受体相互作用蛋白1(RIP1)对BCG诱导小鼠巨噬细胞凋亡的调控作用

旨在通过构建受体相互作用蛋白1(RIP1)腺病毒干扰载体,研究其对BCG诱导的RAW264.7细胞凋亡相关指标的影响,以探讨其在BCG诱导RAW264.7凋亡过程中的调控作用。笔者构建RIP1腺病毒干扰载体,并转染感染BCG的小鼠RAW264.7细胞系,利用流式细胞仪检测各处理细胞凋亡率、细胞线粒体膜电位、细胞活性氧水平及细胞周期等指标,并用Western blot检测凋亡相关蛋白的表达水平。结果显示:BCG感染显著上调了RIP1的蛋白表达水平并提高了小鼠巨噬细胞RAW264.7的凋亡率,当RIP1被干扰后,BCG感染后的RAW264.7细胞凋亡率和活性氧水平显著降低,而促凋亡蛋白Bax表达量显著下调,线粒体膜电位和抑凋亡蛋白表达量上调。同时,BCG感染后细胞周期滞留于G_1期。BCG感染可有效上调RIP1表达量并诱导RAW264.7细胞凋亡。RIP1通过下调BCG感染后RAW264.7细胞的线粒体膜电位,上调活性氧含量并提高凋亡相关蛋白Bax/Bcl-2比值,使细胞周期阻滞于G_1期从而参与诱导细胞凋亡。     

塞内卡病毒A结构蛋白VP1诱导PK-15细胞凋亡

塞内卡病毒A(SVA)作为一种新发病病原,致病机制仍不清楚。通过显微镜观察发现,SVA感染PK-15细胞后能使细胞产生明显的细胞病变(CPE),同时伴随着严重的细胞凋亡。为了深入研究SVA诱导凋亡的情况,在验证SVA各蛋白真核质粒正常表达后,通过Annexin V-FITC/PI双染流式方法检测SVA各蛋白诱导凋亡的情况,Annexin V-FITC/PI和Hoechst染色后显微镜观察磷脂酰丝氨酸和核凝聚程度,通过Western blotting和RT-PCR技术分析SVA-VP1对凋亡通路中主要调控分子Caspase3、Caspase8和Bax蛋白质和mRNA水平的影响,发现SVA-VP1能够显著诱导细胞发生早期和晚期凋亡,并且能够促进凋亡蛋白Caspase3、Caspase8及Bax蛋白和mRNA水平的上调。本研究结果为深入研究SVA调控宿主细胞凋亡的分子机制和致病机制奠定了理论基础。     

Effects of methionine partially replaced by methionyl‐methionine dipeptide on intestinal function in methionine‐deficient pregnant mice

This study was to compare the effects of parenteral supplementation of methionyl‐methionine (Met‐Met) or Met on intestinal barrier function in Met‐deficient pregnant mice. Pregnant mice were randomly divided into three groups. The Control group was provided a diet containing Met and received i.p. injection of saline. The Met group was fed the same diet but without Met and received daily i.p. injection of 35% of the Met contained in the control diet. The Met‐Met group was treated the same as the Met group, except that 25% of the Met injected was replaced with Met‐Met. Met‐Met promoted villus surface area in ileum compared with Met alone. In addition, the mRNA abundance of amino acid and glucose transporters in the small intestine was altered with Met‐Met. Moreover, Met‐Met increased tight junction protein and decreased apoptosis‐related proteins expression in the jejunum and ileum. These results suggest that Met‐Met can promote intestinal function over Met alone in Met‐deficient mice.     

miR-18a通过靶向结合CTGF调控猪颗粒细胞凋亡

为探究miR-18a对猪卵巢颗粒细胞凋亡的调控作用,利用生物信息学分析、荧光素酶活性检测和体外培养颗粒细胞试验验证miR-18a对CTGF的靶向作用及其在猪颗粒细胞中对CTGF基因表达的影响,并通过流式细胞技术检测其对猪卵巢颗粒细胞凋亡的调控作用。生物信息学分析结果表明,CTGF是miR-18a的潜在靶基因,荧光素酶活性检测进一步验证了miR-18a与CTGF的结合。在培养的颗粒细胞中转染miR-18a模拟物后,qRT-PCR和Western blot结果显示CTGF的mRNA和蛋白水平均显著降低,流式细胞技术检测表明miR-18a显著促进颗粒细胞的凋亡。而转染miR-18a抑制剂后,猪颗粒细胞的凋亡率显著降低,共转染miR-18a抑制剂和CTGF的干扰RNA后,颗粒细胞的凋亡率呈现回升。试验结果表明miR-18a通过靶向结合CTGF基因调控猪颗粒细胞的凋亡。     

Over-expression of P21 attenuates cisplatin-induced HK-2 cells injury

AIM:To investigate the effect of P21 on cisplatin-induced renal tubular epithelial cells injury.METHODS:The expression of P21 at mRNA and protein levels in cisplatin treated human renal tubular epithelial cells (HK-2) cells was determined by RT-qPCR and Western blot. Over-expression of P21in the HK-2 cells was induced by the transfection of pcDNA3-P21. The cell viability and cell apoptosis were detected by CCK-8 assay and flow cytometry, respectively. Furthermore, the protein expression of kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), caspase-3, glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP), and phosphorylation level of eucaryotic translation initiation factor 2α (eIF2α) and protein kinase R-like endoplasmic reticulum kinase (PERK) were detected by Western blot.RESULTS:Cisplatin increased the mRNA and protein levels of P21 in a time-and concentration-dependent manner in the HK-2 cells. Over-expression of P21 inhibited cisplatin-induced cell apoptosis, and down-regulated the expression of KIM-1 and NGAL. Furthermore, Over-expression of P21 decreased the protein levels of GRP78, p-PERK, p-eIF2α, CHOP and cleaved caspase-3.CONCLUSION:Over-expression of P21 attenuates cisplatin-induced HK-2 cells injury, and the mechanism may be related to the modulation of endoplasmic reticulum stress pathway and inhibition of cell apoptosis.     

Anticancer effects of resveratrol in canine hemangiosarcoma cell lines

Hemangiosarcoma (HSA) is a highly malignant tumour with aggressive biological behaviour. HSAs are more common in dogs than other domestic animals. The median survival time of dogs with HSA remains short, even with chemotherapy and surgery. Therefore, there is a critical need to improve the adjuvant chemotherapeutic regimens to improve clinical outcomes in dogs with HSA. Resveratrol has been shown to possess strong anti‐proliferative and/or pro‐apoptotic properties in human cancer cell lines. Nevertheless, the potential anticancer effects of resveratrol have not been reported in canine HSAs. The objective of this study is to determine the growth inhibitory effects of resveratrol in HSA cells when used alone or in combination with doxorubicin, a commonly used chemotherapeutic agent. Frog and DD‐1 canine HSA cell lines were treated with varying concentrations of resveratrol with and without doxorubicin. Cell viability was measured by the MTT assay. The expression of apoptotic proteins, activation of p38 mitogen‐activated protein kinase (MAPK), AMP‐activated protein kinase (AMPK) and extracellular signal‐regulated kinase 1/2 (ERK1/2) were assessed by western blotting. Similar to human cancer cell lines, resveratrol markedly inhibited the growth and induced apoptosis in both HSA cell lines. Mechanistically, resveratrol activated p38 MAPK, but did not affect the AMPK or the ERK1/2 pathways. Additional experiments showed that resveratrol augmented the growth‐inhibitory and apoptotic effects of doxorubicin in both HSA cell lines. These findings suggest that resveratrol has pro‐apoptotic effects in canine HSA cells; therefore, its use as a potential adjunct therapy in canine HSA patients warrants further investigation.     

Effect of garlicin on viral myocarditis in mice

AIM: To investigate the effect of garlicin on mouse viral myocarditis and to explore the possible mechanisms. METHODS: Forty BALB/c mice were randomly divided into 4 groups: control group, viral myocarditis group, low-dose garlicin group and high-dose garlicin group. The morphological changes of the myocardium were observed with HE staining. The levels of lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and creatine kinase (CK) were measured by colorimetric method. The protein levels of Bcl-2, Bax, cleaved-caspase-9, cleaved-caspase-3, cleaved-PARP and cleaved-caspase-8 in the myocardial tissues were determined by Western blot. RESULTS: The morphological observation with HE staining showed that garlicin inhibited the necrosis of the myocardium and infiltration of inflammatory cells. Compared with viral myocarditis group, garlicin dose-dependently decreased the levels of LDL, AST and CK. Moreover, garlicin treatment decreased the protein levels of Bax, cleaved-caspase-9, cleaved-caspase-3 and cleaved-PARP, accompanied with an increase in Bcl-2 expression. However, garlicin had no effect on the protein level of cleaved-caspase-8. CONCLUSION: Garlicin effectively attenuates the myocardial damage in mice with viral myocarditis through inhibition of intrinsic apoptosis pathway.     

猪繁殖与呼吸综合征病毒实验感染SPF猪外周血单核细胞和肺泡巨噬细胞早期凋亡细胞的观察

采用Ａｎｎｅｘｉｎ Ｖ－ＦＩＴＣ／ＰＩ双染色法,用流式细胞仪检测了猪繁殖与呼吸综合征病毒（ＰＲＲＳＶ）实验感染ＳＰＦ猪不同时期外周血单核细胞和肺泡巨噬细胞感染Ａｎｎｅｘｉｎ Ｖ－ＦＩＴＣ＾＋／ＰＩ＾－细胞群（早期凋亡细胞群）。结果显示,ＰＲＲＳＶ感染猪外周血单核细胞和肺泡巨噬细胞Ａｎｎｅｘｉｎ Ｖ－ＦＩＴＣ＾＋／ＰＩ＾－细胞群的表达率均明显高于正常对照猪,感染后２４ｈ表达率达最高值。     

TRAIL与5-FU联合诱导白血病细胞凋亡的分子机理研究

用凋亡诱导配体（TRAIL）及5-氟尿嘧啶（5-FU）联合刺激Jurkat细胞36h,以诱导细胞凋亡;用流式细胞术和MTS比色法检测细胞存活率,计算细胞凋亡率;用免疫印迹（western b-lotting）检测p53的表达和胱天肽酶-3、胱天肽酶-8的变化。结果表明：5-FU能有效增加TRAIL诱导的Jurkat细胞凋亡,并伴随p53表达增加及胱天肽酶-3和胱天肽酶-8的活性增加,提示TRAIL和5-FU联合诱导的T淋巴白血病细胞凋亡的分子机制与p53及胱天肽酶-3、胱天肽酶-8有关,为TRAIL和5-FU联合用于治疗白血病提供理论依据。     

dusp1敲降对低温诱导斑马鱼ZF4细胞凋亡的作用

为了研究双特异性磷酸酶(dual-specificity phosphatase 1,dusp1)基因与鱼类低温诱导的细胞凋亡之间的关系,本研究经Eco RI和Age1酶切构建plko.1-dusp1-sh RNA1,plko.1-dusp1-shRNA2,plko.1-negative control(nc),plko.1-EGFP的重组质粒,并分别与慢病毒包装质粒pCMV-DR8.9.1、pCMV-VSVG共同转染至293T细胞中,经293T细胞包装的病毒,感染斑马鱼(Danio rerio)胚胎成纤维样细胞(zebrafish embryos fibroblast like cell line,ZF4),RT-PCR检测表明dusp1成功被敲降。经嘌呤霉素筛选获得稳定表达细胞系,将细胞于28℃培养(正常对照组)或经10℃低温处理10 d,使用annexin V-FITC/Propidium lodide(PI)双色标记流式细胞术检测ZF4细胞凋亡情况。结果显示:低温胁迫下dusp1敲降的细胞凋亡(dusp1-shRNA1敲降,dusp1-kd1;dusp1-shRNA2敲降,dusp1-kd2)比例较nc组显著上调,其中早期凋亡和晚期凋亡细胞比例dusp1-kd1显著高于nc组,并且dusp1-kd1活细胞数显著低于nc组(P0.05),进一步证明dusp1参与鱼类冷应激过程,并在低温情况下保护细胞。该研究为后期对斑马鱼细胞低温胁迫实验奠定了基础,其中dusp1基因沉默型斑马鱼细胞在10℃低温处理10 d凋亡数显著大于对照组,证明dusp1在细胞凋亡信号通路中起到重要调控作用,为低温下研究斑马鱼基因的分子机制提供新的思路。