Paraffin-embedded tissue as a source of RNA for gene expression analysis in oral malignancy

OBJECTIVE: To assess the feasibility of using archival oral mucosal tissue to examine gene expression at the ribonucleic acid (RNA) level.
MATERIALS AND METHODS: We describe the isolation of RNA from 8 nm sections of formalin-fixed paraffin-embedded oral mucosal tissue. RNA was reverse transcribed and three candidate genes amplified by polymerase chain reaction (PCR). The ribosomal protein S14 gene is a housekeeping gene which has been used as an internal standard in several quantitative PCR protocols. The thymidine kinase (TK) gene is expressed at low levels in most tissues and, with a well-documented genomic organisation, is a useful tool for discrimination between genomic DNA and cDNA. The RIa gene is reported to be overexpressed in many cancer cell lines, in malignant tissue and in vitro transformed cellS. RESULTS: The S14 gene, the TK gene and the RIα gene of the cAMP-dependent protein kinase (PKA) were amplified successfully from formalin-fixed paraffin-embedded tissue sections. The TK primer pair is a useful additional tool in the unambiguous identification of RNA-derived species.
CONCLUSION: RNA suitable for reverse transcribed (RT)-PCR was extracted from archival oral mucosal tissue. This should permit rapid sequence analysis of transcribed tumor suppressor genes and oncogenes in this material. Furthermore, the RT-PCR approach described may allow quantification of gene expression in oral mucosal archival material processed in a standard fashion.     

The molecular basis of renal amyloidosis in Irish-American and Polish- Canadian kindreds

Hereditary amyloidosis of an unusual form has been reported in two separate kindreds; one was Polish-Canadian and the other was Irish- American (Am J Med 1975; 59:121 and Trans Assoc Am Physicians 1981; 94:211). In both kindreds, affected members developed hypertension and nephrotic syndrome due to amyloidosis in their forties or fifties, but the genetic background responsible for the condition has been left undetermined. To identify the genetic defect in these kindreds, a portion of exon 5 of the fibrinogen alpha-chain gene in members of these kindreds was examined for a mutation by single-strand conformation polymorphism analysis and direct DNA sequencing. DNA analyses revealed an A-->T transversion at the second base of codon 526 of the fibrinogen alpha-chain gene in both of these kindreds. Analysis of DNA polymorphisms in the fibrinogen alpha-chain gene locus (TCTT repeat in intron 3, Rsal site in exon 5, and Taql site in the 3' flanking region of the gene) showed the haplotype B5-Rsal(+)-Taql(-) for the Val 526 mutant gene in both kindreds studied here, as well as in two kindreds previously described (J Clin Invest 1994; 93:731). The fibrinogen alpha-chain gene mutation (Val 526) is the genetic defect responsible for hereditary renal amyloidosis in these two kindreds, and the mutant genes in the Val 526 kindreds may have been derived from a single founder.      

Demographic characteristics and prevalence of serologic markers among donors who use the confidential unit exclusion process: the Retrovirus Epidemiology Donor Study

BACKGROUND: Most blood centers utilize a confidential unit exclusion (CUE) process, intended to reduce the risk of transfusion-associated infectious diseases by allowing high-risk donors confidentially to exclude their blood from use for transfusion. The effectiveness of this method remains controversial. STUDY DESIGN AND METHODS: Confirmatory or supplemental test results for antibodies to human immunodeficiency virus, human T-lymphotropic virus type I, and hepatitis C virus, as well as hepatitis B surface antigen and syphilis and screening test results for antibodies to hepatitis B core (antigen) and alanine aminotransferase levels were obtained for approximately 1.8 million units donated during 1991 and 1992 at five blood centers within the United States. The prevalences of these infectious disease markers in units that the donors confidentially excluded (CUE+) and units that the donors did not exclude (CUE-) were calculated and examined within demographic subgroups. RESULTS: Units that were CUE+ were 8 to 41 times more likely to be seropositive for antibodies to human immunodeficiency virus and hepatitis C virus, hepatitis B surface antigen, and syphilis and three to four times more likely to react for antibody to hepatitis B core (antigen) or to have elevated alanine aminotransferase levels than units that were CUE- (p < 0.001). The positive predictive value of CUE (the percentage of CUE+ units that were confirmed seropositive for any marker) was 3.5 percent, and the sensitivity of CUE (the percentage of confirmed-seropositive units that were CUE+) was 2.3 percent. CONCLUSION: The current CUE process has low sensitivity and apparently low positive predictive value, and in many cases, it appeared that donors misunderstood it. Yet, CUE was not a “random process,” as CUE+ units were more likely to be seropositive for any infectious disease marker than CUE- units. This suggests that efforts to improve the CUE system may be warranted. As risk factors for transfusion-transmitted infection become more difficult to identify by history-based screening, however, such efforts may have limited effect.     

转脑啡肽基因移植细胞微囊化对大鼠慢性周围神经损伤的镇痛作用

目的：观察微囊化转大鼠脑啡肽原基因（pENK）细胞移植对慢性脊神经压榨性损伤大鼠的镇痛效应. 方法：实验于2006-03/2007-04在郑州大学医学重点实验室完成.①实验方法：通过反转录-聚合酶链反应技术可获得大鼠pENK基因,Hind Ⅲ,ClaⅠ双酶切后,同相应双酶切的pLNCX2载体大片段连接,构建成pENK基因反转录病毒载体pLNCX2-Enk,然后用脂质体法将该载体转染PT67细胞,G418筛选,获得携带pENK基因高滴度反转录病毒产毒细胞系.用海藻酸钠-多聚赖氨酸-海藻酸钠微囊包埋后,进行体外培养,定期检测微囊化细胞活性和pENK分泌量变化.同时,将转基因细胞移植于慢性脊神经压榨性损伤大鼠的蛛网膜下腔.②实验分组：SD大鼠60只,其中53只按照Bennett和Xie法制作大鼠慢性左侧坐骨神经压迫性损伤模型,其中42只造模成功.术后1周,将动物按随机数字表法分为3组,每组16只：微囊化转基因细胞移植组、空囊移植组和阴性对照组.微囊化转基因细胞移植组、空囊移植组分别植入微囊化细胞悬液80 μL（约300个微囊）、空微囊悬液80 μL（约300个空囊）,阴性对照组不注射任何悬液.③实验评估：术后2周和8周行甲醛实验,在大鼠一侧后爪掌侧皮下注射体积分数为0.05的甲醛50 μL,观察其注射后1 h内的痛反应.采用Abbott等所推荐的以缩腿及舔爪时间之和作为行为学反应的指标.注射甲醛后,立即记录大鼠1 h内每5 min的缩腿及舔爪时间. 结果：①术后2周甲醛实验：空囊移植组第一时相的急性痛阶段（注射后5 min）和第二时相的慢性痛阶段（注射后41～45 min）大鼠的缩腿及舔爪时间都少于微囊化转基因细胞移植组（P＜0.01）.而在静息期,3组大鼠的缩腿及舔爪时间差异无显著性意义（P＞0.05）.②术后8周甲醛实验：3组大鼠的痛觉行为都呈明显的双时相反应.除了静息期（注射后5～15 min）,微囊化转基因细胞移植组在各时间点上大鼠的缩腿及舔爪时间均低于空囊移植组（P＜0.05）.微囊化转基因细胞移植组大鼠的缩腿及舔爪时间在第一时相的急性痛阶段和第二时相的慢性痛阶段都低于空囊移植组（P＜0.05）. 结论：微囊化转pENK基因细胞移植对慢性神经痛大鼠有一定的镇痛作用,有望成为运动员慢性疼痛治疗的新方法.     

角质形成细胞的培养及其表皮生长因子受体表达

目的:在成功分离人皮肤角质形成细胞的基础上,观察表皮生长因子受体在人皮肤角质形成细胞中的表达情况。方法:实验于2006-3/10在北京大学深圳医院中心实验室进行。采用dispase Ⅱ-trypsin两步消化法获取表皮基底层细胞,用小鼠皮肤成纤维母细胞滋养层和黄素腺嘌呤二核苷酸培养液进行培养。小鼠皮肤成纤维母细胞的预处理:向对数生长期的小鼠皮肤成纤维母细胞培养液中加入丝裂霉素C至终浓度为4mg/L,37℃下培养4h,弃去培养液,用D-Hank’s液洗3次,加入浓度为0.25g/L的胰蛋白酶消化,分离出细胞,离心(200g,5min),用黄素腺嘌呤二核苷酸培养液悬浮细胞,计数,以5.0×104/cm2的密度种于培养皿内,37℃、体积分数0.05的CO2培养箱下培养。角质形成细胞的培养:将分离的角质形成细胞悬浮在黄素腺嘌呤二核苷酸培养液中,以2.0×104/cm2的密度接种在前1天经丝裂霉素C处理的小鼠皮肤成纤维母细胞滋养层上,37℃、体积分数0.05的CO2培养箱下培养。24h换液,以后每3d换1次液。采用免疫细胞化学的方法检测表皮生长因子受体的表达,采用复合逆转录聚合酶链反应检测角质形成细胞中表皮生长因子受体mRNA的表达。结果:采用dispaseⅡ消化法分离了真皮和表皮,获得较多的角质形成细胞,可以避免真皮成纤维细胞的污染。人皮肤角质形成细胞在黄素腺嘌呤二核苷酸培养液中培养5d可见明显的集落,约10d可长满单层。免疫细胞化学显示表皮生长因子受体在细胞表面有明显的表达,复合逆转录聚合酶链反应显示表皮生长因子受体mRNA有明显的表达。结论:用小鼠皮肤成纤维母细胞滋养层和黄素腺嘌呤二核苷酸培养液可以较好地培养原代人皮肤角质形成细胞,表皮生长因子受体在细胞表面有明显的表达,这些结果为与表皮生长因子受体相关的皮肤病(如银屑病)的研究奠定了基础。     

胶原海绵复合新生大鼠原代心肌细胞构建工程化心肌组织

目的:探索以胶原海绵为支架、新生大鼠原代心肌细胞为种子细胞,于体外构建工程化心肌组织的方法。方法:实验于2005-12/2006-11在解放军第四军医大学西京医院心内科实验室完成。Ⅰ型胶原海绵剪切成方形片状(2.0cm×1.4cm×0.2cm),经60Co照射消毒,于DMEM培养液中水化1h左右。另取1d龄SD大鼠心脏,剪成小碎块,然后用2.5g/L胰蛋白酶于37℃中消化,吸取上清至含胎牛血清的DMEM中,重复消化四五次,用差速贴壁法除去大部分成纤维细胞,将细胞沉淀用DMEM培养液以2×109L-1的密度悬浮备用。将上述的心肌细胞悬液1mL缓慢滴注于玻璃模型中的胶原海绵上,然后置于细胞培养中培养。肉眼及显微镜主要观察工程化心肌组织在培养期间的自发收缩情况,包括收缩的部位、强度、频率、一致性以及收缩随时间变化的情况。苏木精-伊红染色观察工程化心肌组织内胶原纤维的变化,细胞形态,胞核的形状及细胞之间的连接。免疫组织化学染色和透射电镜观察工程化心肌组织片的形态和功能。结果:①细胞接种于胶原海绵上1d后,细胞/胶原复合物的凝胶化过程基本完毕,体积保持恒定,维持至培养结束,第3天细胞/胶原复合物局部出现点片状自发收缩,第5天整个细胞/胶原复合物出现同步化自发收缩,收缩频率61～199次/min。2周后37.5%的工程化心肌组织的自发收缩活动减弱,但75%的工程化心肌组织的自发收缩活动持续至培养结束。②苏木精-伊红染色、免疫组织化学染色和透射电子显微镜显示,工程化心肌组织内细胞间连接广泛存在,细胞多呈纵向分布,胞核呈长圆形,胞浆内α-肌节肌动蛋白阳性,胞内肌原纤维排列整齐,可见到心肌特异性的肌小节结构和Z线,多数细胞具有分化的心肌细胞表型。结论:用新生大鼠原代心肌细胞为种子细胞、以Ⅰ型胶原海绵为支架材料,构建出的工程化心肌组织,于体外可长时间持续自发收缩,该细胞/胶原复合物的形态结构与生理功能均类似于成熟大鼠心肌组织。     

The efficacy of subcutaneous recombinant human erythropoietin in the correction of phlebotomy-induced anemia in autologous blood donors

The efficacy of subcutaneous recombinant human erythropoietin (rhEPO) (500 U/kg; administered twice a week during the 3 weeks before surgery) in the recovery of preoperative hemoglobin concentrations within a 3- week period was studied in 40 patients, each of whom donated 2 units (900 mL) of blood for their own use before total hip replacement surgery. Twenty autologous blood donors received rhEPO (EPO group) and 20 were not treated (control group). The initial hemoglobin concentration (14.0 +/− 1.0 g/dL [140 +/− 10 g/L]) was completely recovered before surgery (14.0 +/− 1.6 g/dL [140 +/− 16 g/L]) in the EPO group, while a decrease from 13.8 +/− 1.1 to 12.2 +/− 1.3 g per dL (138 +/− 11 to 122 +/− 13 g/L) was observed in the control group. The preoperative reticulocyte count showed more than sixfold increase in the EPO group, whereas a twofold to threefold increase was found in the control group. Serum ferritin concentration fell to 42 +/− 29 micrograms per L in the EPO group and to 54 +/− 35 micrograms per L in the control group. The postoperative serum erythropoietin concentration in the EPO group was significantly lower than that in the control group, but it did not differ from the pretreatment value and was attended by a higher hemoglobin concentration after surgery. Only transient flu-like symptoms were mentioned by patients who were treated with rhEPO. Changes in blood pressure or platelet count or other adverse events were not observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

贞芪扶正颗粒和复方阿胶浆干预再生障碍性贫血模型小鼠外周血及骨髓象的变化

目的:观察贞芪扶正颗粒和复方阿胶浆对苯油溶液致小鼠再生障碍性贫血模型的疗效。方法:实验于2005-06/09在河南中医学院药理实验室完成。①选用昆明种成年雄性小鼠90只。按随机数字表法将小鼠分为9组,每组10只:大、中、小剂量贞芪扶正颗粒组:按15,10,5g/kg剂量灌服贞芪扶正颗粒混悬液(主要成分:黄芪、女贞子;甘肃扶正药业科技股份有限公司生产;批号040803,15g/袋)。大、中、小剂量复方阿胶浆组:分别按10,20,30mL/kg剂量灌胃复方阿胶浆(主要成分:阿胶、熟地黄、党参、山楂、人参、蔗糖;山东东阿阿胶股份有限公司生产,批号050446,250mL/瓶),司坦唑醇组:按4mg/kg剂量灌胃司坦唑醇混悬液(司坦唑醇片,广西南宁百会药业集团有限公司生产,批号050306,30mg/片)。空白组和模型组:灌胃同体积的生理盐水(20mL/kg)。每天给药1次,连续给药14d。除空白组外,其余组小鼠皮下注射体积分数0.25苯的玉米油溶液4mL/kg复制苯油溶液致小鼠再生障碍性贫血模型,空白组皮下注射同体积玉米油。②各组小鼠分别于末次给药后24h,进行血常规检测。取右侧股骨,冲出骨髓细胞,采用BI-2000医学图像分析仪计数骨髓有核细胞数。③计量资料符合正态分布、方差齐者用t检验;方差不齐者用t’检验。结果:小鼠90只均进入结果分析。①血细胞测定结果比较:模型组小鼠血红细胞、白细胞、血小板计数和血红蛋白水平明显低于空白组(t=3.39～11.89,P<0.01)。司坦唑醇组3项血细胞计数和血红蛋白水平明显高于模型组(t=4.94～6.73,P<0.01)。大、中剂量贞芪扶正颗粒组和各剂量复方阿胶浆组血白细胞计数明显高于模型组(t=2.32～3.03,P<0.05￣0.01)。中剂量贞芪扶正颗粒组和各剂量复方阿胶浆组小鼠血红细胞计数明显高于模型组(t=2.15～4.84,P<0.05￣0.01)。大、中剂量贞芪扶正颗粒组和各剂量复方阿胶浆组血红蛋白水平明显高于模型组(t=2.33～4.45,P<0.05￣0.01)。大、中剂量贞芪扶正颗粒组和各剂量复方阿胶浆组血小板计数明显高于模型组(t=4.06～6.24,P<0.01)。②骨髓有核细胞数:模型组明显低于空白组(t=8.99,P<0.01)。司坦唑醇组、中剂量贞芪扶正颗粒组和小剂量复方阿胶浆组明显高于模型组(t=2.39～2.82,P<0.05)。结论:贞芪扶正颗粒、复方阿胶浆对皮下注射苯所致小鼠再生障碍性贫血模型血细胞状况及骨髓象均有较好的改善作用,以贞芪扶正颗粒有较为明显的剂量依赖关系。     

A study of confidential unit exclusion

The effectiveness of the confidential unit exclusion (CUE) procedure recommended by the Food and Drug Administration has been questioned by the blood banking community. The purpose of this study was to determine whether donors were informing the blood center correctly regarding the disposition (transfuse or do not transfuse) of their donated blood. A letter explaining the confidential study and requesting permission to send the participant a questionnaire noting his or her self-exclusion choice was mailed to 230 donors who had chosen transfuse and 276 donors who had chosen do not transfuse. After consent was obtained, participants were sent a second packet and asked to indicate whether they had chosen correctly and, if not, to identify reasons for that incorrect choice. A seven-word terminology quiz made up of words from the CUE form was also enclosed. All participants who had chosen transfuse indicated that this was the correct choice. Approximately 50 percent of those who had chosen do not transfuse indicated that this was an incorrect choice; the most common reason was that "I was not paying attention." The most frequently misunderstood term was "confidential." Donors who chose do not transfuse had a significantly higher rate of error on the terminology quiz (p less than 0.01) than did those who chose transfuse.     

鼠尾静脉流体力学转染技术对绿色荧光蛋白表达质粒器官靶向分布的影响

目的:观察流体力学尾静脉注射对绿色荧光蛋白基因器官靶向性的影响,为今后质粒载体的基因治疗和功能研究寻找潜在的靶器官。方法:实验于2005-12/2006-04在江西省分子医学重点实验室完成。选用健康雄性昆明鼠40只,将32只小鼠按随机数字表法分为流体力学注射和常规注射两大组,每大组再分为转染组和对照组两个小组(n=8),并设正常对照组(n=8)。①流体力学转染组将100μg/只绿色荧光蛋白表达质粒溶液2mL在5s内快速注入尾静脉;对照组仅在5s内注入林格氏液2mL。②常规注射组则将2mL林格氏液或绿色荧光蛋白表达质粒溶液在30s左右注入尾静脉。注射结束后24h采集各组小鼠血清检测转氨酶,并采集肝、脾、心、肾、肺和脑组织进行冰冻切片,部分肝组织采用多聚甲醛固定后切片,荧光显微镜下观察。结果:40只小鼠全部进入结果分析,无脱失。①流体力学注射组和常规注射组小鼠血清转氨酶与正常对照组比较差异均无显著性意义(P>0.05)。②常规尾静脉注射引起少数肾小球细胞表达绿色荧光蛋白,而肝、脾、心、肺及脑等组织未见明显绿色荧光蛋白表达。③流体力学注射引起肝内绿色荧光蛋白高水平表达,肝细胞表达率接近45%,其他组织则无绿色荧光蛋白表达。结论:流体力学方法是肝靶向性的活体基因转染方法,绿色荧光蛋白可作为该方法进行目的基因研究的一个可靠和方便的示踪剂。