Transfer of Bt-toxin protein gene into maize by high-velocity microprojectile bombardments and regeneration of transgenic plants

Bt-toxin protein gene was successfully transferred into maize by the microprojectile bombard-ments of cell suspension,embryogenic calli and immature embryos with a Chinese-made particle gun(JQ-700).Although the bombarded embryogenic calli and immature embryos produced less mean transformants per dishthan the cell suspensions,they were the suitable materials for maize transformation because their culture andregeneration have been achieved in most maize cultivars.The evaluation on the resistance of transgenic plantsto corn borer shows the significant difference between them,from highly resistant to susceptible.

Diphtheria Toxin/Human B-Cell Activating Factor Fusion Protein Kills Human Acute Lymphoblastic Leukemia BALL-1 Cells: An Experimental Study

Objective： This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor （DT388sBAFF） in Escherichia coli （E. coli） and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells （BALL-1）. Methods： A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction （PCR） and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside （IPTG）. The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot, and then purified by Ni2＋-NTA affinity chromatography. The expression level of B cell-activating factor receptor （BAFF-R） on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide （MTT） assay. Results： The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion： The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.

Zonula occludin toxin,a microtubule binding protein

AIM To investigate the interaction of Zot withmicrotubule.METHODS Zot affinity column was applied topurify Zot-binding protein（s）from crudeintestinal cell lysates.After incubation at roomtemperature,the column was washed and theproteins bound to the Zot affinity column wereeluted by step gradient with NaCl(0.3 mol·L-1-0.5mol·L-1).The fractions were subjected to6.0%-15.0%（w/v）gradient SDS-PAGE andthen transferred to PVDF membrane for N-terminal sequencing.Purified Zot and tauprotein were blotted by using anti-Zot or anti-tauantibodies.Finally,purified Zot was tested in anin vitro tubulin binding assay.RESULTS Fractions from Zot affinity columnyielded two protein bands with a Mr of 60 kU and45kU respectively.The N-terminal sequence ofthe 60 kU band resulted identical to β-tubulin.Zot also cross-reacts with anti-tau antibodies.Inthe in vitro tubulin binding assay,Zot co-precipitate with Mt,further suggesting that Zotpossesses tubulin-binding properties.CONCLUSION Taken together,these resultssuggest that Zot regulates the permeability ofintestinal tight junctions by binding tointracellular Mt,with the subsequent activationof the intracellular signaling leading to thepermeabilization of intercellular tight junctions.

基于Bt毒素的杀虫蛋白理性设计与创新应用策略

Bt毒素是源于苏云金芽孢杆菌(Bacillusthuringiensis)的具有特殊杀虫功能的大分子蛋白,其制剂和转基因作物已广泛用于害虫防治,产生了巨大的经济和社会生态效益。围绕Bt毒素挖掘和提升其应用价值是持续研究的热点,特别是随着Bt毒素结构功能和作用机制日趋明晰,为其功能修饰和创新应用创造了条件,相关研究蓬勃发展,成效显著。大量研究表明,采用定点突变、结构域替换或融合以及抗独特型抗体模拟等策略,是理性设计活性更高、稳定性更强、杀虫谱更广、非靶标生物安全性更高甚至是可用于害虫抗药性治理的有别于母体Bt毒素的突变体、结构杂合体乃至功能效应物抗体等新型杀虫蛋白的有效手段;此外,采用催化毒素活化、驱动毒素靶向受体结合、促进毒素表达以及同源或异源杀虫材料复配或共表达的协同促效等创新增效策略,也是助推Bt毒素应用价值的重要手段。本文总结了Bt毒素结构功能和作用机制,梳理了基于Bt毒素功能修饰的突变体、结构杂合体以及功能效应物抗体等新型杀虫蛋白理性设计和基于Bt毒素功能增效的创新应用策略等相关研究进展,并结合作者团队在模拟Bt毒素杀虫功能效应物抗体靶向设计研发方面的最新成果,探讨了基于Bt毒素的杀虫蛋白理性设计与创新应用策略未来发展动向及潜在可行捷径,为相关研究提供较为全面的最新有价值的文献资料和启发思路。

尿液中蛋白结合毒素水平与上尿路结石的相关性研究

目的·探讨尿液中蛋白结合毒素(protein-bound uremic toxin,PBUT)水平与上尿路结石形成的关系。方法·以海南省儋州市海头镇社区18~80岁居民为研究对象。记录研究对象基本信息和近3 d饮食情况,收集空腹血清和24 h尿液标本,所有对象接受肾脏和输尿管B超检查。将B超检查发现有上尿路结石或有明确上尿路结石病史的对象作为结石组,其他作为非结石组。检测血、尿中结石形成相关的生化指标,通过高效液相色谱检测血、尿中的PBUT(硫酸吲哚酚、3-吲哚乙酸和硫酸对甲酚),以及尿草酸和尿枸橼酸水平。通过多因素Logistic回归分析上尿路结石形成的相关因素。采用Spearman相关性检验分析尿PBUT与尿尿酸、草酸和枸橼酸的相关性。结果·经筛选,共纳入117例研究对象,其中结石组54例,非结石组63例。2组的性别、年龄、血清指标,以及高血压、糖尿病、高尿酸血症/痛风等合并症比例方面差异均无统计学意义。结石组的24 h尿液pH值、钙、尿酸和氯均显著高于非结石组(均P<0.05),而硫酸吲哚酚显著低于非结石组(P<0.05)。多因素Logistic回归分析结果显示,除尿钙外,尿硫酸吲哚酚水平(OR=0.929,95%CI 0.875~0.986,P=0.016)也是上尿路结石形成的独立相关因素。Spearman相关性分析结果显示,24 h尿液中3-吲哚乙酸(r=0.420,P=0.000)和硫酸对甲酚(r=0.307,P=0.001)水平均与草酸呈正相关,硫酸对甲酚水平与尿酸呈正相关(r=0.297,P=0.002),硫酸吲哚酚水平与枸橼酸呈正相关(r=0.289,P=0.002)。结论·在该研究人群中,尿硫酸吲哚酚水平降低可能是上尿路结石形成的独立危险因素,且尿液中PBUT水平与尿酸、草酸、枸橼酸的水平相关。

Study on Spatial-temporal Dynamics of Bt Toxic Protein Expression in Insect-resistant Transgenic Cotton and Its Degradation in Soil

[Objective] This study aimed to investigate the spatial-temporal dynamics of Bt toxic protein expression in insect-resistant transgenic cotton and its degradation in soil. [Method] Btcry1Ac toxic protein expression in roots, stems and leaves of transgenic cotton Guoshen GK45 at different developmental stages and the annual average content of BtCry1Ac toxin protein in the topsoil, rhizosphere soil and following cotton-growing area were explored and analyzed by using enzyme linked immuno sorbed assay (ELISA). [Result] The content of exogenous BtCry1Ac toxin protein decreased during the growth process of insect-resistant transgenic cotton; to be specific, the content of BtCry1Ac toxin protein in cotton stems and leaves decreased more slowly and always maintained a high level, while that in roots decreased rapidly and reached a minimum level to the following plant growth and development stage. BtCry1Ac toxin protein was detected in topsoil of both non-transgenic and transgenic cotton-growing areas, and the content of BtCry1Ac toxin protein increased in topsoil of following cotton-growing area, which was very low in rhizosphere soil. [Conclusion] Determination of Btcry1Ac toxic protein provides scientific basis for the risk assessment of the cultivation of genetically modified crops and the safety evaluation of soil ecosystem.

儿童肺炎支原体肺炎肺泡灌洗液CARDS TX、HMGB1、TLR2表达及临床意义

目的研究肺炎支原体肺炎(MPP)患儿肺泡灌洗液中社区获得性呼吸窘迫综合征毒素(CARDS TX)、高迁移率族蛋白1(HMGB1)、Toll样受体2(TLR2)表达,探讨其在MPP发病中的临床意义。方法回顾性分析55例MPP病例组及20例对照组临床资料,同时检测两组支气管肺泡灌洗液(BALF)中CARDS TX、HMGB1、TLR2、TLR4、晚期糖基化终末产物受体(RAGE)及髓样分化因子88(MyD88)表达,体外检测不同浓度CARDS TX刺激下HMGB1、TLR2的表达并进行比较。结果与对照组相比,MPP组LYM绝对值计数、PA、CD3^(+)、CD3^(+)CD4^(+)、CD3^(+)CD8^(+)、CD3-CD19^(+)、NK cell、CD19^(+)CD23^(+)明显下降,CRP、LDH、D-二聚体、IgA、IgG、IgM、CARDS TX、HMGB1、TLR2、MyD88均升高,差异有统计学意义(P<0.05或0.001)。CARDS TX刺激后HMGB1、TLR2的表达升高,且呈正相关,差异有统计学意义(P<0.001)。结论CARDS TX可能通过HMGB1-TLR2-MyD88途径参与肺炎支原体(MP)的致病。

枳实消痞颗粒对脾虚气滞型腹膜透析患者营养不良及蛋白结合毒素的影响

目的观察枳实消痞颗粒对脾虚气滞型腹膜透析患者营养不良及蛋白结合毒素的影响。方法选取2021年10月—2022年10月在陕西中医药大学附属医院肾病二科及门诊随访的维持性腹膜透析营养不良患者60例,将其随机对照原则分为对照组和治疗组各30例。对照组采用西医基础治疗,治疗组在对照组治疗基础上服用枳实消痞颗粒,治疗3个月后观察肾功能指标及透析充分性指标[血肌酐(SCr)、尿素氮(BUN)、尿素清除指数(Kt)]、营养状态指标[血红蛋白(Hb)、血清白蛋白(ALB)、血清前白蛋白(PA)、体质指数(BMI)、肱三头肌皮褶厚度(TSF)、上臂围(AC)、上臂肌围(AMC)]、蛋白结合毒素指标[血清和腹透液中硫酸吲哚酚(IS)、硫酸对甲酚(pCS)水平]、中医证候积分、改良主观综合性营养评估表(MQSGA)评分的变化。结果治疗3个月后,治疗组SCr水平、BUN水平、血清和腹透液中IS水平、血清pCS水平及中医证候积分、MQSGA评分均较治疗前明显降低(P均<0.05),Kt及Hb、ALB、PA水平均较治疗前明显提高(P均<0.05),其余指标变化不明显(P均>0.05);对照组仅SCr水平明显降低(P<0.05);治疗后治疗组ALB、PA水平均明显高于对照组(P均<0.05),血清pCS水平及中医证候积分均明显低于对照组(P均<0.05)。结论枳实消痞颗粒能够改善脾虚气滞型腹膜透析患者的营养状态,降低血清及腹透液中IS水平和血清中pCS水平,具有保护肾脏和腹膜作用。

不同血液灌流治疗频率对维持性血液透析患者蛋白质能量消耗的影响

目的 探究不同血液灌流(hemoperfusion,HP)频率对维持性血液透析(maintenance hemodialysis,MHD)患者蛋白质能量消耗(protein-energy wasting,PEW)的影响。方法共纳入80例患者,根据HP治疗频率分为无HP(A)组、每月1次HP组(B)组、每半月1次HP(C)组、每周1次HP(D)组,每组20例患者,随访半年。比较4组患者治疗前后PEW患病率、营养不良炎症评分(malnutrition-inflammation score,MIS)及相关生化指标。结果治疗前,4组患者MIS评分(F=2.194,P=0.096)、PEW患病率(χ^(2)=0.579,P=0.901)、前白蛋白(PA)(F=0.258,P=0.855)、白蛋白(Alb)(F=0.187,P=0.905)、甲状旁腺激素(PTH)(F=0.780,P=0.509)、血红蛋白(Hb)(F=1.823,P=0.150)、β2-微球蛋白(β2-MG)(F=1.833,P=0.148)、C反应蛋白(CRP)(F=0.432,P=0.731)水平比较均无统计学差异;治疗后,D组有15例患者退出试验,C组MIS评分(C-A:t=4.391,P≤0.001;C-B:t=-2.871,P=0.006)、PTH(C-A:t=4.098,P=0.001;C-B:t=-2.551,P=0.047)、β2-MG(C-A:t=5.688,P<0.001;C-B:t=-3.207,P=0.002)、CRP(C-A:t=4.293,P<0.001;C-B:t=-2.843,P=0.006)水平低于A、B组;C组PA(C-A:t=-3.177,P=0.002;C-B:t=2.196,P=0.032)、Alb(C-A:t=-5.540,P<0.001;C-B:t=2.196,P=0.010)水平高于A、B组;C组Hb(t=-3.733,P<0.001)水平高于A组,与B组比较无统计学差异(t=1.451,P=0.152);C组PEW患病率(χ^(2)=6.465,P=0.011)低于A组,A、B 2组间PEW患病率无统计学差异(χ^(2)=2.506,P=0.113)。结论半月1次的HP治疗可以明显改善MHD患者的营养状况,降低PEW的患病率。

Secretory Expression of Recombinant Diphtheria ToxinMutants in B. Subtilis

Three diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-El48S-K516A-F530A were cloned in B- Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS118, but higher yield of 7. 1 mg/L was observed in SMS300 compared with 2. 1 mg/L in SMS118. Western blot showed that the recombinant protein could be effectively secreted into the culture medium as a 58 ku peptide, and could be de-graded into two peptides of 37ku and 21ku.

具杀虫功能的蛋白类生物材料研究进展

害虫是威胁农作物生长发育及产品品质的主要风险因素,对其有效防治是保障农作物稳定生产的重要环节。传统依赖化学农药对害虫防治效果显著,但化学农药本身对非靶标生物以及生态环境所造成的持久性危害风险也同样突出。害虫绿色防治是农业可持续发展的必然要求,其中具杀虫功能的蛋白类生物材料因兼具安全性高、可量产药剂及可在转基因抗虫作物应用的多重优势,是害虫绿色防治创新研发和应用的热点。如具备杀虫功能的Bt毒素、凝集素、动物毒素、防御素、蛋白酶抑制剂等蛋白类生物材料在害虫防治上已有深入研究和广泛应用;此外,近年来包括杀虫抗体在内的一些新型杀虫蛋白和相应创新技术也不断涌现,发展极为迅猛。基于此,本文在系统梳理当前成熟的具杀虫功能的蛋白类生物材料研发和应用现状的基础上,结合作者团队在杀虫抗体靶向设计上取得的原创性研究成果和经验,对蛋白类生物杀虫材料潜在的创新研发与应用进行展望,旨在为农业害虫绿色防治研究提供最新文献资料和技术参考。

Functional verification of the diphtheria toxin A gene in a recombinant system

Diphtheria toxin A （DTA）, a segment of the diphtheria toxin （tox）, inhibits protein synthesis in cells. When released from a cell, DTA is nontoxic and cannot enter other cells independently without the help of diphtheria toxin B. In this study, we artificially synthesized the DTA gene sequence and cloned it into pEGFP-N1 to generate the recombinant vector pEGFP-N1-DTA. This recombinant vector was then transfected into 293T cells to observe the effect of DTA protein expression on enhanced green fluorescent protein （EGFP） protein expression and the proliferation of 293T cells. After 48 h, high levels of EGFP expression were seen in control pEGFP-Nl-transfected cells, whereas very low levels were seen in cells transfected with pEGFP-N1-DTA. Reverse transcription polymerase chain reaction confirmed the expression of the DTA gene in cells transfected with pEGFP-N^-DTA. Further, the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （M-I-I-） assay revealed a significant difference in cell proliferation between the control group and the pEGFP-N1-DTA-transfected group. Using the expression of EGFP expression as an indicator, this study revealed that DTA expression can inhibit intracellular protein synthesis and cell proliferation.

Reporter Gene Assay for Detection of Shellfish Toxins

Objective To explore the potential reporter gene assay for the detection of sodium channel-specific toxins in shellfish as an alternative for screening harmful algal bloom (HAB) toxins, considering the fact that the existing methods including HPLC and bioassay are inappropriate for identifying HAB toxins which poses a serious problem on human health and shellfish industry. Methods A reporter plasmid pEGFP-c-fos containing c-fos promoter and EGFP was constructed and transfected into T24 cells using LipofectAMINE 2000. Positive transfcctants were screened by G418 to produce a pEGFP-c-fos-T24 cell line. After addition of increasing neurotoxic shellfish poison (NSP) or GTX2,3, primary components of paralytic shellfish poison (PSP), changes in expression of EGFP in the cell line were observed under a laser scanning confocal microscope and quantified with Image-pro Plus software. Results Dose-dependent changes in the intensity of green fluorescence were observed for NSP in a range from 0 to 10 ng/mL and for GTX2,3 from 0 to 16 ng/mL. Conclusion pEGFP-c-fos-T24 can be applied in detecting HAB toxins, and cell-based assay can be used as an alternative for screening sodium channel-specific HAB toxins.

基于NLRP3/IL-1β、IL-18通路探讨CARDS Tx在儿童重症肺炎支原体肺炎中的作用

目的研究社区获得性呼吸窘迫综合征毒素(community-acquired respiratory distress syndrome toxin,CARDS Tx)与Nod样受体蛋白3(Nod-like receptor protein 3,NLRP3)/白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-18(interleukin-18,IL-18)通路在儿童重症肺炎支原体肺炎(severe mycoplasma pneumoniae pneumonia,SMPP)中的作用,并评价血清CARDS Tx、NLRP3、IL-1β、IL-18水平对SMPP的预测价值。方法选取2020年10月~2021年9月兰州大学第二医院收治的肺炎支原体肺炎(mycoplasma pneumoniae pneumonia,MPP)患儿为研究对象,根据疾病的严重程度将其分为MPP组与SMPP组,并选择同期至兰州大学第二医院体检的健康儿童为对照组。采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测3组受检者血清中CARDS Tx、NLRP3、IL-1β、IL-18的水平;收集SMPP组中患儿的患侧与对侧支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF),采用ELISA法检测BALF中CARDS Tx、NLRP3、IL-1β、IL-18的水平。结果血清、BALF中CARDS Tx与NLRP3均呈显著正相关(r>0.45,P<0.01)。患侧组BALF中的CARDS Tx、NLRP3、IL-1β、IL-18水平显著高于对侧组(P<0.001)。血清中CARDS Tx、NLRP3、IL-1β、IL-18水平均为SMPP组>MPP组>对照组(P<0.001)。血清中CARDS Tx、NLRP3、IL-1β、IL-18预测SMPP的受试者操作特征(receiver operating characteristic,ROC)的曲线下面积(area under the curve,AUC)均大于0.7(P<0.001)。结论CARDS Tx不仅存在于损伤肺局部,还可进入血液循环,通过激活机体内NLRP3/IL-1β、IL-18通路介导SMPP的发生与进展,并参与SMPP患儿的局部肺损伤机制。血清CARDS Tx、NLRP3、IL-1β、IL-18水平可有效预测SMPP,动态监测其变化情况对于SMPP的疗效评估可能具有重要价值。

细胞致死膨胀毒素研究进展

细胞致死膨胀毒素(CDT)是细菌毒素家族的一种蛋白毒素,是多种革兰氏阴性菌的重要毒力因子,如空肠弯曲菌、幽门螺杆菌、伴放线杆菌等。该毒素的致病机理是通过破坏靶细胞DNA造成细胞损伤,最终导致细胞的分裂周期停滞甚至死亡。论文主要总结了近几年来细胞致死膨胀毒素的相关研究,对其结构、生物学功能、转运机制及应用前景等研究进展进行总结及讨论,旨在为该毒素的相关研究提供一定参考。

苏州地区幽门螺杆菌抗体分型在体检人群中的分布

目的回顾性分析苏州地区幽门螺杆菌抗体分型在体检人群中的流行病学特征。方法选取2018年1—12月苏州市上海交通大学医学院苏州九龙医院的6588名健康体检人群的临床资料,采用免疫印迹法定性检测人血清中多种幽门螺杆菌(helicobacter pylori,HP)IgG抗体,包括细胞毒素相关蛋白A(CagA)、空泡毒素相关蛋白A(VacA)、尿素酶亚单位A(UreA)和尿素酶亚单位B(UreB),观察体检人群幽门螺杆菌抗体分布情况。结果不同性别体检人群幽门螺杆菌感染率比较,差异无统计学意义(P>0.05)。Ⅰ型Hp抗体阳性率(29.6%)高于Ⅱ型Hp抗体阳性率(26.7%)。女性Ⅰ型Hp抗体阳性率显著高于男性,差异有统计学意义(χ^(2)=4.857,P=0.028)。不同性别Ⅱ型Hp抗体阳性率比较,差异无统计学意义(P>0.05)。31~40岁之间男性体检人群幽门螺杆菌抗体分型分布阳性率(Ⅰ型26.12%,Ⅱ型26.95%)与同年龄女性体检人群(Ⅰ型30.23%,Ⅱ型24.23%)比较,差异有统计学意义(χ^(2)=4.207,P=0.040);41~50岁之间男性体检人群幽门螺杆菌抗体分型分布阳性率(Ⅰ型28.89%,Ⅱ型28.41%)与同年龄女性体检人群(Ⅰ型35.10%,Ⅱ型24.20%)比较,差异有统计学意义(χ^(2)=7.531,P=0.006)。结论苏州地区体检人群幽门螺杆菌抗体阳性率较高,达56.30%,但不同性别之间无差异性;31~40岁与41~50岁两个年龄段之间女性Ⅰ型Hp抗体阳性率高于男性,Ⅱ型Hp抗体阳性率低于男性。

高纯度白喉毒素突变体CRM197的制备及鉴定

为了制备高纯度的白喉毒素无毒突变体CRM197,可作为载体蛋白用于细菌性结合疫苗开发,对CRM197基因进行大肠杆菌密码子优化,并克隆至表达载体pET28a(+)中,将鉴定正确的重组质粒pET28a-CRM197转化到大肠杆菌BL21(DE3),经IPTG诱导表达,并分析其表达形式及表达条件的优化。再对表达的重组CRM197蛋白进行纯化,最后对纯化的CRM197进行WB、纯度、分子量和圆二色谱等检测项目的初步鉴定分析。PCR酶切和测序鉴定结果表明重组质粒pET28a-CRM197构建成功,将其转化到大肠杆菌BL21(DE3)中,获得重组工程菌(E.coli(DE3/p28a/197))。该重组工程菌发酵的最佳接种量为5%~10%(v/v),且主要以包涵体形式表达,收获的菌体在高压均质机破碎压力为1000 bar的条件下进行破碎,然后利用6 mol/L盐酸胍进行溶解变性,复性及经30 kD超滤膜包超滤后上纯化系统AKTA pure150 M,经一步阴离子交换层析进行纯化,其纯度可达98%以上,WB、分子量及圆二色谱等鉴定结果均与标准品一致。综上所述,成功建立大肠菌表达系统CRM197制备方法,具有无标签、产量高及纯度高等特点,可用于该蛋白的规模化生产。

基于高效液相色谱-三重四极杆质谱联用方法评价透析器对蛋白结合类尿毒症毒素的清除效果

建立了一种评价透析器对硫酸吲哚酚、硫酸对甲酸和马尿酸3种蛋白结合类尿毒症毒素(PBUTs)清除效果的高效液相色谱-三重四极杆质谱联用方法。配制与透析患者体内PBUTs浓度相当的模拟液代替血液,进行透析模拟治疗,取透析前后的模拟液作为待测样品。样品经乙腈沉淀蛋白和超滤离心处理后,采用Hypersil GOLD VANQUISH C18(100 mm×2.1 mm,1.9μm)色谱柱分离,以5 mmol/L乙酸铵溶液和甲醇为流动相进行梯度洗脱,负离子选择反应监测(SRM)模式扫描,内标法进行定量分析。3种PBUTs在25~1 000 ng/mL范围内线性关系良好,相关系数(r)均大于0.99,定量下限为25 ng/mL;在75、250、750 ng/mL加标水平下,回收率为92.5%~114%,日内、日间相对标准偏差(RSD)均小于5.0%,基质效应为80.6%~105%。该方法成本低、前处理简单、分析速度快、回收率高,适用于透析器对PBUTs清除效果的评价。

透明质酸联合注射血小板血浆及A型肉毒毒素对猪真皮Ⅰ型胶原蛋白表达的影响

目的探讨透明质酸联合A型肉毒毒素(botulinum toxin type-A,Botox A)和富含血小板血浆(platelet rich plasma,PRP)进行猪真皮内注射,检测真皮层成纤维细胞数目及Ⅰ型胶原蛋白mRNA分泌代谢的变化。方法采用巴马猪建立动物模型,使用交联透明质酸(cross-linked hyaluronic acid,HA)和非交联透明质酸(non-cross-linked hyaluronic acid,N-HA)分别与Botox A、PRP联合作为实验组(HA组、HA+Botox A组、HA+PRP组、N-HA组、N-HA+Botox A组、N-HA+PRP组),并与生理盐水组和正常组比较。于注射第1和4周后,通过组织切片染色、实时荧光定量聚合酶链反应(PCR)等方法,定性定量观察、比较真皮层成纤维细胞数量变化,真皮层Ⅰ型胶原蛋白mRNA分泌表达变化。结果HA组的成纤维细胞数量、Ⅰ型胶原蛋白mRNA表达量在第1周和第4周时比N-HA组高,并且HA+PRP组的成纤维细胞数量、Ⅰ型胶原蛋白mRNA表达水平显著高于同期HA+Botox A组、生理盐水组和正常组(P<0.05)。结论应用HA与PRP联合注射更易促进成纤维细胞的增殖和Ⅰ型胶原蛋白mRNA的表达,其主要机制可能是通过改变细胞外基质的理化性质进而促进真皮层细胞因子的合成,从而影响胶原合成。

转Bt基因水稻Cry1Ab杀虫蛋白在水稻土中的降解

室内研究了转 Bt基因水稻克螟稻 2号和华池B6 叶片中的Cry1Ab杀虫蛋白在稻田水稻土中的降解动态。分别将克螟稻 2号的粉碎叶片和华池B6 的整张叶片埋入 3种土壤 (即老黄筋泥田、青紫泥田和黄松田 )中 ,此后每隔 6～ 10d测定土壤或叶片中的Cry1Ab含量。克螟稻 2号粉碎叶片中的Cry1Ab均以处理后的前 36d,尤其是前 6d的降解较快 ,其中在青紫泥田中最快 ,黄松田中最慢。在处理后 6～ 36d内 ,不同土壤中的Cry1Ab残留量有显著差异 ,黄松田中的明显最高 ,老黄筋泥田中的次之 ,青紫泥田中的为最低 ;此后土壤间的残留量差异逐渐缩小 ,至处理后 78d差异已不明显。土壤淹水可显著加快华池B6 叶片中Cry1Ab的降解 ,且淹水后其降解动态在不同土壤之间十分相似 ;淹水对Cry1Ab降解的促进作用仅发生在前 12d之内 ,此后多数时间内残留量在淹水与非淹水处理间无显著差异。建立了各处理中Cry1Ab降解的动力学指数方程 ,相应地得出了其降解的半衰期。还讨论了土壤有机质和微生物等因子对土壤中Cry1Ab降解的影响。