum and follicular fluid anti Mullerian hormone (AMH) level tube,number of retrieved egg,the number of cleavage rate,fertilization oocytes,the number of available embryos and total embryo on in vitro fertilization and embryo transfer (IVF-ET) of different reaction groups,to explore the relationship between AMH and pregnancy outcome of IVF-EH.Methods Eighty-one cases of IVF-ET assisted pregnancy were divided into low ovarian response group,normal ovarian response group and high ovarian response group according to the response of the super ovulation.Serum sex hormone and AMH of all patients were detected on the third day of menstruation,on the day of HCG injection,oocyte pick-up (OPU) and embryo transfer (ET),AMH in FF were detected too.The difference of oocytes,cleavage rate,embryo quality and the indicators mentioned above among the three groups were compared.All of the above indicators were compared between pregnancy group and unpregnancy group.Results (1) The level of AMH on the third day of menstruation ((1.76 ± 0.47) μg/L,(3.45 ± 1.01) μg/L,(6.34 ± 1.29) μg/L,F =2.435,P＜0.001),HCG injection ((1.09±0.13)μg/L,(1.29±0.29)μg/L,(2.64±0.51)μg/L,F=1.542,P=0.001) and embryo planting((1.32±0.33) μg/L,(1.62±0.39) μg/L,(1o 91±0.41) μg/L,F =1.573,P ＜0.001) all were statistically significant in three groups.On day of OPU,the levels of serum AMH ((0.95±0.21)μg/L,(1.15±0.29) μg/L,(1.74±0.41) μg/L,F =12.573,P＜0.001) and AMH in FF((5.82±1.19) μg/L,(6.92±1.05) μg/L,(7.79±1.39) μg/L,F =9.83,P＜0.001) were statistically differences in the three groups.(2)The number of oocytes were positively correlated with AFC,based AMH,levels of E2,P and AMH on the day of HCG injection and OPU,E2 and AMH on the day of ET and AMH in FF(P＜0.05).(3)AMH in FF in pregnancy group was (6.22±0.82)μ g/L,significantly higher than those without pregnancy group ((5.31 ±0.71)μg/L,P =0.037).Conclusions Levels of AMH in serum and follicular fluid are good predictors of ovarian response in IVF-ET.The level of serum AMH can only predict ovarian response,but can not effectively predict pregnancy outcome.The level of AMH in FF is a good predictor of pregnancy outcome.

Objective To study the association between three single nucleotide polymorphisms (SNP; rs2241766,rs1501299 and rs12495941) variants of the adiponectin gene (ADIPOQ) and polycystic ovary syndrome (PCOS) in PCOS family trios.Methods A total of 224 unrelated PCOS probands,their biological parents were recruited.Anthropometric variables such as waist circumference (WC),hip circumference (HC),height and weight were measured in all subjects during the first visit to the outpatient department.Body mass index (BMI) and waist-hip ratio (WHR) were calculated.Serum fasting glucose (FBG),fasting insulin (FINS),total cholesterol (TC),triglycerides (TG),low-density lipoprotein and highdensity lipoprotein cholesterol (HDL) levels were measured.PCOS patients were divided into two groups based on BMh group A (BMI＜25 kg/m2) and group B (BMI≥25 kg/m2).Parents of PCOS patients were accordingly categorized into group C,D (fathers) and group E,F (mothers).The transmission disequilibrium test (TDT) was used to analyze the association between three SNP of ADIPOQ and PCOS.Results (1) A significant positive association was detected between SNP rs1501299 and PCOS (x2=7.093,P=0.008).However we failed to find significant overtransmission of the other two SNP rs2241766 and rs12495941 from parents to PCOS offsprings (x2=1.620,P=0.203; x2=0.713,P=0.398).(2) Linkage disequilibrium (LD) was analyzed in the subjects,rs1501299 and rs2241766 were in weak LD (r2=0.063,D'=0.621).(3) The levels of WC,HC,WHR,testosterone,TG,HDL and FINS were significantly differences between obese and lean PCOS patients (P＜0.05).While in fathers we only found WC,HC,TC levels being statistically different (P＜ 0.05).Mothers of obese PCOS patients had increased levels of FINS compared with mothers of lean PCOS patients (P＜0.05).The genotype frequencies of the three SNP were not different in obese and lean PCOS patients and their parents (P＞0.05).Conclusions TDT confirms that SNP rs1501299 in the ADIPOQ is significantly associated with the risk of PCOS in the Chinese Han population.The three SNP of the ADIPOQ were not associated with the obesity of PCOS.

Objective To evaluate the role of nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway in mitigation of anoxia/reoxygenation (A/R)-induced damage to rat cardiomyocytes by emulsified isoflurane postconditioning.Methods Primarily cultured cardiomyocytes obtained from Sprague-Dawley rats,aged 16-20 weeks,were seeded into the laminin pre-treated 6-well plates at a density of 104/cm2 and cultured for 20 h.The cells were randomly divided into 3 groups (n =12 each) using a random number table:control group (group C),group A/R,and emulsified isoflurane postconditioning group (group EI).The cells were cultured for 110 min without any treatment in group C.The cells were exposed to 45 min anoxia followed by 60 min reoxygenation in A/R and EI groups.After 45 min of hypoxia,the cells were cultured with emulsified isoflurane 1.68 mmol/L for 5 min before onset of reoxygenation in group EI.At the end of reoxygenation,transmission electron microscope was used to observe the ultrastructure of myocardial cells,and mitochondrial function was scored.The mRNA and protein expression of Nrf2,heme oxygenase-1 (HO-1),superoxide dismutase 1 (SOD1),and quinone oxidoreductase 1 (NQO1) was detected by real-time PCR and Western blot.Laser scanning confocal microscopy was used to detect Nrf2 nuclear translocation at the end of incubation,and Nrf2 activity was recorded in the nucleus of myocardial cells.Results Compared with group C,the mitochondrial function score was significantly increased,the mRNA and protein expression of Nrf2,HO-1,SOD1 and NQO1 was down-regulated,and Nrf2 activity in the nucleus was enhanced in A/R and EI groups.Compared with group A/R,the mitochondrial function score was significantly decreased,the mRNA and protein expression of Nrf2,HO-1,SOD1 and NQO1 was up-regulated,and Nrf2 activity in the nucleus was enhanced in group EI.Conclusion The mechanism by which emulsified isoflurane postconditioning mitigates A/R-induced damage to rat cardiomyocytes is related to induced nuclear translocation of Nrf2 and activated Nrf2-ARE signaling pathway.

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.

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.

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 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 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.