Objective To explore the expression of tumor necrosis factor (TNF) mRNA, TNF and TNFR in the gallbladder mucosa which developed from hyperplasia, dysplasia to carcinoma, and to further discuss the relationship between TNF and pathogenesis of gallbladder carcinoma. Methods In situ hybridization and immunohistochemistry were used to determine TNF mRNA, TNF protein and TNFR protein expression in hyperplasia, dysplasia and carcinoma of gallbladder. Results ①No one of 20 cases of gallbladder hyperplasia was found to express TNF mRNA, while 4 of 20 (20%) cases of dysplasia and 18 of 20 (90%) cases of carcinoma were found to express TNF mRNA (P＜0.05). ②For the expression of TNF mRNA in mononuclear cells (MNC), positive staining was found in 15% of gallbladder hyperplasia, 85% of dysplasia and 90% of carcinoma, respectively (P＜0.05). The cell numbers of positive staining MNC were 4.85±1.50, 6.00±2.71 and 9.33±3.07, respectively (P＜0.05). ③In gallbladder carcinoma, the cell number of carcinoma and MNC with positive TNF mRNA expression was correlated with clinical stage (P＜0.05). The higher the clinical stage, the more the positive staining cell numbers. The positive staining cell numbers of carcinoma in stage Ⅰ-Ⅲ and Ⅳ-Ⅴ were 9.13±4.39 and 14.80±4.02, respectively (P＜0.01), and the positive staining cell numbers of MNC were 7.13±2.53 and 11.10±2.23, respectively (P＜0.05). ④The cell numbers of carcinoma and MNC with TNF mRNA expression increased with tumor size. In tumors with diameter over 2 cm and less than 2 cm, the positive staining cell numbers of carcinoma were 14.00±4.20 and 8.83±4.96, respectively (P＜0.05), and that of MNC were 10.50±2.54 and 7.00±2.83, respectively (P＜0.05). ⑤The region of TNF protein expression was similar to that of TNF mRNA, but TNF protein expression was more frequent and wider than that of TNF mRNA. ⑥The tumor necrosis factor receptor was expressed in tumoral vascular endothelial cells and MNC in all cases of carcinoma, but was negatively stained in mucosa epithelial cells and tumor cells of all cases. ⑦There was positive linear correlation in TNF mRNA between tumor cell and MNC (r=0.687, P＜0.01), same as that in TNF protein expression (r=0.742, P＜0.01); and there was positive linear correlation in tumor cell between TNF mRNA and TNF protein expression (r=0.847, P＜0.01), same as that in MNC (r=0.643, P＜0.01). Conclusion The TNF mRNA and TNF protein expression are increasing during the development of gallbladder mucosa epithelial from hyperplasia, dysplasia to carcinoma, and increasing with tumor stage. It suggests that TNF may contribute to carcinogenesis of gallbladder carcinoma induced by gallstone, and related to the progression of gallbladder carcinoma.
【Abstract】Objective To study the relationship of the expression of CD44v6 mRNA and nm23H1 mRNA with the clinical pathology parameter and prognosis of breast cancer, and to investigate the correlation of the expression of CD44v6 mRNA and nm23H1 mRNA. Methods In situ hybridization and CSA immunohistochemistry method were used to detect the expression of CD44v6 mRNA and nm23H1 mRNA in 94 cases of breast cancer. Results The positive expression of CD44v6 mRNA and the negative expression of nm23H1 mRNA were positively correlated with the grading, clinical stage, lymph node metastasis, and recurrence and prognosis of breast cancer. CD44v6 mRNA expression and nm23H1 mRNA were negatively correlated in breast cancer. Patients who had positive expression of CD44v6 mRNA and negative expression of nm23H1 mRNA had a higher lymph node metastatic rate and a lower survival rate. Conclusion Several genes were involved in the occurrence and development of breast cancer in which the expression of CD44v6 mRNA has synergistic action in negative regulation with that of nm23H1 mRNA. Combined detection of the expression of these two mRNA is helpful to judge the metastasis, recurrence and prognosis of breast cancer.
Objective To investigate the association of the expression of CD15 mRNA with the invasion and prognosis of hepatocellular carcinoma (HCC) and the expression of nm23H1 mRNA. Methods In situ hybridization and immunohistochemistry methods were used to detect the expression of CD15 mRNA and protein nm23H1 mRNA in HCC.Results In 99 cases of HCC, the positive rate of CD15 mRNA,its protein and nm23H1 mRNA were 38.4%, 36.4% and 76.8%, respectively. The expression of CD15 mRNA was consistent with its protein and negatively correlated with the expression of nm23H1 mRNA. The expression of CD15 mRNA and its protein, nm23H1 mRNA were associated with the invasiveness and metastasis of HCC and the prognosis of HCC patients. Conclusion The detection of CD15 expression could be a new pathological biology index to judge the metastasis and prognosis of HCC.
Effect of radical operation on expression of interleukin-2(IL-2)mRNA and production of IL-2 were markedly reduced preoperatively and four weeks after operation,expression of IL-2 mRNA significantly enhanced,but it was still lower than that in the normal group.Production of IL-2 nearly reached normal level,When PBL was activated by phytohemagglutinin(PHA),expresseion of IL-2 mRNA and production of IL-2 were much higher than that in non-activated PBL.These results suggested that expression of IL-2 were much higher than that in non-activated PBL.These results suggested that expression of IL-2 mRNA and production of IL-2 are dificient in gastric cancer patients,and radical surgery will help them to recover and they can also be improved through activation with PHA.
Objective It is reported that transforming growth factor β1 (TGF-β1) has the protective effects on the articular cartilage in osteoarthritis (OA). To investigate the significance of the expressions of matrix metalloproteinase 9 (MMP-9), TGF-β1 mRNA and corresponding proteins in OA. Methods The specimens of articular cartilage and synovium were collected from voluntary donators, including 60 cases of OA (experimental group) and 20 cases of traumatic amputation,cruciate l igament rupture, discoid cartilage injury, and menisci injury (normal control group). The pathological changes were observed by HE staining. MMP-9 and TGF-β1 protein expressions were detected by immunohistochemical technique, and the mRNA expressions of MMP-9 and TGF-β1 were detected through in situ hybridization technique; and their correlation was analysed. Results HE staining showed: shrinkage, necrosis, and irregular arrange of the articular chondrocytes, extracellular matrix fracture, hypertrophy and hyperplasia synovium, infiltration of lymphoid and mononuclear cells and prol iferation of many small blood vessels in the experimental group; regular arrangement of the articular chondrocytes, the homogeneously staining matrix, and synovial tissue without chronic inflammation and significant prol iferation in the normal control group. The mRNA and protein expressions of MMP-9 and TGF-β1 were positive in 2 groups. The positive-stained cells included chondrocytes, synovial l ining cells, and vascular endothel ial cells, fibroblasts, and inflammatory infiltrated cells in subsynovial layer. The expressions of mRNA and corresponding protein of MMP-9 and TGF-β1 in the experimental group were significantly higher than those in the normal control group (P lt; 0.01). There was a positive correlation between MMP-9 mRNA and protein expression (r=0.924, P=0.000), and between TGF-β1 mRNA and protein expression (r=0.941, P=0.000) in the experimental group. There was a negative correlation between the expression of MMP-9 protein and TGF-β1 protein (r= — 0.762, P=0.000), and between the expression of MMP-9 mRNA and TGF-β1 mRNA (r= — 0.681, P=0.000) in the experimental group. Conclusion The higher expression of TGF-β1 can protect articular cartilage by down-regulating the expression of MMP-9 of chondrocytes and synoviocytes in OA, which may delay the biological behavior of OA such as occurrence and progress, etc.
Objective To investigate the changes in the expression level of PDGF in the bone callus of rats with femoral fracture and brain injury to explore the effect of brain injury on the fracture heal ing and the related mechanism. Methods Sixty-four 12-week-old SD rats weighing (356 ± 25) g were randomly divided into 8 groups with 8 rats in each. The rats in groups A1, B1, C1 and D1 had a femoral fracture and a brain injury for 1, 2, 3 and 4 weeks, respectively; the rats in groups A2, B2, C2 and D2 had a mere fracture without a brain injury for 1, 2, 3 and 4 weeks, respectively. After the CR films were taken, the bone callus was obtained 1, 2, 3 and 4 weeks after operation, respectively. Then, the bone callus and its histology were examined by HE staining, the expressions and changes in the level of PDGF were examined by the immunohistochemical staining, and the level of PDGF mRNA was measured by in situ hybridization. Results The CR films showed that the callus formation in the A1-D1 groups was earl ier and greater than that in the A2-D2 groups at the same time point. The HE staining indicated that more fibroblasts and early-stage chondrocytes were found in group A1; some fibroblasts in the fracture interspace and few early-stage chondrocytes were found in group A2; some newly-formed trabecular bones were found at the end of the fracture in group B1; but no trabecular bone formation was found in group B2; woven bone formation and a few chondrocytes between trabecular bones in the fracture interspace were found in group C1; only a few trabecular bones in the fracture interspace were found in group C2；woven bones turned to lamellar bones in group D1；and more immature trabecular bones in the fracture interspace were found in group D2. The positive expression of PDGF and PDGF mRNA was b in the cytoplasms of fibroblasts, mesenchymal cells, vascular endothel ial cells, early-stage chondrocytes, osteoblasts and osteoclasts. The percentage of the positive cells for PDGF and PDGF mRNA in the callus was significantly higher in groups A1-D1 than in groups A2-D2 at the same time point (P lt; 0.05). Conclusion Brain injury can promote the fracture heal ing process, which is probably related to an increase in the expression level of PDGF after the brain injury.
Objective To resolve the tough problem of how to observe the growing cells in an opaque vector. Methods The urethral epithelial cells from a young male New Zealand rabbit were inoculated, and were primarily cultured in vitro and subcultured for 3 passages. Then, the urethralepithelial cells were cultured in the collagen chitosan complex for 3, 7, 14 and 21 days. The cells were dyed with 6-carboxyfluorescein diacetateacetoxymethyl ester and propidium iodine, respectively. Then, Interactive Laser Cytometer was used to detect the growing cells. Results The urethral epithelial cells grew and proliferated very well in the collagen chitosan complex vector. After the urethral epithelial cells grew in the collagen-chitosan complex vector for 3 and 7 days, the fluorescent density amount of the surviving cells were(1.09±0.13)×10.8 and (2.04±0.13)×10.8, respectively. However, after 14and 21 days, the fluorescent density amount of the surviving cells was (0.55± 0.09)×10.8 and (0.47±0.03)×108, respectively. There was a significant difference when compared with the amount of the surviving cells at 3 and 7 days（P＜0.05）.Conclusion Using Interactive Laser Cytometer for measurement of the green and red fluorescent densities of different waves, the activity of the cultured urethral epithelial cells in vitro can be rapidlymeasured with the in situ quantitation method. This method solves a difficult problem of observing the growing cells in an opaque vector. The dynamic growing state of the engineering tissues can be observed.
For observation of the change of transforming growth factor-beta 1 (TGF-beta 1) gene expression in the process of skin wound healing, the following experiments were performed. Sixteen Wistar rats were chosen. At each side of the rat’s back, a 1 cm x 1.5 cm middle-thick skin wound was made. After 3, 6, 9 and 12 days, the specimens were taken from the wounds. For each specimen, half of it was used for RNA extraction, and underwent dot blotting; and the other half was frozen immediately and underwent in situ hybridization. The probes were dig-labeled PDGF-BB cDNA probe and TGF-beta 1 probe. The results showed that TGF-beta 1 gene was expressed mainly in fibroblast, epithelial cell and capillary endothelial cell. The peak of TGF-beta 1 mRNA content was in the 6th day postoperatively. After that, the content of TGF-beta 1 decreased to normal. It was suggested that TGF-beta 1 gene expression was in close relation with healing process. TGF-beta 1 may play an important regulatory role in the skin wound healing.
ObjectiveTo investigate the expression and relation of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in rats with diabetic retinopathy.MethodFifty-five Wistar rats were randomly divided into the control group (10 rats), and 1, 3, and 5-month-diabetes group (15 rats in each diabetes group), and the diabetic models were set up. The expressions of VEGF and bFGF were detected by situ hybridation and immunohistochemistry on retinal paraffin sections.ResultsThe results of situ hybridation showed that expression of bFGF was found in 3-month-deatbtes group with the percentage of 77.8%, and 88.9% in 5-month-deatbtes group; the positive expression of VEGF was not found in 3-month-deatbtes group but in 5-month-deatbtes group with the percentage of 66.7%. Immunohistochemistry indicated that the positive expression of bFGF started in 3-month-deatbtes group with the percentage of 55.6%, and 88.9% in 5-month-deatbtes group; the percentage of the expression of VEGF was 33.3% in 3-month-deatbtes group and 88.9% in 5-month-deatbtes group.ConclusionThe expression of VEGF occurs after the expression of bFGF in rats with DR.(Chin J Ocul Fundus Dis, 2005,21:37-40)
Objective To investigate the influnce of L-arginine (L-Arg) and L-nitro-arginine-methyl-ester(L-NAME) to purified retinal ganglion cells(RGCs) apoptosis of rats cultured in different consistencies of L-Arg and L-NAME. Method RGCs from Sprague Dawley (SD) neonatal rats(postnatal 1~5 day) were cultured in assimilative culture solution in vitro and RGCs were purified by Thy1.1 with sheep anti rat FITC monoclonal antibody. RGCs were cultured in different consistencies of L-Arg and L-NAME: 1×10-6, 1×10-5,1×10-4, 1×10-3, 1×10-2 and 1×10-1 mol/L for 24 hours and 48 hours, respectively. The changes of bcl-2, bax and p53 mRNA in RGCs in different consistencies of L-Arg and L-NAME were demonstrated qualitatively and quantitatively by in situ hybridization, and their apoptosis were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) method, respectively. Results After 24 hours in vitro, the purification rate of RGCs in the experiment arrived at 97 %. After 48 hours, there were a few apoptotic cells expression in the control group. Apoptotic cells expression in L-Arg≥1×10-3 mol/L and L-NAME≥1×10-1 mol/L groups increased that had a significant difference with the control group (Plt;0.05). In the group of L-Arg≥1×10-3 mol/L and L-NAME≥1×10-1 mol/L, the expression of bcl-2 mRNA in RGCs became weaker and weaker as the consistencies were increased, but the expression of bax and p53 mRNA in RGCs became higher and higher and had a significant difference with control group (Plt;0.05). Conclusion Lower concentration of L-Arg can promote the growth of purified RGCs in vitro and higher concentration of L-Arg can promote the apoptosis of RGCs. (Chin J Ocul Fundus Dis, 2002, 18: 137-139)