Spectroscopic characterization and microscopic imaging of extracted and in situ cutaneous collagen and elastic tissue components under two-photon excitation

Background/purposes: Understanding the two-photon excitation spectral characteristics and microscopic morphology of cutaneous collagen and elastic tissue components is important for applying multiphoton microscopy (MPM) in basic skin biology research and for clinical diagnosis.
Methods: We developed a system for two-photon excitation spectral measurements at various excitation wavelengths. The microscopic morphology was studied using a commercial multiphoton microscope.
Results: We obtained two-photon excitation fluorescence (TPEF) excitation–emission matrices (EEM), for the first time, of purified collagen and elastin samples, as well as in situ collagen and elastic fibers within excised human dermis. The EEM of the dermis was found to be similar to that of elastin. The excitation spectra for second harmonic generation (SHG) from purified collagen and excised dermis were also studied and were found to have similar spectral shapes.
Conclusion: This study, using the EEM spectroscopic approach, confirmed a previous imaging study inference that in the dermis, TPEF predominantly originates from elastic fibers, while SHG originates solely from collagen fibers. The EEM data and SHG excitation spectra obtained in this study can be used to guide the selection of excitation wavelengths for MPM applications in basic skin biology research and for clinical diagnosis.     

TPEF在大肠癌中的表达及其意义

目的 探讨含表皮生长因子和卵泡抑素结构域的跨膜蛋白(TPEF)在大肠癌中的表达水平及其意义。方法 采用SYBR Green I 荧光定量RT-PCR技术对40例大肠癌组织和相应正常大肠黏膜组织中的TPEF mRNA的表达进行检测,并分析其表达情况与临床病理资料的相关性。结果 癌组织中TPEF mRNA的相对表达量明显低于正常组织（P<0.05）;合并淋巴结转移的组织中表达量亦低于无淋巴结转移的组织(P<0.05)。结论 TPEF基因在大肠癌组织中低表达,与大肠癌的转移相关,可能作为大肠癌恶性潜能的检测指标之一。     

Methylation of the TPEF- and PAX6-promoters is increased in early bladder cancer and in normal mucosa adjacent to pTa tumours

OBJECTIVE

To evaluate CpG island methylation patterns of cancer‐associated genes for their applicability as molecular biomarkers for the detection of superficial bladder cancer and for the discrimination of invasive from noninvasive tumours.

PATIENTS AND METHODS

We analysed the methylation status of CpG islands in the promoter region of the cancer‐associated genes GSTP1, DAPK, MDR1, TPEF, PAX6, and TSLC1 in primary papillary bladder cancer specimens from 39 patients (pT1 10, pTis one, pTa 20, pT2 five). Tumour‐adjacent normal mucosa served as the control. The DNAs were bisulphite‐treated and submitted to methylation‐specific real‐time polymerase chain reactions.

RESULTS

Only TPEF and PAX6 had substantial CpG island methylation percentages. The TPEF‐ and PAX6‐promoters also had significantly higher methylation rates in tumour tissue compared with the normal tumour‐adjacent tissue. Interestingly, the methylation rates of the TPEF‐ and the PAX6‐promoter were higher in adjacent normal tissues from bladders with pTa then in those with pT1 tumours.

CONCLUSION

Our results shed a critical light on the hypothesis that CpG island hypermethylation of the GSTP1‐, DAPK‐, MDR1‐ and TSLC1‐promoter could represent molecular biomarkers for bladder cancer diagnosis and detection. However, methylated PAX6‐ or TPEF‐promoters could represent biomarkers for this disease. Additional studies are needed to evaluate whether methylation rates of these genes in normal bladder tissues are applicable as accessory markers for the tumour state or its invasive behaviour.     

TPEF和STAT1在大肠癌中的表达及其意义

目的：探讨含表皮生长因子和卵泡抑素结构域的跨膜蛋白（TPEF）、信号转导子和转录激活子1（STATI）在大肠癌中的表达水平及其意义。方法：采用SYBR GreenI荧光定量RT—PCR技术对50例大肠癌组织和相应正常大肠黏膜组织中的TPEFmRNA及STAT1 mRNA的表达进行检测,并分析其表达情况与临床病理资料的相关性。结果：癌组织中TPEFmRNA及STAT1 mRNA的相对表达水平明显低于相应正常组织（P〈0．05）;肿瘤TNM分期中T3／T4癌组织表达水平明显低于TI／T2癌组织（P〈0．05）;合并淋巴结转移的组织中表达水平亦低于无淋巴结转移者（P〈0．05）;TPEFmRNA与STAT1mRNA的表达水平呈正相关（r癌组织=0．537,r正常组织=0．851,P〈0．001）。结论：TPEF、STAT1基因在大肠癌组织中低表达,与大肠癌的转移相关,可作为大肠癌恶性潜能的检测指标之一。     

大肠癌患者TPEF基因甲基化状态分析

目的 分析大肠癌患者含表皮生长因子和卵泡抑索结构域的跨膜蛋白（TPEF）基因甲基化状态，寻找新的早期诊断大肠癌分子标志物。方法 常规方法提取大肠癌标本DNA，采用甲基化特异性PCR技术分析TPEF基因甲基化状态。结果 32例大肠癌手术切除标本，24例检测到异常TPEF基冈甲基化（75％），而10例正常组织标本无一例检测到异常甲基化。结论 大肠癌患者TPEF基因甲基化为一频繁发生事件，可望成为大肠癌早期诊断的一个标志物。     

42例大肠癌患者血浆TPEF异常甲基化及K-ras基因突变检测结果分析

目的:测定大肠癌患者外周血血浆游离DNA中含表皮生长因子和卵泡抑素结构域的跨膜蛋白(TPEF)基因启动子区异常甲基化率及K-ras基因12密码子突变率,评估联合检测此二个分子标记非损伤性筛选和诊断大肠癌的可行性.方法:提取大肠癌患者外周血血浆DNA,采用突变富集PCR-RFLP法检测K-ras基因突变,甲基化特异性PCR法检测TPEF基因甲基化异常.结果:42例大肠癌患者血浆标本中,27例检测到TPEF甲基化异常,16例检测到K-ras基因12密码子突变,联合二个分子标记,大肠癌患者诊断符合率为76.2%.结论:联合检测K-ras、TPEF基因异常将可能是一种非损伤性筛选和诊断大肠癌的有效方法.