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31.
本文采用PCR技术对石家庄地区216名无关个体DIS80(pMCT118)位点扩增片段长度多态性的等位基因频率进行调查。PCR扩增产物采用聚丙烯酰胺凝胶电泳分型及溴化乙锭染色,共检出70种基因型,25个等位基因,其中以18、24、30三个等位基因频率最高。将本文结果与日本人群在DIS80(pMCT118)位点多态性结果进行比较,石家庄地区汉族人群DIS80(pMCT118)位点基因频率分布符合HardyWeinberg平衡定律,该位点非父排除率为0.71,个体识别率为0.9,杂合度为0.85。 相似文献
32.
33.
套式PCR检测细菌16SrRNA基因 总被引:2,自引:0,他引:2
目的 应用套式PCR建立快速检查方法来检测血液中是否含有细菌。方法 采用细菌 1 6SrRNA基因通用引物 ,通过套式PCR方法对细菌进行扩增 ,并对其灵敏度、特异性作一评价。结果 临床常见细菌扩增反应为阳性 ,其他病原微生物及人类基因组DNA为阴性。结论 本法具有敏感、快速、特异、准确的优点 ,是一种可靠的实验手段 相似文献
34.
人肿瘤特异抗原MAGE-3基因疫苗的构建和表达 总被引:8,自引:1,他引:7
目的:克隆肿瘤特异性共享抗原mage-3基因,制备基于α-病毒复制酶的高效黑色素瘤特异性基因疫苗。方法:用RT-PCR方法从黑色素瘤细胞系LB373中制备mage-3基因,以含α-病毒复制酶的哺乳细胞高效表达质粒pSMART2a为载体,构建重组DNA疫苗。重组子用载体中的T7和T3启动子序列为测序引物进行自动测序。再将鉴定过的重组质粒用脂质体法转化293细胞,用免疫印迹法和免疫组化法鉴定转化细胞中mage-3蛋白的表达。结果:正确构建了mage-3/pSMART2a重组质粒,并且在转化此质粒的293细胞中检测出了mage-3蛋白的表达。结论:此重组mage-3/pSMART2a质粒可以作为肿瘤特异的DNA疫苗,可进行下一步的肿瘤动物模型的疫苗接种及相关免疫学效应研究。 相似文献
35.
36.
L.J. Mackintosh† M.N.C. de Koning‡ W.G.V. Quint‡ J. ter Schegget‡ I.M. Morgan R.M. Herd† M.S. Campo 《The British journal of dermatology》2009,161(1):56-62
Background Nonmelanoma skin cancer (NMSC) has been linked to cutaneous human papillomaviruses of the genus beta (betaPV).
Objectives We sought to assess the presence of betaPV in NMSC biopsies from a group of Scottish skin cancer patients, both immunocompetent (IC) patients and immunosuppressed (IS) organ transplant recipients.
Methods One hundred and twenty-one paraffin-embedded skin tumours (27 actinic keratosis, 41 intraepidermal carcinoma, 53 squamous cell carcinoma) and 11 normal skin samples were analysed for the presence of betaPV by a polymerase chain reaction–reverse hybridization assay designed to detect the presence of the 25 known betaPV genotypes.
Results In IC patients, betaPV was detected in 30 of 59 (51%) tumours and two of 11 (18%) normal skin samples ( P = 0·046). In IS patients, betaPV was found in 27 of 62 (44%) tumours; no normal skin samples were available for comparison. The most frequently found genotypes were HPV-24, HPV-15 and HPV-38. Of those tumours infected with betaPV, 28 of 57 (49%) were infected with more than one genotype (range 2–8). Tumours from IS patients were from a younger age group (mean age 57·4 years) than IC patients (mean age 73·8 years). Multiple infections were more common in tumours from IC patients (21 of 30; 70%) compared with those from IS patients (seven of 27; 26%) ( P < 0·001). In the IC group, age did not appear to influence the distribution of single and multiple infections whereas in IS patients the proportion of multiple infections to single infections increased with age. There were no multiple infections in normal skin.
Conclusions A wide spectrum of betaPV types was detected in our samples. Further characterization of betaPV in vivo is needed in order to determine the mechanisms by which the virus contributes to cutaneous carcinogenesis. 相似文献
Objectives We sought to assess the presence of betaPV in NMSC biopsies from a group of Scottish skin cancer patients, both immunocompetent (IC) patients and immunosuppressed (IS) organ transplant recipients.
Methods One hundred and twenty-one paraffin-embedded skin tumours (27 actinic keratosis, 41 intraepidermal carcinoma, 53 squamous cell carcinoma) and 11 normal skin samples were analysed for the presence of betaPV by a polymerase chain reaction–reverse hybridization assay designed to detect the presence of the 25 known betaPV genotypes.
Results In IC patients, betaPV was detected in 30 of 59 (51%) tumours and two of 11 (18%) normal skin samples ( P = 0·046). In IS patients, betaPV was found in 27 of 62 (44%) tumours; no normal skin samples were available for comparison. The most frequently found genotypes were HPV-24, HPV-15 and HPV-38. Of those tumours infected with betaPV, 28 of 57 (49%) were infected with more than one genotype (range 2–8). Tumours from IS patients were from a younger age group (mean age 57·4 years) than IC patients (mean age 73·8 years). Multiple infections were more common in tumours from IC patients (21 of 30; 70%) compared with those from IS patients (seven of 27; 26%) ( P < 0·001). In the IC group, age did not appear to influence the distribution of single and multiple infections whereas in IS patients the proportion of multiple infections to single infections increased with age. There were no multiple infections in normal skin.
Conclusions A wide spectrum of betaPV types was detected in our samples. Further characterization of betaPV in vivo is needed in order to determine the mechanisms by which the virus contributes to cutaneous carcinogenesis. 相似文献
37.
PTH has diverse effects on bone metabolism: anabolic when given intermittently, catabolic when given continuously. The cellular
mechanisms underlying the varying target cell response are not clear yet. PTH induces RGS-2, a member of the Regulator of
G-protein Signaling protein family, via cAMP/PKA, and inactivates PKC-mediated signaling. To investigate intracellular signaling
pathways with different PTH concentration-time patterns, we treated UMR 106-01 osteoblast-like cells in a perfusion system.
PTH was administered intermittently (4 min/h, 10−7 M) or continuously at an equivalent cumulative dose (6.6 × 10−9 M). cAMP was measured using radioimmunoassay, mRNA levels using real-time rtPCR and ribonuclease protection assay, and protein
levels using Western immunoblotting. A single PTH pulse transiently increased cAMP levels by 2000% ± 1200%. In contrast to
continuous PTH exposure, cAMP induction remained unchanged with intermittent PTH, ruling out desensitization of the PTH receptor.
In continuously perfused cells, RGS-2 abundance was three to five times higher than in cells intermittently exposed to PTH
for up to 12 h. MKP-1 and -3 were significantly less induced with pulsatile PTH; exposure-mode-dependent differences in MMP-13
and IGFBP-5 were small. Pulsatile but not continuous PTH administration prevents PTHrP receptor desensitization and accumulation
of RGS-2 in osteoblasts, which should preserve PKC-dependent signaling. 相似文献
38.
39.
S. Vemuri 《Chemical biology & drug design》2005,65(4):433-439
Abstract: Precise determination of the peptide content in drug substance samples depends highly upon the particular peptide compound and methodology used. Four independent methods were evaluated and compared to determine which would produce the best experimental precision for analysis of thymalfasin (thymosin α‐1). Four different methods were evaluated including elemental analysis (CHN), quantitative amino acid analysis (AAA), high‐performance liquid chromatography (HPLC), and Kjeldahl. This study demonstrates that the AAA method is highly variable in one laboratory while quite precise in another laboratory. Similarly, HPLC results depended on the laboratory conducting the study with more precise values obtained under cGMP. On the contrary, the CHN method yielded highly precise [i.e. <2% coefficient of variation (CV)] values. As precise knowledge of protein content is fundamental for the compounding of final pharmaceutical product of a specific potency, the CHN analysis is recommended for peptide content determination of the drug substance thymalfasin. 相似文献
40.
M. Diez L. Favaloro A. Bertolotti J. M. Burgos C. Vigliano M. P. Lastra M. J. Levin A. Arnedo C. Nagel A. G. Schijman R. R. Favaloro 《American journal of transplantation》2007,7(6):1633-1640
Heart transplantation (HTx) is a useful therapy for end‐stage Chaga? cardiomyopathy; however, Chagas reactivation remains a mayor complication. Parasitological methods offer poor diagnostic sensitivity, and use of more sensitive tools such as the Polymerase chain reaction (PCR) is usually necessary. In the present study, reactivation incidence and PCR usefulness for early reactivation diagnosis, as well as for treatment response evaluation during follow‐up, were analyzed using Strout parasite detection test, in 10 of 222 consecutive HTx patients suffering Chagas cardiomyopathy. PCR strategies targeted to minicircle sequences (kDNA, detection limit 1 parasite/ 10 mL blood) and miniexon genes (SL‐DNA, 200 parasite/10 mL) were performed to compare parasite burdens between samples. No patients received prophylactic antiprotozoal therapy (benznidazole). Five patients (50%) exhibited clinical reactivation within a mean period of 71.6 days; positive Strout results were observed in most cases presenting clinical manifestations. kDNA‐PCR was positive 38–85 days before reactivation, whereas SLDNA‐PCR became positive only 7–21 days later, revealing post‐HTx parasitic load enhancement present prior to clinical reactivation development. Reactivations were successfully treated with benznidazole and generated negative PCR results. Results observed in this study indicate the value of PCR testing for an early diagnosis of Chagas reactivation as well as for monitoring treatment efficacy. 相似文献