Estradiol stimulated proliferation and differentiation of prostatic stromal cells through regulation of BPH-1 paracrine

AIM: To characterize the effect of estradiol on proliferation, differentiation and extracellular matrix (ECM) accumulation in stromal cells through regulation of BPH-1 paracrine. METHODS: BPH-1 cells were stimulated with different concentrations of estradiol. Conditioned media (CM) were harvested and their effects on stromal cell cultures were tested. Cell proliferation was determined by MTT assay. mRNA of smoothelin, fibronectin, collagen Ⅳ and transforming growth factor β₁(TGF-β₁) were analyzed by real-time RT-PCR. Western blotting was used to determine smooth muscle myosin heavy chain (SMMHC). ELISA and radioimmunoassay were respectively used to measure fibronectin, TGF-β₁ and collagen Ⅳ protein expressions.RESULTS: Estrodiol stimulated the expression and secretion of TGF-β₁ in BPH-1 cells. The proliferation of stromal cells increased when they were cultured with CM harvested from estrogen treated BPH-1 cells. The mRNA levels of collagen Ⅳ and smoothelin increased in stromal cells treated with CM from BPH-1 cells. The results of radioimmunoassay also showed that the collagen Ⅳ protein level up-regulated in the supernatants and cell extracts of CM-treated stromal cells. A neutralizing antibody to TGF-β₁ inhibited the stimulation of collagen Ⅳ and SMMHC by BPH-1 CM. The expression of fibronectin was only marginally changed in stromal cells cultured in the presence of BPH-1 CM. CONCLUSION: The BPH-1 cells increase ECM accumulation and differentiation of stromal cells through TGF-β₁. Estradiol stimulate differentiation of stromal cells by induction of TGF-β₁ expression. Estradiol stimulate proliferation by influencing the factors secreted from prostatic epithelial cells.     

Caspase 3 inhibitor Ac-DEVD-CHO attenuates 10-hydroxycamptothecin-induced activation of NF-κB in Bcap37 cells

AIM:To investigate the role of caspase 3 inhibitor Ac-DEVD-CHO in caspase 3 signaling pathway and NF-κB activation induced by 10-hydroxycamptothecin (HCPT) in human breast carcinoma cells. METHODS:The cell growth inhibition was measured by MTT assay. Agarose gel electrophoresis was performed for detecting cell apoptosis. Western blotting was used for determining protein expression. DIG-EMSA was conducted to measure the DNA-binding activation of NF-κB. RESULTS:Caspase 3 inhibitor Ac-DEVD-CHO attenuated HCPT-induced apoptosis in human breast carcinoma. Ac-DEVD-CHO also suppressed the degradation of caspase 3 and IκBα,and arrested the activation of NF-κB. CONCLUSION:Caspase 3 inhibitor Ac-DEVD-CHO regulates the activation of caspase 3 and NF-κB,and attenuates apoptosis in Bcap37 cell line induced by HCPT.     

Characteristics of ventricular electrophysiology in a right ventricular rapid pacing-induced canine heart failure model

AIM: To research the characteristics of ventricular electrophysiology in right ventricular rapid pacing-induced congestive heart failure (CHF) dogs.METHODS: Dogs (n=16) were randomly divided into 2 groups: the control (n=7) and the CHF group (n=9) induced by rapid right ventricular pacing at 240 pulse·min-1 for 4 to 5 weeks.The electrophysiologic parameters were evaluated by the technique of standard electric stimulation and monophasic action potential (MAP) recording.RESULTS: (1) Ventricular effective refractory period (VERP),ventricular MAP duration (MAPD90),ventricular late repolarization duration (VLRD) and intra-ventricular conduction time (IVCT) were prolonged by 26% (P<0.01),43% (P<0.01),318% (P<0.05),and 19% (P<0.01),respectively in CHF group.(2)The ratio of VERP to MAPD₉₀ (VERP/MAPD₉₀) was decreased by 13% (P<0.05) in CHF group.(3) The dispersion of ventricular recovery time (VRT-D) was increased by 185% (P<0.01) in CHF group.(4) The ventricular fibrillation threshold (VFT) was decreased by 48% (P<0.01) in CHF group.CONCLUSION: The abnormal electrophysiological changes in the CHF condition may be contributing factors of lethal ventricular arrhythmias and sudden cardiac deaths in CHF.     

Effect of different PRA serum levels from patients with β-thalassemia on the proliferation and differentiation potential of the hematopoietic stem cells /progenitor cells of cord blood

AIM: To probe the effect of different panel reactive antibody(PRA) serum levels from patients with β-thalassemia on the proliferation and differentiation potential of the hematopoietic stem /progenitor cell of cord blood.METHODS: 1×105 mononuclear cells (MNCs) isolated from umbilical cord blood were incubated with different PRA serum levels (0 μL,50 μL,100 μL) respectively and complement,inoculated into the methylcellulose cultural system.The proliferation and differentiation potential of the hematopoietic stem /progenitor cell of cord blood by the colony formation assay were detected on day 7 and day 14,respectively.RESULTS: After culture of 7 days,the total colonies and CFU-GM were 88.20±9.41,79.00±11.39 in group A and 88.60±9.12,79.20±10.44 in group B,which were significantly higher than those of 20.60±7.39,15.20±4.66 in group C and those of 4.00±2.05,1.40±0.51 in group D (P<0.01).Meanwhile compared with group A and group E,no significant difference was found (P>0.05).After culture for 14 days,the total colonies and CFU-GM were 216.00±31.10,117.40±24.80 in group A and 213.20±31.06,116.00±19.75 in group B,which were significantly higher than those of 97.80±14.43,32.80±8.10 in group C and those of 31.40±13.41,8.40±4.30 in group D (P<0.01).The CFU-GEMMs were 45.60±8.51 in group A and 42.60±7.03 in group B,which were significantly higher than those of 20.80±6.96 in group C and those of 7.80±6.06 in group D (P<0.05).The BFU-MK was 12.80±4.42 in group A and 11.00±2.74 in group B respectively,which were significantly higher than that of 1.00±0.55 in group D (P<0.05).The CFU-E in group B 17.20±4.03 was significantly higher than that of 5.60±2.87 in group D (P<0.05).Meanwhile compared with group A and group E,no significant difference was found (P>0.05).By the Kendall test,there were negative correlations between the level of PRA serum and the total colonies,CFU-GM on day 7,the total colonies,CFU-GM,CFU-GEMM,BFU-E,BFU-MK on day 14 (tau-b=-0.793,-0.849,-0.808,-0.804,-0.645,-0.674,-0.624,P<0.01).There was a negative correlation between the level of PRA serum and CFU-MK on day 14 (tau-b=-0.466，P<0.05).CONCLUSION: PRA sera inhibit the colony in the colony cultures of the hematopoietic stem cells/progenitor cells in cord blood.The inhibition depends on the level of PRA sera.The higher the level of PRA sera,the stronger the inhibition is observed in our study.     

Sequence analysis and prokaryotic expression system construction of PIA genes isolated from Neisseria gonorrhoeae

AIM: To analyze the nucleotide and putative amino acid sequences of PIA genes isolated from N.gonorrhoeae and to construct the prokaryotic expression system of PIA gene.METHODS: The entire PIA genes from 9 strains of N.gonorrhoeae were amplified by using high fidelity PCR.The target amplification fragments were sequenced after T-A cloning.Homology comparison of the nucleotide and putative amino acid sequences of PIA genes from the isolates with the reported sequences in GenBank was then performed.A prokaryotic expression system of PIA gene was constructed.Different dosages of IPTG were applied to induce the expression of the target recombinant protein (rPIA) and 10% SDS-PAGE plus Bio-Rad Agarose Image Analysor was used to determine the expression level of rPIA.rPIA was extracted using Ni-NTA affinity chromatography and the purified effect was detected by SDS-PAGE.RESULTS: In comparison with the reported PIA gene sequences (GenBank No: L19962),the homologies of nucleotide and putative amino acid sequences of PIA genes from the isolates were 99.6%-100% and 99.1%-100%,respectively,which indicated that all the isolates were belonging to serovars IA6.Output of rPIA was as high as 50.1% of the total bacterial proteins.The purified rPIA only showed a single target protein fragment in gel.CONCLUSION: Serovar IA6 is dominant in the local N.gonorrhoeae isolates and sequences of the encoding gene are relatively conserved.The constructed prokaryotic expression system is able to express rPIA with high efficiency,which may lay a foundation for further development of serological detection kit and vaccine of N.gonorrhoeae.     

不同装液量对金顶侧耳液体摇瓶培养的影响

以食用菌金顶侧耳为试验研究对象;用综合PDA(马铃薯20 g,葡萄糖20 g,硫酸镁1.5g,磷酸二氢钾3.0g,水1 000 mL,pH自然)为液体培养基;以(25±1)℃,180 r/min为气浴恒温振荡器的温度和振荡速率.设定了60、80、100、120、140 mL 5个不同的装液量.经过7 d的摇瓶培养,过滤发酵液,得到菌丝体和粗酶液.再通过测定菌丝体烘干后的生物量,以及测定粗酶液中多酚氧化酶和漆酶活性,得出了:装液量为100 mL,菌丝体烘干生物量明显大于其他装液量;装液量为140 mL,有利于多酚氧化酶的分泌,酶的活力较强,并且其活力随装液量的递增而变强.装液量为100mL,有利于漆酶的分泌,酶的活力较强,不同装液量的菌丝生长都在第6天出现了高峰期,其中以装液量为100mL/250mL时,其菌丝球数量较多.     

氮肥运筹对杂交苏丹草产量饲用品质及再生系数的影响

采用大田小区栽培试验,研究了氮肥对杂交苏丹草产量和饲用营养品质及再生系数的影响。结果表明,杂交苏丹草干物质总产量氮肥处理显著高于对照,N4处理最高,相同的施氮量分次施用比一次性施用增产效果好。氮肥处理粗蛋白(CP)含量均高于对照,头茬草CP含量最高,随收获次数增加,CP含量降低,分次施肥处理施氮量增加,CP含量降低。对照处理粗灰分(CA)含量最高,随收获次数增加CA和酸性洗涤纤维(ADF)含量降低,无氮浸出物(NFE)含量升高。杂交苏丹草5大营养成分产量均是N4处理最高,对照最低,CP产量顺序是N4>N1>N2>N3>N0,3次收获的粗脂肪(EE)、CA、ADF、NFE总产量,均是肥料分次施用比一次性施用的处理高。各氮肥处理总能量(GE)均高于对照,其中N4处理最高,其次是N2处理,分次施肥的处理有利于总能量的增加。再生系数表现为相同施氮量分次施用处理高于一次性施用的处理,N4处理可使杂交苏丹草再生系数达到最大。比较发现,生产上采用N4(600 kg/hm2,底肥∶追肥=1∶2)处理可获得高产优质的牧草。