Objective To construct vectors that express phosphatidylinositol-3-kinase, catalytic, beta polypeptide （PIK3cb） shRNA in eukaryon plasmid catalyzed by PI3K in rat, then test their effects on intimal hyperplasia in transplanted vein graft. Methods One hundred and fifty SD rats were randomly divided into six groups （n=25, in each group）: blank (25% Pluronic F-127), shRNA-1, shRNA-2, 1/2 （shRNA-1+shRNA-2）, negative control (pGenesil-1 scramble shRNA) and positive control （wortmannin） group. The jugular vein in rats were interpositioned autologously into the common carotid artery. shRNA and 25% Pluronic F-127 were mixed and coated around the transplanted vein in three PIK3cb shRNA groups. Every 5 samples were removed according to the time point (1, 3, 7, 14 and 28 days after operation), respectively. The thickness of intima and neointima area were calculated and analyzed by computer system. The PCNA expression was detected by Western blot and SP immunohistochemistry. Results The intimal thickness of three PIK3cb shRNA groups were lower than those in the blank group and negative control group on day 3, 7, 14, 28 after operation (P＜0.05); The neointima area in three PIK3cb shRNA groups (except shRNA-2 group on day 3, 7) began to decrease significantly from day one (P＜0.05). The protein expression of PCNA in three PIK3cb shRNA groups on day 3 after operation were decreased compared with blank group and negative group (P＜0.05). The percentage of the PCNA positive cells area in three PIK3cb shRNA groups were significantly lower than those in blank group and negative control group in each time point (Plt;0.05). There were no significant differences between blank and negative control group in different time points (Pgt;0.05). Conclusion The PIK3cb shRNA can effectively inhibit the proliferation of vascular smooth muscle cell, which may provide a new gene therapy for the prevention of vein graft restenosis after bypass grafting.
To determine the state of fibroblast during the process of development of hypertrophic scar (HS), 40 specimens of HS in different periods were collected. The expressions of prolifrating cell nuclear antigen (PCNA) and Ag-protein in nucleolar organizer regions (Ag NORs) as well as the content of total amino acids in the tissues were examined. The hypertrophic scar of 1st and 3rd month old, the expression of PCND and Ag NORs were the highest. In the 9th and 12th month old, althrough PCNA was nearly negative, but the expression of Ag NORs was low. The content of total amino acid was increased gradually as HS developed but the increase of amount of hydroxyproline was markedly slowed down in 9 month old HS. It was suggested that: (1) in the developing process of HS the proecess of overproliferation of fibroblasts was short and limitted in 1-3 months period in the process of wound lealing; (2) the synthesis of collagen was nearly stopped at 6 months, but that of other extracellular matrix such as fibronectin and proteoglycan might be continued to aggregate after 12 months.
ObjectiveTo observe the longterm effect of suramin on the inhibition of proliferation of human retinal pigment epithelial (RPE) cells in vitro. MethodsRPE cells grown in 9 pieces of 96well plate (12 wells each plate) were divided into experimental and control group, with 6 wells in each group. The concentration of 0.1 ml RPE cells in each well is 5×104 cells/ml. After the change of the medium, RPE cells were treated with suramin (250 μg/ml) in experimental group while treated with nothing in the control group. The medium of the 2 groups were changed to the normal medium after 4 days. At the 1st, 2nd, and 4thday after the addition of suramin and at the 1st, 2nd, 3rd, 5th, 6th, 7th, 9th , 11th and 13th day after removing suramin, 1 plate was randomly selected to stop culturing, and the proliferation of RPE cells were detected by methyl thiazolyl tetrazolium (MTT) assay. ResultsUnder reversed microscope, RPE cells in control group were fused completely at the 7th day after inoculation. The extracellular space of RPE cells in experimental groups was larger than that in the control group, and remained unfused at the 13th day after inoculation. The inhibitory rate of proliferation of RPE cells at the first day after treated with suramin was 14.85% and increased to the highest 25.79% at the 4th day. The first day after the suramincontaining media was removed, the inhibitory rate decreased to 12.35%, and then raised gradually to over 20% at the 3rd to 5th day. Finally, the rate drop to 14.71%. ConclusionSuramin has the long-term effect on the inhibition of RPE cells induced by serum, especially the inhibitive effect after the remove of suramin, which indicates the specific double-peak inhibition during the whole process.(Chin J Ocul Fundus Dis, 2005,21:25-27)
Objective To evaluate the effect of vascular endothelial cell growth factor (VEGF) antisense oligodeoxynucleotides (ASODNs) on the expression of VEGF in rats with oxygen-induced retinopathy. Methods Thirty newborn Sprague-Dawley (SD) rats were randomly divided into 3 groups:normal control group, disposal group and non-disposed group, The animal models with oxygen-induced proliferative retinopathy were established by raising the rats in hyperoxic environment. Retrobulbar injection was performed with VEGF ASODNs or normal saline on the rats in 3 groups respectively. The intraocular tissues (all the tissues except the cornea, sclera, and lens) and serum were collected, and the expressions of VEGF were determined by using competitive enzyme immunoassay.Results The expressions of VEGF in intraocular tissues of rats in disposal group were significantly lower than those in non-disposed group (P＜0.05), and there was no statistical difference between the disposal and normal control group (P＞0.05). There was no significant difference of the expressions of VEGF in serum of rats between the disposal and non-disposed group (P＞0.05), which were both lower than those in the normal control group (P＜0.05). Conclusion VEGF ASODNs could significantly inhibit the expression of VEGF in intraocular tissues. (Chin J Ocul Fundus Dis，2003，19：172-174)
ObjectiveTo investigate the relationship between topical reactive lymphoid hyperplasia and postoperative recurrence and survival of gastric cancer patients. MethodsThe clinical and pathological data of gastric cancer patients who underwent D2 radical gastrectomy from January 2007 to July 2009 were retrospectively analyzed. Based on the number of reactive lymph nodes, cases were divided in to topical reactive lymphoid hyperplasia group (RLH, n=18) and non-RLH group (n=43) by using a median method. The postoperative disease-free survival (DFS) and overall survival (OS) rates of patients in different groups were compared using Kaplan-Meier method and log-rank test, respectively. ResultsThere were no significant difference between the two groups in age, gender, pathological stage, surgical approach, extent of surgery or methods of postoperative chemotherapy (P > 0.05). The median disease-free survival time was 50 months in RLH group, and the median disease-free survival time was 39 months in non-RLH group. DFS of patients in RLH group was significant higher than non-RLH group (66.7% vs. 34.9%, P=0.048). The median survival time was 53.6 months and 52.3 months, respectively, in RLH group and non-RLH group. No difference was found in OS between the two groups (72.2% vs. 60.5%, P=0.338). ConclusionTopical reactive lymphoid hyperplasia reactive the immunity of gastric cancer patients and contact postoperative DFS rate.
Proliferative vitreoretinopathy (PVR) is a common complication and major cause of blindness of ocular trauma. Many cytokines, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), participate in the process of the pathogenesis of traumatic PVR. VEGF competitively inhibits binding of PDGF to its receptor (PDGFRα), enables indirect activation of PDGFRα by non-PDGF ligands, resulting in reduced p53 expression, cell proliferation and migration, which is a key point in the pathogenesis of traumatic PVR.
Objective To explore the change of gene expression of stress activated protein kinase (SAPK) and its upstream signalregulated molecule ——mitogen activated protein kinases(MAPKs) (MKK4 and MKK7) in hypertrophic scar and autocontrol normal skin. Methods The total RNA was isolated from 8 hypertrophic scars and 8 auto-control skin, and then mRNA was purified. The gene expressions of MKK4, MKK7 and SAPK were examined with reverse transcriptionpolymerase chain reaction(RT-PCR) method. Results In hypertrophic scar, both MKK7 and SAPK genes weakly expressed. In auto-control skin, the expression of these 2 genes was significantly elevated in comparison with hypertrophic scar (Plt;0.01). The expression levelsof these 2 genes were 1.5 times and 2.6 times as long as those of hypertrophic scar, respectively. Gene expression of MKK4 had no significant difference between autocontrol skin and hypertrophic scar (Pgt;0.05). Conclusion Decreased gene expression of MKK7 and SAPK which results in reducing cell apoptosis might be one of the mechanisms for controlling the formation of hypertrophic scar.
Purpose To study the possibility of prevention of proliferative vitreoretinopathy（PVR） by transduction of exogenous gene in vivo． Methods PVR model of rabbits was induced by intravitreal injection of fibroblasts．beta;-galactosidase (lacZ) gene as a reporter gene was transfered into the vitreous of PVR model eyes mediated by retroviral vector, and the expression of the gene in eye tissues was determined ． Gene transfection was done on the 6th day after fibroblasts injection,and the dosage of intravitreal injection of reporter gene was 0．1ml PLXSN/lacZ serum-free supernatant (1．1times;106 cfu/ml)． Results lacZ gene expression was seen in proliferative membranes after gene transfection, and the expression was located maily at the surface of PVR membrane．The reporter gene expression lasted at least more than 30 days．No expression was found in retinal tissues． Conclusions Retrovirus mediated gene can be directionally transducted in PVR membrane,and might possess the feasibility of gene therapy for PVR． (Chin J Ocul Fundus Dis, 2001,17:224-226)
Objective To investigate the effects of asiaticoside onthe proliferation and the Smad signal pathway of the hypertrophic scar fibroblasts.Methods The hypertrophic scar fibroblasts were cultured with tissue culture method. The expressions of Smad2 and Smad7 mRNA after asiaticoside treatment were determined by reverse transcriptionpolymerase chain reaction 48 hours later. Thecell cycle, the cell proliferation, the cell apoptosis and the expression of phosphorylated Smad2 and Smad7 with(experimental group) or without(control group) asiaticoside were detected with flow cytometry, immunocytochemistry and Western blot. Results Asiaticoside inhibited the hypertrophic scar fibroblasts from phase S to phase M. The Smad7 content and the expression of Smad7 mRNA were (1.33±1.26)% and (50.80±22.40)% in experimental group, and (9.15±3.36)% and (32.18±17.84)% in control group; there were significant differences between two groups (P＜0.05). While the content and the mRNA expression of Smad2 had no significant difference between two groups. Conclusion Asiaticoside inhibits the scar formation through Smad signal pathway.