CRISPR/Cas9-mediated NlInR2 mutants:Analyses of residual mRNA and truncated proteins

CRISPR/Cas9 technology is a powerful genome manipulation tool in insects.However,little is known about whether mRNA and protein of a target gene are completely cleared in homozygous mutants.This study generated homozygous mutants of the insulin receptor gene 2(NlInR2)in the brown planthopper(Nilaparvata lugens)using CRISPR/Cas9 genome editing.Both frameshift mutants,E5_D17 and E6_I7,differentiated towards long wings,but there were differences in wing morphology,with E5_D17 showing wing deformities.Subsequent investigations revealed the presence of residual expression of NlInR2 mRNA in both mutants,as well as the occurrence of spliceosomes featuring exon skipping splicing in E5_D17.Additionally,the E5_D17 exhibited the detection of N-terminally truncated NlInR2 protein.RNA interference experiments indicated that the knockdown of NlInR2 expression in the E5_D17 mutant line increased the proportion of wing deformities from 11.1 to 65.6%,suggesting that the residual NlInR2 mRNA of the E5_D17 mutant might have retained some genetic functions.Our results imply that systematic characterization of residual protein expression or function in CRISPR/Cas9-generated mutant lines is necessary for phenotypic interpretation.

MN/CA9蛋白在富糖原型肾细胞癌中的表达及其意义

目的:研究MN/CA9蛋白在富糖原型肾细胞癌中的表达。方法:对正常肾组织、肾损伤、肾细胞癌及非肾性富糖原癌中MN/CA9蛋白的表达进行分析。结果:仅在肾细胞癌上可见MN/CA9蛋白的表达,而其它切片中未见表达。结论:MN/CA9蛋白表达可以作为肾细胞癌,尤其是富糖原型肾细胞癌的一种肿瘤标志物,以辅助其早期诊断。

逆转录聚合酶链反应检测小鼠MN/CA9基因的表达

目的:克隆、分析ICR小鼠MN/CA9基因序列,并检测MN/CA9基因在ICR小鼠各组织中的表达。方法:分别取ICR小鼠小肠、肝、肾、脾、胃、胸腺、心脏、卵巢、肺、胰腺、肌肉、皮肤、子宫和膀胱新鲜组织,采用胍尼啶异硫氢酸呱酚氯仿提取法(GIT)提取总RNA。合成cDNA的第1链和第2链,连接EcoRⅠ载体,EcoRⅠ末端磷酸化,XhoⅠ消化,将cDNA构建到ZAPexpressvector进行包装、种植培养,筛选、切取噬菌体,使用引物扩增后进行DNA序列分析。用逆转录聚合酶链反应检测小鼠MN/CA9基因在上述组织中的表达。结果:使用人类MN/CA9基因片段做探针,用放射性同位素32P标记探针,筛选1·47×103大肠埃希菌落,杂交后发现一阳性cDNA信号,序列测定确定其含1671bp核苷酸序列(已在GenBank登录,登录号AB086322),这个序列与人类的MN/CA9(基因序列号Z54349)有69·1%的同源性。在引物P521-P1193区间,MN/CA9基因在小鼠小肠、子宫、肌肉、胰腺、心脏、肺、胸腺、脾、肾、卵巢、胃和膀胱组织表达均较强,在皮肤和肝脏不表达。结论:MN/CA9基因在ICR小鼠组织中的表达与人类基本相同,可以用ICR小鼠进行MN/CA9基因的研究。

MN／CA9和Ep-cam基因检测在良恶性胸腔积液鉴别中的价值

目的探讨MN／CA9和Ep—cam基因检测对良、恶性胸腔积液鉴别诊断的价值。方法选择35例恶性胸腔积液（MPE）和30例良性胸腔积液（BPE），采用SYBRGreenIFQRT—PCR检测胸水沉淀细胞中MN／CA9mRNA、Ep—cammRNA的表达，胸水找脱落细胞，免疫化学发光法检测胸水CEA水平。结果MN／CA9和Ep—cam基因在良、恶性胸腔积液中的表达均有明显差异（均P〈001）。对MPE的诊断，用FQRT—PCR方法检测MN／CA9mRNA和Ep—cammRNA的敏感性（SN）均高于其它两项检查，差异均有统计学意义（均P〈005），而特异性（SP）无统计学差异（均P〉0．05）。细胞学阴性的MPE，MN／CA9诊断的SN为55．6％，SP为100％；Ep—cam诊断的SN为61.5％，SP为100％；两者联合检测的细胞学阴性MPE的SN为76．9％，SP为100％。结论SYBRGreenIFQRT—PCR检测MN／CA9和Ep—cam基因有助于鉴别良、恶性胸腔积液；联合检测MN／CA9和Ep—cam基因的表达有助于提高细胞学阴性MPE诊断的敏感性。

宫颈癌特异抗原MN/CA9的多糖蛋白表达载体的构建及鉴定

目的克隆宫颈癌MN/CA9抗原的多糖蛋白cDNA,并构建表达载体。方法从宫颈癌HeLa细胞中提取总RNA,RT-PCR法扩增MN/CA9抗原的多糖蛋白cDNA,将其插入载体pCA13,转化大肠杆菌JM109,构建MN/CA9抗原的多糖蛋白表达载体pCA13-MN。酶切及测序鉴定。结果酶切及序列分析结果证明,目的基因成功克隆入载体。结论成功构建了MN/CA9多糖蛋白表达载体。

肾细胞癌MN/CA9蛋白的表达及其生物学意义

目的 :研究MN/CA9蛋白的表达和肾细胞癌的关系 ,探讨MN/CA9蛋白在肾细胞癌诊断研究中的应用。方法 :应用免疫组化和免疫印迹的方法 ,对正常肾组织、良性肾损害和肾细胞癌组织的MN/CA9蛋白的表达进行了分析。结果 :正常人肾组织和良性肾损害均为MN/CA9阴性 ,而在 30例肾癌中却有 2 8例为MN/CA9阳性 ,尤其是 2 6例富糖原型RCC均呈现MN/CA9强阳性。结论

Adjusting CA19-9 values to predict malignancy in obstructive jaundice:Influence of bilirubin and C-reactive protein

AIM:To find a possible relationship between inflammation and CA19-9 tumor marker by analyzing data from patients with benign jaundice(BJ) and malignant jaundice(MJ).METHODS:All patients admitted for obstructive jaundice,in the period 2005-2009,were prospectively enrolled in the study,obtaining a total of 102 patients.On admission,all patients underwent complete standard blood test examinations including C-reactive protein(CRP),bilirubin,CA19-9.Patients were considered eligible for the study when they presented obstructive jaundice confirmed by instrumental examinations and increased serum bilirubin levels(total bilirubin > 2.0 mg/dL).The standard cut-off level for CA19-9 was 32 U/mL,whereas for CRP this was 1.5 mg/L.The CA19-9 level was adjusted by dividing it by the value of serum bilirubin or by the CRP value.The patients were divided into 2 groups,MJ and BJ,and after the adjustment a comparison between the 2 groups of patients was performed.Sensitivity,specificity and positive predictive values were calculated before and after the adjustment.RESULTS:Of the 102 patients,51 were affected by BJ and 51 by MJ.Pathologic CA19-9 levels were found in 71.7% of the patients.In the group of 51 BJ patients there were 29(56.9%) males and 22(43.1%) females with a median age of 66 years(range 24-96 years),whereas in the MJ group there were 24(47%) males and 27(53%) females,with a mean age of 70 years(range 30-92 years).Pathologic CA19-9 serum level was found in 82.3% of MJ.CRP levels were pathologic in 66.6% of the patients with BJ and in 49% with MJ.Bilirubin and CA19-9 average levels were significantly higher in MJ compared with BJ(P = 0.000 and P = 0.02),while the CRP level was significantly higher in BJ(P = 0.000).Considering a CA19-9 cut-off level of 32 U/mL,82.3% in the MJ group and 54.9% in the BJ group were positive for CA19-9(P = 0.002).A CA19-9 cut-off of 100 U/mL increases the difference between the two groups:35.3% in BJ and 68.6% in MJ(P = 0.0007).Adjusting the CA19-9 value by dividing it by serum bilirubin level meant that 21.5% in the BJ and 49% in the MJ group remained with a positive CA19-9 value(P = 0.003),while adjusting the CA19-9 value by dividing it by serum CRP value meant that 31.4% in the BJ group and 76.5% in the MJ group still had a positive CA19-9 value(P = 0.000004).Sensitivity,specificity,positive predictive values of CA19-9 > 32 U/mL were 82.3%,45% and 59.1%;when the cutoff was CA19-9 > 100 U/mL they were,respectively,68.6%,64.7% and 66%.When the CA19-9 value was adjusted by dividing it by the bilirubin or CRP values,these became 49%,78.4%,69.4% and 76.5%,68.6%,70.9%,respectively.CONCLUSION:The present study proposes CRP as a new and useful correction factor to improve the diag-nostic value of the CA19-9 tumor marker in patients with cholestatic jaundice.

MN/CA9基因检测与肾细胞癌的诊断

目的 探讨MN CA9基因的检测在诊断肾细胞癌 (RCC)中的价值。方法 本组共有 70例病人 (无临床转移的RCC病人 4 2例 ,有转移的RCC病人 3例 ,其他肾脏疾病病人 2 5例 ) ,采用RT PCR方法检测癌组织、正常肾组织及血液和尿液中MN CA9基因的表达。结果 4 2例RCC病人的癌组织中 38例检测到MN CA9基因的表达 ,其中16例外周血中有MN CA9基因表达 ,6例尿液中检测到该基因 ,3例已有转移病人中 2例血液有该基因表达 ,1例尿液中有表达 ,而其他肾脏疾病的 2 5例肾组织中仅 1例有MN CA9的表达 ,外周血及尿液中未检测到该基因。结论 MN CA9基因是RCC病人的一个有诊断意义的生物指标 。

MN/CA9基因检测与肾细胞癌的意义

目的 :探讨MN/CA9基因的检测在诊断肾细胞癌 (RCC)中的价值。方法 :对无临床转移的RCC患者 4 2例 ,有转移的RCC患者 3例 ,其他肾脏疾病患者 2 5例 ,采用RT PCR方法检测癌组织 ,正常肾组织及血液和尿液中MN/CA9基因的表达。结果 :4 2例无临床转移的RCC患者的癌组织中 38例检测到MN/CA9基因的表达 ,其中 1 6例外周血中有MN/CA9基因表达 ,6例尿液中检测到该基因 ;3例已有肺转移患者中 2例血液有该基因表达 ,1例尿液中有表达。而其他肾脏疾病的 2 5例肾组织中仅 1例有MN/CA9的表达 ,外周血及尿液中未检测到该基因。结论 :MN/CA9基因是RCC患者的一个有诊断意义的生物指标 。

CA199、hsCRP、TSGF在梗阻性黄疸鉴别诊断中的意义

目的探讨临床工作中胆汁和血清CA199、高敏C反应蛋白质(hsCRP)、肿瘤特异性生长因子(TSGF)对良、恶性梗阻性黄疸病因鉴别诊断的意义。方法收集21例恶性梗阻性黄疸疾病患者、17例良性梗阻性黄疸疾病患者和15例无黄疸的胆道良性疾病患者的胆汁及血清标本,检测CA199、hsCRP、TSGF水平,并同期检测各组患者血清总胆红素和直接胆红素水平。结果①三组胆汁中CA199分泌水平均明显高于血清(P<0.05)。恶性组与良性组之间胆汁CA199水平、血清CA199水平差异无统计学意义(P>0.05),恶性组与无黄疸组、良性组与无黄疸组之间的胆汁、血清CA199差异均有统计学意义(P<0.05)。②三组组内胆汁hsCRP均明显低于血清(P<0.05)。三组组间胆汁hsCRP水平、血清hsCRP水平差异均无统计学意义(P>0.05)。③三组组内胆汁CA199/hsCRP比值与血清CA199/hsCRP比值差异有统计学意义(P<0.05),三组组间胆汁、血清CA199/hsCRP比值差异均无统计学意义(P>0.05)。④恶性梗阻性黄疸患者血清CA199/hsCRP比值与D/T比值[直接胆红素(D-bil)与总胆红素(T-bil)的比值]存在高度线性相关关系(R=0.914,P<0.01),D/T比值愈大,CA199/hsCRP值升高愈明显,而在良性组与无黄疸组内均无此现象。⑤三组组内胆汁TSGF水平与血清TSGF水平差异无统计学意义(P>0.05),三组组间胆汁TSGF水平、血清TSGF水平比较差异亦无统计学意义(P>0.05)。结论恶性组血清中CA199/hsCRP比值与D/T比值有高度依存关系,而良性组和无黄疸组中无此现象。单独检测胆汁或血清CA199、hsCRP、TSGF对良、恶性梗阻性黄疸的鉴别诊断无明显临床意义。

Subculturing cells have no effect on CRISPR/Cas9-mediated cleavage of UL30 gene in pseudorabies virus

CRISPR/Cas9-mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus(PRV) could escape from CRISPR/Cas9-mediated inhibition.In order to elucidate whether the escape of PRV from Cas9-mediated inhibition was due to cell deficiencies, such as genetic instability of sgRNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sgRNA-expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9-mediated cleavage of PRV. Different passages of PX459-PRV cells can stably express sgRNA to facilitate Cas9/sgRNA cleavage on the UL30 gene of PRV, resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.

应用CRISPR/Cas 9系统构建肺癌细胞系获得AMPKα1基因敲除的稳定细胞株

目的利用CRISPR/Cas 9系统在人肺癌细胞系A549和H460中获得敲除腺苷单磷酸活化蛋白激酶α1(adenosine monphosphate-activated protein kinase,AMPKα1)的稳定表达细胞系。方法根据CRISPR/Cas 9靶点设计原则,设计三条引导RNA(sgRNA),分别构建AMPKα1-CRISPR/Cas9 KO及其HDR质粒。将质粒转染至A549和H460细胞后,筛选稳定的单克隆细胞株。用Western blot鉴定筛选获得的肺癌细胞株是否表达AMPKα1。实验被分为原始对照组和敲除组,采用MTT法检测AMPKα1的肺癌细胞增殖情况。结果经过CRISPR/Cas 9系统处理后,筛选获得的A549和H460敲除AMPKα1稳定细胞株与原始细胞株A549和H460相比,Western blot检测不到AMPKα1蛋白质的表达,差异具有统计学意义(t=137.7,79.17,均P<0.0001)。MTT结果显示,筛选获得的A549和H460敲除AMPKα1的稳定细胞株与原始细胞株A549和H460相比,敲除细胞株的增殖速度明显减弱,差异具有统计学意义(t=3.956~28.16,均P<0.05)。结论CRISPR/Cas 9系统成功建立敲除AMPKα1表达的稳定肺癌细胞株,为进一步研究AMPKα1在肺癌发生发展中的作用提供细胞模型。

The Role of Metal Ions in Protein and Fatty Acids Biosynthesis in Soybean under Micronutrients Application to Soil

The present study is part of our ongoing investigation to study the role of trace elements on soybean seed composition (protein, oil, and fatty acids). This study was conducted to study the effects of five trace elements (Mn, Cu, Zn, Mo, B). The treatments of Mn, Cu, Zn, Mo, and B were chlorides, except Mo as oxide, and B as boric acid. The treatments were Mn, Cu, Zn, Mo, and B alone and in combination with the chelating agent citric acid (CA), for example Mn + CA, Cu + CA, and Zn + CA. Soybean cultivar (Bolivar with maturity group V) was grown in a repeated greenhouse experiment in a randomized complete block design. The compounds were applied to three-week-old soybean plants at V3 (vegetative) and at R3 (beginning of seed-pod initiation) stages. The plants were allowed to grow until maturity under greenhouse conditions. The harvested seeds were analyzed for mineral, protein, and fatty acid contents. Results showed that Mn, Cu, and B treatments increased seed protein, while Zn, Mo, Cu + CA, and B + CA decreased the protein. Treatments of Zn, Mo, CA, Cu + CA, Zn + CA, Mo + CA, and B + CA increased the oil. Treatments of Mn and Cu decreased the oil. The Cu and B treatments increased oleic acid by 8.0% and 7.4%, respectively for Cu and B. Treatments of Mn, Mo, CA, and Mn + CA, Cu + CA, Zn + CA, Mo + CA, and B + CA decreased oleic acid by 0.6% to 14.4%. Treatments of Cu, Zn, Mo, B, CA, Mn and their combination with CA increased linoleic acid by 1.3% to 6.5%. Our goal was to identify the trace elements that would make desirable alteration in the seed composition qualities.

Review: Plant Genome Editing Targeted by RNA-Guided DNA Endonuclease CRISPR/Cas9

This review chronicles the development of the research on CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9) during the last 30 years from the discovery of CRISPR sequence, of biological significance and of the molecular mechanism for adaptive bacterial immunity. It describes recent works on structural and functional diversity of CRISPR/Cas systems, and on three-dimensional structure-based improvements of on-target specificity of CRISPR/Cas9 and Cpf1 endonucleases. The review ends with the application of CRISPR/Cas9 to targeted editing of plant genomes. Importantly, plant commodities modified by CRISPR-Cas9 have not been considered as genetically modified organisms (GMO) as long as foreign DNAs from plant pests were not introduced, according to the recent determination by the USDA.

化瘀消斑汤对颈动脉粥样硬化兔模型脂代谢及CRP、MMP-9的影响

目的:观察化瘀消斑汤对颈动脉粥样硬化兔模型血脂、C反应蛋白(C reactive protein,CRP)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)表达及动脉粥样硬化进展的影响。方法:将40只新西兰大耳兔随机分为空白组5只、模型组15只、化瘀消斑汤组10只和阿托伐他汀钙组10只。除空白组外各组大耳兔均给予高脂饲料及静注牛血清白蛋白造模,空白组及模型组给予生理盐水灌胃;化瘀消斑汤组每日予化瘀消斑汤16.35 m L·kg^(-1)灌胃;阿托伐他汀钙组予以阿托伐他汀钙片灌胃,阿托伐他汀钙片研磨成粉状,换算成大耳兔用量按1.09 mg·kg^(-1)标准灌胃。用ELISA法测定血清中血脂水平、CRP及MMP-9含量,光镜下观察病理切片标本。结果:与空白组比较,模型组、化瘀消斑汤组、阿托伐他汀钙组血脂水平、CRP及MMP-9含量明显升高,差异具有统计学意义(P<0.05);与模型组比较,化瘀消斑汤组、阿托伐他汀钙组血脂水平、CRP及MMP-9含量明显降低,差异具有统计学意义(P<0.05);化瘀消斑汤组与阿托伐他汀钙组血脂水平、CRP及MMP-9含量比较,差异无统计学意义(P>0.05)。结论:化瘀消斑汤可改善颈动脉粥样硬化兔血脂水平,减少炎性因子CRP、MMP-9的表达,进而干预颈动脉粥样硬化的进展。

2型糖尿病患者锰超氧化物歧化酶Ala-9Val基因多态性与颈动脉粥样硬化的相关性研究

目的探讨2型糖尿病(type 2 diabetes mellitus,T2DM)患者锰超氧化物歧化酶(manganese-superoxide dismutase,Mn-SOD)Ala-9Val基因多态性与颈动脉粥样硬化(carotid atherosclerosis,CAS)的关系。方法选取九八七医院2017年1月~2019年1月的310例T2DM患者为T2DM组,根据颈动脉超声检查结果分为无CAS亚组和CAS亚组。另随机选取同期160例健康体检的志愿者作为对照组,采用聚合酶链反应-限制性片段长度多态性法(polymerase chain reaction restriction fragment length polymorphism,PCR-RFLP)检测Mn-SOD基因Ala-9Val位点基因型。分别比较各组间Ala-9Val位点基因型和等位基因分布频率。采用Logistic多因素回归分析法探究T2DM患者发生CAS的独立危险因素。结果对照组和T2DM组的Ala-9Val位点基因型分布频率差异无统计学意义(χ^2=4.210,P=0.122),两组的等位基因分布频率差异具有统计学意义(χ^2=4.487,P=0.034)。CAS亚组与无CAS亚组的Ala-9Val位点基因型和等位基因分布频率差异均具有统计学意义(χ^2=12.283,P=0.002;χ^2=10.984,P=0.001)。Logistics多因素回归分析结果显示,Val/Val基因型是T2DM患者发生CAS的独立危险因素(OR=1.865,95%CI 1.086~3.200,P=0.007)。结论T2DM患者Mn-SOD基因Ala-9Val位点多态性与CAS存在一定程度的相关性。

转录因子HNF1A、HNF4A和FOXA2调节肝细胞蛋白质N-糖基化

Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulatory transcriptional loop.The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes.Our in silico analysis of HNF1A,HNF4A.and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrich-ment of these transcription factors specifically in the liver.Our previous studies identified HNF1A as a master regulator of fucosylation,glycan branching,and galactosylation of plasma glycoproteins.Here,we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype.We used the state-of-the-art clus-tered regularly interspaced short palindromic repeats/dead Cas9(CRISPR/dCas9)molecular tool for the downregulation of the HNF1A,HNF4A,and FOXA2 genes in HepG2 cells-a human liver cancer cell line.The results show that the downregulation of all three genes individually and in pairs affects the transcrip-tional activity of many glyco-genes,although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures.The effect is better seen as an overall change in the total HepG2 N-glycome,primarily due to the extension of biantennary glycans.We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure.We also propose a model showing feedback loops with the mutual activation of HNF1A-FOXA2 and HNF4A-FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.

CA19-9、CA125和CP2在卵巢黏液性肿瘤诊断和监测中的价值

目的探讨血清肿瘤标志物CA19-9、CA125及CP2在卵巢黏液性肿瘤诊断和监测中的价值。方法对北京大学人民医院1999年1月至2007年6月间收治的273例卵巢肿瘤患者的临床资料进行回顾性分析，探讨血清肿瘤标志物CA19-9、CA125及CP2在50例卵巢黏液性肿瘤诊断和监测中的价值，并与223例卵巢非黏液性肿瘤进行比较。结果（1）卵巢黏液性肿瘤中，CA19-9的曲线下面积最大（为0．95），其次是CA125（为0．90）；而卵巢非黏液肿瘤中，CA125和CP2的曲线下面积最大（均为0．90）。（2）卵巢黏液性肿瘤患者联合检测CA19-9和CA125时，其敏感度（93．8％）较单项检测（CA19-9和CA125分别为75．0％和66．7％）明显提高（P〈0．05），而特异度（分别为86．1％、86．6％和90．2％）无明显变化（P〉0．05）。卵巢非黏液性肿瘤患者联合检测CA125和CP2时的敏感度（85．0％），较CP2（70．6％）单项检测明显提高，差异有统计学意义（P〈0．05）；较CA125（80．7％）单项检测虽有提高，但差异无统计学意义（P〉0．05）；3者的特异度（分别为90．2％、88．5％和93．9％）比较，差异无统计学意义（P〉0．05）。（3）82例卵巢恶性肿瘤术前血清肿瘤标志物阳性患者中。可行满意的肿瘤细胞减灭术患者[70％（57／82）]的血清肿瘤标志物于术后2个月内降为正常的百分率高于未能行满意肿瘤细胞减灭术者（分别为75％和28％），差异有统计学意义（P〈0．05）；且其术后血清肿瘤标志物再次上升的平均时间延长（分别为18．2和16．4个月），但差异无统计学意义（P〉0．05）；复发率（分别为35％和56％）及死亡率（分别为14％和32％）降低，差异有统计学意义（P〈0．05）。20例术前血清肿瘤标志物阴性患者均可行满意的肿瘤细胞减灭术，其中复发患者仅2例（10％）。（4）卵巢黏液性肿瘤患者术后复发时多为血清CA19-9水平上升，而卵巢非黏液性肿瘤术后复发时主要为血清CA125水平上升，部分患者血清CP2水平也上升。（5）术前血清肿瘤标志物阳性患者较阴性患者生存率明显下降，其中CA125（＋）与CA125（-）、CP2（＋）与CP2（-）患者间生存率比较，差异有统计学意义（P〈0．05）；而CA19-9（＋）与CA19-9（-）患者间生存率比较，差异则无统计学意义（P〉0．05）。结论CA19-9是诊断卵巢黏液性肿瘤的敏感指标，与CA125联合检测可提高对卵巢黏液性肿瘤诊断的敏感度，并对术后监测有重要临床意义。CA125和CP2联合检测则对诊断卵巢非黏液肿瘤更敏感。

艾灸促进胃黏膜细胞HSP70表达上调对细胞凋亡线粒体信号转导途径的影响

目的:探讨艾灸预处理抑制细胞凋亡、保护胃黏膜损伤的机制.方法:32只健康Wister大鼠随机分为4组,即捆绑组(A组)、模型组(B组)、艾灸穴位组(C组)和艾灸非穴组(D组),每组8只.艾灸穴位组和艾灸非穴组大鼠艾灸预处理8d,捆绑组和模型组大鼠只捆绑,不艾灸.除捆绑组外,其余各组采用无水乙醇灌胃法制备大鼠急性胃黏膜损伤模型.采用Western blot法检测大鼠胃黏膜细胞色素C(Cyt-C)的表达,免疫组织化学方法检测大鼠胃黏膜HSP70、胃黏膜细胞凋亡、凋亡活化因子1(Apaf-1)、Caspase-9和Caspase-3的表达.结果:与A组相比,B组大鼠胃黏膜HSP70表达,胃黏膜细胞凋亡指数(AI),胞质Cyt-C、Apaf-1、Caspase-9和Caspase-3含量明显增加(2.93±0.29vs2.51±0.22,3.52±0.77vs2.25±0.53,1.63±0.36vs0.75±0.23,4.32±0.67vs3.44±0.86,4.05±1.01vs3.76±0.82,3.55±0.86vs2.35±0.71,P<0.01或0.05).与B组相比,C组大鼠胃黏膜HSP70表达(3.93±0.36)明显增加(P<0.01),而细胞凋亡指数(1.53±0.45),胞质Cyt-C(0.97±0.26)、Apaf-1(2.24±0.49)、Caspase-9(2.43±0.73)、Caspase-3(1.97±0.61)均显著降低(P<0.01).与C组相比,D组大鼠胃黏膜HSP70表达(3.35±0.34)显著降低(P<0.01),而凋亡指数(3.06±0.81),Cyt-C(1.45±0.29),Apaf-1(3.16±0.66),Caspase-9(3.33±0.76),Caspase-3(2.98±0.86),显著升高(P<0.01或0.05).结论:艾灸通过上调大鼠胃黏膜细胞HSP70表达,继而作用于凋亡线粒体信号转导途径相关靶点,由此抑制胃黏膜细胞凋亡,达到保护胃黏膜损伤的作用,且这种保护作用具有一定的穴位特异性.

双靶向溶瘤腺病毒MD55对肿瘤细胞与正常细胞的杀伤作用比较

应用分子克隆和同源重组技术构建双靶向溶瘤腺病毒MD55。分析MD55病毒在细胞中的复制能力,并用MTT和结晶紫方法检测MD55对肿瘤细胞和正常细胞生长的影响,West-ern印迹检测病毒感染后细胞中E1A蛋白和多聚ADP核糖聚合酶(PARP)片断的表达。结果显示,MD55能在MN/CA9阳性的肿瘤细胞SW620和OSRC-2中特异性表达E1A蛋白,并且在这些细胞中的复制能力和对这些细胞的杀伤性与对照病毒ZD55基本相同,而其在正常细胞中的复制能力很弱,对正常细胞的伤害很小;同时MD55可诱导肿瘤细胞SW620和OSRC-2的凋亡,而对正常细胞的凋亡无影响。实验结果表明双靶向溶瘤腺病毒MD55靶向性和安全性比单靶向病毒ZD55更高,有望成为一种很好的肿瘤靶向基因-病毒治疗载体。