A Proteinase from Mung Bean Sprouts That Inactivaties Soybean Trypsin Inhibitor

By 30% - 60% (NH4)(2)SO4 fractional precipitation, anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatography on Waters AP-1 column (Protein(PM)-Pak DEAE 15HR), a proteinase which can inactivate soybean trypsin inhibitor (STI) was purified from mung bean ( Vigna rabiata (L.) Wilezek) sprouts. Its molecular weight was estimated to be 29.8 kD by SDS-PAGE, and its K-m and V-max for STI were 769.2 N-alpha -benzoyl-L-arginine ethyl ester BAEE/mL and 115.3 BAEE . mL(-1) . min(-1) respectively. This proteinase was stable at temperatures lower than 50 degreesC and pH 6.5 - 8.5, and 90.91% STI activity of defatted soybean powder was inactivated by this preparation, with proteolytic activity 5 000 BAEE/mL at 50 degreesC and pH 8.0 in 4 h.

Correlation of Inhibitor Proteinase in Varieties and Lines of Cotton(Gossypium hirsutum L.) to Different Geographic Population of Verticillium dathliae Klebahn

Breeding for wilt resistance and its theoretical basis are primarily responsible for increases in cotton yield and fiber quality. Breeding for immunity is the most efficient method in our struggle with infectious diseases.

Molecular Cloning of a Thiol Proteinase Inhibitor Gene and Its Expression in E.coli

A cDNA library was constructed with 1.5×10~6 pfu from rice immature seeds,fromwhich a cDNA clone for rice thiol proteinase inhibitor,oryzacystatin(OC),was isolated byscreening with synthesized oligodeoxynucleotide probe,which contained a 309bp open read-ing frame,84bp 5′-end noncoding region and a poly(A)signal AATAAA at the 3′-end fol-lowed by 31Nt poly(A).Then the coding region of OC was amplified and inserted into thedownstream of λP_RP_L promoter for thermal-inducible expression in E.coli.Shifting the cul-ture temperature from 30℃ to 42℃ led to a high level expression of OC,which exhibited adistinct band of 12.0 kDa and accounted for at least 10% of the total soluble proteins fromSDS-PAGE.The papain-inhibitory activity of the expressed OC was further confirmed.

On the Activities of Pancreatic Proteases and Alpha-1 Proteinase Inhibitor in Meat-Type Chicken

The study was aimed at the evaluation of the effects of breed, age, different digestion stimulators, and dietary crude protein (CP) level on the activities of proteolytic enzymes in pancreatic tissue and duodenal chymus (in vivo), serum trypsin and α1-proteinase inhibitor (A1PI) concentrations in meat-type chicks. The study of age dynamics of trypsin and A1PI concentrations was performed on the chicks of hybrid cross “Smena-8”and two parental lines (Plymouth Rock and Cornish) fed standard commercial corn-wheat-SBM diets. Twenty birds per breed were euthanized at 1, 7, 14, 21, 28 and 35 days of age to obtain blood samples and pancreatic homogenate. Experiments on the effects of different digestion promotors (probiotic, acidifier, phytobiotic, enzymatic preparation) and different CP levels (finisher diet, CP 20%, vs. ground corn, CP 8.5%) were performed on 12 hybrid chicks with fistulated duodenum from 14 to 50 days of age. The following conclusions were made: 1) At 1 day of age high proteolytic activity in pancreatic tissue and maximal serum concentrations of trypsin and A1PI were found in both hybrid and parental lines. Since 7 to 35 days of age A1PI concentration was nearly constant, serum trypsin concentration decreased while proteolytic activity in pancreatic tissue exhibited undulate increase;2) Proteolytic activity in pancreatic tissue was higher in hybrids compared to the parental lines from 7 to 35 days of age (p 0.05);3) Supplementation of diet with exogenous enzymes stimulated the digestion due to the increase in protease activity in duodenal chymus by 9.1% compared to unsupplemented control (p 0.05);4) Proteolytic activity in duodenal chymus significantly responded to the substitution of ground corn for the complete diet by 2-fold decrease while serum trypsin concentration responded by 2.5-fold increase (p 0.001). This fact can indicate that physiological functions of digestive proteases are not confined to the digestive processes.

Cloning and Sequence Analysis of a Cysteine Proteinase Inhibitor Gene from Seedless Litchi

[Objective] This study aimed to clone and analyze the cysteine proteinase inhibitor gene from seedless litchi. [Method] According to the EST se- quence of cysteine proteinase inhibitor in constructed SSH snhtraetive library of seedless litchi abortion, nucleotide sequence of the cysteine proteinase inhibitor gene was obtained by using RACE technology and analyzed by using bioinformatics software. [ Result ] A cysteine protease inhibitor gene was obtained with the sequence of 635 bp containing a 321 bp open reading frame. It was predicted that the erlcoded protein contained 106 amino acids with conserved domain of cysteine proteinase inhibitor and had relatively high homology with the cysteine proteinase inhibitor gene of several species, [ Conclusion] This study laid the foundation for further ex- ploring the physiological functions of this cysteine proteinase inhibitor gene in plants.

Studies on Introduction of Arrowhead Proteinase Inhibitor Gene into N． tobacco Protoplasts

The Arrowhead Proteinase Inhibitor(API)gene was introduced into the protoplasts or mesophyll cells of N.tobacco by PEG-mediated.The transformed protoplasts undergoing dirrerentiation of callus and regeneration of plantlet have been growing into transgenic plants. Restriction endonuclease analysis of products amplificated by PCR indicates the existence of the API gene in the transformed plantlet.The extract of the leaves from the transformed plants shows trypsin inhibitory activity,which indicates the expression of the introduced API gene and the transformed plants can accumulate the inhibitor. However, the variation of the inhlbitory activity of the transformed plants reveals the importance of the integration site of the API gene in the genome.

A Kunitz trypsin inhibitor from chickpea (<i>Cicer arietinum</i>L.) that exerts an antimicrobial effect on Fusarium oxysporum f.sp. ciceris

Fusarium oxysporum f.sp. ciceris (Foc) is one of the most important fungal pathogens of chickpea and is regarded as a constant threat in tropical and subtropical countries. In order to correlate Fusarium wilt resistance/susceptibility in Cicer arietinum to the presence or absence of trypsin inhibitor (TI) in the crude extract, trypsin inhibitory assay (TIA) and in vitro activity of TI against Foc were studied. In the present study, a 20 kDa trypsin inhibitor was purified from Fusarium wilt resistant cultivar (viz. JG 2001-12) by ammonium sulfate precipitation, dialysis and chromatographies with Sephadex G-100 and Diethyl aminoethyl cellulose (DEAE-cellulose-52) ion-exchange column. Results of pathogenecity assay were found to be in correlation to the trypsin inhibitor assay where the Fusarium wilt resistant cultivar showed high trypsin inhibitory activity (99%) in the presence of trypsin enzyme using both natural and synthetic substrates. Preliminary studies using crude extracts of JG 2001-12 showed a decrease in radial growth of Foc. A 45%-82% reduction in conidium germination at 20 μg&middotmL-1?Cicer arietinum trypsin inhibitor (CaTI) concentration was observed, thereby, indicating the use of CaTI in suppression of pathogen and in its deployment through transgenic plants for the management of Fusarium wilt.

老年高危心血管病患者应用依洛尤单抗的有效性及安全性研究

目的探讨老年高危心血管病患者应用依洛尤单抗治疗的有效性和安全性。方法纳入2019年11月至2022年11月在解放军总医院第一、二、六、八医学中心心内科住院的经常规他汀类药物治疗后血脂控制不达标的心血管病高危患者153例,根据患者年龄分为非老年组(<60岁)46例,60~74岁老年组66例,≥75岁老年组41例,均按指南应用依洛尤单抗治疗;选取同期在解放军总医院第一、二、六、八医学中心心内科住院血脂控制不达标的年龄≥75岁心血管病高危未应用依洛尤单抗患者50例为常规治疗组,应用一种他汀类药物联合依折麦布治疗。比较各组患者临床基线资料及用药第4、12周的血液指标和12周内药物不良反应及主要不良心血管事件(MACE)发生情况。结果非老年组、60~74岁老年组和≥75岁老年组用药第4、12周低密度脂蛋白胆固醇(LDL-C)和总胆固醇(TC)水平较基线降低,差异有统计学意义(P<0.05,P<0.01);3组用药第12周LDL-C、TC水平比较无显著差异(P>0.05)。非老年组、60~74岁老年组、≥75岁老年组用药12周内不良事件发生率比较无显著差异(2.2%vs 3.0%vs 2.4%,P>0.05)。≥75岁老年组和常规治疗组用药第12周LDL-C、TC水平显著低于基线(P<0.05,P>0.01);≥75岁老年组用药第12周LDL-C水平显著低于常规治疗组[(1.36±0.44)mmol/L vs(1.87±0.56)mmol/L,P<0.01]。≥75岁老年组和常规治疗组用药12周内MACE发生率比较无显著差异(12.2%vs 16.0%,P>0.05),2组生存率比较无显著差异(P=0.576)。结论各年龄组患者应用依洛尤单抗治疗均可在短期内取得良好疗效,75岁以上老年患者应用依洛尤单抗治疗同样具有良好的有效性和安全性。

GhWRKY75 positively regulates GhPR6-5b via binding to a W-box TTGAC(C/T)to orchestrate cotton resistance to Verticillium dahliae

Verticillium dahliae is an important fungal pathogen affecting cotton yield and quality.Therefore,the mining of V.dahlia-resistance genes is urgently needed.Proteases and protease inhibitors play crucial roles in plant defense responses.However,the functions and regulatory mechanisms of the protease inhibitor PR6 gene family remain largely unknown.This study provides a comprehensive analysis of the PR6 gene family in the cotton genome.We performed genome-wide identification and functional characterization of the cotton GhPR6 gene family,which belongs to the potato protease inhibitor I family of inhibitors.Thirty-nine PR6s were identified in Gossypium arboreum,G.raimondii,G.barbadense,and G.hirsutum,and they were clustered into four groups.Based on the analysis of pathogen-induced and Ghlmm transcriptome data,Gh PR6-5b was identified as the key gene for V.dahliae resistance.Virus-induced gene silencing experiments revealed that cotton was more sensitive to V.dahliae V991after PR6-5b silencing.The present study established that GhWRKY75 plays an important role in resistance to Verticillium wilt in cotton by positively regulating GhPR6-5b expression by directly binding to the W-box TTGAC(T/C).Our findings established that GhWRKY75 is a potential candidate for improving cotton resistance to V.dahliae,and provide primary information for further investigations and the development of specific strategies to bolster the defense mechanisms of cotton against V.dahliae.

双肾血流阻力指数联合脂肪特异性丝氨酸蛋白酶抑制剂预测2型糖尿病肾脏疾病患者肾功能的价值

目的探讨双肾血流阻力指数联合脂肪特异性丝氨酸蛋白酶抑制剂(visceral adipose tissue-derived serine protease inhibitor,Vaspin)预测2型糖尿病肾脏疾病患者肾功能的价值。方法选取2021年8月至2022年8月秦皇岛市第一医院收治的108例2型糖尿病肾脏疾病患者,入院后进行彩色多普勒超声检查,测量患者双肾血流阻力指数,将患者分为肾血流阻力指数升高组40例和肾血流阻力指数正常组68例。采用酶联免疫吸附试验检测血清Vaspin水平,并搜集患者临床资料;采用单因素和多因素Logistic回归分析影响肾血流阻力指数升高的危险因素。采用Pearson分析肾血流阻力指数、Vaspin与临床资料的关系,根据肾血流阻力指数、Vaspin绘制受试者工作特征曲线,以曲线下面积评估其对2型糖尿病肾脏疾病患者肾功能的预测价值。结果两组患者性别、糖尿病肾脏疾病分期、尿蛋白定量、血白蛋白、糖化血红蛋白、血尿酸、尿白蛋白与肌酐比值、尿α1微球蛋白、尿β2微球蛋白等一般资料比较差异均无统计学意义(P>0.05);肾血流阻力指数升高组患者年龄[(56.33±5.19)岁比(50.78±5.24)岁]、糖尿病病程[(12.58±3.42)年比(10.13±3.25)年]、高血压(87.50%比61.76%)高于肾血流阻力指数正常组,而三酰甘油[(1.72±0.45)mmol/L比(2.11±0.58)mmol/L]、低密度脂蛋白[(2.33±0.57)mmol/L比(2.61±0.62)mmol/L]、血红蛋白[(102.39±21.64)g/L比(113.76±28.45)g/L]、左肾体积[(76.58±15.33)cm^(3)比(81.35±17.16)cm^(3)]、右肾体积[(71.62±12.39)cm^(3)比(80.47±16.51)cm^(3)]、估算肾小球滤过率[(40.37±12.58)mL·min^(-1)·(1.73 m^(2))-1比(52.14±13.26)mL·min^(-1)·(1.73 m^(2))^(-1)]、血清Vaspin水平[(1.52±0.38)μg/L比(1.74±0.43)μg/L]低于肾血流阻力指数正常组(P<0.05)。Pearson相关性分析结果显示,肾血流阻力指数与低密度脂蛋白、估算肾小球滤过率、血清Vaspin呈负相关关系(P<0.05),与血肌酐、尿白蛋白与肌酐比值呈正相关关系(P<0.05)。多因素Logistic回归分析结果显示,年龄≥55岁(OR=1.061,95%CI:1.036~1.086)、血肌酐升高(OR=1.012,95%CI:1.004~1.020)、估算肾小球滤过率升高(OR=1.037,95%CI:1.007~1.068)、血清Vaspin下降(OR=1.132,95%CI:1.053~1.217)是影响肾血流阻力指数升高的独立危险因素(P<0.05)。双肾血流阻力指数联合Vaspin预测2型糖尿病肾脏疾病患者肾功能进展的曲线下面积为0.79,敏感性和特异性分别为82.49%、68.73%。结论双肾血流阻力指数升高、血清Vaspin水平降低是影响2型糖尿病肾脏疾病患者肾功能的独立危险因素,二者联合检测对预测2型糖尿病肾脏疾病患者肾功能进展具有较好预测价值。

Cloning of a Potato Proteinase Inhibitor Gene PINII-2x from Diploid Potato(Solanum phurejia L.) and Transgenic Investigation of Its Potential to Confer Insect Resistance in Rice

Both cDNA and a genomic DNA fragment encoding a new potato proteinase inhibitor Ⅱ were isolated from a diploid potato IVP101 （Solanum phurejia L.） and named PINII-2x. Nucleotlde sequencing confirmed that the DNA fragment of PINll-2xwas 580 bp, including a 115-bp intron and two exons. The deduced PINII-2x proteln contained an Intact signal peptide and two active sites. The PINII-2x gene and its deduced PINII-2x protein had 88% and 93% homology with another tetraploid potato proteinase inhibitor Ⅱ, respectively. Northern blotting analysis Indicated that the mRNA of PINII-2x gene was wound induced in potato leaves. Binary vector pNAR301 and pNAR302 were constructed for rice transformation, in which the PINII-2x cDNA was driven, respectively, by rice actin I promoter （Actl） and maize ubiquitin promoter （Ubll）. Via an Agrobacteriummediated method, these two constructs were transferred into japonica rice cv. Xiushui 63. PCR and Southern blotUng analysis for transgenic rice revealed the integration of the PINII-2x gene. Northern blotting analysis also confirmed transcripts of the PINII.2x gene in transgenic rice plants. Insect bloassays using stripe stem borer （Chilo auppressalis Walker） demonstrated that the average weight and body length of larvae In transgenic plants were only nearly 50% and 61% of those of larvae in control plants, respectively. These results Indicate that the PINII-2x gene should be an effective insect-resistance gene and could be valuable for application in crop breeding for insect resistance.

Novel alleles among soybean Bowman-Birk proteinase inhibitor gene families

Trypsin inhibitors have been found in various animals, plants and microorganisms.There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors(BBI) and Kunitz in-hibitors(KTI).The different BBI genes from wild soybean(G.soja) and cultivated soybean(G.max) formed a multigene family.We constructed a cDNA library of cultivar 'SuiNong 14' seed at the R7 growth stage using the SMART Kit.Seventeen contigs or singletons were highly homologous to soy-bean protease inhibitors.Contigs of 5, 35, 8 and 9 were highly homologous to BBI family members BBI-A1, BBI-A2, BBI-C and BBI-D, respectively.Sequence analyses showed there were novel allelic varia-tions among the 4 BBI members in SuiNong 14.Based on the comparison of soybean seed cDNA li-braries from different developmental stages, it was apparent that the expression of trypsin inhibitors increased during seed development in soybean.Phylogenetic analysis of BBI gene sequences among dicotyledonous and monocotyledonous plants demonstrated that these genes shared a common pro-genitor.

Isolation of tomato proteinase inhibitor Ⅱ gene and the function of its intron

The genomic DNA sequence of tomato pro-teinase inhibitor II gene (named tin2i, whose accession number in GenBank is AF007240) was isolated by PCR techniques. The intron sequence (TPI), with a length of 109 bp, owns typical structures of GT/AG dinucieotides at both ends and high content of AT base pairs which accounts for 80.7% of the total nucleotides. As shown by recombination experiment, the TPI sequence could efficiently promote the expression of the reporter gene gusA and this effect was independent of the position and orientation of the intron, thus showing its role as an enhancer. Such experiments as gel retardation assays, GUS histochemical staining and GUS fluorometric assays further demonstrated that TPI sequence maybe has promoter-like activity.

Co-expression of the proteinase inhibitors oryzacystatin I and oryzacystatin II in transgenic potato alters Colorado potato beetle larval development

Colorado potato beetle （CPB; Leptinotarsa decemlineata Say, Coleoptera： Chrysomelidae） has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II （OCI and OCII） have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato （Solanum tuberosum L.） cultivars Desiree, Draga6evka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition ofcysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long-term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae.

INHIBITORY PROPERTY CHARACTERIZATION AND REACTIVE SITE EXPLORATION OF THE ARROWHEAD PROTEINASE INHIBITOR

Affinity chromatography was used to separate two components A and B of the crystalline arrowhead proteinase inhibitor. Both A and B are double-headed and multifunctional proteinase inhibitors. Inhibitor A is capable of inhibiting equimolarly trypsin and chymo-trypsin simultaneously, and has a weak inhibitory activity toward kallikrein; whereas inhibitor B can inhibit two molecules of trypsin simultaneously, and shows rather higher inhibitory activity toward kallikrein than inhibitor A, but its inhibitory activity toward chymo-trypsin is much weaker than that of inhibitor A. The results of chemical modification and the competitive binding of trypsin and chymotrypsin with inhibitor A showed that the two reactive sites of both inhibitors A and B are Lys and Arg residues. Among them the Lys reactive site is specific for inhibiting mainly trypsin, whereas the active domain composed of the Arg reactive site appears to be multifunctional and capable of inhibiting many different Ser proteinases. Based on the structural characteristics of inhibitors A and B, it was predicated that the two reactive sites should be located in the positions Lys-Ser (44-45) and Arg-Tyr-Lys (76-78), respectively. In inhibitor A, there exists another hydrophobic residue involved in inhibiting chymotrypsin. This residue might be situated in the reactive region composed of the Arg reactive site.

尿胰蛋白酶抑制剂分离纯化研究进展

尿胰蛋白酶抑制剂(Urinary Trypsin Inhibitor,UTI)是从人类尿液中分离纯化得到的一种具有丝氨酸蛋白酶抑制活性的硫酸软骨素蛋白聚糖,其对胰蛋白酶、透明质酸酶、粒细胞弹性蛋白酶、纤溶酶等蛋白酶均具有良好抑制作用,被广泛用于临床治疗急性胰腺炎和作为急性循环衰竭的抢救辅助用药。本文主要综述了尿胰蛋白酶抑制剂的来源、结构、生理功能、传统分离纯化工艺以及基因工程技术生产新方法等,旨在为尿胰蛋白酶抑制剂的高质量生产以及应用提供参考。

Salicylic acid and ethylene signaling pathways are involved in production of rice trypsin proteinase inhibitors induced by the leaf folder Cnaphalocrocis medinalis(Guenée)

The roles of signaling pathways in the production of trypsin proteinase inhibitors(TrypPIs)in rice infested by the leaf folder(LF) Cnaphalocrocis medinalis were studied.Infestation by LF increased TrypPI levels in the leaves of rice plants at the tillering,booting and flowering stages but decreased TrypPI levels at the ripening stage;TrypPI levels in rice stems did not increase at any developmental stage.Infestation by LF at the tillering stage systemically increased TrypPI levels in leaves but not in stems;it also enhanced salicylic acid(SA)levels in leaves and stems,and the ethylene level released from plants.However,LF infestation did not increase JA concentrations.Exogenous application of SA or ethylene enhanced TrypPI levels in the leaves and stems of plants at the tillering stage,whereas treatment with both SA and ethylene induced lower levels of TrypPIs than treatment with SA or ethylene alone,suggesting an antagonistic effect of SA and ethylene on TrypPIs induction.The results suggest that both SA and ethylene signaling pathways are involved in the production of TrypPIs in rice induced by LF;moreover,the antagonistic effect of SA and ethylene may explain the changes in TrypPI levels seen at different plant developmental stages and in different organs.

冠心病患者血清RBP4、CysC水平与肠道菌群的关系

目的:探讨冠心病患者血清视黄醇结合蛋白4(RBP4)、胱抑素C(CysC)的水平及其与肠道菌群的关系。方法:选择2019年12月至2020年12月在我院重症医学科就诊的97例冠心病患者作为冠心病组,99名同期健康体检者作为对照组。比较两组血清RBP4、CysC水平及肠道菌群阳性率及数量。以冠心病患者血清RBP4、CysC平均水平为界,将其分为血清RBP4高水平组(RBP4≥35.97 ng/ml)53例和低水平组(RBP4<35.97 ng/ml)44例,血清CysC高水平组(CysC≥1.49 ng/ml)49例和低水平组(CysC<1.49 ng/ml)48例。比较不同水平组菌群数量,采用Pearson法分析RBP4、CysC水平与菌群数量的相关性。结果:与对照组比较,冠心病组RBP4、CysC水平均显著升高,双歧杆菌属、厚壁菌、乳酸杆菌属、变形杆菌的培养阳性率及菌群数量均显著降低(P均<0.001),葡萄球菌、大肠埃希菌的培养阳性率及菌群数量均显著升高(P均<0.001)。与RBP4低水平组比较,RBP4高水平组双歧杆菌、厚壁菌、乳酸杆菌、变形杆菌数量均显著减少,葡萄球菌、大肠埃希菌数量显著增加(P均<0.001);与CysC低水平组比较,CysC高水平组双歧杆菌、厚壁菌、乳酸杆菌、变形杆菌数量显著减少,葡萄球菌、大肠埃希菌数量显著增加(P均<0.001)。Pearson相关分析显示,RBP4水平与双歧杆菌属、厚壁菌、乳酸杆菌属、变形杆菌数量呈显著负相关(r=-0.626~-0.482,P均<0.001),与葡萄球菌、大肠埃希菌数量显著正相关(r=0.302、0.337,P均<0.01);CysC水平与双歧杆菌属、厚壁菌、乳酸杆菌属、变形杆菌数量呈显著负相关(r=-0.621~-0.502,P均<0.001),与葡萄球菌、大肠埃希菌数量呈显著正相关(r=0.308、0.340,P均<0.01)。结论:冠心病患者血清RBP4、CysC水平升高,且与肠道菌群组成密切相关。

SERPINE1影响结肠癌细胞RKO和LoVo的增殖与凋亡

目的探究丝氨酸蛋白酶抑制剂1(serine proteinase inhibitor family E member 1,SERPINE1)在结肠癌(colon adenocarcinoma,COAD)中的表达、意义及对COAD细胞增殖与凋亡的影响,了解其在COAD进展中发挥的生物学功能。方法通过Gene Expression Profilling Interactive Analysis2.0(GEPIA2.0)数据库分析SERPINE1在COAD中的表达与分期及预后关系。收集作者医院5例COAD患者癌组织与对应的正常结肠组织标本,应用Western blot实验验证SERPINE1在COAD中的表达;接着构建SERPINE1过表达与干扰表达的慢病毒载体,进行细胞转染,通过实时荧光定量逆转录聚合酶链反应(real-time reverse transcription quantitative polymerase chain reaction,RT-qPCR)和Western blot对实验细胞筛选并验证过表达和干扰表达的效率;进而通过细胞计数试剂盒(cell counting kit 8,CCK-8)和Hoechst细胞核染色检测细胞增殖与凋亡;最后通过Western blot实验了解其对增殖标志物Ki-67(marker of proliferation Ki-67,MKI67)、Bcl-2关联X蛋白(Bcl-2 associated X protein,Bax)与B细胞淋巴瘤2基因(B cell lymphoma 2,Bcl-2)比值的影响。结果通过GEPIA2.0数据库查找并分析COAD中SERPINE1表达与临床分期和预后关系发现,SERPINE1在COAD中表达水平高于正常结肠组织,SERPINE1的表达水平与临床分期正相关,而与预后生存总生存期负相关(P均<0.05)。CCK-8实验结果表明,oe-RKO组和oe-LoVo组COAD细胞在48h时间点吸光度(optical density,OD)值大于oe-RKO-NC组和oe-LoVo-NC组,sh-RKO-822组和sh-LoVo-822组COAD细胞在48 h时间点OD值小于sh-RKO-NC和sh-LoVo-NC组(P均<0.05)。Hoechst凋亡细胞核染色结果表明,oe-RKO组和oe-LoVo组COAD细胞凋亡数量低于oe-RKO-NC组和oe-LoVo-NC组,sh-RKO-822组和sh-LoVo-822组COAD细胞凋亡数量明显高于sh-RKO-NC组和sh-LoVo-NC组(P均<0.05)。Western blot结果显示,oe-RKO组和oe-LoVo组COAD细胞MKI67蛋白表达明显高于oe-RKO-NC组和oe-LoVo-NC组,Bax/Bcl-2明显低于oe-RKO-NC组和oe-LoVo-NC组;sh-RKO-822组和sh-LoVo-822组COAD细胞MKI67蛋白表达明显低于sh-RKO-NC组和sh-LoVo-NC组,Bax/Bcl-2明显高于sh-RKO-NC组和sh-LoVo-NC组(P均<0.05)。结论SERPINE1在COAD中高表达、与临床分期正相关,而与患者预后负相关;SERPINE1可能通过影响COAD细胞MKI67表达和Bax/Bcl-2促进COAD RKO和LoVo细胞的增殖,抑制其凋亡。

Transgenic Tobacco Plants by Cotransformation With Proteinase Inhibitor Ⅱ and δ-endotoxin Genes

In recent years, great progress has been made in. producing plants resistant to insect attack by genetic engineering of B. t δ-endotoxin gene or plant proteinase inhibitor genes, and how to avoid the emergence of the insect resistance to B. t δ-endotoxin produced by transgenic plants has been paid attention to. To combine one B. t δ-endotoxin gene with an incompatible B. t δ-endotoxin gene or with other genes such as proteinase inhibitor gene might alleviate the insect resistance problem. In this note, we study the