Objective To study the influence of transforming growth factor-β1(TGF-β1), dentin non-collagen proteins(dNCPs) and their complexon tissue engineering pulp system. Methods Collagen I and dentin powder were used to construct the system of pulp cells in 3dimensional culture, dentin powder was added in the gel. The tissue engineering pulp were divided TGF-β1 group, dNCPs group, TGF-β1/dNCPsgroup and control group.After3, 6 and 14 days, the appearance and the differentiation of pulp cells were observed by HE staining and immunohistochemical staining -respectively. Results Collagen I could form netted collagen gel construction. Growing condition of pulp cells in gel was similar to that of pulp cells in vivo. After the TGF-β1 and dNCPswere added, the pulp cells had some characteristics of odontoblasts and had unilateral cell process after culture 6 days. Pulp cells arranged with parallel columnar and form dentin-pulp-like complex after 14 days. Immunohistochemical staining showed dentin salivary protein(DSP) began to express in some cells.The number of positive cell was most in the TGF-β1 group. No positive cells were detected in the control group. Conclusion The transforming growth factor-β1 and noncollagen proteins can stimulate the pulp cells to transform into odontoblasts to some extent, which promote the formation of tissue engineering pulp.
Objective To study the relationship between the expression of sonic hedgehog (Shh) and vascular endothelial growth factor (VEGF) in hypoxic human retinal pigment epithelial (hRPE) cells. Methods Cultured hRPE-19 cells (3rd - 6th generations) were used in this experiment. hRPE-19 cells were divided into three groups including the control group, the hypoxia experimental group (100 μmol/L CoCl2) and the inhibition group (pretreatment with 20 μmol/L cyclopamine 1 hour before hypoxia). After culturing for 4, 8, 12 and 24 hours, the mRNA level of Shh and VEGF genes in these cells were measured by fluorescence quantitative polymerase chain reaction, and the protein level of Shh and VEGF in the supernatants were measure by enzyme-linked immunosorbent assay. The relationship between the expression of Shh and VEGF was analyzed by Pearson correlation analysis. Results The control group expressed low levels of Shh and VEGF mRNA/protein. The expression of Shh and VEGF mRNA/protein in the hypoxia experimental group was significantly higher than that in the control group (F=178.364, 183.732, 77.456, 91.572; P＜0.01). The expression of Shh and VEGF mRNA in the inhibition group was significantly lower than that in the hypoxia experimental group (F=68.745, 121.834; P＜0.01). In the hypoxia experimental group, the expression of VEGF protein was positively correlated with the expression of Shh protein (r=0.942, P＜0.05); and the expression of VEGF and Shh mRNA was positively correlated (r=0.970, P＜0.01). However, there was no significant correlation in the expression of VEGF and Shh mRNA in the inhibition group (r=0.915, P＞0.05). Conclusion There is a positive correlation between the expression of Shh and VEGF in hypoxic hRPE cells.
Objective To investigate the inhibitory effects of IBI302 on experimental choroidal neovascularization (CNV). Methods Affinity of IBI302 to vascular endothelial growth factor (VEGF) family cytokines (including VEGF-A165, VEGF-A121 and placental growth factor PlGF) and complements (C3b, C4b) was determined by enzyme-linked immunosorbent assay (ELISA). The antagonist effect of IBI302 on VEGF was measured by proliferation, migration and tube formation tests of human umbilical vein endothelial cells (HUVEC). The anti-complement activity of IBI302 was measured by hemolysis test mediated by complement classical pathway and alternative pathway. Rhesus laser-induced CNV model was divided into 5 groups including model control group, bevacizumab group, IBI302 0.25 mg group, IBI302 0.50 mg group and IBI302 1.25 mg group. Fluorescein angiography and optical coherence tomography were performed on these monkeys at 14 and 28 days after drug delivery to observe the fluorescein leakage area and retinal thickness. The aqueous VEGF concentration was measured at 29 days after drug delivery. Results IBI302 showed good affinity to VEGF-A165, VEGF-A121 and PlGF, as well as C3b and C4b. IBI302 significantly inhibited the proliferation, migration and tube formation of HUVEC induced by VEGF-A165. IBI302 inhibited the hemolysis induced by complements obviously. At 14 and 28 days after drug delivery, the area of fluorescein leakage and retinal thickness in IBI302 0.25 mg group, IBI302 0.50 mg group, IBI302 1.25 mg group were reduced. The differences of the area of fluorescein leakage and retinal thickness in three IBI302 groups were not significant (P > 0.05). At 29 days after drug delivery, the VEGF concentration in the aqueous of rhesus monkey in bevacizumab group [(38.644±6.521) pg/ml] was decreased than that in model control group [(94.203±17.360) pg/ml], the difference was significant (P < 0.05). The VEGF concentration in the aqueous of rhesus monkey in three IBI302 groups were less than 31.300 pg/ml. Conclusion IBI302 inhibited experimental CNV through blocking the activity of VEGF and complement.
Objective To clarify the relationship between inhibition of proliferation and cxpression of Ki-67 in cultured human retinal pigment epithelial(RPE) cells. Methods The cultured human RPE cells were treated with daunoblastina at a dose of 180 mu;g/L for 12h.Twenty-four hours later,DNA inhibiting rate was studied by using tritium-labelled thymidine deoxyribose(3H-TdR)incorporation assay.The expression of Ki-67 was evaluated by immunocytochemical staining technique and image analysis system.Flow cytometry was used to analyse cell cycle. Results DNA inhibiting rate was directly proportional to the dosage of daunoblastina.The proportion of the cells positive staining to Ki-67 in the control and the daunoblastina-treated group were 89.3% and 45.6%(Plt;0. 01),and the integral optical density values for expression of Ki-67 in the two groups were 68.1plusmn;6.2 and 27.3plusmn;5.5(Plt;0.01),respectively.The percen tage of cells in G2 phase of cell cycle increased from 8.9% to 29.5%. Conclusion G2 block was induced and poliferation was inhibited by daunoblastina in cultured human RPE cells.There is a relatively good correlation between Ki-67 immunostaining and inhibition of RPE cell proliferation. (Chin J Ocul Fundus Dis，2000，16：1-70)
Objective To review the recent advances in transforming growth factor-β(TGF-β) super family study and its role in new bone formation. Methods The latest original articles related to this subject were retrieved extensively,especially the effect of TGF-β, bone morphogenetic proteins(BMPs) and activin(ACT) on distractionosteogenesis. Results TGF-β, BMPs and ACT play important roles in prompting new bone formation and each of them has different effects. Among them, TGF-β can stimulate the proliferation of osteoblast and synthesis ofextra cellular medium; BMPs can initiate the differentiation of interstitial cell toosteocyte; then ACT displays the combine effect of above two factors. Conclusion TGF-β superfamily can regulate new bone formation and thus shorten the course of mandibular distraction osteogenesis.
ObjectiveTo observe the expression of Toll-like receptor 4 (TLR4) and inflammatory cytokines, leucocytic density and permeability in retina of diabetic rat. MethodsA total of 106 Brown Norway rats were randomly divided into experimental group and control group with 53 rats in each group. Diabetic model was established in experimental group by intraperitoneal injection of streptozotocin, and control rats received intraperitoneal injection of an equal volume of citric acid-sodium citrate buffer. Four weeks later, the retinas were collected for further analysis. TLR4 RNA and protein expression were measured by quantitative polymerase chain reaction and Western blot. Inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemo-attractant protein-1 (MCP-1), were measured by enzyme-linked immunosorbent assay in rat retina homogenate. Leukocyte density in the retina was measured by acridine orange fundus angiography. The retinal permeability was evaluated by Evans blue (EB) staining. ResultsTLR4 expression was significantly increased in diabetic rats of experimental group compared with non-diabetic rats of control group (F=1.606, 0.789; P < 0.05). Inflammatory cytokines (TNF-α, IL-1β and MCP-1) were significantly increased in retina of diabetic rats of experimental group versus non-diabetic rat of control group (F=24.622, 5.758, 4.829; P < 0.05). The retinal leukocyte density was (6.2±0.5)×10-5, (2.2±0.3)×10-5 cells/pixel2 in experimental and control group respectively, the difference was statistically significant (F=2.025, P < 0.05). The amount of retinal EB leakage was (23.41±4.47), (13.22±3.59) ng/mg in experimental and control group respectively, the difference was statistically significant (F=21.08, P < 0.05). ConclusionTLR4 and inflammatory cytokines expression, leucocytic density and permeability increased significantly in retina of diabetic rat.
Objective To observe the effect of shRNA interference lentivirus vector targeting rat Sirt1 gene on the expression of Sirt1 in retinal ganglion cell (RGC). Methods Four short hairpin (sh) RNA interference sequences targeting rat Sirt1 gene were designed. The target sequences of Oligo DNA were synthesized and annealed to double strand DNA, which was subsequently connected with pGLV3 lentivirus vector to build the lentiviral vector. The positive clones were identified by polymerase chain reaction (PCR) and DNA sequencing. The lentiviral vector construct and lentiviral packaging plasmids were co-transfected into 293T cells, then the titer of lentivirus were determined. The RGC were divided into 6 groups including blank group, negative control group and si-Sirt1-1, si-Sirt1-2, si-Sirt1-3, si-Sirt1-4 groups. Real-time PCR and Western blotting were used to detect the expression of Sirt1 mRNA and protein in the RGC cells. Results PCR and DNA sequencing analysis confirmed that the shRNA sequence was successfully inserted into the lentivirus vector. The concentrated titer of virus suspension was 8×108 TU/ml after the recombinant lentiviral vector successfully transfected and harvested in 293T cells. Comparing with NC group, the expression of Sirt1 mRNA and protein were significantly decreased in the si-Sirt1-1, si-Sirt1-2, si-Sirt1-3 and si-Sirt1-4 groups (F=27.682, 1 185.206; P=0.000, 0.000). The si-Sirt1-2 group had the strongest effect in reducing the expression of Sirt1 mRNA and protein. Conclusion The 4 lentiviral vectors harboring RNAi targeting rat Sirt1 gene can effectively down regulate the expression of Sirt1 mRNA and protein in RGC cells.
ObjectiveTo observe the influence of heat shock protein 27 (HSP27) sensibilization to retinal ganglion cells (RGC) apoptosis of rats. MethodsThirty-five female Wistar rats were randomly divided into HSP27 sensibilization group (15 rats), borate buffer solution (BBS) control group (15 rats) and normal group (5 rats). The rats in HSP27 sensibilization group were received hypodermic injection in rear limb with 100 μg HSP27 and complete freund adjuvant, intraperitoneal injection with 1 μg pertussis toxin. The BBS control group received the same volume of BBS at the same site. The normal group received no intervention. The intraocular pressure was measured 3 days before injection and 1, 2, 4, 6, 8 weeks after injection. Four, 6 and 8 weeks after injection, the retinal frozen sections was made to observe RGC apoptosis by terminal-deoxynucleoitidyl transferase mediated nick end labeling. The anti-HSP27 level in serum and cerebrospinal fluid were detected by enzyme linked immunosorbent assay. ResultsThere was no obvious change of intraocular pressure in rats in 3 groups before injection (P>0.05). RGC apoptosis was observed in HSP27 sensibilization group 4 weeks after injection, and increased significantly at 6 weeks after injection. There was no RGC apoptosis in BBS control group and normal group. The level of anti-HSP27 in serum and cerebrospinal fluid of HSP27 sensibilization group occurred at 4 and 6 weeks after injection respectively, decreased with prolongation of injection time. Compared with BBS control group and normal group, the RGC apoptosis rate, anti-HSP27 level in serum and cerebrospinal fluid of HSP27 sensibilization group were significantly increased (P<0.05). There was no significant difference of the RGC apoptosis rate, anti-HSP27 level in serum and cerebrospinal fluid between BBS control group and normal group (P>0.05). ConclusionsHSP27 sensibilization could promote the RGC apoptosis. The variation trend of anti-HSP27 level in cerebrospinal fluid is consistent with the RGC apoptosis.
Objective To investigate the effect of lung volume reduction surgery （LVRS） on messenger RNA expression levels of cytoskeletal proteins in diaphragmatic muscle tissues of emphysematous rabbits. Methods A total of 40 rabbits were randomly divided into 4 groups （10 rabbits in each group） ：normal control group, emphysema group, sham operation group and LVRS group. Rabbits in control group were intratracheally administered with 0.9% normal sodium, but those in other groups were intratracheally administered with 0.4% papain at the dose of 0.5 ml/kg and inhaled cigarette smoke to induce emphysema model. Then, rabbits in emphysema group were fed routinely, however, after median sternotomy , bilateral LVRS was performed in LVRS group but not in sham operation group. The mRNA expression levels of titin and nebulin in the diaphragmatic muscles of rabbits in each group were detected by reverse transcription-polymerase chain reaction（RT-PCR）. Results Compared with control group, the mRNA expression levels of titin and nebulin in the rabbit diaphragm of emphysema groups and sham operation group decreased significantly （P〈0.01 ）, so did those in LVRS group （P〈0.05）. But it increased significantly in LVRS group compared with emphysema group and sham operation group （P〈0.05）. Conclusion LVRS can increase the mRNA expression levels of titin and nebulin in diaphragmatic muscle tissues of emphysematous rabbits, which may be the associated mechanisms at the molecular level in restoring the functions of the emphysematous diaphragm by LVRS.
Objective To explore the relationship between the diabetic retinopathy (DR) and the changes of erythrocyte deformability(ED),erythrocyte membrane phospholipid and spectrin. Methods One hundred and eight patients with non-insulin dependent diabetes mellitus were divided into DR group(55 cases)and nonDR(NDR)group(53 cases).The changes of erythrocyte filtration index(EFI),erythrocyte membrane phospholipid and spectrin dimers(SP-D)and spectrin tetramers (SP-T)were measured in patients of DR and NDR groups and compared with the results of 53 cases of normal control group. Results The EFI,SP-D, SP-D/SP-T,sphingomyelin (SM) /phophatidylcholine(PC)were higher,and SPT,SM,PC,phophatidylserine(PS)and phatidylethanolamine(PE)were lower in patients with DR than those in control and NDR patients (F=8.467～18.925,q=6.845～12.627,Plt;0.001).The changes of all indicators in proliferative DR(PDR) patients were more obvious than those in background DR(BDR) patients(t=5,825-15.443,Plt;0.001).The EFI in DR patients was positively correlated to SM/PC,SP-D and SP-D/SP-T(Plt;0.01)，negatively correlated to SM,PC,PE,PS and SP-T(Plt;0.01). Conclusions The decrease of ED caused by the abnormalities of erythrocyte membrane phospholipid and spectrin might participate in the occurance and development of DR,and correlated to the degree of pathologic changes. (Chin J Ocul Fundus Dis, 1999, 15: 160-162)