OBJECTIVE:To evaluate the expression of thymidylate synthase (TS) in myoepithelial cells (MECs) of salivary adenoid tissues and explore its clinical significance.METHODS:Immunohistochemical staining EnVision method was used to detect the expression of TS, P63, Calponin, CK5/6 and S-100 in 32 salivary gland specimens, including 10 non-neoplastic and salivary inflammation specimens, 11 mixed tumor specimens, 5 basal cell carcinoma specimens and 6 adenoid cyst carcinoma specimens. The specificity and sensitivity of TS as a specific molecular marker of salivary muscle epithelial cells were evaluated in comparison with P63, Calponin, CK5/6 and S-100.RESULTS:The expression pattern of TS in all the salivary gland tissue specimens was identical with that of p63. TS and P63 both showed strong immunohistochemical expressions in MECs of salivary adenoid tissue specimens. Calponin, CK5/6, and S-100 showed cytoplasmic/membranous expressions in the MECs. In addition, TS exhibited weak or moderate cytoplasmic expression in a few salivary gland epithelial cells, cancer cells and scattered stromal cells, with negative expression in the cell nuclei. The expression of TS in the MECs of all the salivary adenoid specimens was highly consistent with those of P63, Calponin, CK5/6 and S-100 (P>0.05) Except for CK5/6 expression in Salivary inflammation and Salivary gland specimens. Kappa>0.75. The specificity and sensitivity of TS as a molecular marker of MECs were both 100%.CONCLUSIONS:TS is a new specific marker of MECs for differential diagnosis of salivary gland tumors.

目的 评估胸苷酸合成酶(thymidylate systhase,TS)作为前列腺基底细胞分子标志的敏感性和特异性及其在前列腺癌诊断中的临床价值. 方法应用免疫组化EnVision法检测TS、P63、CK-H(抗体克隆号:34βE12)、P504S在108例前列腺组织样本中的表达,其中正常和前列腺增生样本27例、前列腺非典型上皮内瘤变样本18例、前列腺腺癌样本63例,并比较TS与前列腺基底细胞特异性标志物P63、CK-H之间的一致性. 结果TS能特异性地高表达于前列腺基底细胞胞核内,且其表达模式和强度与P63的表达完全一致,CK-H在基底细胞胞质中呈强阳性表达.统计学分析结果显示,TS在前列腺基底细胞中的表达与P63和CK-H具有高度一致性(Kappa≥0.75),而且,TS作为前列腺基底细胞分子标志的特异性和敏感性均为100％.同时,TS在3例(16.7％)前列腺上皮内瘤变样本中弱阳性表达于腺泡细胞胞质内,TS在25例(39.7％)前列腺腺癌细胞胞质内呈不同强度阳性表达.P63和CK-H在前列腺腺泡细胞和腺癌细胞胞质中不表达.P504S在前列腺腺癌细胞质中呈强阳性表达. 结论胸苷酸合成酶能特异性表达于前列腺基底细胞核内,因而,可作为一种新的前列腺基底细胞的特异性分子标志而用于前列腺良恶性肿瘤的诊断与鉴别诊断.

利用湿壁塔法测量复合胺砜溶液吸收CO2的动力学特性,实验工况包括不同的反应温度(293.15～323.15 K)和不同的溶液浓度(0.5～6.6 mol/L).本文简化了气液传质模拟,计算了不同工况下的吸收速率常数、总反应速率常数、二级反应速率常数及增强因子等动力学参数,同时根据实验结果拟合得到了二级反应速率常数和增强因子的经验公式,并得到了复合胺砜溶液与CO2反应的表观反应活化能为为20.38 kJ/mol.

Objective To investigate the expression of p16INK4a protein in human cancers and analyze its clinical significance. Methods A total of 642 cases surgical specimens of primary human tumors from 2014 to 2015 were underwent operations in the study. Immunohistochemical staining was used to detect and analyze the expression of p16INK4 in tumor tissues. Results Of the 642 tumor samples, 120 were benign and 522 were malignant. The expression rates of p16INK4a negative(-), weak positive(+), moderately positive(++) and strong positive(+ + +) were 17.43％ (91/522), 11.11％ (58/522), 19.16％ (100/522) and 52.30％(273/522) in malignant tumor tissues, respectively; while in benign lesions, the expression rates were 34.17％ (41/120), 23.33％(28/120), 26.67％ (32/120) and 15.83％ (19/120), respectively. The difference was statistically significant (P<0.05). The positive expression rate of p16INK4a protein in different tissues derived tumors was not the same (P<0.05). Using p16INK4a protein as a biological molecules marker of malignant tumor, the sensitivity and specificity and positive predictive value of it were different as choosing different positive cell value. While using moderately positive as positive value, the sensitivity, specificity and positive predictive value were 71.46％, 57.50％ and 87.97％. While using strong positive as positive value, the sensitivity, specificity and positive predictive value were 52.30％, 84.17％ and 93.49％. Conclusion The compensatory high expression of p16INK4A is a tumor specific immune phenotype, which is related to the abnormal cell cycle patterns of tumor, and can be used as a molecular marker for the diagnosis, differential diagnosis and molecular typing of malignant tumors.

Objective To explore the changes of PD-L1 expression in the spleen, kidney and heart of septic mice. Methods Forty C57 mice were randomly divided into:sepsis model group and sham group( n=20 in each group) . Sepsis model was induced by cecal ligation and puncture. The mice were sacrificed at 24 h(n=10) and 48 h(n=10) in two groups, respectively. Spleen, kidney and heart at 24 h and 48 h were obtained to detect the PD-L1 expression by immunohistochemistry. Peripheral blood samples at 24 h and 48 h were taken to detect the PD-L1 expression in monocytes by flow cytometry. Ascites at 24 h and 48 h were collected to detect the en-dotoxin level by enzyme-linked immunosorbent assay. Results Compared with sham group, PD-L1 expression in the spleen and kid-ney was increased at 24 h and 48 h in sepsis model group(P<0. 05). PD-L1 expression was not observed in the heart in sepsis model group and sham group. Positive cells of PD-L1 expression were found in the red pulp, splenic corpuscle and renal tubules in sepsis model group. Compared with sham group, PD-L1 expression in the monocytes was increased in sepsis model group(P<0. 01), and the endotoxin level in ascites was also increased(P<0. 01). Conclusion PD-L1 expression is increased in spleen and kidney of sep-sis mice. Increased PD-L1 expression may be involved in the development of sepsis.

OBJECTIVE:To predict and identify B-cell linear epitopes of hepatitis B e antigen (HBeAg).METHODS:The B-cell linear epitopes of HBeAg were predicted using the software provided by NCBI Database and Immune Epitope Database (IEDB) and synthesized by a solid-phase method followed by conjugation with keyhole limpet hemocyanin (KLH). The KLH conjugates were used for immunization of New Zealand white rabbits, and the immune response of the rabbits was monitored by direct ELISA using a bovine serum albumin conjugate of the predicted epitopes. RESULTS Four new B-cell linear epitopes of HBeAg were identified, namely (1)MDIDPYKEFG(10), (37)LYREALESPEHCSP(50), (74)SNLEDPAS(81) and (127)RTPPAYRPPNAPIL(140). The rabbits immunized with the KLH conjugate showed an antibody titer over 1:512 000. The antisera of B-cell linear epitopes collected could specifically react with HBeAg as shown by ELISA.CONCLUSION:Four B-cell linear epitopes of HBeAg have been confirmed using bioinformatics methods, which provides new evidence for further functional studies of HBeAg in hepatitis B.

Objective:To explore the application value of liquid-based thin-layer cytological detection technology(LCT).Methods:The results of LCT from the hospital from July 2,2008 to January 16,2009 were analyzed,then the results were compared with those of routine cervical smears.Results:The positive rate of LCT(the positive cases including LSIL,HSIL,SCC,ACC,ASC,AGUS,AGC,and HPV infection) was 13.51%(104/770),44 positive cases underwent cervical biopsy,19 cases(48.18%) were found with positive results of cervical histological examination.The positive rate of routine cervical smears was 1.83%(31/1 692),22 positive cases underwent cervical biopsy,11 cases(50.00%) were found with positive results of cervical histological examination.The analysis on grouping of degrees of lesions of the positive cases showed that there was no significant difference in the total detection rate of cervical lesions of high grade(including HSIL,SCC,and ACC) between LCT(1.17%,9/770) and routine cervical smears(1.30%,22/1 692)(P0.05).The proportions of cases with cervical lesions under LSIL(including LSIL,ASC,AGUS,and HPV infection) accounted for 91.35%(95/104) in LCT positive group and 29.03 %(9/31) in routine cervical smears positive group,there was significant difference between the two groups(P0.05).The proportions of cases under forty years old accounted for 55.77% in LCT positive group and 25.81% in routine cervical smears positive group,there was significant difference between the two groups(P0.05).Conclusion:LCT technology can provide high-quality cytological smears,increase the positive rate of cervical cytological detection,especially for the young patients with cervical lesions under LSIL,LCT plays an important role in early detection and diagnosis of cervical cancer.

Objective To design and develop a reliable,ease-to-use and cheap tissue microarryer for making tissue microarrays,and discuss its characteristics.Methods According to the facture procedure and principle of tissue microarray construction,HT-1 tissue microarrayer was designed and developed.The tissue microarrayer consisted of a recipient paraffin block molding machine,a punch needle,a negative-pressure embedding instrument,and a special manipulator.Using HT-1 tissue microarrayer,the array holes in recipient paraffin block could be punched by single-shaping technique in one action in several seconds,while no chapping was guaranteed.During the TMAs paraffin block embedding process,the remnant air bubble between the tissue cylinders and array holes in recipient paraffin block could be exhausted rapidly and completely.Results Using HT-1 tissue microarrayer,an array holes recipient paraffin block(several to several hundreds holes)could be made in several seconds.Several TMAs blocks with 56 tissue cylinders(1.5 mm in diameter)were constructed easily and quickly within 20 min.Under HE and immunostaining procedures,the tissue cores were well aligned,and orientation was properly done.The tissue cores on the slide maintained intact histological structure.The tissue structure and background of the HE and immunostaining were clear.There was less sample loss(the loss rate was less than 1.0%±1.1%).Conclusion HT-1 tissue microarrayer is a simple,economical and high efficiency/cost(E/C)and easy-to-use device.

In order to explore the activity of a peptide containing rat sodium pump α2 subunit M1-M2 extramembrane fragment (RES2 derivative) in vitro, the peptide (Leu-Ala-Ala-Met-Glu-Asp-Glu-Pro-Ser-Asn-Asp-Asn-Gly-Gly-Gly-Ser) was synthesized by peptide synthesizer with Fmoc method and purified by high performance liquid chromatography (HPLC). Its binding activity was identified by radioligand-receptor binding assay (RRA) and its bioactivity was measured by erythrocyte (86)Rb uptake. The results of saturation binding experiment and competitive binding experiment showed that the synthesized RES2 derivative had the capability to bind to (3)H-ouabain. The dissociation constant (K(d)) was 38.46 nmol/L and IC(50) was 6.353 nmol/L. Erythrocyte (86)Rb uptake experiment showed that the RES2 derivative blocked the inhibitory effect of ouabain on the sodium pump on erythrocyte membrane in a dose-dependent manner. The results showed that the RES2 derivative is capable of binding to ouabain and improving the activity of sodium pump on erythrocyte membrane, suggesting that the RES2 derivative might become an effective antihypertensive drug in the future.

OBJECTIVE To prepare highly specific chicken egg yolk IgY antibody against human papillomavirus 16 type L1 main capsid protein (HPV16L1) for detection of HPV16L1. METHODS Purified HPV16L1 protein was used to immunize the hens, from which the eggs were collected since one week after the first immunization. The egg yolk was separated and the IgY antibody purified by PEG-6000 method. The bioactivity of the antibody was tested using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was performed to detect the HPV16L1 in the CHO cells transfected with the recombinant pcDNA-EGFP-HPV16L1 plasmid (containing EGFP-HPV16L1 fusion gene) for assessing the specific affinity of IgY to HPV16L1. RESULTS After 3 immunizations of the hens, the titer of the purified IgY antibody against HPV16L1 from the egg yolk reached 1:10240. The IgY bound specifically to the EGFP-HPV16L1 protein expressed in the transfected CHO cells. CONCLUSION High titer IgY can be prepared by immunization of the hens with HPV16L1 protein, and the prepared IgY can be used for HPV16L1 detection at the cellular level.

AIM:To improve specificity and accuracy of endogenous ouabain measurement assay.METHODS:Anti-ouabain polyclonal antibody egg yolk (IgY) and anti-ouabain rabbit antibody (IgG) were prepared respectively. In the presence of two kinds of antibody, then the specificity and accuracy of enzyme-linked immunosorbent assay (ELISA) were compared.RESULTS:The ELISA, in the presence of IgY, provided a sensitivity of the average intraassay coefficient of variation(CV) was 2.03%, and the inter-assay CV was 2.34% respectively. In contrast, IgG were 2.83% and 3.29%. No significant interferences were observed with hydrocortisone and dexamethasone. There was 3.45% vs. 5.95%, 3.20% vs. 5.20% of crossreaction with cedilanid and digoxin.CONCLUSION:The specificity and accuracy of ELISA, in which IgY was used, were more better than IgG.

OBJECTIVE:To prepare highly specific anti-ouabain polyclonal antibody for detecting endogenous ouabain in tissues.METHODS:Ouabain-BSA compound was used to immunize hens, and the eggs were collected one week after the first immunization. The IgY antibodies in the egg yolk were separated and purified by PEG-6000 Method, and analyzed by 12% SDS-PAGE and enzyme-linked immunosorbent assay (ELISA) for titration. The IgY antibodies obtained were applied subsequently in ELISA and immunohistochemistry.RESULTS:The IgY titer increased rapidly after the second immunization, with the highest titer of 1:10240 that lasted for at least 4 weeks. Competitive ELISA for IgY detection showed an average intraassay coefficient of variation (CV) of 2.03% and an inter-assay CV of 2.34%. Immunohistochemistry visualized the location of the endogenous ouabain mainly in the cytoplasm of the zona reticularis of rat adrenal cortex.CONCLUSION:Immunization of hens allows efficient preparation of IgY antibody which can be used in routine immunoassays.

BACKGROUND & OBJECTIVE:Intestinal metaplasia (IM) is thought as the precancerous lesion of gastric carcinoma, and CDX2 gene plays important roles in development and differentiation of intestinal epithelium, and maintenance of intestinal phenotype. Recent studies found that CDX2 were expressed aberrantly in IM of chronic atrophic gastritis (CAG) and some gastric carcinomas, which implied that CDX2 may play an important role in IM formation and gastric carcinogenesis. This study was to investigate the roles of CDX2 in the development and progression of IM and gastric carcinogenesis, and determine the correlation of IM to gastric carcinogenesis. METHODS:A tissue microarray containing 46 cases of CAG with IM, 40 cases of gastric carcinoma, and 32 cases of IM foci in paracancerous tissues was constructed. High iron diamine/alcian blue (HID/AB) and HE staining were used to classify IM and gastric carcinoma, and the expression of CDX2 protein and mRNA in different gastric lesions was assessed with immunohistochemistry and in situ hybridization, respectively. RESULTS:The proportion of type III IM was significantly higher in IM foci in paracancerous tissues than in CAG with IM (56.25% vs. 21.74%, P<0.01). The positive rates of CDX2 protein were 69.56% in IM foci in CAG, 53.13% in IM foci in paracancerous tissues, and 42.50% in gastric carcinomas, and the positive rates of CDX2 mRNA were 63.04%, 46.87%, and 35.00%, respectively. The positive rates were significantly lower in gastric cancer than in IM in CAG (P<0.01), but there was no significant difference between gastric cancer and IM foci in paracancerous tissues (P>0.05). The expression of CDX2 protein and mRNA was significantly higher in intestinal-type gastric cancer than in diffuse-type gastric cancer (54.55% vs. 27.78%, 45.45% vs. 22.22%, P<0.05). The expression of CDX2 protein was significantly lower in type III IM than in type I IM (46.42% vs. 79.31%, P<0.05). CONCLUSIONS:CDX2 may play important roles in the development and progression of IM and gastric carcinogenesis.

Objective: To prepare highly selective anti-ouabain polyclonal antibody to further improve the sensitivity and specificity level of the detection of endogenous ouabain. Methods: Different doses of ouabain-BSA conjugate (0.5 mg/kg; 1.0 mg/kg) was used to immunize hens. Eggs were collected one week after the first immunization. The egg yolks were separated and purified by PEG-6 000 method. The purity was analyzed by 12% SDS-PAGE, and the bioactivity was tested by ELISA. Results: IgY could be tested one week after first immunization, but the titer was very low. The titer was increased fast after the second immunization. The highest titer was 1:10 240. And this level lasted for at least 4 weeks. At the same time, the results showed that the more antigen was used, the longer time the high titer IgY lasted. Conclusion: High titer IgY can be prepared by multiple immunizations of hens. The method is simple and effective, and the highest titer IgY lasts longer. It is probably an effective way to prepare anti-ouabain IgY.

目的探讨组织芯片技术在免疫组化试验中的可靠性和提高组织芯片可信性的方法.方法选择82例人乳腺癌标本石蜡蜡块,利用传统制片技术和自行研制的专用器具分别制作常规石蜡切片和组织芯片,采用免疫组化技术检测雌激素受体(ER)、孕激素受体(PR)的表达.结果采用自行研制的专用器具制备组织芯片,所得样本的可分析率均在92.9%以上,且随着对同一标本取材数量的增加,其可分析标本率也明显上升.采用传统方法检测的82例乳腺癌标本中ER和PR的阳性率分别为61%和58.5%;采用组织芯片技术,一式一份取材,其阳性率分别为51.9%和50.0%,一式两份方式取材,其阳性率分别为53.8%和53.8%,采用一式三份方式取材,其阳性率分别为57.1%和60.7%,且三种不同取材方式的检测结果与传统制片法之间并无统计学差异(P>0.75).结论在采用统一制备标准并保证组织芯片质量的前提下,挖取直径1.5mm的组织样本制备组织芯片,尽管组织芯片技术与传统方法检测的结果的一致率仍随着对同一标本取材次数的增加而提高,但是三种不同取材方式的检测结果之间并无统计学差异.因此,一式一份取材制备组织芯片完全可替代传统制片技术用于免疫组织化学乃至原位杂交、荧光原位杂交技术的研究,且可保证组织芯片的可信性和高通量特征.

AIM:To investigate the roles of mucin histochemistry, cytokeratin 7/20 (CK7/20) immunoreactivity, clinical characteristics and endoscopy to distinguish short-segment Barrett's esophageal (SSBE) from cardiac intestinal metaplasia (CIM).METHODS:High iron diamine/Alcian blue (HID/AB) mucin-histochemical staining and immunohistochemical staining were used to classify intestinal metaplasia (IM) and to determine CK7/20 immunoreactivity pattern in SSBE and CIM, respectively, and these results were compared with endoscopical diagnosis and the positive rate of gastroesophageal reflux disease (GERD) symptoms and H pylori infection. Long-segment Barrett's esophageal and IM of gastric antrum were designed as control.RESULTS:The prevalence of type III IM was significantly higher in SSBE than in CIM (63.33% vs 23.08%, P< 0.005). The CK7/20 immunoreactivity in SSBE showed mainly Barrett's pattern (76.66%), and the GERD symptoms in most cases which showed Barrett's pattern were positive, whereas H pylori infection was negative. However, the CK7/20 immunoreactivity in CIM was gastric pattern preponderantly (61.54%), but there were 23.08% cases that showed Barrett's pattern. H pylori infection in all cases which showed gastric pattern was significantly higher than those which showed Barrett's pattern (63.83% vs 19.30%, P< 0.005), whereas the GERD symptoms in gastric pattern were significantly lower than that in Barrett's pattern (21.28% vs 85.96%, P< 0.005).CONCLUSION:Distinction of SSBE from CIM should not be based on a single method; however, the combination of clinical characteristics, histology, mucin histochemistry, CK7/20 immunoreactivity, and endoscopic biopsy should be applied. Type III IM, presence of GERD symptoms, and Barrett's CK7/20 immunoreactivity pattern may support the diagnosis of SSBE, whereas non-type III IM, positive H pylori infection, and gastric CK7/20 immunoreactivity pattern may imply CIM.

Objective To e xpl ore t he cli nical , p at hol ogic a nd i mmunohist oc he mical c ha r act e ristics of gast r oi nt esti nal st r omal t umor ( GIS T) a nd st r omal t umor beyond t he gast r oi nt esti nal t r act . Metho ds Exp ressi on of CD117 , CD34 , SMA , Des mi n a nd S2100 p r ot ei n i n 53 GIS T we re det ect e d usi ng i mmunohist oc he mical S2P met hod , a nd t he n a nalyze d. Re sult s Of 53 cases , 38 cases we re det ect e d as spi ndle t yp e , 6 as epit heli oi d t yp e a nd 9 as mi xt ure t yp e ; 9 cases we re be nign , 20 we re bor de rli ne a nd 24 we re maligna nt . Of t he 9 be nign cases , 8 origi nat e d i n st omac h , w hile only one origi nat e d i n i nt esti ne a nd beyond t he gast r oi nt esti nal t r act . The r at e of i mmunohist oc he mical e xp ressi on was s howe d as f oll ows : CD117 92. 45 % a nd CD34 77. 36 %. Conclusion GIS T has its uni que morp h ol ogical c ha r act e ristics . To det ect CD117 a nd CD34 si mult a ne ously ma y be helpf ul t o t he diagnosis . The maligna nt p ot e ntial of st r omal t umor i n i nt esti ne a nd beyond t he gast r oi nt esti nal t r act is highe r t ha n t hat i n st omac h . The esti mati on of be nigna ncy a nd maligna ncy dep e nds on t he sit e , t he size a nd nuclei mit oses of t he

目的探讨胃肠道及胃肠外间质瘤(GIST)的临床、病理和免疫组化特征.方法用免疫组化S-P法观察了53例GIST的CD117、CD34、SMA、Desmin和S-100蛋白表达并分析其与临床病理的关系.结果 53例间质瘤中梭形细胞型38例,上皮样细胞型6例,混合型9例;良性9例,交界性20例,恶性24例;9例良性间质瘤,8例发生在胃,而肠和胃肠外间质瘤仅1例良性.CD117、CD34免疫组化阳性表达率分别为92.45%及77.36%.结论 GIST有其独特的形态特征;CD117和CD34同时检测有助于GIST的诊断;肠和胃肠外GIST的恶性潜力较胃GIST高;GIST良恶性的评估取决于肿瘤的部位、大小和核分裂的多少.

免疫组化及原位杂交技术广泛应用医学科学实验之中,在免疫组化染色中多数是经过DAB显色后用苏木素复染,阳性表达呈棕黄色颗粒状,细胞核呈蓝色.但在皮肤组织及富有黑色素细胞的病变中,其阳性表达的棕黄色颗粒与黑色素颗粒及某些棕褐色或棕黄色的外源性和内源性色素,在光镜下均可呈棕黄色颗粒与免疫组化的阳性颗粒色泽相似，难于区别,往往给实验结果的判断造成较大困难.因此,我们在免疫组化过程中采用蓝色DAB显色剂显色,阳性表达呈紫蓝色与色素棕黄色颗粒区别对比明显,效果满意,可广泛应用于皮肤免疫组化及原位杂交检测之中.

目的探讨尿液中碱性成纤维细胞生长因子(bFGF)浓度,以鉴别婴幼儿血管瘤和血管畸形并对血管瘤进行分期.方法采用ELISA法检测29例增生期血管瘤、24例消退期血管瘤、12例血管畸形患儿和10例对照婴幼儿尿液中bFGF浓度.结果增生期血管瘤患儿尿液中bFGF浓度高于消退期血管瘤、血管畸形和对照组,差别有显著性意义(P<0.05),消退期血管瘤、血管畸形与对照组之间尿液中bFGF浓度的差别亦有显著性意义(P<0.05).结论用ELISA法测定尿液中bFGF简便、易行、可靠,可为临床鉴别血管瘤和血管畸形,并为划分血管瘤分期及监测治疗效果提供客观的依据.