Objective To investigate the clinical significance of c-myc、Bcl-2 protein expression, DNA ploidy and their relationship in breast cancer. MethodsWT5”BZ The expression of c-myc、Bcl-2 protein in 146 breast tumor tissues was examined by using immunohistochemical methods(S-P)and DNA ploidy in 72 cases of breast carcinoma by flow cytometry. ResultsKG1 Nuclear expression of c-myc protein was detected in 9 6% of tumors, and it was related to the DNA aneuploidy and prognosis. Cytoplasmic expression of c-myc protein was present in 91 8% of the carcinomas. Moderate to strong c-myc protein expression in cytoplasmic was associated with the positive status of ER. Bcl-2 protein was positive in 78 8% of cancers. Overexpression of Bcl-2 protein was associated with ER(+) status and the lack of axillary lymph node metastasis. DNA aneuploidy was found in 51 4% of 72 cancers, it was related to nuclear expression of c-myc protein and prognosis.WT5”HZConclusion Nuclear expression of c-myc protein and DNA ploidy are important prognostic factors.

BACKGROUND:Ligament flavum hypertrophy is the main cause of lumbar spinal stenosis,which is the result of multiple pathological factors working together.Currently,the molecular mechanism and pathway of action of ligament flavum hypertrophy are unclear,and there is a lack of effective non-surgical treatment options. OBJECTIVE:To investigate the molecular mechanisms of ligament flavum hypertrophy using methylation sequencing and transcriptome integration analysis methods. METHODS:Five normal ligament flavum tissue samples and five hypertrophic ligament flavum tissue samples were collected.Abnormal methylation sites and methylation status were recorded by methylation sequencing and abnormally expressed genes were recorded by transcriptome integration analysis.The genes that showed a negative correlation between methylation level and expression level at the intersection of the two were selected.GO and KEGG enrichment analyses were used to study the major functional pathways and molecular functions of differentially expressed genes.Key genes regulating ligamentum flavum hypertrophy were screened using protein-protein interaction analysis. RESULTS AND CONCLUSION:Methylation sequencing of the two groups of the ligament flavum showed 37 173 hypermethylation sites and 10 583 low methylation sites.Transcriptome integration analysis found 720 abnormally expressed genes,of which 463 were upregulated and 257 were down-regulated.There were 383 overlapping genes,of which 192 genes showed a negative correlation between the methylation level and the expression level.GO functional pathway analysis results showed that molecular function was enriched to 10 terms,cellular component was enriched to 15 terms,and biological process(BP)was enriched to 30 terms.The results of KEGG pathway enrichment analysis showed that 192 genes were mainly enriched to 9 pathways,such as PI3K-Akt signaling pathway,Rap1 signaling pathway,and focal adhesion signaling pathway.The protein-protein interaction analysis identified five genes,PPARG,EGFR,CNR1,TNF and COL11A2,which may be the key genes that regulate ligamentum flavum hypertrophy,and they can influence the occurrence and development of ligamentum flavum hypertrophy mainly through the regulation of tissue fibrosis,cell proliferation and differentiation,inflammatory factor levels,and collagen fiber expression.

Objective: To investigate the effect of transforming growth factor β 1 (TGF-β 1) induced proliferation of ligamentum flavum cells and ligamentum flavum hypertrophy and its effect on connective tissue growth factor (CTGF) expression. Methods: The ligamentum flavum tissue in lumbar intervertebral disc herniation was extracted and the ligamentum flavum cells were isolated and cultured by collagenase pre-digestion method. Morphological observation, immunofluorescence staining observation, and MTT assay were used for cell identification. The 3rd generation ligamentum flavum cells were divided into 5 groups. The cells of groups A, B, C, and D were respectively sealed with 3 ng/mL TGF-β 1, 50 ng/mL CTGF, 3 ng/mL TGF-β 1+CTGF neutralizing antibody, and 50 ng/mL CTGF+CTGF neutralizing antibody. Serum free DMEM was added to group E as the control. MTT assay was used to detect the effects of TGF-β 1 and CTGF on the proliferation of ligamentum flavum cells. Western blot was used to detect the expression of CTGF protein. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of collagen type Ⅰ, collagen type Ⅲ, and CTGF genes. Results: The morphological diversity of cultured ligamentum flavum cells showed typical phenotype of ligamentum flavum fibroblasts; all cells expressed collagen type Ⅰ and vimentin, and some cells expressed collagen type Ⅲ; MTT identification showed that with the prolongation of culture time, the absorbance ( A) value of each generation of cells increased gradually, and the A value of the same generation of cells at each time point was significantly different ( P<0.05), there was no significant difference in A value between the cells of each generation at the same time point ( P>0.05). After cultured for 24 hours, MTT assay showed that the A value of cells in groups A and B was significantly higher than that of group E ( P<0.05). After adding CTGF neutralizing antibody, the A value of cells in groups C and D decreased, but it was still higher than that of group E ( P<0.05). There were also significant differences among groups A, C and groups B, D ( P<0.05). Western blot analysis showed that the relative expression of CTGF protein in groups A and B was significantly higher than that in group E ( P<0.05), while the relative expression of CTGF protein in groups C and D was significantly lower than that in group E ( P<0.05), and the difference between groups A, C and groups B, D was also significant ( P<0.05). qRT-PCR detection showed that the mRNA relative expression of CTGF, collagen type Ⅰ, and collagen type Ⅲ in group A was significantly higher than that in group E ( P<0.05). After adding neutralizing antibody, the mRNA relative expression of genes in group C was inhibited and were significantly lower than that in group A, but still significantly higher than that in group E ( P<0.05). The mRNA relative expressions of collagen type Ⅰ and collagen type Ⅲ in group B was significantly higher than that in group E ( P<0.05), but the mRNA relative expression of CTGF was not significantly different from that in group E ( P>0.05); after neutralizing antibody was added, the mRNA relative expression of collagen type Ⅰ and collagen type Ⅲ in group D was inhibited and was significantly lower than that in group B, but still significantly higher than that in group E ( P<0.05); there was no significant difference in the mRNA relative expression of CTGF between group D and groups B, E ( P>0.05). Conclusion: TGF-β 1 can promote CTGF, collagen typeⅠ, collagen type Ⅲ gene level and protein expression in ligamentum flavum cells, and TGF-β 1 can synergistically promote proliferation of ligamentum flavum cells through CTGF.

Objective: To investigate the mechanism of p38 mitogen activated protein kinase (MAPK) signaling pathway in regulating the hyperplasia and hypertrophy of human lumbar ligamentum flavum via transforming growth factor β 1 (TGF-β 1)/connective tissue growth factor (CTGF). Methods: The lumbar ligamentum flavum tissue taken from patient with lumbar intervertebral disc herniation was isolated by collagenase-predigested explant cultures. The ligamentum flavum cells were treated with the extracellular regulated protein kinase pathway blocker PD98059, c-Jun N-terminal kinase pathway blocker SP600125, and p38 pathway blocker SB203580, and then the mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescence quantitative PCR (qRT-PCR). The ligamentum flavum cells were divided into 4 groups, and transfected with small interfering RNA (siRNA), p38 siRNA, siRNA+3 ng/mL TGF-β 1, and p38 siRNA+3 ng/mL TGF-β 1 in groups A, B, C, and D, respectively. After 24 hours of transfection, immunofluorescence staining was performed to observe the expressions of p38 and phosphorylation p38 (p-p38); the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ in each group were detected by qRT-PCR; the protein expression of CTGF in each group was detected by Western blot. Results: p38 pathway blocker SB203580 could significantly reduce the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ ( P<0.05). After 24 hours of transfection, immunofluorescence staining showed positive staining with p38 and p-p38 expressions in groups A, C, and D and negative staining in group B. Compared with group A, the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ and relative protein expression of CTGF in group B decreased significantly ( P<0.05), while those in groups C and D increased significantly ( P<0.05); and those indicators significantly increased in group C than in group D ( P<0.05). Conclusion: TGF-β 1/CTGF based on the p38 MAPK signaling pathway play an important role in the occurance and development of hypertrophy of human lumbar ligamentum flavum.

OBJECTIVETo study the influence of heat-induced epitope retrieval (HIER) on endogenous avidin-binding activity (EABA) and to establish an effective way to block EABA in immunohistochemistry.

METHODSSystematically screening EABA in 164 (679 samples) formalin-fixed and paraffin-embedded human tissues including 76 (102 samples) normal tissues and 88 (577 samples) tumor tissues as well as 4 (80 samples) formalin-fixed and paraffin-embedded rat normal tissues using tissue array (tissue chip), HIER, immunohistochemistry and egg white solution blocking. In addition, EABA was also examined in 9 (15 samples) human frozen tissues.

RESULTS(1) EABA was detected in frozen tissues. (2) No staining for EABA was seen in formalin-fixed and paraffin-embedded tissues. (3) EABA was revealed after the tissues treated with microwave HIER. (4) The density of signal for EABA was variable from tissue to tissue and cell to cell. (5) The signals of EABA expressed in scatter or diffuse in tissues and in granular form in cytoplasm. (6) EABA was found in a wide range of epithelial tissues, especially in gland epithelia of normal and tumor tissues. These included kidney, adrenal cortex, liver, C cells of thyroid gland, oxyphil cells of parathyroid, fundal gland of stomach, sebaceous gland of skin, duct of salivary; oncocytoma and papillary adenocarcinoma of kidney and thyroid gland, adenolymphoma of parotid, carcinoma of liver cell, adenocarcinoma of stomach, colon, prostate, gall bladder and endometrium, and so on. (7) EABA was easier revealed by higher pH value buffer (EGTA pH 9.0) than that with lower pH value (EDTA pH 8.0 and citrate pH 6.0). (8) The revealed EABA could be effectively blocked using 20% egg white solution.

CONCLUSIONSHIER could unmask EABA in formalin-fixed and paraffin-embedded tissues. The unmasked EABA present in a wide range of human normal and tumor tissues as well as in rat normal tissues. The EABA could influence routine immunohistochemistry staining when using (strept)avidin-horseradish peroxidase detective system. The egg white solution could effectively block EABA and eliminate the influence of EABA on immunohistochemistry.

Animals ; Avidin ; antagonists & inhibitors ; metabolism ; Biotin ; metabolism ; Cells, Cultured ; Egg Proteins ; pharmacology ; Epithelial Cells ; metabolism ; Epitopes ; Female ; Hot Temperature ; Humans ; Immunohistochemistry ; Male ; Neoplasms ; metabolism ; pathology ; Rats