Effect of DNA methylation of miR-30a-5p promoter region on hepatic injury induced by homocysteine

AIM: To explore the role of DNA methylation of microRNA-30a-5p(miR-30a-5p) promoter region in hepatic injury. METHODS: Four-week-old normal mice and cystathionine β-synthase (CBS) single gene knockout mice were used and divided into normal (CBS^+/+, n=12) group and single gene knockout (CBS^+/-, n=12) group, and the mice were fed with high methionine diet for 8 weeks. HL-7702 hepatic cells were routinely cultured in vitro and divided into control group, homocysteine (Hcy) group and Hcy+5-azacytidne (AZC) group. Serum Hcy, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by automatic biochemical analyzer. The levels of ALT and AST in the cells culture medium were determined by the microplate method. Hepatic injury in the mice were observed with HE staining. Cell viability staining was used to measure the viability of hepatocytes. RT-qPCR was used to detect the expression of miR-30a-5p in the liver tissues and hepatocytes. The correlation between the expression of miR-30a-5p and serum ALT and AST levels was analyzed by Pearson correlation analysis. DNA methylation level of miR-30a-5p promoter region in the liver tissues and hepatocytes was detected by nested landing methylation-specific PCR (nMS-PCR). RESULTS: Compared with the CBS^+/+ mice, the serum levels of Hcy, ALT and AST in the CBS^+/- mice were significantly increased (P < 0.05). HE staining showed the hepatocyte swelling and nuclear fragmentation and dissolution. The expression level of miR-30a-5p in the liver tissues was decreased (P < 0.01). Besides, the expression level of miR-30a-5p in the mice was negatively correlated with serum ALT and AST levels (r²=0.4557, P=0.0003, r²=0.4626, P=0.0003), and the DNA methylation of miR-30a-5p promoter region was increased (P < 0.01). In the HL-7702 cells, compared with control group,the ALT and AST levels were increased in Hcy group (P < 0.05, P < 0.01), and the cell viability was remarkablely decreased. DNA methylation of miR-30a-5p promoter region was increased (P < 0.01), which decreased after treated the cells with AZC (P < 0.05), while the expression level of miR-30a-5p in the cells was increased (P < 0.05). CONCLUSION: Hypermethylation of miR-30a-5p promoter region may play an important role in hepatic injury.     

基于GlobeLand 30的哈密市2000-2020年土地利用格局变化研究

[目的]研究新疆维吾尔自治区哈密市近20 a的土地利用变化,为该地区土地利用规划及生态环境保护提供有力支撑。[方法]基于2000,2010,2020年的GlobeLand 30数据集,通过利用转移矩阵、坡度分布指数、标准差椭圆以及土地利用重心迁移,分析哈密市2000-2020年土地利用的时空变化特征。[结果]①哈密市2000-2020年土地利用变化比较明显。2020年各土地利用类型面积比例为:未利用地>草地>耕地>林地>建设用地>水域。哈密市虽然面积大但能利用的绿洲面积较小。2020年草地面积为10226.39 km²,水域面积为295.17 km²。2000-2020年土地利用类型变化量中,草地的减少量最大,流失面积为671.48 km²,主要转换成未利用地、林地和少量耕地;建设用地增量最大,面积高达405.60 km²。2000-2020年其他土地利用类型中林地和耕地面积呈增长趋势;未利用地和水域呈减少趋势,其中水域面积的减少量很小,基本保持稳定。②2000-2020年草地在5个坡度梯度上分布比较均匀;而耕地和建设用地基本分布在Ⅰ级和Ⅱ级坡度梯度,耕地在Ⅰ级坡度梯度上显优势分布;林地在Ⅰ级坡度梯度上面积最大;未利用地分布面积随着坡度梯度的增加而减少。③2010-2020年土地利用变化与2000-2010时段的变化相比方向性较明显,范围更广,重心向西北方向迁移。[结论]2000-2020年哈密市的土地利用变化有着明显的时空特征,坡度对各地类分布的影响较为明显。     

Pathogenicity of emerging Japanese type 1 porcine reproductive and respiratory syndrome virus in experimentally infected pigs

To clarify the pathogenicity of Japanese type 1 porcine reproductive and respiratory syndrome virus (PRRSV) isolate in experimentally infected pigs, we evaluated clinical signs and monitored viremia for 21 days post-inoculation (dpi). Lungs were mottled, tanned and reddish in appearance; had lesions predominantly in the cranial, middle and accessory lobes; and failed to collapse at 10 dpi. Although microscopic lesions of lungs were reproduced using the Japanese emerging type 1 PRRSV isolate under experimental conditions, no significant differences were noted between the challenge and control groups regarding mean rectal temperature and daily weight gain. These results provide useful insights into the limited pathogenicity of single infection with the Japanese type 1 PRRSV isolate in piglets, which differ from findings in reported field cases.     

“猪康散”保健用药对人工感染猪繁殖与呼吸综合征病毒仔猪血清细胞因子含量的影响

选取28日龄健康杜长大三元杂交仔猪40头,随机分为4组,每组10头。36~39日龄中药组(Ⅲ、Ⅳ组)分别在基础日粮中添加0.5%、1.0%"猪康散",40日龄除阴性对照组(Ⅰ组)外均滴鼻接种PRRSV SCM株1.0mL。于40、45、50、55、60日龄每组选取4头仔猪,前腔静脉采血测定血清细胞因子(IL-2、IL-6、IFN-γ、TNF-α)的变化。结果显示,在整个试验期间,除0.5%中药组40日龄仔猪血清中IL-2含量与阴性对照组差异不显著外(P0.05),0.5%和1.0%中药组仔猪血清中IL-2、IL-6、IFN-γ、TNF-α的含量均显著高于阴性对照组和阳性对照组(P0.01或0.05);除两中药组40日龄仔猪血清中IL-6含量差异不显著外(P0.05),1.0%中药组仔猪血清中IL-2、IL-6、IFN-γ、TNF-α含量均显著高于0.5%中药组(P0.01或0.05)。结果表明,在断奶仔猪基础日粮中添加0.5%和1.0%质量分数的"猪康散"能明显提高人工感染PRRSV仔猪血清中IL-2、IL-6、IFN-γ、TNF-α的含量,从而提高仔猪机体免疫功能,达到抗PRRSV的能力。     

Construction and Identification of PRRSV ORF5 Gene Prokaryotic Expression Vector

The present study was designed to construct recombinant plasmids,which could express porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 gene.RNA was extracted from spleen and lung samples of the suspected pigs which were infected with PRRSV.According to PRRSV ORF5 gene,a pair of primers was designed for RT-PCR amplification.The ORF5 target gene was cloned into pMD19-T vector and then the recombinant pMD19-ORF5 was achieved.According to the sequencing results and the characteristics of expression vectors,a pair of primers with NcoⅠand XbaⅠenzyme cleavage sites was designed.Target fragment dORF5 was amplified and then connected to pProEXHTb and pNZ8149 vectors,respectively.And recombinant HTb-dORF5/DE3 and pNZ8149-dORF5/NZ3900 was induced by IPTG and Nisin,respectively,and analyzed by SDS-PAGE and Western blotting.Recombinant HTb-dORF5/DE3 induced by 1.5 mmol/L IPTG was expressed in the highest quantity.There were specific band at about 22 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.Recombinant pNZ8149-dORF5/NZ3900 induced by 20 ng/mL Nisin was expressed in the highest quantity.There were specific band at about 19 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.The IFA result showed specific green fluorescence.This study successfully constructed recombinant plasmids HTb-dORF5 and pNZ8149-dORF5 and expressed,the result laid a solid foundation for further development of PRRS vaccines.     

Evaluation of two commercial PRRSV antibody ELISA kits with samples of known status and singleton reactors

Two commercial PRRSV ELISA kits (IDEXX and Bionote) were evaluated for their sensitivity and specificity using 476 PRRS-positive serum samples collected from 7 animal challenge experiments and 1,000 PRRS-negative sera. Both ELISA kits exhibited 100% sensitivity with sera collected 14 to 42 days post-infection, and the results from the kits were highly correlated (R²=0.9207). The specificity of IDEXX or Bionote kit was 99.9% or 99.7%, respectively. In addition, the Bionote ELISA kit was used to examine 100 sera that were determined to be falsely positive either by IDEXX 2XR or 3XR ELISA, and only 7 of these samples were found to be positive. These results indicate that both ELISA kits exhibited similar levels of sensitivity and specificity and would complement one another for the verification of false-positive samples.     

30℃条件下玉米自呼吸对储藏环境中气体浓度变化的影响研究

通过对3种不同水分(偏低水分11.8％、安全水分13.3％和偏高水分16.3％)的玉米在30℃条件下,密闭储藏环境内N2、O2和CO2百分浓度变化的测定,研究密闭储藏环境条件下玉米粮粒周围环境气体成分浓度的变化规律.20 L规模的试验室研究结果表明:不同水分玉米在密闭储藏时环境中N2浓度随时间的变化均不大;不同水分玉米在密闭储藏时环境中O2浓度与储藏时间呈负相关;不同水分玉米在密闭储藏时环境中CO2浓度与储藏时间呈正相关.在气调储藏时,对偏低水分和安全水分的玉米可充分利用粮粒的自呼吸使环境中的O2浓度在21 d内迅速下降,后期可通过粮粒的自呼吸使O2浓度维持在5％左右,偏高水分的玉米在11 d内即可使O2浓度迅速下降,但后期O2浓度接近0％,无氧呼吸加剧.通过对玉米密闭储藏环境中气体浓度以及耗氧量和CO2累积量随时间的变化趋势线进行回归分析,得到30℃条件下不同水分玉米密闭环境中N2、O2和CO2的回归方程以及耗氧量和CO2累积量的回归方程,利用相应回归方程,可获得玉米在密闭储藏时环境中不同储藏时间段的气体浓度,为气调储藏时玉米粮粒自呼吸的合理利用提供基础技术参数和数据模型.     

转Bt基因抗虫棉中棉所30的碳、氮代谢特征

本试验在田间条件下研究了转Bt基因抗虫棉中棉所30的碳、氮代谢特征,于各主要生育期测定了棉株地上部营养器官的全N、非蛋白质N和可溶性糖及淀粉含量.研究结果表明,与轮回亲本中棉所16相比,中棉所30苗蕾期的C/N较低,非蛋白质N含量较高,N代谢比较旺盛;初花期至盛花期的C/N则较高,C代谢明显加强;进入结铃早期,中棉所30叶片中可溶性糖、淀粉及营养体中非蛋白质N的含量均下降,反映出其"根源”和"叶源”的功能已减弱,表现出早衰迹象.为了改善转Bt基因抗虫棉中棉所30的生育状况,提高其产量和品质,"促源”是根本.     

降雨侵蚀力新算法在豫西山区应用的初步研究

降雨侵蚀力是定量监测评价一个地区土壤侵蚀状况的重要因子之一,找到适宜简便的计算方法十分重要.卜兆宏提出的降雨侵蚀力新算法具有简便易用的优点.本文利用位于豫西山区鲁山县的3个水文雨量站10年185次的自记降雨过程资料,以经典算法年R值为基准,对新算法在该区的适用性进行了分析评价.结果表明:新算法结果与经典值存在高度的一致性,一致性高达90.2%,模型有效系数为96.4%,相对误差为7.7%,说明新算法能够在该区应用,可以采取此法对该区的降雨侵蚀力进行深入的分析研究;并同时对资料摘读时应该注意的问题进行了讨论.