Effects of hypoxia-inducible factor-1 signal pathway on proliferation in hypoxic pulmonary artery smooth muscle cells

AIM: To investigate the effect of hypoxia on the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMC); and to evaluate the role of hypoxia-inducible factor-1α(HIF-1α), iNOS, P-ERK1/2 protein expression in hypoxic pulmonary hypertension (HPH) pathogenesis.METHODS: Cultured rat PASMC were divided into normoxic group; hypoxic group; hypoxia+ADM(adrenomedulin) group, hypoxia+L-NAME(iNOS inhibitor) group; hypoxia+PD98059 group. Proliferation was investigated by MTT and PCNA. Apoptosis was examined by flow-cytometry. Westen blotting was used to measure protein expression of HIF-1α, P-ERK1/2 and iNOS. RESULTS: (1) A value of 24 h-hypoxia was significantly higher than that in the normoxic group (P<0.01). In the hypoxia+PD98059 group, ADM was significantly lower than that in hypoxia group, whereas A value of the hypoxia+L-NAME was significantly higher than that in hypoxic group and normoxic group (P<0.01). (2) PCNA was positive in PASMC after 24 h hypoxia (P<0.01). PD98059, ADM inhibited the expression of PCNA significantly (P<0.01), whereas L-NAME increased the expression of PCNA significantly (P<0.01). (3) Apoptosis index was not significantly difference among the different groups (P>0.05). (4) HIF-1α, iNOS and P-ERK1/2 expression was poorly positive in normoxic group, positive after hypoxia for 4h (P<0.01), reaching its peak at 8 h hypoxia (P<0.01), HIF-1α, P-ERK1/2 expression declined after 24 h hypoxia. L-NAME promoted the expression of HIF-1α, PD98059 inhibited the expression of HIF-1α, iNOS and P-ERK1/2 partly. ADM inhibited the expression of HIF-1α partly, promoted the expression of iNOS. CONCLUSION: (1) Hypoxia stimulates the proliferation of PASMC, and has no obvious effects on the apoptosis of PASMC. (2) HIF-1 plays an importent role in the proliferation of hypoxic PASMC.     

灵芝孢子对肿瘤细胞生长的抑制效果研究

应用3种肿瘤细胞模型对未破壁灵芝孢子、机械破壁灵芝孢子和全灵芝剥壁孢子进行抗肿瘤试验研究。结果表明,未破壁及不同破壁方式的灵芝孢子对人恶性乳腺癌细胞(MT-1)、人恶性淋巴癌细胞株(Jurkat)、人恶性脑肿瘤细胞(U343)均有不同程度的抑制作用,抑制效果强弱为全灵芝剥壁孢子机械破壁灵芝孢子未破壁灵芝孢子,表明破壁的确能提高灵芝孢子对肿瘤细胞的抑制作用,而且选择破壁的方法也很重要,生物酶法破壁能使灵芝孢子的生物活性成分得到充分释放,所以大大提高灵芝孢子的功效。     

Hemin induces and sustains the phosphorylation of Erk1/2 in human umbilical vascular endothelial cells

AIM: To investigate the relationship between hemin and Erk1/2 activation in human umbilical vascular endothelial cells (HUVECs). METHODS: Cultured HUVECs were separately incubated with hemin or H₂O₂ for different times. Subsequently Erk1/2 phosphorylation and total Erk1/2 were determined by Western blotting assay. Flow cytometry was employed to determine the cell cycle distribution. RESULTS: Hemin at the concentration of 1-10 μmol/L induced the phosphorylation of Erk1/2 in HUVECs, and sustained the phosphorylation of Erk1/2 for three hours. The duration of phospho-Erk1/2 induced by hemin was much longer than that in H₂O₂ control (3 h vs 30 min). CONCLUSION: Hemin induces and sustains the phosphorylation of Erk1/2 in HUVECs, which indicates that the effect of hemin on the Erk1/2 activation may be one of pharmacological target of hemin.     

Effects of baicalin on CA46 cell xenografts in nude mice

AIM: To study the effects of baicalin on CA46 cell xenografts in nude mice. METHODS: The nude mice with CA46 cell xenografts were treated with drugs via intraperitoneal injection daily, and were divided into 5 groups: negative control group, 15 mg/kg baicalin group, 30 mg/kg baicalin group, 60 mg/kg baicalin group and 4 mg/kg etoposide (VP-16) positive control group. After 12-day treatment, the weight of CA46 cell xenografts stripped from some nude mice in the 5 groups was used to evaluate the effect of baicalin on xenograft growth in the nude mice. The apoptosis, necrosis and pathological changes of the xenograft cells were examined under light microscope and transmission electronic microscope respectively. The expression levels of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway-related proteins extracted from xenografts were determined by Western blotting. The other nude mice with CA46 cell xenografts in the 5 groups continued to be treated with the drugs until death in order to evaluate the effect of balcalin on survival time of the nude mice with CA46 cell xenografts. RESULTS: Baicalin remarkably inhibited the growth of CA46 cell xenografts, induced apoptosis and necrosis of xenograft cells, and reduced the protein expression of phospho-Akt (p-Akt), nuclear factor-kappa B (NF-κB), mammalian target of rapamycin (mTOR) and phospho-mTOR (p-mTOR) in the xenografts after 12-day treatment. Furthermore, baicalin prolonged the survival time of the nude mice with CA46 cell xenografts in a dose-dependent manner. CONCLUSION: Baicalin inhibits the growth and induces apoptosis of CA46 cell xenografts in the nude mice, and prolongs the survival time of the nude mice with CA46 cell xenografts through the mechanism of down-regulating PI3K/Akt/NF-κB and PI3K/Akt/ mTOR signaling pathways.     

Effects of exosomes derived from hypoxia-preconditioned umbilical cord mesenchymal stem cells on function of endothelial cells

AIM To investigate the effect of exosomes derived from hypoxia-preconditioned human umbilical cord mesenchymal stem cells (hUCMSCs) on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs). METHODS hUCMSCs and HUVECs were isolated, cultured and identified. Exosomes derived from hUCMSCs were extracted by ultracentrifugation. The morphological change of exosomes was observed under transmission electron microscope. The particle size and concentration of exosomes were detected by nanoparticle tracking analysis, and the surface specific marker proteins of exosomes were determined by Western blot. hUCMSCs were divided into normoxia group and hypoxia group. The viability of hUCMSCs was measured by CCK-8 assay. HUVECs were divided into control group, normoxic exosome group and hypoxic exosome group. The proliferation of HUVECs was detected by EdU assay. The migration ability was detected by cell scratch assay and Transwell experiment. Tube formation ability was evaluated by tube formation experiment. RESULTS Compared with normoxia group, hypoxia pretreatment enhanced the viability and exosome release of hUCMSCs. Compared with normoxic exosome group, hypoxic exosomes enhanced the proliferation, migration and tube formation of HUVECs. CONCLUSION Exosomes derived from hUCMSCs under hypoxia enhances the proliferation, migration and tube formation of HUVECs.     

Effects of Nrf2 overexpression on proliferation,activation and collagen type I synthesis in cultured hepatic stellate cells stimulated by ethanol

AIM：To investigate the effects of nuclear factor E2-related factor 2 (Nrf2) overexpression on the proliferation, cell cycle distribution, collagen type I (Col I) synthesis and alpha-smooth muscle actin (α-SMA) expression in cultured hepatic stellate cell line HSC-T6 stimulated by ethanol. METHODS：Cultured HSC-T6 cells were transfected with pEGFP-Nrf2 or pEGFP-N1 (empty vector) plasmid by liposome transient transfection. The cells were divided into control group, ethanol group, ethanol+pEGFP-Nrf2 group and ethanol+pEGFP-N1 group. The mRNA expression of Nrf2, α-SMA and Col I was determined by RT-PCR, and their protein expression was detected by Western blotting. Cell proliferation was assessed by MTT assay, and cell cycle was detected by flow cytometry. RESULTS：The pEGFP-Nrf2 plasmid was successfully transfected into HSC-T6 cells, and the mRNA and protein expression of Nrf2 was higher than other three groups 48 h after transfection (P<0.05). Compared with control group, the cell proliferation and the mRNA and protein expression of α-SMA and Col I in ethanol group and ethanol+pEGFP-N1 group were significantly increased (P<0.05), and the numbers of HSC-T6 cells were decreased in G1 phase and increased in S phase (P<0.05), without significant differences between the two groups (P>0.05). Meanwhile, the cells in ethanol+pEGFP-Nrf2 group showed significantly decreased proliferation level, down-regulated mRNA and protein expression of α-SMA and Col I, higher numbers in G1 phase and lower numbers in S phase compared with ethanol group and ethanol+pEGFP-N1 group (P<0.05). CONCLUSION：Nrf2 overexpression could significantly down-regulate the expression of α-SMA and Col I and cause G1/S phase arrest in HSC-T6 cells cultured with ethanol, thus inhibiting the proliferation and activation of the cells.     

禽致病性大肠杆菌Hcp2b对雏鸡气管黏膜细胞因子-细胞因子受体相互作用通路的影响

T6SS(type Ⅵ secretion system)是革兰阴性菌中常见的一种分泌系统,其效应蛋白Hcp2b作用机制迄今仍未明晰.本研究以禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)Hcp2b蛋白为研究主体,旨在探究Hcp2b蛋白在APEC感染鸡气管黏膜过程中发挥...     

第十九期鸡胚原始生殖细胞的冷冻保存

采用Ficoll密度梯度离心,提取第 19期(孵化 72h)性腺中的PGCs,对其应用不同的冷冻保护液和不同的平衡方法进行冷冻保存,并于复苏后进行体外培养。复苏后的PGCS用台盼蓝染色检测其存活率,结果发现:从第 19期性腺中获取的PGCs在同一种冷冻保护液下,采用不同的平衡方法进行冷冻,对PGCs的存活率有显著影响(P<0.05)或极显著影响(P<0.01);平衡方法相同,在不同冷冻保护液之间存在显著(P<0.05)或极显著 (P<0.01)差异。PGCs经体外培养 24h后再进行冷冻保存,复苏后其存活率、体外培养存活时间均极显著(P<0.01)短于分离后直接冷冻的PGCs。     

High‐level exogenous trans10, cis12 conjugated linoleic acid plays an anti‐lipogenesis role in bovine mammary epithelial cells

Primary bovine mammary epithelial cells (BMECs) were treated by 0, 37.5, 75, 112.5, 150 μmol/L trans10, cis12 conjugated linoleic acid (CLA) to evaluate the effects of different level trans10, cis12 CLA on lipogenesis in BMEC. Addition of 75–150 μmol/L trans10, cis12 CLA reduced significantly the triacylglycerol (TAG) content (P < 0.05), but did not have inhibiting action on cell proliferation (P > 0.05). Treatment with 150 μmol/L trans10, cis12 CLA for 48 h resulted in a 17.1% reduction (P < 0.0001) of medium chain fatty acids (MCFA, C14 < C < C16), a 26.5% reduction (P < 0.0001) of unsaturated fatty acids (UFA) and a corresponding reduction of the mRNA abundance of acetyl coenzyme A (acetylCoA) carboxylase (ACC) (P = 0.046), fatty acid synthase (FAS) (P = 0.017) and stearoylCoA desaturase1 (SCD1) (P = 0.002). Another finding was that trans10, cis12 CLA elevated expression of diacylglycerol acyltransferase2 (DGAT2) (P = 0.020) and long chain acylCoA synthetases (ACSL) (P = 0.032). In conclusion, higher trans10, cis12 CLA, not low trans10, cis12 CLA, inhibited milk fat synthesis and changed fatty acid composition by regulating the expression of FAS, ACC, SCD1, DGAT2 and ACSL.     

猪丁型冠状病毒在悬浮培养猪肾细胞LLC-PK1上的增殖特性分析

旨在分析猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)在悬浮培养的猪肾细胞LLC-PK1上的增殖特性,为PDCoV灭活疫苗的规模化生产提供细胞材料。采用逐步降血清法优化LLC-PK1细胞悬浮培养工艺;利用有限稀释法筛选PDCoV适应性细胞株;利用间接免疫荧光法鉴定PDCoV对LLC-PK1细胞的感染性;分别对PDCoV接种LLC-PK1悬浮细胞的初始密度、MOI、收毒时间、TPCK胰酶浓度等参数进行优化,确定最佳悬浮培养条件。成功筛选出可高效增殖PDCoV的单克隆悬浮细胞株LLC-PK1Sa,且利用其增殖的PDCoV可特异性的感染LLC-PK1细胞;PDCoV按MOI为10^-3接种于密度为2×10⁶ cells·mL^-1的LLC-PK1Sa细胞,当TPCK胰酶终浓度达到7.5 μg·mL^-1时,接毒后48 h收获的病毒液滴度最高。本研究首次实现了PDCoV在LLC-PK1Sa悬浮细胞中的高效增殖,并对悬浮培养条件进行了初步优化,可为PDCoV灭活疫苗的规模化生产提供理论参考。