The correlation between ultrasonographic features, bFGF, and the local invasiveness of thyroid papillary carcinoma.

The present study aimed to investigate the correlation between ultrasonographic features, basic fibroblast growth factor (bFGF), and the local invasiveness of papillary thyroid carcinoma (PTC).A total of 350 samples of thyroid nodules were collected. Routine ultrasonography was performed before the operation and routine pathological diagnosis and bFGF detection were performed after the operation.'These 350 samples of thyroid nodules included 90 samples of nodular goiter, 36 samples of focal thyroiditis, and 224 samples of PTC. A total of 326 thyroid nodules were examined for bFGF. The results revealed that the difference in the expression of bFGF between the benign and malignant groups was statistically significant (P < .05) and the difference in the positive expression of bFGF between the invasive and non-invasive PTC groups was statistically significant (P < .05).Whether the shape of PTC is regular or not and whether there is micro-calcification in PTC and other ultrasonographic features, the size and location of the lesions and the age of the patient help make a preliminary prediction of local invasiveness before the operation. Postoperative detection of bFGF is helpful for further risk assessments of PTC.

[Blocking of Notch signaling decreased scar formation in rabbit ear hypertrophic scar model].

OBJECTIVE: To investigate the effects of Notch signaling on scars in a rabbit ear model of hypertrophic scarring. METHODS: The hypertrophic scar of rabbits' ears was reproduced. The left rabbit's ear wounds as the N-[N-(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester (DAPT) treated group were treated intradermally with the gamma-secretase inhibitor DAPT to inhibit the activation of Notch at 1, 3, 7 and 14 day time points. The right ears as the control group were treated with normal saline at the same time points. Experimental and control wounds were harvested on days 14, 21, 28 and 35 post wounding, and then examined histologically to quantify hypertrophic index and fibroblasts. The expression of epidermal differentiation markers-keratin 14 (K14), keratin 19 (K19), Involucrin and Notch downstream molecules-P21, P63 were examined and analyzed with immunohistochemistry staining. RESULTS: Both hypertrophic index (1.93 +/- 0.32, 1.82 +/- 0.36, 1.79 +/- 0.25) and number of fibroblasts [(4.08 +/- 0.88), (3.30 +/- 0.53), (3.19 +/- 0.73) x 10(3)/mm(2)] in the DAPT treated group were significantly reduced on days 21, 28 and 35, compared with the control group [2.56 +/- 0.29, 2.61 +/- 0.30, 2.58 +/- 0.39, and (5.45 +/- 0.99), (4.80 +/- 1.13), (4.43 +/- 1.17) x 10(3)/mm(2), all P < 0.01)]. The K19, K14 and P63 increased their expression in the DAPT treated group (28.6% +/- 5.7%, 53.1% +/- 4.5%, 57.0% +/- 5.8%) relative to the control group (10.1% +/- 2.8%, 30.8% +/- 4.9%, 16.5% +/- 2.2%, all P < 0.01) on day 14 post wounding, while the Involucrin and P21 decreased their expression in the DAPT treated group (12.3% +/- 1.9%, 11.0% +/- 1.7%) relative to the control group (29.3% +/- 4.6%, 44.3% +/- 3.5%, both P < 0.01). CONCLUSION: Inactivation of Notch signaling will inhibit scar epidermis to over-differentiation, and thereby inhibit proliferation of hypertrophic scars in the rabbit ears.

[Expression of Notch receptors, ligands and downstream target genes in epidermis of hypertrophic scar].

OBJECTIVE: To study the expression of Notch receptors, ligands and downstream target genes in hypertrophic scar and normal skin, and to investigate its role in the development of hypertrophic scar. METHODS: By immunohistochemistry, the expression of epidermal differentiation markers- beta1 integrin, keratin 14 (K14) and keratin 19 (K19), as well as Notch 1-4 and Jagged1 were examined in hypertrophic scars and normal skins. The expression of Notch downstream genes- P21 and P63 was analyzed with real-time quantitative PCR and immunohistochemistry staining. RESULTS: Histological analysis revealed a significant epidermal thickening in the hypertrophic scars, with excessive cell layers above the basal layer. Compared to the normal epidermis, the expression of beta1 integrin, K19 and K14 decreased in hypertrophic scars (P <0.05). Positive expression rate of Notch1 and Jagged1 in keratinocytes was significantly higher in hypertrophic scar than in normal skin (P < 0.05), while there was no difference in Notch2 and 3 positive expression rate. Furthermore, the expression of P21 was significantly up-regulated, while the expression of P63 was down-regulated in keratinocytes of hypertrophic scar (P < 0.05). CONCLUSIONS: Notch signal may play an important role in hypertrophic scar pathogenesis. Over-differentiation of Keratinocytes in hypertrophic scar may be related to the overexpression of Notch1 and Jagged1, up-regulation of P21 gene and down-regulation of P63 gene.

[Effect of METH1 gene transfection on the proliferation of rabbit's ear scar].

OBJECTIVE: To investigate the effect of METH1 gene transfection on fibroblast proliferation and I, III collagen synthesis in rabbit ear scar. METHODS: The hypertrophic scar model on the rabbit ears was reproduced. 10 days after epithelization, Ad-METH1 was injected into the scar tissue. 30 days later, the effect of METH1 gene transfection on the angiogenesis, fibroblast proliferation and the ratio of collagen I/III in the scar tissue was detected by microcirculation microscope, AgNOR particle count and collagen dyeing. RESULTS: 30 days after injection of Ad-METH1, angiogenesis, fibroblast proliferation and the ratio of collagen I/III in the scar tissue were obviously suppressed. CONCLUSION: Early application of Ad-METH1 after epithelization can markedly inhibit the formation of the hypertrophic scar.

[The study on reversing mechanism of multidrug resistance of K562/A02 cell line by curcumin and erythromycin].

OBJECTIVE: To investigate the effects of curcumin (Cur) and erythromycin (EM) on multidrug resistance (MDR) reversal of K562/A02 cell line and their mechanism. METHODS: MTT assay was employed to determine the sensitivity of Cur, EM-treated K562/A02 cells to adriamycin (ADM). Flow cytometry was used to measure intracellular mean fluorescence intensity (MFI) of daunorubicin (DNR). P-gp expression was determined by immunohistochemistry. RT-PCR technique was used to examine the mdr1 mRNA level. RESULTS: IC(50) of ADM in K562/A02 cells was decreased when treated with Cur or EM, and the reversal times (RvT) was 4.9, 3.7 respectively. The RvT reached to 11.3 when treated with Cur (2.5 microg/ml) combined with EM (120 microg/ml). The DNR MFI in K562/A02 cells was significantly lower than that in K562 cells (P < 0.01), and was increased significantly when treated with Cur (2.5 microg/ml) or EM (120 microg/ml) (P < 0.05). There was no significant difference between DNR MFI of K562/A02 cells treated with Cur (2.5 microg/ml) or EM (120 microg/ml). Immunohistochemistry showed that P-gp expression was significantly higher in K562/A02 cells than in K562 cells (P < 0.01), and was reduced in K562/A02 cells treated with each (P < 0.01), though being still higher than that in K562 cells (P < 0.01). P-gp expression of K562/A02 cells treated with each drug for 5 days were lower than that for 3 days (P < 0.01), and lowered further when treated with Cur and EM together (P < 0.01). Mdr1 mRNA level in K562/A02 cells was higher than in K562 cells (P < 0.01), and was decreased when treated with each of the drugs (P < 0.01). The mdr1 mRNA level of K562/A02 cells treated with Cur (2.5 microg/ml) plus EM (120 microg/ml) was decreased most significantly than that treated with other group of drugs. After 5 day treatment the mdr1 mRNA level of K562/A02 cells with Cur (2.5 microg/ml) was lower than that with EM 120 microg/ml (P < 0.01). CONCLUSIONS: Either Cur or EM can partly reverse the multidrug resistance of K562/A02 cells and decrease the expression and function of P-gp in a time-dependent way. MDR reversing effect of Cur combined with EM is stronger than that of Cur or EM alone.

Survivin expression and its relation with proliferation, apoptosis, and angiogenesis in brain gliomas.

BACKGROUND: An unbalance of cell proliferation and cell apoptosis is an important mechanism in carcinogenesis, and angiogenesis also plays a crucial role in tumorigenesis. Recently, survivin has been identified as an important member of the inhibitor of apoptosis protein (IAP) family. Although it has been shown that survivin is highly expressed in gliomas, and is associated with tumorigenesis, progression, and poor prognosis of gliomas, as yet the relation of survivin expression with proliferation, apoptosis, and angiogenesis of gliomas it is still unclear. METHODS: Eighty-three cases of brain glioma were chosen and protein expressions of survivin and proliferating cell nuclear antigen (PCNA) in glioma cells and Factor VIII-related antigen (FVIII-RAg) in vascular endothelial cells were investigated by immunohistochemistry. Apoptotic cells of brain glioma were screened by TdT-mediated dUTP nick end-labeling (TUNEL), and survivin immunoreactivity score (IRS), proliferative index (PI), apoptotic index (AI), overall daily growth (ODG), and microvessel density (MVD) in brain gliomas were measured. RESULTS: The survivin IRS, PI, AI, ODG, and MVD of brain gliomas were 3.75 +/- 3.89, 28.39 +/- 19.49%, 1.00 +/- 0.80%, 12.19 +/- 10.21%, and 62.75 +/- 31.50, respectively, and all of them increased markedly with an increase in the pathologic grade of brain gliomas (P < 0.001 for all). PI, ODG, and MVD in the survivin-positive group were significantly higher than those in the survivin-negative group (P < 0.001 for all). PI, ODG, and MVD were positively correlated with survivin IRS (P < 0.001 for all). Although there was no significant difference between AI in the survivin-positive group or in the survivin-negative group (P = 0.108), AI was inversely correlated with survivin IRS (P = 0.005). CONCLUSIONS: Survivin is overexpressed in brain gliomas, which may play an important role in malignant proliferation, antiapoptosis, and angiogenesis of brain gliomas.

[Application of histochemical staining in diagnosis of osteosarcomas].

OBJECTIVE: To study the histochemical staining in the diagnosis of osteosarcoma. METHODS: To compare the effectiveness of picrosirius red, improved Ponceau trichrome and Masson trichrome staining methods on bone formation tissues in conventional osteosarcoma, paraosteal osteosarcoma, periosteal osteosarcoma, extraskeletal osteosarcoma, inflammatory myofibroblastic tumour, malignant fibrohistiocytoma, chondrosarcoma, fibrosis with ossification and calcification. RESULTS: With modified Ponceau trichrome staining, bone formation tissues showed a homogenous, orange-red interblended with blue in color. From osteoid to mature bone the color changed from orange-red, light blue to dark blue. Fibrotic tissue was stained blue in color with striated appearance. Cartilage was not stained. Picrosirius red method gave bone formation tissues homogenous staining. Along with bone maturation, from osteoid tissue to mineralized bones, the color showed changes from light red, yellow, orange-red, red to dark purple. The cartilage demonstrated homogenous light red in color. Fibrous tissue stained red interblended with yellow in color, striated in shape. With Masson trichrome staining osteoid displayed pale blue and mineralized bone showed dark blue in color. Fibrotic tissue showed a striated blue staining. CONCLUSION: The modified Ponceau trichrome and Picrosirius red staining methods are better than Masson trichrome to demonstrate bone formation tissue in osteosarcoma. The former two methods could be also used in study on bone formation.

[Research on relationship of survivin gene expression with malignant proliferation and apoptosis of brain glioma].

OBJECTIVE: To investigate the expression level of inhibitor of apoptosis protein survivin gene in human brain glioma and its role in malignant proliferation and antiapoptosis of brain glioma. METHODS: Eighty-three cases of brain glioma specimen was chosen, protein expression of survivin and proliferating cell nuclear antigen (PCNA) was investigated by immunohistochemistry streptavidin-biotin complex (SABC) method, the immunoreactivity score (IRS) of survivin and the proliferative index (PI) were counted. Apoptotic cells were screened by TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method, and the apoptotic index (AI) of brain glioma was calculated. RESULTS: The survivin IRS, PI and AI of brain glioma were 3.8 +/- 3.9, (28.4 +/- 19.5)% and (1.0 +/- 0.8)% respectively, and all of them were elevated with the increase of pathological grade of brain glioma (P < 0.01 for all). PI in survivin positive group was significantly higher than that in survivin negative group (P < 0.01), and PI was positively correlated with survivin IRS (r = 0.740, P < 0.01). There was no significant difference between AI in survivin positive group and that in survivin negative group (P > 0.05), however, AI was negatively correlated with survivin IRS (r = -0.307, P < 0.01). CONCLUSIONS: Survivin is overexpressed in brain glioma, and which may play important roles in malignant proliferation and antiapoptosis of brain glioma.

[Retrospective analysis of 1905 patients with skin cancer from two general hospitals in western China from 1981 to 2000].

OBJECTIVE: To investigate the clinical features and changes in the incidence of skin cancer in two hospitals located in western China. METHODS: The patients diagnosed pathologically as skin cancer from 1981 to 2000 were retrospectively collected from the two hospitals. Clinical data of patients with skin cancer were collected and analyzed. RESULTS: (1) Of the 1 905 patients with skin cancer, squamous cell carcinoma accounted for 29.4%(560 patients), basal cell carcinoma 28.0% (534), and cutaneous malignant melanoma(CMM) 16.0% (305). (2) There were 591 patients with skin cancer diagnosed between 1980 and 1990, and 1 314 between 1991 and 2000, and accounted for 0.34% and 0.58% of all biopsy cases, respectively. The number of total biopsy patients increased 1.6% every year during the 20 years. The number of biopsy patients with skin cancer and with CMM increased 3.5% and 3.9% every year,respectively. (3) Of the 305 CMM patients, 63.3% located on the acra. These patients were elder, and have a higher rate of trauma and a higher incidence in the male than that in patients with CMM located on the other sites. (4) Of the 305 CMM patients, 64 (21%) had history of trauma at the primary onset sites, and 47 (15.4%) had history of small congenital nevi at the primary sites. CONCLUSION: There are some differences in the clinical features such as location and age between the skin cancer patients in our study and those in white population. The incidence of skin cancer in the two hospitals had been increasing in the 20 years (between 1981 and 2000). Both trauma and small congenital nevi are important risk factors of CMM.

Expression of COX-2, iNOS, p53 and Ki-67 in gastric mucosa-associated lymphoid tissue lymphoma.

AIM: To assess the expression of cyclooxygenase-2 (COX-2), nitric oxide synthase (iNOS), p53 and Ki-67 in gastric mucosa-associated lymphoid tissue (MALT) lymphoma and clarify the relationship between COX-2 expression and iNOS or p53 expression in these patients. METHODS: The expressions of COX-2, iNOS, p53 and Ki-67 were detected in 32 gastric MALT lymphoma specimens and 10 adjacent mucosal specimens by immunohistochemical Envision method. RESULTS: COX-2 and iNOS expressions were significantly higher in gastric MALT lymphoma tissues than those in adjacent normal tissues. The expression of COX-2 was observed in 22 of 32 cases of MALT lymphoma tissues (68.8%). A positive cytoplasmic immunoreactivity for iNOS was detected in 17 of 31 cases (53.1%). COX-2 expression in gastric MALT lymphoma tissues was positively correlated with iNOS expression (r=0.448, P=0.010) and cell proliferative activity analyzed by Ki-67 labeling index (r=0.410, P=0.020). The expression of COX-2 protein did not correlate with age, sex, stage of disease, lymph node metastasis or differentiation. The accumulation of p53 nuclear phosphoprotein was detected in 19(59.4%) of tumors. p53 protein was expressed in 11 of 23 assessed LG tumors and in 8 of 9 assessed HG tumors. The difference of p53 positivity was found statistically significant between LG and HG cases (P=0.0302). The p53 accumulation correlated with advanced clinical stage (stage III+IV vs stage I+II, P=0.017). There was a significant positive correlation between COX-2 expression and p53 accumulation status (r=0.403, P=0.022). The mean PI of Ki-67 in each grade group were 36.0+/-7.73% in HG and 27.4+/-9.21% in LG. High-proliferation rate correlated with HG tumors (r=0.419, P=0.017). The correlation coefficient showed a significant positive correlation between PI and COX-2 expression in MALT lymphoma patients (r=0.410, P=0.020). CONCLUSION: COX-2 expresses in the majority of gastric MALT lymphoma tissues and correlates with cellular proliferation and iNOS expression. COX-2 overexpression is closely associated with p53 accumulation status. iNOS and COX-2 may play a synergistic role in the pathogenesis of gastric MALT lymphoma.

Primary targeting of recombinant Fv-immunotoxin hscFv(25)-mTNFalpha against hepatocellular carcinoma.

AIM: To obtain human recombinant Fv-immunotoxin hscFv(25)-mTNFalpha (mutant human TNFalpha fused to human scFv(25)) against hepatocellular carcinoma (HCC). METHODS: Two relevant sites of enzymatic digestion were added to mTNFalpha by PCR. MTNFalpha was linked to the 3' end of hscFv(25) in pGEX4T-1 vector. This anti-HCC recombinant Fv-immunotoxin hscFv(25)-mTNFalpha was expressed in Escherichia coli and purified from inclusions. After purified by glutathione-S-transferase affinity chromatography and thrombin digestion, it was identified by electrophoresis and Western blot. And then, the purified recombinant Fv-immunotoxin was injected into nude mice with HCC xenografts through their tail veins. MTNFalpha protein and PBS were used as control at the same time. After treated for two weeks, nude mice were executed. The bulk and weight of tumors were observed. The tumor tissues were stained by immunohistochemical method with TNFalpha antibody. RESULTS: The expression ratio of recombinant Fv-immunotoxin hscFv(25)-mTNFalpha was 12% of bacterial protein. The result of tumor restraining trials of hscFv(25)-mTNFalpha showed 2/5 complete remission and 3/5 partial remission. mTNFalpha restraining trials showed 5/5 partial remission. The therapeutic result of hscFv(25)-mTNFalpha was better than that of mTNFalpha (F=8.70, P<0.05). The hscFv(25)-mTNFalpha remedial tumor tissues were positive for TNFalpha by immunohistochemical staining. The positive granules mainly existed in the cytoplasm of tumor cell. CONCLUSION: Recombinant Fv-immunotoxin hscFv(25)-mTNFalpha has better therapeutic effect than mTNFalpha. It can inhibit the cellular growth of HCC and has some potential of clinical application.

[Construction, expression and targeting therapeutic of single-chain immunotoxin against hepatocellular carcinoma].

OBJECTIVES: To obtain high therapeutic effect and low toxicity single-chain immunotoxin against hepatocellular carcinoma (HCC). METHODS: Human mutant tumor necrosis factor-alpha (mTNFalpha) was linked with the 3' end of humanized single-chain Fv against HCC (hscFv25) in pGEX4T-1 vector. The anti-HCC immunotoxin was expressed in Escherichia coli and identified by western blot. The primary tumor regression trial in nude mice bearing HCC was evaluated the targeting therapeutic value of hscFv25-mTNFalpha. The tumor tissues were stained by immunohistochemical with TNFalpha antibody. RESULTS: The expression of single-chain immunotoxin hscFv25-mTNFalpha was 12% of total bacteria proteins. The tumor regression trials of hscFv25-mTNFalpha showed 5/5 effective. It had 2/5 completely remission and 3/5 partly remission. The therapeutic result of hscFv25-mTNFalpha was better than that of mTNFalpha (F=8.70, 0.05). The HCC tissue treated by hscFv25-mTNFalpha expressed TNFalpha positive reaction. The positive granule mainly existed in HCC cytoplasm. CONCLUSION: The single-chain immunotoxin hscFv25-mTNFalpha has high therapeutic effect and low toxicity. It has potentialities for clinical application.

Clinicopathologic study of mucosa-associated lymphoid tissue lymphoma in gastroscopic biopsy.

AIM: To explore and discuss the clinicopathologic characteristics of mucosa-associated lymphoid tissue (MALT) lymphoma in gastroscopic biopsy specimen. METHODS: A retrospective study of 26 cases of lymphoma diagnosed by gastroscopic biopsy during 1999 to 2001 from gastroscopy files of Xijing Hospital was made. The diagnostic criteria were adopted according to the new classification of non-Hodgkin's lymphoma. RESULTS: Twenty-six cases of primary gastric lymphoma consisting of 15 men and 11 women, aged between 23 to 76 years were recruited from 6 225 cases who received gastroscopy. All of them were diagnosed by both endoscopic findings and histological examinations. Histologically, 23 cases were MALToma (low grade) and 3 cases lymphoblastic lymphoma (high grade). Immunohistochemically, all cases were CD20 positive, while CK and EMA were negative. CONCLUSION: The majority of the cases of primary low-grade gastric lymphoma have morphologic and clinical features that justify their inclusion in the category of low-grade lymphoma of mucosa associated lymphoid tissue.

Expression of RNase H of human hepatitis B virus polymerase in Escherichia coli.

AIM: To amplify HBV-RNase H gene fragment and expression of RNase H for further use in the studies of HBV associated liver diseases. METHODS: The encoding gene of HBV-RNase H was separately amplified for the first half and second half (H1 and H2) by PCR from full length HBV gene and cloned into pT7Blue-T vector. Clones were first screened by digestion with XbaI and Hind III enzyme for the correct size, and analyzed further by DNA sequencing. The RNase H1 and H2 fragments isolated from XbaI and Hind III digestion products of pT7 Blue-RNase H plasmid were ligated to the GSTag expressing vectors separately, and expressed in E.coli BL21. The expressed proteins were checked by PAGE gel and Western blot. RESULTS: Both H1 and H2 nucleotide seqences consisting of known genes and proteins, in correct size, were further confirmed by Western blot to be the GST and RNase H1 or H2 fusion proteins. CONCLUSION: The successful cloning and expression of HBV-RNase H will contribute to further research and application in HBV-associated diseases.

Expression and significance of a new tumor suppression gene PTEN in primary renal cell carcinoma.

BACKGROUND & OBJECTIVE: PTEN gene is a newly discovered tumor suppressor gene, that maps to chromosome 10q23, and of longstanding interest to those studying somatic mutations in human tumors. Mutation and deletion of PTEN gene probably resulted in a new signal transduction pathway related to human malignant tumors. It was reported that PTEN gene mutated and deleted in renal cell carcinoma, however, there was no report concerning its expression in renal cell carcinoma. This study was designed to investigate expression and significance of PTEN gene in primary renal cell carcinoma. METHODS: Immunohistochemical peroxidase-conjugated streptavidin (SP) method was used to detect expression of PTEN gene in 40 cases of primary renal cell carcinoma, 18 with renal tissues closely adjacent to carcinoma and 5 case of normal renal tissues. The relationship between expression of tumor suppressor gene of PTEN and the percentage of lymph node metastasis of renal cell carcinoma was analyzed. RESULT: It was showed that PTEN gene was expressed in all 5 case of normal renal tissues and 18 cases of renal tissues closely adjacent to carcinoma. The intensity was relatively strong and it was localized mainly in cytoplasm of epithelium cells of renal tubular. Expression of PTEN in 40 cases of renal cell carcinoma were different, 12.5% were negative, 17.5% were weak positive, and 70% were strong positive. PTEN protein was localized in cytoplasm of carcinoma cells. The percentage of lymph node metastasis of renal cell carcinoma negative or weak positive of PTEN protein was 80% and 51.74%, respectively. The percentage of lymph node metastasis of renal cell carcinoma positive of PTEN protein was 10.71%, comparing with those of renal cell carcinoma negative or weak positive of PTEN protein, the difference was significant (P < 0.05). CONCLUSION: This study suggested that PTEN gene was deleted or weakly expressed in primary renal significant cell carcinoma, which is probably related to tumorigenesis and development of renal cell carcinoma.