Objective To investigate the effect of the 8-bromum-cyclic adenosine monophosphate (8-Br-cAMP) on the telomerase activity and changes of cell cycle in retinoblastoma （RB） cells. Methods The cultured RB cells were divided into the experimental group (8-Br-cAMP) and control group. After cultured for 24, 48 and 72 hours in vitro, the telomerase activity of RB cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and the changes of cell cycle were detected by flow-cytometry. Results The difference of telomerase activity was significant between the experimental groups and control group (Plt;0.01). There was a negative correlation between the A value of absorbance and the time in the experimental groups (r=-0.778 9, F=33.936, Plt;0.01). The changes of the cell cycle were that the percentages increased in G1 phase and decreased in S phases. Conclusion 8-Br-cAMP may weaken telomerase activity, affect the cell cycle, and inhibit the proliferation of RB cells. (Chin J Ocul Fundus Dis，2004，20：358-360)
OBJECTIVE: To explore the effect of Fas/Apo-1 and Bcl-2 gene expression on mechanism of scar formation. METHODS: Immunohistochemical method was applied to defect the expression of Fas and Bcl-2 protein in fibroblasts from 10 cases with normal skin, 10 cases with hypertrophic scar and 10 cases with keloid. RESULTS: The positive expression rate of Bcl-2 protein in keloid was 83.2%, significantly higher than that in hypertrophic scar (38.6%), (P lt; 0.01), and the positive expression rate in hypertrophic scar and keloid was higher than that in normal skin (6.78%), (P lt; 0.01). But the positive expression rate of Fas/Apo-1 protein was 78.4% in normal skin 80.4% in hypertrophic scar, 84.4% in keloid respectively, which showed no significant difference among them (P gt; 0.05). CONCLUSION: Bcl-2 gene but Fas gene may take part in the formation of pathologic scar.
OBJECTIVE The effect of platelet-derived wound healing factor (PDWHF) on wound healing in diabetic rats was studied. METHODS Forty-four male SD rats were randomly divided into 2 groups. Thirty-two rats of experimental group accepted intraperitoneal injection of alloxan (1.5 mg/10 g body weight). Within one or two days after injection, while the blood sugar of the rats was higher than 180 mg/dl, the animal model of diabetic rat should have been established. Then a dorsal incision was given to every rat. After the addition of PDWHF (the experimental group) or bovine albumin (the control group), the incision was sutured up. Seven, ten and fourteen days after operation, the breaking strength of the wound was measured. On another hand, specimen from the wound was taken for the culture of fibroblasts. When the cultured fibroblasts have been incubated with 10% PDWHF for 4, 8 and 12 hours, the procollagen I (alpha 1) mRNA levels were examined respectively, and compared with those of control. RESULTS Significant difference in wound breaking strength had been observed between PDWHF-treated incisions and the control on 7, 10 and 14 days after wounding (P lt; 0.01). Experiment in vitro demonstrated that the procollagen I (alpha 1) mRNA levels in wound fibroblasts incubated with 10% PDWHF for 4, 8 and 12 hours were 0.9, 3.7 and 2.2 folds higher than those in fibroblasts in control. CONCLUSION It was suggested that direct stimulation of procollagen I (alpha 1) gene expression was one of the ways that PDWHF played its role in accelerating wound healing.
Objective To investigate the expression of T cell receptor (TCR) Vβ8.3 gene on CD4+ T lymphocytes in the rats with experimental autoimmune uveoretinitis (EAU). Methods Eighteen Lewis rats were divided into EAU, complete Freund′s adjuvant, and the control group. Inter photoreceptor retinoid-binding protein (IRBP) R16 peptide was synthesized using Fmoc procedure for induction of EAU. Magnetic absorption cell sorting (MACS) me thod was used to isolate the CD4+T lymphocytes from the spleen of the rats. Flow cytometry was used to monitor the efficiency of isolation. The expression of TCR Vβ8.3 gene segment on CD4+T lymphocytes was determined by fluorescent quantitative polymerase chain reaction. Results EAU was successfully induced in the Lewis rats immunized with IRBP R16 peptide. The proportion of CD4+T lymphocytes isolated by means of MACS was statistically higher than that before isolation (P＜0.001). The expression of TCR Vβ8.3 gene segment on CD4+ T lymphocytes in EAU rats was significantly higher than that in the control (P＜0.05). Conclusions There is a predominant usage of antigen-specific TCR Vβ 8.3 gene in EAU rats induced by IR BP R16 peptide, which may serve as a target for immunotherapy of EAU. (Chin J Ocul Fundus Dis，2004，20：165-167)
OBJECTIVE: To construct a co-expressing vector of human bone morphogenetic protein 2 (BMP-2) and osteoprotegerin (OPG) and to determine the expression of BMP-2 and OPG in myoblast C2C12. METHODS: Using the isolated total RNA from osteosacoma cell line MG63 as a template, the cDNA encoding region of human OPG was amplified by reverse transcription-polymerase chain reaction (RT-PCT) method and cloned into sites EcoR 1 and BamH I of mammalian expressing vector pIRES2-EGFP, and the cDNA encoding region of human BMP-2 was cloned into endonucleases site BstX I. Then the recombinant plasmid pIRES2-BMP-2-OPG was transformed into C2C12 cell line, the expression of OPG and BMP-2 were determined by Western blot assay. RESULTS: The sequence of OPG cDNA obtained was the same as that reported, recombinant plasmid pIRES2-BMP-2-OPG was constructed successfully. Human OPG and BMP-2 co-expression cell line C2C12 was selected and confirmed by Western blot analysis. CONCLUSION: The co-expressing vector of OPG and BMP-2 is constructed and can expressed stably in myoblast C2C12. The co-expression of human OPG and BMP-2 may be logical approach for treatment of osteoporosis and bone metastasis.
Objective To evaluate the effect of copper-ion on the prol iferation and differentiation of human umbil ical vein endothel ial cell (HUVEC). Methods HUVEC were cultured and passaged in vitro. HUVEC were inoculated into 96-well plate with density of 5 × 103/well. All the cells were divided into 3 groups randomly according to different culture mediums: group A (5 μmol/L CuSO4), group B (25 μmol/L CuSO4), group C (control group). Every group had 4 wells, and the basic culture medium was MCDB131. The cell growth curves of 3 groups were drawn by using MTT. HUVEC were inoculated into 6-well plate with density of 2 × 105/well. Grouping of the cells was the same as the above. The gene expressions of endothel ial nitric oxide synthase (eNOS) and tyrosine kinase with immunoglobul in-l ike and EGF-l ike domain 1 (Tie-1) were detected by real-time RT-PCR. Results The growth curves revealed that the exponential growth time was the first 3 days, plateau growth time begun on the 4th day. The prol iferation of group A was ber than that of groups B and C from the 3rd day, within 2 days, the prol iferation of group B was ber than that of group C; however, it decreased and was weaker than group C from the 4th day, all showing statistically significant difference (P lt; 0.05). The results of real-time RT-PCR revealed that the expressions of eNOS in groups A, B and C were 7.294 ± 1.488, 0.149 ± 0.044 and 1.000 ± 0.253; and the expressions of Tie-1 in groups A, B and C were 1.481 ± 0.137, 1.131 ± 0.191 and 1.000 ± 0.177. Group A compared with groups B and C, both of 2 genes were up-regulated (P lt; 0.05). Group B compared with group C, eNOS was down-regulated (P lt; 0.05) and the difference of Tie-1 expression was not statistically significant (P gt; 0.05). Conclusion 5 μmol/L copper-ion can promote the prol iferation and differentiation of HUVEC effectively.
Purpose To investigate nucleoside diphosphate kinase (NDPK ) expression of tumor metastasis suppressor gene nm23 in heterotransplanted model of retinoblastoma(RB) in nude mice，and analyse the correlation between the expression of nm23 gene and the formation and progression of heterotran splanted RB． Methods SP immunohistochemical method was used to detect the expression of nm23 gene product NDPK in 20 tumors of heter otransplanted RB model and normal retinal tissue． Results The negative staining of nm23/ NDPK was found in normal retinal tissue , whereas 100% expression rate in RB tumors with positive number of 48.73plusmn;2.37． No statistical significance of the expression of nm23/ NDPK was observed between the intraocular growth phase （I～Ⅲ grade） and invasive phase （ Ⅳ～Ⅴ grade）in heterotransplantedRB tumors． Conclusion The function of nm23 gene as a tumor metastasis suppressor in heterotra nsplanted RB tumors was less prominent ,but it may play a role in carcinogen esis and progrssion of RB and may predict poor prognosis． (Chin J Ocul Fundus Dis, 2001.17:47-49)
Objective To search for the significant early diagnostic and prognostic indicators in cholangiocarcinoma. Methods Literatures about the variation of various kinds of genes in cholangiocarcinoma and their clinical significances were reviewed.Results There are several genes mutation, deletion and overexpression in cholangiocarcinoma, K-ras gene was most deeply investigated. Conclusion K-ras mutation detection may be of help in the early finding and prognosis of cholangiocarcinoma.
Objective To observe and evaluate the expression and significance of Nogo66 receptor (NgR) mRNA in adult ratsprime;optic nerve. Methods Optic and sciatic nerves of 8 adult rats were used to make the sections, which were divided into 3 groups: optic-nerve experimental group, sciatic-nerve control group, and optic-nerve negative control group. In situ hybridization was used to observe the expression of NgR mRNA in optic nerve and sciatic nerve. Results The expression of NgR mRNA in the 8 rats was positive in optic nerve and negative in sciatic nerve. The positive signals were arranged along the long axis of optic nerve. Conclusion The expression of NgR mRNA is positive in optic nerve while negative in sciatic nerve in adult rats, which suggests that the positive expression and distribution of NgR may be related to the poor regenerate ability of optic nerves. (Chin J Ocul Fundus Dis, 2005,21:246-248)
Objective To investigate the development and metastasis of malignant choroidal melanoma cell strain OCM-1-gfp modified with green fluorescent protein(GFP) and the factors which affected the tumor biological behaviors. Methods GFP was transfected into malignant melanoma cell strain OCM-1.Melanoma cells with high and stable expression of GFP were injected into subretinal space and the subcutaneous space of hind leg of Balb/c nude mouse respectively in order to establish orthotopic and heterotopic transplanted tumor models.The development and metastasis process of orthotopic tumor models was observed directly by fluorescence microscope，and the size of the hypodermal tumor was measured by vernier.The expressions of 13 genes in melanoma were detected by means of immunohistochemistry staining. Results Malignant choroidal melanoma cell strain OCM-1 stably expressed GFP and preserved the characteristics of parental generation，OCM-1-gfp may develop melanoma and continue to metastasize in nude mouse.Positive expression of most of the antibodies，including Rb，p53，p21，E2F，NFkappa;B，cyclin D1，proliferation cellular nuclear antigen（PCNA），bcl2、bclXL/S，bax，and epithelial growth factor（EGF）and its receptor（EGFR），was found.While the staining of inhibition gene p16 was negative. Conclusions GFP is the marker for observing the development and metastasis of malignant choroidal melanoma in vivo.The rate of tumor formation and development process in orthotopic models does not differs much from which in heterotopic models of malignant choroidal melanoma.The expressions of lots of genes in malignant choroidal melanoma developed from OCM-1-gfp including p16、p53、NFkappa;B，cyclin D，PCNA，EGF，and EGFR are abnormal. (Chin J Ocul Fundus Dis, 2006, 22: 170-173)