Objective: To apply chromogenic in situ hybridization (CISH for detection of HER2 gene amplification in breast cancer tissues and to discuss some modifications of the CISH method. Methods: HER2 gene amplification was detected by CISH in 60 breast cancer specimens with an immunohistochemical score over 2+. The correlation between the results of IHC and CISH was analyzed. Our experience in CISH manipulation was summarized and optimization to CISH was discussed. Results: CISH identified gene amplification in 91% (40/44) specimens with an IHC score of 3+ and in 50% (8/16) specimens with an IHC score of 2+. The total concordance rate between IHC and CISH was 80% (48/60, P<0.01). The thickness of sections should be controlled within 4-5 μm; the denaturation should be complete; and the post-hybridization washing temperature and time were also very important and the temperature should be controlled at 70-75 °C. The dyeing time of hematoxylin should also be restrictedly controlled. Positive control should be set up in the experiment for high quality of the experiment. Conclusion: CISH has high concordance rate with IHC in examining HER2 amplification and it may be a new method for detection of HER2 gene. The thickness of the sections, the post hybridization washing temperature and time, and the time of hematoxylin dyeing should be strictly controlled.

Objective: To verify whether hot PBS washing after the enzymatic reaction between tyramine and horseradish peroxidase can improve the sensitivity and specificity of catalyzed signal amplification (CSA). Methods: Using 7 different types of primary antibodies and 2 tumor microarray system, we carried out En Vision, LSAB, Standard CSA, and modified CSA staining and compared their sensitivities and specificities. Results: The modified CSA method was the most sensitive one among the 4 methods, followed by standard CSA, En Vision, and LsAB method. The sensitivity of modified CSA method was 2-3 times higher than that of standard CSA. The 4 methods had similar background staining and had exact localization of cells. Conclusion: The modified CSA is more stable and sensitive than traditional CSA staining method.

Adult ; Aged ; Aged, 80 and over ; Breast Neoplasms ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; metabolism ; pathology ; Female ; Homeodomain Proteins ; biosynthesis ; genetics ; Humans ; Middle Aged ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Estrogen ; metabolism ; Transcription Factors ; biosynthesis ; genetics

Objective To investigate the nerve recanalization and the motor function of hind legs after transplantation of peripheral nerve grafts treated with microsurgical technique at chronic spinal cord injury (SCI) in rats.Methods The SD rats were established into SCI model with improved Allen method.The rats were divided into two parts 12 weeks after the injury.In experimental group:by microsurgieal technique. the sural nerves were removed epineufium and transplanted into SCI lesion,control group rats were treated without any operation.Retrograde HRP tracing through sciatic nerve were practiced at 1 month,2 month,3 month after transplantation of peripheral nerve grafts.The morphological changes were observed at section of spinal cord and the motor functions of both hind legs of rat were detected.Results The morphology of the injured spinal cord sections turned better.Retrograde HRP tracing through sciatic nerve showed some HRP positive markers at the site of near rostral end of the nearly injured part at one month after transplantation and increased with the time going by.Motor function of hind legs of rats recovered significantly in transplantation groups.Conclusion Peripheral nerve grafts treated with mierosurgical technique have repairing effect on chronic spinal cord injury in rats.

Actins ; analysis ; Antigens, CD34 ; analysis ; Carcinoma, Papillary ; classification ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Keratin-19 ; analysis ; Keratin-20 ; analysis ; Muscle, Smooth ; chemistry ; Pancreatic Neoplasms ; classification ; metabolism ; pathology ; Proto-Oncogene Proteins c-kit ; analysis ; Receptors, Estrogen ; analysis ; Receptors, Progesterone ; analysis

0.05).Conclusion MK mRNA overexpresses in the breast cancer tissues.It might be considered to be a reference indicator for deter- mining the angiogenesis and invasion of breast carcinoma.

Objective To observe the effects of culture medium of a mn iotic cells on NO and NOS in retinal tissues of rabbits in vitro in order to pro vide a protective method for antioxidation in retina transplantation. M ethods Thirty adult healthy rabbits (30 right eyes) were divided into 3 groups. Group Ⅰ: fresh retinal tissue; group Ⅱ: routine culture medium; group Ⅲ: culture medium of amniotic cells. The retinal tissues in group Ⅱ and Ⅲ we re cultured in the corresponding culture medium for 1 week. The content of NO an d NOS in retinal tissues in the 3 groups were determined. Results Compared with group I, the content of NO and NOS of group Ⅱ increased obvio usly (t=3.821, 3.854; P0.05) . Conclusion Culture medium of amniotic cells may remove free r adicals and enhance the ability of antioxidation.

OBJECTIVETo investigate the expression of Fas/FasL in infantile hemangiomas and discuss the role of Fas/FasL in the pathologic evolution of infantile hemangioma.

METHODThe EnVision immunohistochemical stain and RT-PCR technique was used to examine the expression of Fas/FasL protein and mRNA in the infantile hemangiomas.

RESULTS(1) In the early and middle proliferating stage, a number of infantile hemangioma cells expressed Fas. In the late proliferating stage, the number of positive cells increased obviously and the expression of Fas mRNA was reaching the strongest level. In the early regressing stage the Fas still existed in some cells and after that the expression decreased quickly. (2) Up to the middle proliferating stage, there were a few of FasL(+) cells foound. In the late proliferating stage, the number of FasL(+) cells increased significantly. From the early regressing stage, the number of FasL(+) cells decreased rapidly and disappeared.

CONCLUSIONThere may exist significant correlation between the expression of Fas/FasL and the development of the infantile hemangioma cells. The apoptosis of the infantile hemangioma cells mediated by Fas/ FasL may be the major reason of the spontaneous involution of infantile hemangioma.

Apoptosis ; Child ; Child, Preschool ; Fas Ligand Protein ; metabolism ; Hemangioma ; metabolism ; pathology ; Humans ; Hyperplasia ; Infant ; RNA, Messenger ; metabolism ; Signal Transduction ; fas Receptor ; metabolism

OBJECTIVETo investigate the expression of five kinds of cyclins in hepatocellular carcinoma (HCC) and their association with degree of tumor differentiation, metastasis and infection of hepatitis B virus (HBV).

METHODSThe HCC tissue microarrays were composed of those from 273 cases of HCC tissues, 144 surrounding-tumor liver tissues and 10 normal liver tissues obtained from autopsy. The diameter of each specimens on tissue microarrays was 2.0 mm. Immunohistochemistry was used to detect the expression of cyclin A, cyclin B, cyclin D1, cyclin D3 and cyclin E on HCC tissue microarrays. The association of the expression of these cyclins with the infection rate of HBV was also analyzed.

RESULTSThree paraffin-embedded HCC tissue microarrays were successfully constructed, including 136, 143 and 148 tissue spots, respectively. The positive rates of cyclins in 273 cases of HCC were cyclin A 52.7%, cyclin B 45.4%, cyclin D1 35.9%, cyclin D3 44.3% and cyclin E 23.1%; while the figures in 144 surrounding-tumor tissues were 8.3%, 5.6%, 4.9%, 6.3% and 1.4%, respectively. In 10 normal liver tissues these cyclins exhibited negative staining, with the exception that cyclin D1 was positive in one case of normal liver tissue. The positive rate of cyclins in HCC were significant higher than those in surrounding-tumor liver tissues (P < 0.01), in HCC tissues with histological grade II and III, the cyclins expression were stronger than that in grade I (P < 0.05). The positive rates of cyclins, except cyclin A in HCC with portal vein invasion were higher than those without portal vein invasion (P < 0.01). Infection of HBV did not have significant relationship with the expression of cyclins (P > 0.05).

CONCLUSIONCyclins in different cell cycles overexpressed at varied levels in hepatocellular carcinoma, and the increased expression of cyclins may shorten the tumor cell cycle phase, accelerate cell proliferation, and have a close relationship with HCC aggressiveness.

Carcinoma, Hepatocellular ; chemistry ; Cyclin A ; analysis ; Cyclin B ; analysis ; Cyclin D1 ; analysis ; Cyclin D3 ; Cyclin E ; analysis ; Cyclins ; analysis ; Hepatitis B ; metabolism ; Humans ; Immunohistochemistry ; Liver Neoplasms ; chemistry

OBJECTIVETo explore the effects of hepatitis B virus X gene and p53 on hepatocellular growth.

METHODSTwo kinds of plasmids containing sense and antisense human wild p53 gene respectively were constructed. SMMU-7721 cells were transfected with HBx, sense-wtp53 antisense-wtp53 separately or cotransfected with either HBx and sense-wtp53 or HBx and antisense-wtp53. Flow cytometry was adopted to measure the apoptosis rates and the effects of HBx on cell cycle progression. The activity of p21(Waf1) promoter-luciferase construct was detected. Growth curves for SMMU-7721 stably transfected with pcDNA3 and pcDNA3HBx were analyzed.

RESULTSAfter doxorubicin administration, HBx was noticed able to initiate apoptosis of the liver cells. The apoptosis rate was 5.32% in the pcDNA3 transfected and 12.66% in the pcDNA3HBx transfected groups respectively. HBx could also abrogate p53-mediated apoptosis. The apoptosis rate in groups transfected with pcDNA3, pcDNA3wtp53 and pcDNA3HBx + pcDNA3wtp53 was 5.32%, 11.72% and 4.67% respectively. In compared with the normal group, the number of cells in transiently HBx-expressed group and HBx-transfected group decreased 4.79% and 10.25% respectively. HBx inhibited the activity of p21(Waf1) promoter-luciferase constructed (P < 0.05) and promoted cell growth. The growth rate of HBx expression cells was faster.

CONCLUSIONUnder DNA damage, HBx reduced expression of p21(Waf1) by repressing the activity of p53 protein, followed by disturbing the regulation of G(0)-G(1) cell cycle checkpoint, and promoted the growth rate of hepatoma cells.

Apoptosis ; Carcinoma, Hepatocellular ; pathology ; virology ; Cell Division ; Cell Line, Tumor ; Genes, p53 ; Hepatitis B Antigens ; biosynthesis ; genetics ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; pathology ; virology ; Trans-Activators ; biosynthesis ; genetics ; Transfection ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics