云南省德宏和昆明地区高效抗逆转录病毒治疗后HIV-1流行毒株pol区基因变异分析

Objective To investigate the variations in the pol region of HIV-1 strain in treatment failed patients in Yunnan province's Dehong prefecture and Kunming. Methods Blood samples were collected from 139 patients who experienced treatment failure ( HAART treatment ＞ 1 years and HIV-1 RNA Viral load ＞ 1 000 copies/ml). HIV-1 RNA was extracted from plasma, and nested-PCR was performed for amplification of PR and RT genes on the HIV-1 pol region. The PCR products were then sequenced and submitted to Stanford HIV Drug Resistance Database for comparison. The evolution tree was built up with MEGA 4. 1 system, combined with patients' demographics. Results The most prevalent mutation in Kunming patients were T215F/N/Y/I, M41L/M, and T69G/N/I/S/A/D, the mutation rates were 39%(24/62), 27% (17/62) and 27% (17/62) , respectively, which were higher than the corresponding mutations in the Dehong prefecture [16% ( 11/69), 13% (9/69) and 9% (6/69)]. The rate differences were statistically significant ( x2 = 8.646, 4.242 and 7. 909, all P ＜ 0.05 ). The most common HIV-1 pol region subtype in the Dehong patients were CRF01_AE subtype (32%, 22/69), followed by C subtype (25% ,17/69), and B subtype ( 19%, 13/69). Major subtypes in Kunming patients were 08_BC (60%,37/62 ), CRF01_AE subtype(21% , 13/62 ) and 07_BC ( 15% ,9/62). Conclusions Partial differences of the point mutations of the HIV-1 strain pol region and frequency of their occurrences exist among Dehong and Kunming patients, HIV-1 strains in Dehong prefecture for the NNRTIs mutations at the T215 Y/N/T, M41L and T69G/N/I/S/A/D are significantly higher than those in Kunming. Six isoforms are found respectively:CRF01_AE, B, C, BC, 08_BC and 07_BC from the epidemic strains of HIV-1 pol region subtype in Dehong and Kunming areas.     

云南省德宏和昆明地区高效抗逆转录病毒治疗后HIV-1流行毒株pol区基因变异分析

目的 调查云南省德宏和昆明两个地区HIV-1流行毒株pol区基因在接受HAART后的变异情况.方法 选择接受HAART 1年以上血浆HIV-1病毒载量＞1 000拷贝/ml的139例HIV/AIDS患者,提取血浆中HIV-1 RNA,采用RT-nested-PCR扩增HIV-1的pol区基因蛋白酶(PR)和逆转录酶(RT)基因片段,测定DNA序列.经拼接整理后与美国斯坦福大学网站提供的HIV耐药数据库(Stanford HIV Drug Resistance Database)进行比对,分析耐药突变位点,采用MEGA 4.1构建系统进化树以确定HIV的亚型,结合HIV/AIDS患者流行病学资料进行综合分析.结果 昆明地区患者HIV以T215F/NY/I、M41L/M和T69G/N/I/S/A/D突变较多,突变率分别为39%(24/62)、27%(17/62)和27%(17/62),高于德宏地区患者HIV的相应突变率[分别为16%(11/69)、13%(9/69)和9%(6/69)],差异有统计学意义(x2值分别为8.646、4.242和7.909,P均＜0.05).德宏地区HIV-1流行毒株pol区基因亚型主要为CRF01_AE亚型为32%(22/69)、C亚型为25%(17/69)、B亚型19%(13/69).而昆明地区主要是08_BC亚型60%(37/62)、CRF01_AE亚型21%(13/62)和07_BC亚型15%(9/62).结论 云南省德宏、昆明两地区HIV-1流行毒株pol区基因PR、RT基因突变频率存在部分差异,昆明地区T215F/N/Y/I、M41L/M和T69G/N/I/S/A/D突变率高于德宏地区.两地共检出HIV-1流行毒株pol区基因CRF01_AE、B、C、BC、08_BC和07_BC共6种基因亚型.

Abstract：

Objective To investigate the variations in the pol region of HIV-1 strain in treatment failed patients in Yunnan province's Dehong prefecture and Kunming. Methods Blood samples were collected from 139 patients who experienced treatment failure ( HAART treatment ＞ 1 years and HIV-1 RNA Viral load ＞ 1 000 copies/ml). HIV-1 RNA was extracted from plasma, and nested-PCR was performed for amplification of PR and RT genes on the HIV-1 pol region. The PCR products were then sequenced and submitted to Stanford HIV Drug Resistance Database for comparison. The evolution tree was built up with MEGA 4. 1 system, combined with patients' demographics. Results The most prevalent mutation in Kunming patients were T215F/N/Y/I, M41L/M, and T69G/N/I/S/A/D, the mutation rates were 39%(24/62), 27% (17/62) and 27% (17/62) , respectively, which were higher than the corresponding mutations in the Dehong prefecture [16% ( 11/69), 13% (9/69) and 9% (6/69)]. The rate differences were statistically significant ( x2 = 8.646, 4.242 and 7. 909, all P ＜ 0.05 ). The most common HIV-1 pol region subtype in the Dehong patients were CRF01_AE subtype (32%, 22/69), followed by C subtype (25% ,17/69), and B subtype ( 19%, 13/69). Major subtypes in Kunming patients were 08_BC (60%,37/62 ), CRF01_AE subtype(21% , 13/62 ) and 07_BC ( 15% ,9/62). Conclusions Partial differences of the point mutations of the HIV-1 strain pol region and frequency of their occurrences exist among Dehong and Kunming patients, HIV-1 strains in Dehong prefecture for the NNRTIs mutations at the T215 Y/N/T, M41L and T69G/N/I/S/A/D are significantly higher than those in Kunming. Six isoforms are found respectively:CRF01_AE, B, C, BC, 08_BC and 07_BC from the epidemic strains of HIV-1 pol region subtype in Dehong and Kunming areas.     

A Study on the D-loop Region of Mitochondrial DNA (mtDNA) Mutation in Cervical Carcinomas

Objective Background-study on genesis and development of tumor is mainly concentrated on gene mutation in nucleus.In recent years,however,the role of mitochondrial DNA(mtDNA) mutation in tumor genesis has been given more and more attention,which is the only extra-nucleus DNA in cells of higher animals.Carcinoma of the uterine cervix is a common tumor in gynecology,but there are few reports of mtDNA mutation in this area.The focus of this study was to investigate the mtDNA mutation in tumor tissues of cervical carcinomas and their relationship to tumorigenesis and tumor development.Methods The D-loop region of 24 cervical carcinomas together with the adjacent normal tissues were amplified by PCR and sequenced.Results Among the 24 cervical carcinomas,30 mutations in 9 patients′ specimen were identified with the mutations rate of 37.5%(9/24).There were 8 microsatellite instabilities among the mutations and 13 new polymorphisms which were not reported previously in the Genbank.Conclusions The D-loop region of mitochondrial DNA is a highly polymorphoric and mutable region and the mutation rate is relatively high in patients with cervical carcinomas.     

酶切富集联合DHPLC检测肿瘤患者外周血EGFR和KRAS基因突变及其临床应用

Objective To establish a REDE-DHPLC method for detecting the EGFR and KRAS mutations in plasma DNA from tumor patients, and investigate its clinical significance. Methods Restriction endonucleases Mse Ⅰ , Msc Ⅰ , BstN Ⅰ and Bgl Ⅰ were used to digest the wild type fragments of exon 19,exon 21 of EGFR gene and coden 12, 13 of KRAS gene for enriching the mutation fragments, and REDE-DHPLC method was established to detect EGFR and KRAS mutations. The sensitivities of REDE-DHPLC and conventional DHPLC were analyzed by using a series of plasmids containing 50%, 10%, 5%, 1% and 0. 1% mutation genes. Then, Plasma samples and paraffin-embedded tissue samples of 120 NSCLC patients and 120 colorectal cancer patients were detected by REDE-DHPLC. Compared with conventional DHPLC and sequencing, the diagnostic efficiency of REDE-DHPLC method was evaluated by detecting the mutation status of 2 genes in plasma of NSCLC and colorectal cancer patients. Results The sensitivity values of REDE-DHPLC and conventional DHPLC for detecting mutations in 4 loci were 0. 1% and 1%respectively. Plasmid DNA containing 0.1% mutation gene was detected to be positive continually for 2 to 3 times by REDE-DHPLC. EGFR mutation rates of 120 plasma from NSCLC patients detected by REDE-DHPLC, conventional DHPLC and sequencing methods were 27. 5%, 16. 7% and 12.5% respectively, and KRAS mutation rates of 120 plasma from colorectal cancer patients were 38. 3%, 25. 8% and 16. 7%,respectively. The positive rates of EGFR and KRAS mutation detected by REDE-DHPLC were significantly higher than conventional DHPLC(x2 = 4. 092, 4. 301, all P ＜ 0. 05 ) and sequencing method (x2= 8. 438,14. 127,all P ＜ 0. 05 ). In comparison with conventional DHPLC, the sensitivities of REDE-DHPLC for detecting EGFR and KRAS mutation were 100% (20/20,31/31), the specificities were 87. 0% (87/100)and 83. 2% (74/89). In comparison with sequencing method, the sensitivities of REDE-DHPLC were 100%( 15/15,20/20), the specificities were 82.9% (87/105)and 74. 0% (74/100). The coincidence rate of the two methods for detecting EGFR and KRAS mutation were 89. 2% ( 107/120, Kappa = 0. 690, P ＜ 0. 05 ) and 87.5% ( 105/120, Kappa= 0. 718, P ＜ 0. 05 ). The Consistency of EGFR and KRAS mutation status in plasma and tissues detected by REDE-DHPLC were 91.7% (33/36, Kappa =0. 939,P ＜0. 05)and 90. 2 %(46/51, Kappa = 0. 914, P ＜ 0. 05 ), respectively. Conclusions The REDE-DHPLC method is highly sensitive and specific for detecting EGFR and KRAS mutations in plasma DNA from tumor patients. The results are easy to be interpreted without missing homozygous point mutation, which indicate that the detection of EGFR and KRAS mutations in plasma DNA by REDE-DHPLC could therefore extend to be usedin clinical laboratory.     

酶切富集联合DHPLC检测肿瘤患者外周血EGFR和KRAS基因突变及其临床应用

Objective To establish a REDE-DHPLC method for detecting the EGFR and KRAS mutations in plasma DNA from tumor patients, and investigate its clinical significance. Methods Restriction endonucleases Mse Ⅰ , Msc Ⅰ , BstN Ⅰ and Bgl Ⅰ were used to digest the wild type fragments of exon 19,exon 21 of EGFR gene and coden 12, 13 of KRAS gene for enriching the mutation fragments, and REDE-DHPLC method was established to detect EGFR and KRAS mutations. The sensitivities of REDE-DHPLC and conventional DHPLC were analyzed by using a series of plasmids containing 50%, 10%, 5%, 1% and 0. 1% mutation genes. Then, Plasma samples and paraffin-embedded tissue samples of 120 NSCLC patients and 120 colorectal cancer patients were detected by REDE-DHPLC. Compared with conventional DHPLC and sequencing, the diagnostic efficiency of REDE-DHPLC method was evaluated by detecting the mutation status of 2 genes in plasma of NSCLC and colorectal cancer patients. Results The sensitivity values of REDE-DHPLC and conventional DHPLC for detecting mutations in 4 loci were 0. 1% and 1%respectively. Plasmid DNA containing 0.1% mutation gene was detected to be positive continually for 2 to 3 times by REDE-DHPLC. EGFR mutation rates of 120 plasma from NSCLC patients detected by REDE-DHPLC, conventional DHPLC and sequencing methods were 27. 5%, 16. 7% and 12.5% respectively, and KRAS mutation rates of 120 plasma from colorectal cancer patients were 38. 3%, 25. 8% and 16. 7%,respectively. The positive rates of EGFR and KRAS mutation detected by REDE-DHPLC were significantly higher than conventional DHPLC(x2 = 4. 092, 4. 301, all P ＜ 0. 05 ) and sequencing method (x2= 8. 438,14. 127,all P ＜ 0. 05 ). In comparison with conventional DHPLC, the sensitivities of REDE-DHPLC for detecting EGFR and KRAS mutation were 100% (20/20,31/31), the specificities were 87. 0% (87/100)and 83. 2% (74/89). In comparison with sequencing method, the sensitivities of REDE-DHPLC were 100%( 15/15,20/20), the specificities were 82.9% (87/105)and 74. 0% (74/100). The coincidence rate of the two methods for detecting EGFR and KRAS mutation were 89. 2% ( 107/120, Kappa = 0. 690, P ＜ 0. 05 ) and 87.5% ( 105/120, Kappa= 0. 718, P ＜ 0. 05 ). The Consistency of EGFR and KRAS mutation status in plasma and tissues detected by REDE-DHPLC were 91.7% (33/36, Kappa =0. 939,P ＜0. 05)and 90. 2 %(46/51, Kappa = 0. 914, P ＜ 0. 05 ), respectively. Conclusions The REDE-DHPLC method is highly sensitive and specific for detecting EGFR and KRAS mutations in plasma DNA from tumor patients. The results are easy to be interpreted without missing homozygous point mutation, which indicate that the detection of EGFR and KRAS mutations in plasma DNA by REDE-DHPLC could therefore extend to be usedin clinical laboratory.     

A Study on the D-loop Region of Mitochondrial DNA （mtDNA） Mutation in Cervical Carcinomas

Objective Background-study on genesis and development of tumor is mainly concentrated on gene mutation in nucleus. In recent years, however, the role of mitochondrial DNA （mtDNA） mutation in tumor genesis has been given more and more attention, which is the only extra-nucleus DNA in cells of higher animals. Carcinoma of the uterine cervix is a common tumor in gynecology, but there are few reports of mtDNA mutation in this area. The focus of this study was to investigate the mtDNA mutation in tumor tissues of cervical carcinomas and their relationship to tumorigenesis and tumor development. Methods The D-loop region of 24 cervical carcinomas together with the adjacent normal tissues were amplified by PCR and sequenced. Results Among the 24 cervical carcinomas, 30 mutations in 9 patients＇ specimen were identified with the mutations rate of 37.5 96 （9/24）. There were 8 microsatellite instabilities among the mutations and 13 new polymorphisms which were not reported previously in the Genbank. Conclusions The D-loop region of mitochondrial DNA is a highly polymorphoric and mutable region and the mutation rate is relatively high in patients with cervical carcinomas.     

泉州市流感疫情实验室检测及H3N2亚型基因特性分析

目的 了解2009年泉州地区流感流行及病毒株的变异情况,探讨流感病毒基因的变异与流感流行的关系.方法 对泉州市198例流感患者的咽拭子采用MDCK细胞培养进行病毒分离,经血清学试验鉴定分型和实时荧光RT-PCR方法检测病毒核酸.对其中4株毒株提取病毒RNA,采用RT-PCR扩增病毒HA1基因,纯化产物进行核苷酸序列测定,用DNAstar megalign软件分析基因.结果 198份咽拭子中有98份为H3N2亚型流感病毒核酸阳性,分离到62株H3N2亚型流感病毒,HA1基因经核苷酸序列测定显示,其基因特性更接近于A/Ningbo/333/2008,核苷酸同源性为98.7%,与A/Xiamen/70/2004的同源性为96.8%,由HA1基因核苷酸序列推导的氨基酸系列与疫苗株A/Brisbane/10/2007相比,有7个氨基酸位点发生变异,其中有1个位点位于抗原决定簇A区(144),有2个位点位于抗原决定簇B区(158、189),种系发生树分析也证实HA1基因存在一定差异.结论引起2009年泉州市流感在部分集体单位流行的病毒为H3N2亚型,其基因特性和抗原性与疫苗株相比均发生了一定变异.

Abstract：

Objective To obtain the information of the 2009 influenza outbreak and the variations of influenza virus strains in quanzhou, and explore the relationship between the genetic variation of influenza virus and influenza epidemic. Methods During the influenza outbreak in quanzhou,one hundred and ninetyeight throat swabs specimens from the patients with influenza were collected. Viruses were isolated with MDCK cells and identified with serological test, followed by real-time RT-PCR. RNA of four influenza virus strains were extracted, then HA1 gene was amplified by RT-PCR. The purified PCR products were sequenced. The data were analyzed with the software DNAstar megalign. Results Total 98 pieces of H3N2 subtype influenza virus nucleic acid were detected in 198 throat swabs specimens,among which 62 influenza virus strains were identified as subtype influenza A( H3N2 ). The sequencing results of HA1 gene in these positive strains showed that their genetic characterization were more closed to strains A/Ningbo/333/2008 with a nucleotide homology of 98.7%, which was 96.8% as compared with A/Xiamen/70/2004. The amino acids sequences deduced from the nucleotide sequences in HA1 region of the isolated strain had 7 mutant sites compared with A/Brisbane/10/2007 vaccine strain. One variant amino acids were found located in the antigenic determinant sites A( 144 ), two were in the sites B( 158,189 ). Phylogenetic analysis also confirmed the difference in HAl domain. Conclusion The influenza virus strains causing the flu outbreak among some communities of quanzhou in 2009 are subtype influenza A ( H3N2 ), whose genetic characterization and antigenicity were different from the vaccine strain.     

katG基因二个不同区域基因变异与结核分枝杆菌异烟肼耐药的相关性研究

目的 分析结核分枝杆菌katG基因2个不同区域的基因变异,并确定与INH耐药的相关性.方法 从痰液分离并鉴定结核分枝杆菌耐INH菌株53株,用PCR扩增katG基因的2个区域:区域1为第1位密码子至150位密码子,区域2为第227位密码子至470位密码子,并分别测序.结果 3株对INH耐药但2个区域都不发生突变.14株区域1存在突变,其中5株只在区域1存在突变,5株在区域1出现缺失突变,并呈现高度耐药.点突变是区域2的主要特点,特别是S315位密码子,60.4%(32/53)S315发生突变,最常见的是S315N(AGC→AAC)(18株);katG S315在高度INH耐药和低度INH耐药的结核分枝杆菌中突变率分别是84.4%(27/32)、15.6%(5/32),两组间差异有统计学意义(x2=30.25,P＜0.01).27株S315突变呈高度耐药,占S315突变菌株总数的84.4%,其余18株至少有一个非S315点突变的耐药株中高度耐药只有5株,占27.7%,两组间差异有统计学意义(x2=16.02,P＜0.01).对INH耐药的结核分枝杆菌区域2的突变发生率为84.9%.5株只在区域1存在突变,通过检测基因突变诊断INH耐药的检出率上升至94.3%.结论 S315突变发生率最高,突变类型和位置与耐药程度密切相关,分析区域1能使检出率提高9.4%.

Abstract：

Objective To analyze and compare the mutations in two different regions of the katG gene and study the relevance of Mycobacterium tuberculosis isoniazid-resistance and mutations in two different regions of the katG gene. Methods Fifty-three INH-resistant Mycobacterium tuberculosis strains isolated in cultures of sputum samples obtained from Zhejiang province were analyzed. PCR was used to amplify two regions of the katG gene (GenBank accession no. U06258) region 1 (from codon 1 to codon 150) and region 2 ( from codon 227 to codon 470) which were then sequenced in order to identify mutations. Results Three strains resistant to INH did not contain mutations in either region. Fourteen strains carried mutations in region 1. Among them 5 strains barbered deletions, and showed high-level resistance to isoniazid. Five strains had mutations only in region 1. Region 2 carried multiple point mutations, especially at codon 315, and there were S315 N ( AGC→AAC ) substitution in 18 of those cases. The frequency of mutations in the katG S315 of high-level INH-resistance isolates ( 84. 4%, 27/32) was significantly higher than those of low-level INH-resistance isolates( 15.6%, 5/32 ), there was statistically significant difference (x2 = 30. 25, P ＜ 0. 01 ).katG S315 mutations in high-level INH-resistance frequency (84. 4%, 27/32) was significantly higher than the other mutations of katG gene of high-level INH-resistance frequency (27. 7%, 5/18 ), there was significant difference (x2 = 16.02, P ＜ 0. 01 ). The analysis of region 2 allowed INH resistance to be diagnosed in 84. 9% of the strains. Five strains had mutations only in region 1 ,which allowed the proportion of INH-resistant strains identified to be increased to 94. 3%. Conclusions The number of mutations at codon 315 was high. Mutation type and location closely related with drug resistance and the analysis of region 1 resulted in a 9. 4% increase in the rate at which mutations were identified.     

2011年浙江省台州地区新发现HIV-1感染者分子流行病学调查

目的 了解2011年浙江省台州地区新发现艾滋病病毒（HIV）感染者的基本情况以及不同HIV-1基因亚型的分布情况。方法 选取台州市2011年新发现未经抗病毒治疗的HIV感染者抗凝全血标本140份,提取血浆中的病毒RNA,通过RT-PCR扩增HIV-1 pol 区的蛋白酶基因全序列与反转录酶基因部分序列,使用HIV-Blast软件判定亚型。结果 2011年台州地区新发现的未经抗病毒治疗的HIV感染者男女性别比为3.24:1,感染者中92.86%为性传播感染,3.57%为注射毒品感染;共检测出5种HIV-1基因亚型,分别为CRF01_AE、CRF07_BC、CRF08_BC、B和C亚型。性传播感染人群中57.14%为CRF01_AE亚型,注射毒品人群中以CRF07_BC亚型为主。结论 台州地区艾滋病患者以性传播途径感染为主,HIV-1基因亚型呈多样性特点,CRF01_AE亚型流行于性传播感染人群,而CRF07_BC亚型流行于毒品注射感染人群。     

hLRH-1不同变异体在人胚胎组织的表达谱

目的分析hLRH-1不同变异体在人胚胎组织的表达谱。方法通过设计特异引物，并经常规RT-PCR方法检测hLRH-1v1和hLRH-1在三例引产胎儿各组织中的表达情况：同时以实时定量RT-PCR法分析总hLRH-1的mRNA的相对表达谱。结果检测的结果显示，hLRH-1v1在人类各不同发育阶段的胎儿组织广泛表达，而hLRH-1则仅局限于肝、胃、小肠和胰腺组织。结论hLRH-1v1和hLRH-1在人胎儿组织表达谱的差异提示它们在早期胚胎发育中可能起不同的作用。     

复发转移性乳腺癌患者外周血BRCA1、CDH1、DKK1和SFRP1甲基化检测意义的研究

目的 探讨BRCA1、CDH1、DKK1和SFRP1基因甲基化与乳腺癌患者肿瘤激素受体状态、复发转移的关系.方法 利用甲基化特异性PCR法(MSP)检测115例复发转移乳腺癌患者外周血BRCA1、CDH1、DKK1和SFRP1的甲基化情况,与65例健康对照组进行比较.结果 BRCA1、CDH1和SFRP1的甲基化状态在...     

直肠癌中肿瘤/睾丸抗原相关基因的表达

目的检测MAGE-1、NY-ESO-1、SCP-1基因在直肠癌中的表达情况,探索其在直肠癌免疫治疗中的应用价值。方法应用MAGE-1、NY-ESO-1、SCP-1单克隆抗体,对68例直肠癌组织进行免疫组织化学法（SP法）检测,SPSS统计软件分析所得数据。结果在检测的68例直肠癌标本中,MAGE-1、NY-ESO-1、SCP-1的阳性表达率分别为30.9%（21/68）、23.5%（16/68）、17.6%（12/68）。多个CTA基因可在直肠癌中同时表达,至少表达一种CTA的频率为67.7%（46/68）,同时表达两种或两种以上CTA的频率是20.6%（14/68）,同时表达三种CTA的频率为5.9%（4/68）。阳性颗粒均位于细胞质。MAGE-1、NY-ESO-1、SCP-1基因的表达主要集中在肿瘤细胞分化较好Ⅰ级直肠腺癌,阳性率有显著性差异（P〈0.05）,与年龄、性别、肿瘤大小、Dukes分期和淋巴结转移无显著相关性（P〉0.05）。结论 CT抗原（MAGE-1、NY-ESO-1、SCP-1）在直肠癌组织中有高特异的表达,这使得用这些CTA编码的蛋白作为疫苗用于直肠癌的免疫治疗成为可能。     

过表达XBP-1的不同剪切体对骨髓瘤细胞分化的影响

本研究探讨XBP-1的两种不同剪切体XBP-1u,XBP-1s在骨髓瘤诱导分化过程中的作用机制。分别构建过表达质粒pcDNA3.1-C-XBP-1u,pcDNA3.1-C-XBP-1s,转染进入骨髓瘤细胞系U266和RPMI-8226细胞。光学显微镜观察细胞形态,流式细胞术检测细胞表面CD49e的表达率,ELISA检测上清液中轻链蛋白含量的改变以判断细胞的分化程度,Western blot检测XBP-1u和XBP-1s表达水平的变化。结果表明:过表达XBP-1u可以促进骨髓瘤细胞的分化,在形态学上显示浆细胞更加成熟;在U266和RPMI-8226细胞中,细胞表面CD49e的阳性表达率也明显上调,分别由对照的(9.02±0.3)%,(5.17±0.92)%增加到(27.7±1.14)%,(13.97±1.79)%(p0.01)。U266和RPMI8226细胞培养上清中的轻链蛋白含量由对照的(474.75±19.52)ng/ml,(289.44±6.19)ng/ml增加到(692.34±21.17)ng/ml,(401.55±13.7)ng/ml(p0.01,p0.05),而在过表达XBP-1s的骨髓细胞中则没有明显的改变,表明过表达XBP-1s对骨髓瘤细胞的分化没有促进作用。结论:XBP-1u表达水平的增高对于骨髓瘤细胞的分化具有重要作用。     

人参皂苷单体Rb1及Rg1对白血病细胞K562增殖的影响

背景:国内外研究发现人参皂苷及其单体在抑制白血病细胞增殖、增加化疗药物的敏感性和逆转耐药等方面均有疗效,目前主要集中于对实体瘤和急性白血病的研究,涉及慢性白血病的报道非常少.目的:探讨人参皂苷单体Rb1,Rg1对人慢性粒细胞白血病细胞K562增殖的影响.设计、时间及地点:细胞学体外观察,于2008-03/2009-03在重庆医科大学干细胞与组织工程研究室和眼科实验室完成.材料:K562细胞由重庆医科大学临床检验系提供.纯度为98.6%的人参皂苷单体Rb1,Rg1由南京艾斯替么中药研究所提供.方法:取对数生长期的K562细胞,调整密度为7×108L-1,空白对照组予以常规培养;人参皂苷单体Rb1组分别加入20,40,80,160 μmol/L的Rb1:人参皂苷单体Rg1组分别加入5,10,20,40,80 μmol/L的Rg1.主要观察指标:MTT比色法、锥虫蓝拒染法检测K562细胞增殖情况,流式细胞仪测定细胞周期分布的变化.结果:人参皂苷单体Rb1,Rg1在体外对K562细胞增殖均有明显的抑制作用,在20～160 μmol/L Rb1和5～20 μmol/L Rg1浓度范围内,其抑制作用呈浓度依赖性,且均在作用48 h时抑制率达高峰.与空白对照组比较,体外培养48 h后160 μmol/LRb1组细胞周期分布无变化;20 μmol/L Rg1组S期细胞比例明显增加(P<0.05),G1期细胞比例明显下降(P<0.05),且160 μmol/L Rb1组、20 μmol/L Rg1组均未出现"亚二倍体"峰.结论:人参皂苷单体Rb1,Rg1均可抑制K562细胞增殖,Rg1增殖抑制作用可能是通过将K562细胞阻滞于S期而实现的,而Rb1的增殖抑制作用与细胞周期的关系有待进一步分析.     

NF-кB活化及WT1、MDR1的表达在急性非淋巴细胞白血病中的临床意义

本研究通过观察初治与难治急性非淋巴细胞白血病中NF-κB活性及WT1、MDR1的表达水平,探讨三者在急性非淋巴细胞白血病疗效中的作用与相互关系,为寻找新的改善难治急性非淋巴细胞白血病疗效的方法提供理论依据.急性非淋巴性白血病患者45例,其中初治组20例(A组),难治组25例(分为B、C两组).以15例单纯缺铁性贫血患者作为对照组.采用凝胶电泳迁移分析(EMSA)法检测各组病人NF-κB的活化水平,用逆转录多聚酶链式反应(RT-PCR)法检测各组病人WT1及MDR1的表达水平.结果发现,在对照组未检测到NF-κB活化及WT1和MDR1表达;在初治组中NF-κB的活化水平、WT1和MDR1表达水平均明显低于难治组,而在难治B、C两组中三者无明显区别;各组病人细胞中NF-κB活化水平与WT1及MDR1的表达水平呈正相关.结论NF-κB的活化,WT1、MDR1的高表达可能是急性非淋巴细胞白血病难治的原因之一,急性非淋巴细胞白血病中NF-κB的活化水平与WT1、MDR1的表达呈正相关.     

HIV-1env、gag与IFNα-2b基因融合蛋白的免疫学研究

目的将编码艾滋病病毒（HIV-1）外膜蛋白的env基因、核蛋白的gag基因与编码干扰素（IFNα-2b）基因分别插入pSFJ16与pSFJ38真核表达载体，观察表达产物诱导机体产生细胞免疫的变化规律。方法利用分子生物学技术构建重组质粒，经质脂体转染与野生型痘苗病毒在Cos-7细胞内同源重组，筛选、纯化获得重组痘苗病毒vJ38gag／IFNα-2b和vJ16env／IFNα-2b。免疫小鼠后，检测小鼠外周血与牌淋巴细胞对ConA及LDs的反应性，用流式细胞仪测定小鼠脾细胞CD4^＋、CD8^＋，细胞计数。结果外周血与脾淋巴细胞对ConA及Lps的反应性，实验组与对照组比较有显著差异（P〈0.05）。CD4^＋T淋巴细胞与对照组比较有显著差异（P〈0．05）。CD8^＋T淋巴细胞与对照组比较无显著差异（P〉0．05），但呈增高趋势。结论重组痘苗病毒能诱导小鼠产生较强的细胞免疫。重组痘苗病毒能在体外与HIV—1 env和HIV-1 gag阳性血清发生特异性反应，具有免疫原性和免疫反应性。     

甲型H1N1流感重症与危重症患者临床特征分析

目的：探讨发展为重症与危重症的甲型H1N1流感患者的临床特征,为临床早期诊断提供依据。方法：回顾分析甲型H1N1流感住院患者的临床资料。以轻症患者（88例）为对照,分析甲型H1N1流感重症患者（19例）和危重症患者（12例）的性别、体质量指数、发病至入院时间、入院时体温、住院期间最高体温、血白蛋白、血钙、心肌酶、血小板、氧合指数、胸部X线片、CT等12项临床诊断指标与急性肺损伤（ALI）、急性呼吸窘迫综合征（ARDS）、感染性休克、多器官功能障碍综合征（MODS）以及死亡的关系。结果：按标准化回归系数绝对值由大到小：氧合指数、CT、入院体温、体质量指数对ALI的影响最大;氧合指数、心肌酶、体质量指数、入院体温、血白蛋白对ARDS的影响最大;白蛋白、氧合指数、心肌酶、入院体温、住院期间最高体温、血小板对感染性休克的影响最大;氧合指数、心肌酶、CT、白蛋白、入院体温、从发病到入院时间对MODS的影响最大;白蛋白、氧合指数、心肌酶、入院体温、住院期间最高体温、血小板对死亡的影响最大。结论：氧合指数、CT、入院体温、体质量指数等临床指标异常可提示ALI;氧合指数、心肌酶、体质量指数、入院体温、血白蛋白等临床指标异常可提示ARDS;血白蛋白、氧合指数、心肌酶、入院体温、住院期间最高体温、血小板等临床指标异常可提示感染性休克;氧合指数、心肌酶、CT、血白蛋白、入院体温异常及从发病到入院时间长可提示MODS;血白蛋白、氧合指数、心肌酶、入院体温、住院期间最高体温、血小板等临床指标异常可提示死亡。密切关注以上指标可以对其进行早期识别与干预。     

EB病毒感染的系统性红斑狼疮患者中BYRF1基因和BcLF1基因的表达与系统性红斑狼疮分期的关系

目的 探讨EB病毒（EBV）感染的系统性红斑狼疮（SLE）患者EBV基因BYRF1和BcLF1表达与SLE的关系。方法 应用聚合酶链反应（PCR）-Southern杂交技术检测44例SLE患者和43名正常对照外周血单个核细胞中EB病毒（EBV）特异性DNA片段，反向（RT）-PCR检测EBV特异性DNA片段阳性患者病毒潜伏期基因BYRF1和增殖期基因BcLF1的表达。结果 SLE患者组EBV特异性DNA片段BamHI-W阳性率为72．73％（32／44），明显高于正常对照组6．98％（3／43）（X^2=39．1779，P=0）。32例EBVBamHI-W片段阳性标本中有20例BYRF1mRNA阳性，其中活动期患者9例，稳定期患者11例，活动期和稳定期患者阳性率的差异无显著性（P=0．2907）。EBV增殖期基因BcLF1mRNA16例阳性，其中活动期患者14例，稳定期患者2例，活动期和稳定期患者之间的差异有显著性（P=0．0002）。结论 BYRF1mRNA和BcLF1mRNA的表达可能在一定程度上参与SLE的发病和病程进展。