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Objective To investigate the effect of p38MAPK gene silencing recombinant adeno-virus on the expression of target gene in different time and to detect the effect of p 38MAPK signal pathway on the upper lip scar hyperplasia at different time to determine the optimal scar treatment time .Methods The adenovirus vector was injected into the scar tissue in 0 week ,1 week and 2 week after cheiloplasty in rabbit .The specimens were harvested in 3 week postoperatively .Four methods in-cluding Sirius red staining ,immunohistochemical staining (IHC) ,Western blotting (WB) ,real-time PC (RT-PCR) were used to quantitatively and quantitatively detect the relative expression levels of p38MAPK and scar-related factors (col Ⅰ ,col Ⅲ ,MM P1 ,TIMP1) .Results Sirius red staining and immunohistochemical staining showed that in 1st week the expression of col Ⅲ and MMP1 in scar tis-sue was significantly higher than that in 0 week and 2 week after operation and the expression of col Ⅰand TIMP1 was significantly less than that in 0 week and 2 week after operation .The results of WB and RT-PCR were consistent with that of IHC .Conclusions After injection into the upper lip scar tis-sue with adenovirus in 1 week ,the degree of scar hyperplasia is the least .
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<p><b>OBJECTIVE</b>RNA interference was applied to knockdown the Dhcr7 gene in mouse embryonic palatal shelves to facilitate understanding of the function of Dhcr7 gene variants in the fusion of palatal shelves.</p><p><b>METHODS</b>The pAdTrack-CMV-siDhcr7 was constructed using the specific siRNA sequence of Dhcr7 from C57BL/6J mouse. The pAdTrack-CMV- siDhcr7 of positive clones was reconstructed in vitro, and the recombinant adenovirus pAdEasy-1-siDhcr7 of kanamycin resistance was screened. The adenovirus vector DNA was then prepared for transfecting the embryonic palatal shelves. Thirty pairs of embryonic palatal shelves at 13.5 d gestational age were harvested and then randomly divided into the following three groups: normal control group (n = 10), which included palatal shelves inculture medium without cholesterol; blank adenovirus control group (n = 10), which included palatal shelves in culture medium without cholesterol and blank adenovirus; and experimental group (n = 10), which included palatal shelves in culture medium without cholesterol and adenovirus encoding Dhcr7 siRNA. At 48 h after in vitro cultivation, the mRNA and protein of the palatal shelves were obtained for scanning electron microscopy (SEM), reverse transcription polymerase chain reaction (RT-PCR), and Western blot analyses.</p><p><b>RESULTS</b>SEM showed that the palatal shelves of the normal control and blank adenovirus control groups fused and formed continuous palates, whereas those of the experimental group was almost undeveloped but exhibited large gaps between the two palatal shelves. RT-PCR and Western blot analyses showed that the mRNA and protein of Dhcr7 in the experimental group decreased compared with those in the normal control group with a significant difference (P < 0.05).</p><p><b>CONCLUSION</b>Results indicate that Dhcr7 gene silencing affects the fusion of palatal shelves. Thus, Dhcr7 gene may serve a function in the normal development of palates.</p>
Subject(s)
Animals , Mice , Cleft Palate , Gene Silencing , Mice, Inbred C57BL , Microscopy, Electron, Scanning , Organ Culture Techniques , Oxidoreductases Acting on CH-CH Group Donors , Palate , RNA, MessengerABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of 7- dehydrocholesterol reductas (Dhcr-7) gene silencing on the palatal development by sonic hedgehog (Shh)-bone morphogenetic protein2(BMP-2) signal pathway in vitro.</p><p><b>METHODS</b>A total of 60 pairs of palatal shelves fromgestation day (GD) 13.5 mouse embryos were divided into three groups (A, B, C) of 20 randomly. In group A (control), palatal shelves were cultured with medium containing no cholesterol.In group B (Dhcr-7-siRNA), palatal shelves were cultured without cholesterol medium but containing Dhcr-7 siRNA adenovirus. After 48h, the culture medium of groups A and B were changed with medium without cholesterol. In group C (cholesterol), palatal shelves were cultured without cholesterol medium but containing Dhcr-7 siRNA adenovirus. After 48h, the culture medium of group C was changed with medium containing 600 mg/L cholesterol. After 72h again, tissues dyeing and scanning electron microscope (SEM) technique were used to observe morphological changes of palates. Both RT-PCR and Western blottingtechniques were used to measure mRNA and protein expressions for Dhcr-7, Shh, and BMP-2, respectively.</p><p><b>RESULTS</b>The tissues dyeing and SEM showedthat the palates fusedin groups A and Candthe palates did not fuse in group B eventually. The expression of both mRNA and proteins for Shh and BMP-2 in group B wasdecreased with the Dhcr-7 reduction. In group B, the mRNA and protein expression of Shh was separately 0.063±0.018 and 0.092±0.065;the mRNA and protein expression quantity of BMP- 2 was separately 0.054±0.018 and 0.049±0.021. In group A, the mRNA and protein expression of Shh was separately 0.667±0.093 and 0.639±0.078;the mRNA and protein expression of BMP-2 was separately 0.591 ± 0.043 and 0.569 ± 0.081. The difference of Shh and BMP- 2 mRNA and protein expression between A and B group were statistically significant separately (P < 0.05). The expression of both mRNA and protein for Dhcr-7 (0.074±0.034 and 0.075±0.028) did not changebasicallyin group C, compared with the Dhcr- 7expression of mRNA and protein (0.083±0.045; 0.067±0.065) in group B, the difference wasnot statistically significant(P > 0.05). In group C, the mRNA and protein expressionof Shh (0.649±0.085 and 0.608±0.092) and BMP-2 (0.578±0.062 and 0.548±0.065) were significantly increased. The difference of Shh and BMP-2 mRNA and protein expression between B and C group were statistically significant separately (P < 0.05).</p><p><b>CONCLUSIONS</b>Dhcr-7 could influence the expression of Shh and BMP-2. Dhcr-7 reductase regulated the palatal development by the Shh-BMP-2 signal pathway.</p>
Subject(s)
Animals , Mice , Bone Morphogenetic Protein 2 , Genetics , Metabolism , Cholesterol , Culture Media, Conditioned , Chemistry , Pharmacology , Hedgehog Proteins , Genetics , Metabolism , Oxidoreductases Acting on CH-CH Group Donors , Metabolism , Palate , Metabolism , RNA, Messenger , Metabolism , RNA, Small Interfering , Metabolism , Random Allocation , Signal TransductionABSTRACT
BACKGROUND:Recombinant bovine basic fibroblast growth factor is a manifold effect cytokine which can promote angiogenesis, wound healing, tissue repair and bone regeneration. Recombinant bovine basic fibroblast growth factor with good histocompatibility is easy to operate and has been widely used in oral and maxil ary surgery. OBJECTIVE:To evaluate the effect of recombinant bovine basic fibroblast growth factor against dry socket syndrome after tooth extraction. METHODS:A total of 160 patients who had been extracted mandibular third molar were selected and randomly divided into two groups. In the experimental group, recombinant bovine basic fibroblast growth factor was put into the sockets after mandibular third molars were extracted, while in the control group, we let the wounds to be healed natural y without any materials. The incidence of dry socket syndrome was observed and compared between two groups at 3 days, 5 days and 1 week after tooth extraction. RESULTS AND CONCLUSION:One patient had dry socket after operation in the experimental group, and the incidence was 1.25%. In the control group, 10 patients suffered from dry socket, and the incidence was 12.5%. There was a significant difference in the incidence of dry socket between the two groups (P<0.01). There was visible granulation tissue within the tooth socket after tooth extraction in the experimental group, and extraction sockets narrowed and were fil ed with granulation tissues, which was 1-2 days earlier than the control group. No al ergies, tissue hyperplasia and other local and systemic reactions occurred in patients receiving implantation of recombinant bovine basic fibroblast growth factor gel. These findings indicate that local implantation of recombinant bovine basic fibroblast growth factor gel after mandibular tooth extractions can speed up the healing of dental extraction wounds.
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BACKGROUND: How to make growth factor plays a role persistently and efficiently is a key in constructing bone tissue engineered bone. Currently, varied microspheres or scaffolds were used as release carriers, however, the delayed release effects needs elevating.OBJECTIVE: To prepare chitosan microosPheres/nano-hydroxyapatite/poly (lactic-co-glycolic acid) (CMs/nHA/PLGA) scaffolds, and to measure its characteristics of delayed release of bovine serum albumin (BSA).METHODS: CMs were prepared by an emulsifying cross linking method with BSA as a model protein. Using ice particulates as porogen, composite CMs/nHA/PLGA scaffolds were prepared by freeze-drying. The characteristic and morphology of the composite were observed by scanning electron microscope, later particle size analyzer, mercury porosimeter and universal testing machine, and the release behavior of BSA was investigated in vitro.RESULTS AND CONCLUSION: The CMs were spherical shape with a regular surface, with diameters of 20-40 μm. The encapsulation efficiency of the CMs was 86.5%, and the loading capacity was 0.8%. With the increase of initial BSA dosage, the loading capacity increased to 2.6%, while the encapsulation efficiency decreased to 74.1%. The CMs can be uniformly distributed in PLGA scaffolds to form CMs/nHA/PLGA scaffolds, which had 100-400 μm pore diameter and over 80% porosity, with 1.1-2.3 pMPa compressive strength, and 26.5% cumulative degradation at 10 weeks. The cumulative release of BSA from nHA/PLGA scaffolds was above 85% at 36 hours, which from CMs was 33.6% at 10 days, and that from CMs/nHA/PLGA scaffolds was 81.5% at 40 days. The results demonstrated that CMs/nHA/PLGA scaffolds have an excellent releasing efficiency for protein drugs with suitable compressive strength and degradation, which would be used as delivery system and tissue engineering scaffolds.