Assessment of the diagnostic value and prognosis of different detection markers in endocervical adenocarcinoma / 中华肿瘤杂志

Objective: To study the diagnostic value of different detection markers in histological categories of endocervical adenocarcinoma (ECA), and their assessment of patient prognosis. Methods: A retrospective study of 54 patients with ECA in the Cancer Hospital, Chinese Academy of Medical Sciences from 2005-2010 were performed. The cases of ECA were classified into two categories, namely human papillomavirus-associated adenocarcinoma (HPVA) and non-human papillomavirus-associated adenocarcinoma (NHPVA), based on the 2018 international endocervical adenocarcinoma criteria and classification (IECC). To detect HR-HPV DNA and HR-HPV E6/E7 mRNA in all patients, we used whole tissue section PCR (WTS-PCR) and HPV E6/E7 mRNA in situ hybridization (ISH) techniques, respectively. Additionally, we performed Laser microdissection PCR (LCM-PCR) on 15 randomly selected HR-HPV DNA-positive cases to confirm the accuracy of the above two assays in identifying ECA lesions. Receiver operating characteristic (ROC) curves were used to analyze the efficacy of markers to identify HPVA and NHPVA. Univariate and multifactorial Cox proportional risk model regression analyses were performed for factors influencing ECA patients' prognoses. Results: Of the 54 patients with ECA, 30 were HPVA and 24 were NHPVA. A total of 96.7% (29/30) of HPVA patients were positive for HR-HPV DNA and 63.3% (19/30) for HR-HPV E6/E7 mRNA, and 33.3% (8/24) of NHPVA patients were positive for HR-HPV DNA and HR-HPV E6/E7 mRNA was not detected (0/24), and the differences were statistically significant (P<0.001). LCM-PCR showed that five patients were positive for HR-HPV DNA in the area of glandular epithelial lesions and others were negative, which was in good agreement with the E6/E7 mRNA ISH assay (Kappa=0.842, P=0.001). Analysis of the ROC results showed that the AUC of HR-HPV DNA, HR-HPV E6/E7 mRNA, and p16 to identify HPVA and NHPVA were 0.817, 0.817, and 0.692, respectively, with sensitivities of 96.7%, 63.3%, and 80.0% and specificities of 66.7%, 100.0%, and 58.3%, respectively. HR-HPV DNA identified HPVA and NHPVA with higher AUC than p16 (P=0.044). The difference in survival rates between HR-HPV DNA (WTS-PCR assay) positive and negative patients was not statistically significant (P=0.156), while the difference in survival rates between HR-HPV E6/E7 mRNA positive and negative patients, and p16 positive and negative patients were statistically significant (both P<0.05). Multifactorial Cox regression analysis showed that International Federation of Obstetrics and Gynecology (FIGO) staging (HR=19.875, 95% CI: 1.526-258.833) and parametrial involvement (HR=14.032, 95% CI: 1.281-153.761) were independent factors influencing the prognosis of patients with ECA. Conclusions: HR-HPV E6/E7 mRNA is more reflective of HPV infection in ECA tissue. The efficacy of HR-HPV E6/E7 mRNA and HR-HPV DNA (WTS-PCR assay) in identifying HPVA and NHPVA is similar, with higher sensitivity of HR-HPV DNA and higher specificity of HR-HPV E6/E7 mRNA. HR-HPV DNA is more effective than p16 in identifying HPVA and NHPVA. HPV E6/E7 mRNA and p16 positive ECA patients have better survival rates than negative.

Analysis of Detection of Mixed Semen Stains with Different Immunological Test Methods and Case Applications / 法医学杂志

Objective To perform the separation and confirmation of mixed semen stains with immunological test method, and find a more effective method for the detection of mixed semen stains. Methods The semens of three volunteers were mixed. The mixed semen stains were processed and tested with prostate-specific antigen （PSA） colloidal gold immunoassay strip method, immunomagnetic beads method and laser capture microdissection, respectively. Statistics of the results of STR were gathered and compared with those of a single semen stain. Results After PSA colloidal gold immunoassay strip method testing, the samples showed a purplish red line in the test area and the control area. The results obtained with the immunomagnetic beads method showed a more complete and effective short tandem repeat （STR） sequence. The mixed semen stains were processed with laser capture microdissection and low volume amplified. The results were summarized and superimposed to obtain a complete single typing, which matched the single semen stain typing, with a typing success rate of 84.00%. Single suspect Y-STR typing was obtained with the application of the method above in actual cases, which provided evidence basis for rapid solving of the case. Conclusion The combination of PSA colloidal gold immunoassay strip method, immunomagnetic beads method and laser capture microdissection can be used to separate and confirm the mixed semen stains.

Methods for mammalian single cell research - a review / 生物工程学报

Basic research in life science and medicine has dug into single cell level in recent years. Single-cell analysis offers to understand life from diverse perspectives and is used to profile cell heterogeneity to investigate mechanism of diseases. Single cell technologies have also found applications in forensic medicine and clinical reproductive medicine, while the techniques are rapidly evolving and have become more and more sophisticated. In this article, we reviewed various single cell isolation techniques and their pros and cons, including manual cell picking, laser capture microdissection and microfluidics, as well as analysis methods for DNA, RNA and protein in single cell. In addition, we summarized major up-to-date single cell research achievements and their potential applications.

Comparative proteome research of epithelial cells and stroma from invasive ductal carcionma patients / 中国药理学通报

Aim To study the difference of proteins expression of epithelial cells and stroma of breast canc-er patients.Methods Formalin-fixed paraffin-embed-ded tissue samples from invasive ductal carcionma pa-tients were stained by improved HE staining methods. And the neoplastic epithelium and stroma were micro-dissected using laser capture microdissection and ana-lysed by mass spectrometry.Results Total 43 1 differ-ent proteins were detected by mass spectrometry and there were respectively 384 and 298 different kinds of proteins identified in epithelial cells and stroma.A-mong them,25 1 proteins were commonly expressed in two samples,while the contents of 69 and 60 proteins in stroma were respectively higher or lower than the ones in epithelial cells.The expression level and local-ization of proteins identified in epithelial cells and stro-ma were associated with their roles in development and progression of tumor cells.Conclusions The differen-tial expression proteins between epithelial cells and stroma of invasive ductal carcionma patients may func-tion as biomarkers for breast cancer screenning,diag-nosis and prognosis.

Study of the microsatellite instability involved in human gastric carcinoma and precancerous lesion / 肿瘤研究与临床

Objective To explore whether microsatellite instability (MSI) is exsist in human gastric carcinoma and precancerous lesion.Methods The MSI was detected in gastric carcinoma and precancerous lesion by tissue laser capture microdissection and denaturing high performance liquid chromatogragh (DHPLC).Results MSI was found in 10.5 ％ intestinal metaplasia,21.12 ％ gastric hyperplasia and 43.75 ％gastric carcinoma respectively.In contrast,there was no microsatellite instability in normol gastric mucosa.It displayed accumulation and alteration from normal to cancerous tissues.Conclusion Pure tumor samples are obtained from tissue laser capture microdissection which could avoid the heteroplasmy of tissue.DHPLC is a high sensitive,fast and semiautomated method for identifying MSI.This experiment showed an increasing tendency of MSI incidence from precancerous lesion to gastric carcinoma.MSI could be used as a marker to predicate the occurrence of gastric carcinoma.

Expression Patterns of Superoxide Dismutase Genes in the Stage-specific Seminiferous Tubules of Mice Excised by a Laser Capture Microdissection / 한국실험동물학회지

Spermatogenesis is a particularly difficult process to study the unique multiple cellular associations within the seminiferous epithelium. Laser capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. The superoxide dismutase (SOD) is the first and most important enzyme of antioxidant defense systems against superoxide anion. The aim of this study was to investigate the quantitative changes of SOD gene expression according to the spermatogenic cycle in mouse testes using LCM and real-time polymerase chain reaction (PCR) techniques. Frozen sections (10 micrometer) were obtained from the testes of 8-weeks-old ICR mice. LCM was used to capture all cells in cross-sectioned seminiferous tubules which were grouped into stages I-V, VII-VIII, and IX-XI. The expression level of cytoplasmic Cu, Zn-SOD (SOD1) mRNA was remarkably higher than those of mitochondrial Mn-SOD (SOD2) and extracellular Cu, Zn-SOD (SOD3) mRNAs in mouse testes. During spermatogenesis, the expressions of SOD1 and SOD2 mRNAs were highest on stages I-V, began to decrease after stage VII, and showed a lowest level on stage IX-XI. However, the expression of SOD3 mRNA was highest on stages VII-VIII. These findings suggest that the subtypes of SOD are expressed differentially in mouse testes during spermatogenesis.

Pathology and gene expression of the sciatic nerve after non-freezing cold injury / 中华物理医学与康复杂志

Objective To investigate the morphological changes in the sciatic nerve and the dorsal root ganglions (DRGs) and also gene expression in DRGs after non-freezing cold injury, and to explore the molecular mechanism of peripheral nerve cold injury and regeneration. Methods Twenty-four male Wistar rats were used. The sciatic nerve on one side was cooled to 4℃ for 2 h, and the sciatic nerve on the opposite side was exposed, but without cooling. Sciatic nerves and L4, L5 and L6 DRGs from both sides were harvested at the 1st, 2nd and 3rd week after cooling. Any pathological changes were observed using light and electron microscopy. Laser capture microdissection (LCM) was used to investigate the DRG neurons' gene expression. The array result was verified with RT-PCR for eight genes. Results Large fiber degeneration was obvious by the 7th day after cooling. Myelinated fiber regeneration had begun by the 14th day, so this time was chosen to explore the neurons' gene expression. Ninety-six genes and expressed sequence tags (ESTs) were up-regulated greater than 2 fold. Their proteins' functions were classified as adaptive response to external stimulus, apoptosis regulation, cell adhesion, immune and inflammation response,nerve regeneration, pain associated molecules, microtubule cytoskeleton, ion-channels, neurotransmitters and receptors, and neuropeptides. Conclusions A complex molecular mechanism is involved in cold injury and regeneration of the sciatic nerve, and many genes are involved. Large scale microarray analysis is a potent means to screen out related genes, thus suggesting future repair strategies.

Comparative proteome analysis of laser capture microdissection for purified primary tumor and lymph node metastatic tumor in human lung squamous carcinoma / 中南大学学报(医学版)

Objective To search for lymph node metastasis-associated proteins in human lung squamous carcinoma (hLSC).Methods Laser capture microdissection (LCM) was used to purify the target cells from lung primary tumor and matched lymph node metastatic tumor in hLSC. Two-dimensional gel electrophoresis (2-DE) was performed to separate the total proteins of microdissected tumor cells from lung primary tumor and matched lymph node metastatic tumor. PDQuest software was applied to analyze 2-DE images. Differential protein spots between the two types of tissues were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). The expression of Rho-GDIα, one of the differential proteins, in the microdissected lung primary tumor cells (LPTC) and matched lymph node metastatic tumor cells (LNMTC) was detected by Western blot. Results In the present study, 2-DE patterns of microdissected LPTC and LNMTC were established, and 22 differential proteins in the above two tissues were identified, of which 14 were down-regulated in LNMTC and 8 were up-regulated in LNMTC.Conclusion The 22 differential proteins may play some roles in the process of lymph node metastasis in hLSC, and the data provide new clues for metastasis-associated biomarker screen and mechanism of hLSC.

Quantitative Proteomics Analysis of LCM Purified Stroma of Nasopharyngeal Carcinoma and Normal Nasopharyngeal Mucosa / 生物化学与生物物理进展

The mechanism of how stroma plays an important role in tumor carcinogenesis is now a hotspot. To delineate the features of stromal protein between nasopharyngeal carcinoma (NPC) and normal nasopharyngeal mucosa(NNM), laser capture microdissection (LCM) was performed to purify stromal cells from the NPC and NNM, respectively. The protein expressed profiles of the stroma of NPC and NNM were compared using fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) and 34 differential protein spots between tumor stroma and normal stroma were chosen to be identified by mass spectrometry (MS). A total of 20 differential proteins were identified, and three differential proteins (CapG, L-plastin and S100A9) were selectively further validated by Western blotting and immunohistochemical analysis to confirm the results of 2D-DIGE. 2D-DIGE patterns of the stroma of NPC and NNM were established for the first time, the results suggested that differentially expressed proteins in the stroma of NPC and NNM may be useful for understanding the relationship between NPC cells and their surrounding microenvironment. Further studying of these proteins will be helpful to elucidate the mechanisms of NPC carcinogenesis and provide new thoughts on therapy of NPC through stroma.

Comparative Proteomics Analysis of LCM Purified Nasopharyngeal Carcinoma and Normal Nasopharyngeal Epithelial Tissue / 生物化学与生物物理进展

To search for nasopharyngeal carcinoma (NPC) biomarkers,laser capture microdissection (LCM) was used to purify the target cells from NPC and normal nasopharyngeal epithelial tissues (NNET). Two-dimensional gel electrophoresis (2-DE) was performed to separate the total proteins of microdissected NPC and NNEC,PDQuest software was applied to analyze 2-DE images,and the differential protein spots between the two types of tissues were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. The expression of cytokeratin 8(CK8),one of the differential proteins,in the microdissected NPC and NNEC as well as 4 NPC cell lines with different differentiated degrees and/or metastatic potentials was detected by Western blot. Immunohistochemistry was also used to detect the expression of CK8 in paraffin-embedded tissues including 63 cases of primary NPC,28 cases of NNET and 20 cases of cervical lymphonode metastasis. In the present study,2-DE patterns of microdissected NPC and NNEC were established,and 29 differential proteins in the above two tissues were identified,of which 15 only expressed or up-regulated in NPC and 14 only expressed or up-regulated in NNET. The expression level of differential protein CK8 between the NPC and NNET was selectively confirmed,and was found to be related to the differentiation and/or metastasis of NPC cell lines. Significant down-regulation of CK8 was observed in NPC compared with NNET,and significant up-regulation of CK8 was also observed in lymphonode metastasis compared with primary NPC. The data suggest that CK8 may be related to the differentiation and lymphonode metastasis of NPC,and may serve as molecular biomarkers for metastasis and differentiation of NPC.

Inhibitory effect of 5-aza-2'-deoxycytidine on human gastric cancer xenografts in nude mice / 上海第二医科大学学报

Objective To observe the effect of 5-aza-2'-deoxycytidine(5-aza-CdR)on the normal epithelial specific-1 gene(NES1)and the growth of human gastric cancer xenografts in nude mice,and to explore the possible anti-tumor mechanisms and search for new treatment for gastric cancer.Methods Human gastric caner xenograft model in nude mice was established and treated with 5-aza-CdR.The growth of xenografts in nude mice was observed,and the status of methylation and protein expression of NES1 gene were detected by MSP and immunohistochemistry respectirely.Results After treatment with 5-aza-CdR,the growth of the xenografts in nude mice was greatly inhibited(P

Application of laser capture microdissection to capture pure cells in lung cancer and their paired normal lung tissues / 西安交通大学学报(医学版)

Objective Microheterogeneity is an important problem in molecularly biological and proteomic research on malignancies.The goal of this study was to investigate the method to purify targeted cells in lung cancer and its paired normal tissues for proteomic study on lung cancer applying laser capture microdissection(LCM).Methods LCM technique was emploged to obtain the cells of lung cancer tissues and their paired normal tissues from frozen sections.Results LCM can be applied to quickly and precisely obtain pure targeted cells subgroup or single cell without interstitium under the microscope.Conclusion LCM can tackle the problem of tissue heterogeneity in molecular analysis successfully.These results indicate that LCM has a potential as a tool in lung cancer proteomic research.

Global Analysis of Estrogen-Regulated Genes in Mouse Uterus using cDNA Microarray and Laser Capture Microdissection / 대한불임학회지

No abstract available.

Global Analysis of Estrogen-Regulated Genes in Mouse Uterus using cDNA Microarray and Laser Capture Microdissection / 대한불임학회지

No abstract available.

Laser capture microdissection combined with functional grouping cDNA microarray analysis in cerebral ischemia / 西安交通大学学报(医学版)

Objective To develop a method to analyze functional grouping cDNA microarray gene expression in microvessels and neurons from rat brain using laser capture microdissection(LCM). Methods Microvessels and neurons were captured using the PixCell ⅡLCM instrument. The total RNA was extracted from the LCM samples according to the manufacturer's protocol of RNAqueous-Micro kit. The total RNA were processed for one round of T7-based RNA amplification; cDNA probe were synthesized using gene-specific primer and labeled for cDNA microarray analysis. Results Amplified RNA from microvessels and neurons allowed us to measure 96 gene expression in functional grouping cDNA microarray. Conclusion LCM allowed us to harvest pure microvessels and neurons from their native tissue environment, combined with methods of T7 RNA amplification and gene-specific primer amplification so that we can analyze functional grouping cDNA profiling in LCM samples.