Objective To observe the proliferation and migration of endothelial cells after 30% total burn surface area (TBSA) of deep partial thickness scald, and the effect of basic fibroblast growth factor (bFGF) on angiogenesis during wound healing.Methods A total of 133 male Wistar ratswere divided randomly into normal control (n=7), injured control group (n=42), bFGF group (n=42) andanti-c-fos group (n=42). The apoptosis expression of fibroblasts was determinedwith in situ hybridization and the changes of proliferation cell nuclear antigen(PCNA), focal adhesion rinase(FAK), c-fos and extracellular signalregulated kinase(ERK) proteins expression were detected with immunohistochemistry staining technique after 3 hours, 6 hours, 1 day, 3 days, 7 days, 14 days and 21 days of scald.Results In injured control group and bFGF group, theproliferation rate of the vascular endothelial had evident changes 7 days and14 days after scald； the expression of FAK was increased 14 days after scald. ERK proteins expression was different between injury control group and bFGF group at initial stage after scald. Stimulation of ERKs by bFGF led to up-regulation of c-fos and b expression of FAK. Conclusion Exogenous bFGF extended the influence on wound healing process by ERK signaling pathway, affecting migration cascade of vascular endothelial cell. The oncogene proteins play an important role on accelerating angiogenesis duringwound healing.
Objective To observe the inhibitory effects of local co-transfection of tissuetype plasminogen activator(tPA) gene and proliferating cell nuclear antigen antisense oligodeoxynucleotides(PCNA-ASODN) on the intima proliferation and restenosis of autograft artery in rabbits. Methods One hundred and twenty male Zelanian rabbits were randomly divided into four groups(n=30, in each group): control group, PCNA-ASODN group, tPA group and tPA+PCNAASODN group. The left and right external iliac arteries (length 1.0 cm) were transplanted reciprocally. The transplanted arteries were respectively soaked in lipofection, PCNAASODN, pBudCE4.1/tPA and pBudCE4.1/tPA+PCNA-ASODN solution about 15 minutes. The transplanted arteries were sutured with 9-0 sutures soaked in PCNA-ASODN and pBudCE4.1/tPA solution. Each group were divided into five subgroups(n=6, in each subgroup) according to the sacrifice time (3 d, 7 d, 14 d, 28 d and 56 d after operation). On every sacrifice time point, the vascular specimens were harvested. The thrombocyte assembling and thrombus forming lining vessel wall were observed by scanning electron microscope. The pathological morphology of transplanted arteries were observed under microscope(HE). The intimal areas and stenosis ratio(%) of transplanted arteries were calculate and analyzed statistically among groups by computer system. The mRNA expression of tPA gene in transplanted ressel wall was detected with vevere transcriptionPCR(RT-PCR). The number of PCNA positive cells in transplanted vessel wall was counted by SP immunochemisty.Results The mRNA expression of tPA gene in the transplanted vessel wall in tPA and tPA+PCNA-ASODN groups was higher than that of the other two groups(P＜0.01).The number of PCNA positive cells in the transplanted arteries in PCNAASODN, tPA and tPA+PCNAASODN groups were significantly lower than that of control group(P＜0.05，P＜0.01). The intimal areas and degrees of luminal stenosis of PCNAASODN, tPA and tPA+PCNAASODN groups were lower than those of control group(P＜0.05，P＜0.01), and those of tPA＋ PCNA-ASODN group were lower than those of PCNA-ASODN and tPA groups(P＜0.05). Scanning electron microscopy showed that there were a few thrombocytes lining the vessel wall of tPA group and tPA+PCNAASODN group and no thrombus, whereas there were abundant thrombocytes and thrombi lining the vessel wall of the control group. Conclusion Co-transfection of tPA gene and PCNA-ASODN can effectively inhibit the proliferation of VSMC, hyperplasia of intima and restenosis of transplanted artery.
Objective To investigate the effect of ursolic acid on the proliferation and apoptosis of human osteosarcoma cell line U2-OS and analyze its mechanism. Methods Human osteosarcoma cell line U2-OS was divided into 4 groups, which was cultured with ursolic acid of 0, 10, 20, and 40 μmol/L, respectively. At 0, 24, 48, and 72 hours after being cultured, the cell proliferation ability was detected by cell counting kit 8 (CCK-8). At 48 hours, the effects of ursolic acid on cell cycle and apoptosis of U2-OS cells were measured by flow cytometry. Besides, the expressions of cyclin D1 and Caspase-3 were detected by real-time fluorescent quantitative PCR and Western blot. Results CCK-8 tests showed that the absorbance (A) value of each group was not significant at 0 and 24 hours (P>0.05); but the differences between groups were significant at 48 and 72 hours (P<0.05). Flow cytometry results showed that, with the ursolic acid concentration increasing, the G1 phase of U2-OS cells increased, the S phase and G2/M phase decreased, and cell apoptosis rate increased gradually. There were significant differences between groups (P<0.05). Compared with the 0 μmol/L group, the relative expressions of cyclin D1 mRNA and protein in 10, 20, and 40 μmol/L groups significantly decreased (P<0.05); whereas, there was no significant difference in relative expression of Caspase-3 mRNA between groups (P>0.05). However, with the ursolic acid concentration increasing, the relative expressions of pro-Caspase-3 protein decreased and the relative expressions of activated Caspase-3 increased; there were significant differences between groups (P<0.05). Conclusion Ursolic acid can effectively inhibit the proliferation of osteosarcoma cell line U2-OS, induce the down-regulation of cyclin D1 expression leading to G0/G1 phase arrest, increase the activation of Caspase-3 and promote cell apoptosis.
Objective To investigate the effect of different concentrations of raloxifene (RAL) on the proliferation and apoptosis of human aortic valve interstitial cells (AVICs) in vitro. Methods AVICs were isolated from human aortic valve by collagenase type Ⅱ, and cultured in different concentrations (0 nmol/L, 0.1 nmol/L, 1 nmol/L,10 nmol/L, 100 nmol/L and 1 000 nmol/L) of RAL. AVICs cultured in 0 nmol/L RAL were treated as the control group and those in other concentrations of RAL as the experiment groups. The proliferation and apoptosis of AVICs were evaluated by Cell Proliferation Assay (MTS assay) on day 0, 3, 5, 7 and 9. Flow cytometry was used to detect the cell cycle and apoptosis of AVICs on day 7. Results MTS results showed that the optical density value at 490 nm was much less in 10 nmol/L RAL and 100 nmol/L RAL groups (P＜0.05) on day 5, 7 and 9 than that in the control group. Flow cytometry results demonstrated that S-phase rate (P＜0.05) and cell apoptosis rate (P＜0.05) on day 7 were lower in the 10 nmol/L and 100 nmol/L RAL groups compared with the control group. Conclusion RAL with suitable concentration can inhibit proliferation and apoptosis of AVICs, which will lay an important foundation for further research of the role of RAL on heart valve diseases.
OBJECTIVE: To observe the effects of hyaluronic acid (HA) and basic fibroblast growth factor (bFGF) on the proliferation of the cells from medial collateral ligament (MCL) and anterior cruciate ligament (ACL) cells. METHODS: The MCL cells and ACL cells of mature New Zealand white rabbit were cultured, while HA, bFGF or HA and bFGF were added to the cell culture media, the cellular proliferation was assayed by MTT method. RESULTS: HA only had no effect on the preoliferation of ACL cells, but had a small stimulatory effect on the proliferation of MCL cells. The addition of 1 ng/ml bFGF enhanced the proliferation of both MCL and ACL cells significantly, and this enhancement was maximal in the concentration of 50 ng/ml. However, the enhancement of proliferation of MCL and ACL cells could be achieved when the combination of HA in concentration of 100 micrograms/ml and bFGF in concentration of 100 ng/ml. CONCLUSION: It is evident that bFGF can enhance the proliferation of the ligament cells. HA can maintain the normal growth of ACL cells with no effect on the proliferation of the cells, while HA has a small stimulatory effect on the proliferation of MCL cells. However, when bFGF is coordinated with HA, more improvement of cellular proliferation can be achieved. HA can be used as a potential carrier for bFGF to enhance the healing of ligamentous tissue injuries.
Objective To investigate the division, prol iferation and differentiation abil ities of nestin+/GFAP+cell after spinal cord injury and to identify whether it has the characteristic of neural stem cells (NSCs). Methods Twelvemale SD rats, aged 8 weeks and weighing 200-250 g, were randomized into 2 groups (n=6 per group): model group inwhich the spinal cord injury model was establ ished by aneurysm cl ip compression method, and control group in which no processing was conducted. At 5 days after model ing, T8 spinal cord segment of rats in each group were obtained and the gray and the white substance of spinal cord outside the ependymal region around central tube were isolated to prepare single cellsuspension. Serum-free NSCs culture medium was adopted to culture and serum NSCs culture medium was appl ied to induce differentiation. Immunohistochemistry detection and flow cytometry were appl ied to observe and analyze the type of cells and their capabil ity of division, prol iferation and differentiation. Results At 3-7 days after injury, the model group witnessed a plenty of nestin+/GFAP+ cells in the single cell suspension, while the control group witnessed few. Cell count of the model and the control group was 5.15 ± 0.71 and 1.12 ± 0.38, respectively, indicating there was a significant difference between two groups (P lt; 0.01). Concerning cell cycle, the proportion of S-phase cell and prol iferation index of the model group (15.49% ± 3.04%, 15.88% ± 2.56%) were obviously higher than those of the control group (5.84% ± 0.28%, 6.47% ± 0.61%), indicating there were significant differences between two groups (P lt; 0.01). In the model group, primary cells gradually formed threedimensional cell clone spheres, which were small in size, smooth in margin, protruding in center and positive for nestin immunofluorescence staining, and large amounts of cell clone spheres were harvested after multi ple passages. While in the control group, no obvious cell clone spheres was observed in the primary and passage culture of single cell suspension. At 5 days after induced differentiation of cloned spheres in the model group, immunofluorescence staining showed there were a number of galactocerebroside (GaLC) -nestin+ cells; at 5-7 days, there were abundance of β-tubul in III-nestin+ and GFAP-nestin+ cells; and at 5-14 days, GaLC+ ol igodendrocyte, β-tubul in II+ neuron and GalC+ cell body and protruding were observed. Conclusion Nestin+/GFAP+ cells obtained by isolating the gray and the white substance of spinal cord outside the ependymal region around central tube after compressive spinal cord injury in adult rat has the abil ity of self-renewal and the potential of multi-polarization and may be a renewable source of NSCs in the central nervous system.
Purpose To observe the expression of proliferating cell nuclear antigen（PCNA）and bcl-2 of cultured human retinal pigment epithelial cells（RPE）． Methods SABC techniques were applied for immunocytochemical staining of cultured RPE with mouse anti-human PCNA monoclonal antibody and rabbit antihuman bcl-2 antibodies． Results 31.2% and 50.6% cultured cells were positive to anti-human PCNA at 24h and 48h after seeding，respectively．The positive staining was mottled in the nucleus．positive staining for bcl was seen in 76%to 90% cells as fine granules scattered within the cytoplasm. Conclusion One half of cultured RPE expressed PCNA，indicating that the cells were in phase S of the cell cycle．Positive staining for bcl-2 appeared in much more RPE cells．These biological markers may be associated with the growth activity of cultured RPE． (Chin J Ocul Fundus Dis,1998,14:26-28)
ObjectiveTo explore the effects of exogenous estrogen receptor β1 (ERβ1) gene on the expression of human telomerase reverse transcriptase (hTERT) as well as the changes of proliferation ability in MDA-MB-231 cell line by transfecting recombinant eukaryotic expressing vector containing ERβ1 cDNA into human breast cancer MDA-MB-231 cell. MethodsRecombinant eukaryotic expressing vector containing ERβ1 cDNA was transfected into human breast cancer MDA-MB-231 cell by using cationic liposome as transfecting agent (acted as pcDNA3.1ERβ1 transfection group), empty vector group and non-transfection group acted as controls. The expression levels in both the mRNA and protein of both the ERβ1 and hTERT were tested by real-time PCR and Western blot, respectively. The change of proliferation ability in MDA-MB-231 cell was displayed by cell growth curve, and the change of cell apoptosis was detected by flow cytometry. ResultsThe expression level of ERβ1 mRNA in the pcDNA3.1-ERβ1 transfection group (0.449±0.077) significantly increased as compared with the nontransfection group (0.153±0.035) or the empty vector group (0.160±0.020), P=0.001 or P=0.000. The expression level of ERβ1 protein in the pcDNA3.1-ERβ1 transfection group (0.847±0.065) significantly increased as compared with the non-transfection group (0.356±0.050) or the empty vector group (0.390±0.030), P=0.001 or P=0.000. The expression level of hTERT mRNA in the pcDNA3.1-ERβ1 transfection group (0.127±0.020) significantly decreased as compared with the non-transfection group (0.283±0.025) or the empty vector group (0.283±0.049), P=0.001 or P=0.002. The expression level of hTERT protein in the pcDNA3.1-ERβ1 transfection group (0.147±0.023) significantly decreased as compared with the non-transfection group (0.783±0.025) or the empty vector group (0.802±0.019), P=0.001 or P=0.002. The rate of cell apoptosis in the pcDNA3.1-ERβ1 transfection group 〔(6.15±0.94)%〕 was higher than that in the non-transfection group 〔(1.41±0.42)%〕， P=0.001. Cell proliferation curve showed that proliferation ability significantly decreased in the pcDNA3.1-ERβ1 transfected groups as compared with the non-transfection group (Plt;0.05). ConclusionERβ1 could inhibit cell growth of human breast cancer MDA-MB-231 cell by down-regulating the expression of hTERT.
Objective To observe the clinical features of congenital hypertrophy of retinal pigment epithelium （CHRPE）. Methods The clinical data of 13 CHRPE patients including visual acuity, slit-lamp microscope examination, indirect ophthalmoscope examination and fundus fluorescein angiography (FFA) were retrospectively analyzed. The patients, 9 males and 4 females, with the mean age of 27.8 years. Results All patients were unilateral, without systemic diseases and no subjective symptoms in majority. Only 30.77% of initial diagnosis was correct, other diagnosis include choroidal nevi, old chorioretinopathy or no diagnosis. The round or oval black lesion was found in ocular fundus of all patients, 7.69% was located on the optic disk, 46.15% was located on the inferior temporal retina, 30.77% was located on the superior temporal retina, 15.39％ was located on the inferior nasal retina. 92.31％ was pigmented CHRPE and 7.69％ was non-pigmented CHRPE. FFA showed blocked fluorescence and transmitted fluorescence in the lesion, few eyes were found dilated capillary vessel and fluorescent leakage on the late stage of FFA, most eyes had normal retinal vessels. Conclusion The isolated CHRPE is round or oval black lesion in ocular fundus which lack of subjective symptoms, mostly located on the peripheral retina; the FFA characteristics showed blocked fluorescence and transmitted fluorescence, and CHRPE often misdiagnosed as other disease, it should be combine the ocular fundus manifestation with the FFA to diagnose properly.
Objective To explore the effect of the platelet-rich plasma (PRP) on proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs) in China goat in vitro. Methods MSCs from the bone marrow of China goat were cultured. The third passage of MSCs were treated with PRP in the PRP group (the experimental group), but the cells were cultured with only the fetal calf serum (FCS) in the FCS group (the control group). The morphology and proliferation of the cells were observed by an inverted phase contrast microscope. The effect of PRP on proliferation of MSCs was examined by the MTT assay at 2,4,6 and 8 days. Furthermore, MSCs were cultured withdexamethasone（DEX）or PRP; alkaline phosphatase (ALP) and the calcium stainingwere used to evaluate the effect of DEX or PRP on osteogenic differatiation of MSCs at 18 days. The results from the PRP group were compared with those from the FCS group. Results The time for the MSCs confluence in the PRP group was earlier than that in the FCS group when observed under the inverted phase contrast microscope. The MTT assay showed that at 2, 4, 6 and 8 days the mean absorbance values were 0.252±0.026, 0.747±0.042, 1.173±0.067, and 1.242±0.056 in the PRP group, but 0.137±0.019, 0.436±0.052, 0.939±0.036, and 1.105±0.070 in the FCS group. The mean absorbance value was significantly higher in the PRP group than in the FCS group at each observation time (P＜0.01). Compared with the FCS group, the positive-ALP cells and the calcium deposition were decreased in the PRP group; however, DEX could increase boththe number of the positiveALP cells and the calcium deposition. Conclusion The PRP can promote proliferation of the MSCs of China goats in vitro but inhibit osteogenic differentiation.