用反相液相色谱分离多肽时峰容量与色谱柱长度关系的研究

在蛋白质组学研究中,多肽混合物的有效分离对蛋白质鉴定和蛋白质之间相互作用的研究起着决定性的影响。基于此,用反相液相色谱研究了在两个不同长度的色谱柱上分离多肽混合物时色谱柱长度与峰容量的关系,同时考察了梯度洗脱时间对峰容量和峰宽的影响。实验结果表明,色谱柱长度对峰容量有显著的影响,而延长梯度洗脱时间不仅可以增加峰容量,而且可以增加峰宽。这说明用毛细管液相色谱 串联质谱联用方法对多肽混合物进行分离鉴定时,采用较长的色谱柱和较长的梯度洗脱时间有利于对更多的多肽进行分析鉴定。

Alterations in the human epidermal growth factor receptor 2-phosphatidylinositol 3-kinase-v-Akt pathway in gastric cancer

AIM:To investigate human epidermal growth factor receptor 2(HER2)-phosphatidylinositol 3-kinase(PI3K)-vAkt murine thymoma viral oncogene homolog signaling pathway.METHODS:We analyzed 231 formalin-fixed,paraffinembedded gastric cancer tissue specimens from Japanese patients who had undergone surgical treatment.The patients' age,sex,tumor location,depth of invasion,pathological type,lymph node metastasis,and pathological stage were determined by a review of the medical records.Expression of HER2 was analyzed by immunohistochemistry(IHC) using the HercepTest TM kit.Standard criteria for HER2 positivity(0,1+,2+,and 3+) were used.Tumors that scored 3+ were considered HER2-positive.Expression of phospho Akt(pAkt) was also analyzed by IHC.Tumors were considered pAkt-positive when the percentage of positive tumor cells was 10% or more.PI3K,catalytic,alpha polypeptide(PIK3CA) mutations in exons 1,9 and 20 were analyzed by pyrosequencing.Epstein-Barr virus(EBV) infection was analyzed by in situ hybridization targeting EBV-encoded small RNA(EBER) with an EBER-RNA probe.Microsatellite instability(MSI) was analyzed by polymerase chain reaction using the mononucleotide markers BAT25 and BAT26.RESULTS:HER2 expression levels of 0,1+,2+ and 3+ were found in 167(72%),32(14%),12(5%) and 20(8.7%) samples,respectively.HER2 overexpression(IHC 3+) significantly correlated with intestinal histological type(15/20 vs 98 /205,P = 0.05).PIK3CA mutations were present in 20 cases(8.7%) and significantly correlated with MSI(10/20 vs 9/211,P < 0.01).The mutation frequency was high(21%) in T4 cancers and very low(6%) in T2 cancers.Mutations in exons 1,9 and 20 were detected in 5(2%),9(4%) and 7(3%) cases,respectively.Two new types of PIK3CA mutation,R88Q and R108H,were found in exon1.All PIK3CA mutations were heterozygous missense singlebase substitutions,the most common being H1047R(6/20,30%) in exon20.Eighteen cancers(8%) were EBV-positive and this positivity significantly correlated with a diffuse histological type(13/18 vs 93/198,P = 0.04).There were 7 cases of lymphoepithelioma-like carcinomas(LELC) and 6 of those cases were EBV-positive(percent/EBV:6/18,33%;percent/all LELC:6/7,86%).pAkt expression was positive in 119(53%) cases but showed no correlation with clinicopathological characteristics.pAkt expression was significantly correlated with HER2 overexpression(16/20 vs 103/211,P < 0.01) but not with PIK3CA mutations(12/20 vs 107/211,P = 0.37) or EBV infection(8/18 vs 103/211,P = 0.69).The frequency of pAkt expression was higher in cancers with exon20 mutations(100%) than in those with exon1(40%) or exon9(56%) mutations.One case showed both HER2 overexpression and EBV infection and 3 cases showed both PIK3CA mutations and EBV infection.However,no cases showed both PIK3CA mutations and HER2 overexpression.One EBVpositive cancer with PIK3CA mutation(H1047R) was MSI-positive.Three of these 4 cases were positive for pAkt expression.In survival analysis,pAkt expression significantly correlated with a poor prognosis(hazard ratio 1.75;95%CI:1.12-2.80,P = 0.02).CONCLUSION:HER2 expression,PIK3CA mutations and EBV infection in gastric cancer were characterized.pAkt expression significantly correlates with HER2 expression and with a poor prognosis.

L-精氨酸及其N衍生物对高血压大鼠离体血管的舒张作用

用L-精氨酸及L-精氨酸N端α衍生物N-乙酰精氨酸、N-苯甲酰精氨酸,对原发性高血压大鼠离体主动脉血管平滑肌的舒张作用进行研究,并与正常血压大鼠的主动脉血管平滑肌做比较。结果发现,L-精氨酸、N-乙酰精氨酸、N-苯甲酰精氨酸、和具有精氨酸-甘氨酸-天冬氨酸,即RGD序列的短肽精氨酸-甘氨酸-天冬氨酸-苯乙酰胺（RGD-PEA）,对于高血压及正常血压大鼠离体主动脉血管平滑肌均有程度不等的舒张作用,且显示剂量依赖性和内皮依赖性。药效动力学分析表明,舒张血管的半数有效浓度除RGD-PEA在10^(-6)mol/L之外,其余的均在10^(-7)mol/L范围;L-Arg及其N衍生物对高血压大鼠主动脉的最大舒张效应在4％～20％范围。

恶性疟原虫杂合抗原基因的表达及其产物免疫功能的研究

化学合成的人恶性疟原虫杂合多肽抗原 AB 基因,编码子孢子 CSP重复序列 NANP、CSPTh2R和红内期spf35.1、spf83.1、spf55.1、spf83.18、pf155/RESA-5’端重复区共7个不同免疫功能表位,与大肠杆菌质粒pWR450-1重组,构建成pWR450-1/AB表达载体。在乳糖操纵子调控下以β-半乳糖苷酶-恶性疟原虫杂合多肽抗原融合蛋白形式,在大肠杆菌中高效表达。表达产物经SDS-PAGE电泳分离纯化后,加福氏佐剂免疫家兔,诱导产生较高滴度的抗血清。抗血清对人恶性疟原虫体外生长有明显的抑制作用,24h抑制率为65.92％;72h的抑制率为72.63％。对照血清无抑制作用。分离纯化的表达产物能与鼠抗恶性疟原虫和疟疾患者抗血清起免疫反应。这些结果表明合成基因的表达产物具有较好的免疫原性。

反流性食管炎患者血浆血管活性肠肽、胃动素变化及其意义

目的:测定反流性食管炎患者血浆血管活性肠肽(VIP)、胃动素浓度,及下食管括约肌压力(LESP),以探讨反流性食管炎的发病机制。方法:采用液体灌注体外传感器法测定28例反流性食管炎患者下食管括约肌压力。采用放射免疫法测定其血浆VIP及胃动素浓度。结果:病例组血浆VIP浓度与对照组差异有显著性(P<0.05)。LESP与血浆VIP浓度呈明显负相关(r=-0.912.P<0.01)。病例组血浆胃动素浓度与对照差异有显著性(P<0.05)。LESP与血浆胃动素浓度无相关性。结论:反流性食管炎患者存在胃肠激素异常,其中血浆VIP升高,胃动素降低。血浆VIP升高可降低LESP,而胃动素降低对LESP无影响。