OBJECTIVE: To observe the protein expression of phosphorylated form of P38 mitogen-activated protein kinase(P38MAPK) and c-Jun in hypertrophic scar skin and to explore their influences on the formation and maturation of hypertrophic scar. METHODS: The expression intensity and distribution of phosphorylated form of P38MAPK and c-Jun were examined with immunohistochemistry and pathological methods in 16 cases of hypertrophic scar skin and 8 cases of normal skin. RESULTS: In normal skin, the positive signals of phosphorylated form of P38MAPK mostly distributed in basal lamina cells of epidermis, while c-Jun was mainly located in epidermal cells and endothelial cells. The positive cellular rates of two proteins were 21.3% +/- 3.6% and 33.4% +/- 3.5% respectively. In proliferative hypertrophic scar skin, the particles of phosphorylated P38MAPK and c-Jun were mainly located in epidermal cells and some fibroblasts. The positive cellular rates of two proteins were significantly elevated to 69.5% +/- 3.3% and 59.6% +/- 4.3% respectively (P lt; 0.01). In mature hypertrophic scar, the expression of these proteins decreased but was still higher than that of normal skin. CONCLUSION: The formation and maturation of hypertrophic scar might be associated with the alteration of phosphorylated P38MAPK and c-Jun protein expression in hypertrophic scar.
ObjectiveTo systematically review the association between expression of osteopontin (OPN) and Chinese population with hepatocellular carcinoma (HCC) and its clinical pathological characteristics. MethodsSuch databases including CBM, CNKI, VIP and WanFang Data were searched from inception to July 2014, for studies about the association between expression of OPN and Chinese population with HCC and its clinical pathological characteristics. Two reviewers independently screened literature according to the exclusion and inclusion criteria, extracted data, and assessed methodological quality of included studies. Then, meta-analysis was performed using RevMan 5.2 software. ResultsA total of 10 case-control studies (involving 723 HCC cases and 102 controls) were included. The results of meta-analysis showed that:OPN expression was higher in HCC group than normal control group (OR=10.25, 95%CI 6.13 to17.14); and higher in imperfect capsular infiltration group than perfect capsular infiltration group (OR=2.71, 95%CI 1.58 to 4.64). However, no significant difference was found in OPN expression between isolated tumour group and multiple tumours group (OR=0.95, 95%CI 0.56 to 1.62); between high differentiation group and low differentiation group (OR=0.60, 95%CI 0.36 to 1.01); and between clinical stages I-Ⅱ group and clinical stages Ⅲ-IV group (OR=0.93, 95%CI 0.53 to 1.63). ConclusionCurrent evidence shows that OPN may take part in the whole course (occurrence and advance) of HCC in Chinese population, but the problem whether it can be used as a factor to evaluate prognosis needs to be further studied.
Objective To investigate the expression of enhancer of zeste homolog 2 （EZH2） mRNA in colorectal cancer tissues， and to explore the relationship with clinicopathological features of it. Methods PCR was used to examine the expression of EZH2 mRNA in colorectal cancer tissues， paracancerous tissues， and normal epithelium tissues of 27 patients with colorectal cancer. Meanwhile， the relationship between the expression of EZH2 mRNA and clinicopathological features of colorectal cancer were analyzed. Results Expression level of EZH2 mRNA in colorectal cancer tissues， paracancerous tissues， and normal epithelium tissues were 3.01±1.29， 1.20±0.69， and 0.87±0.37 respectively， and the expression value of colorectal cancer tissues at the top （P＜0.01）， but there were no significant difference between the paracancerous tissues and normal epithelium tissues （P=0.403）. Expression of EZH2 mRNA were related to TNM stages （P=0.002） and degree of differentiation （P=0.005）， but were not related to age （P=0.388）， gender （P=0.963）， diameter of tumor （P=0.579）， histological type （P=0.945）， location of tumor （P=0.611）， and lymph node metastasis （P=0.449）. Conclusions EZH2 mRNA expresses highly in colorectal cancer tissues， so it may play an essential role in the emergence and development of colorectal cancer. EZH2 mRNA can be used as one of the referenced indexes to determine the malignant degree and process of colorectal cancer.
In order to study the expression change of insulin-like growth factor-1 (IGF-1) and its receptor genes in different generations of tendon cell in culture, Dig-labeled synthesized oligonucleotide probes were used to detect the mRNA expression in primary, 6th and 13th generation of tendon cell. The results showed that IGF-1 receptor mRNA was expressed in all of the 3 above generation tendon cells. IGF-1 mRNA was expressed only in primary and 6th generation cells. Tendon cell of 13th generation did not express IGF-1 mRNA. It might suggest that the absence of IGF-1 mRNA expression be one of the causes which led to the decrease of reproductive ability of 13th generation tendon cell.
Objective To investigate the expression levels of osteoprotegerin (OPG) and receptor activator of NF-κB l igand (RANKL) mRNAs in bone tissues of the femoral head of the patients suffering glucocorticoid-induced osteonecrosisof the femoral head (ONFH), and to discuss the relationship between OPG/RANKL and ONFH. Methods Between March2007 and March 2008, bone tissues of the femoral head were collected as the experimental material from 35 patients suffering ONFH (experimental group) and from 21 patients suffering fracture of femoral neck (control group). The ratio of men to women in both groups was 4 ∶ 3, whose age was 41-70 years old (55.34 on average in the experimental group and 55.33 on average in the control group). The experimental group received over 3 weeks’ glucocorticoid treatment or more than 1 week’ s high-dose glucocorticoid treatment in recent 2 years, while the control group never received more than 1 week’s hormone treatment. In the two groups, the microstructure of bone tissues of the femoral head was detected by HE staining and the bone tissue total RNA was extracted, and then the expression levels of OPG mRNA and RANKL mRNA were examined by realtime quantitative PCR (RTQ-PCR) for each sample. Results HE staining: bone trabeculae and bone units were replaced by interrupted bone fragments, which were surrounded by many inflammatory granulation tissues and few osteocytes were seen in bone lacunae in the experimental group. In the control group, bone trabeculae and bone units were made by complete lamellar bones which surrounded blood vessels and osteocytes were seen in lacunae. RTQ-PCR testing: in the experimental group, OPG mRNA and RANKL mRNA were 1.35 ± 0.42 and 4.36 ± 1.35, respectively, while in the control group they were 1.78 ± 0.63 and 3.49 ± 1.02, respectively. The expression level of OPG mRNA in the experimental group was significantly lower than that in the control group, and the expression level of RANKL mRNA of the former was significantly higher than the latter. The OPG mRNA/ RANKL mRNA ratio in the xperiment group (0.34 ± 0.16) was significantly lower than that in the control group (0.54 ± 0.20), and there was significant difference (P lt; 0.05). Conclusion The glucocorticoid-induced ONFH may be related to the expression levels of OPG mRNA/RANKL mRNA in bone tissues.
Objective To construct the recombined DNA pcDNA3.1-hBMP-2 and transfect into human marrow stromal stem cells (MSCs) in vitro, and to explore theeffects of transfection on cellular proliferation and expression of vascular endothelial growth factor (VEGF). Methods The expression of human bone morphogenetic protein 2(hBMP-2) in these cells after transfection was determined by in situ hybridization and immunohistochemical analysis and Western blot analysis. The changes of cell proliferation were observed by flow cytometry. The effects of BMP-2 gene transfection on expression of VEGF in the cells were analyzed by in situ hybridization of VEGF cDNA probe. Results Stable expressionof hBMP-2 in pcDNA3.1-hBMP-2 transfected MSCs was confirmed in the levels of mRNA and protein.Cellular proportion in S period increased, which indicated that the synthesis of cell DNA increased. The expression of VEGF in the cells increased obviously. Conclusion With the help of lipofectamine, the pcDNA3.1-hBMP-2 were transfected into human MSCs successfully. hBMP-2 plays an important role in promoting cellular proliferation and vascular generation during bone repair.
Objective To observe the expression and localization of cellular homolog FLICE-like inhibitory protein (c-FLIP) in the procedure of benign biliary stricture formation and discuss the significances. Methods The method of in situ hybridization was used in anastomotic tissues from 15 dogs (experimental group) in 2, 3, 4, 5, 6 months after bile duct injury and 15 matching sham operation dogs (sham operation group) for analyzing the expression and localization of c-FLIP and calculating the average integrated optical density of each slice. Stain cells were counted under the magnification field (×400) and at least 5 fields per slice were examined. The cells stained red in the nuclei and (or) the cytoplasm were positive cells. The signals meant: Negative for cells no stained, weak positive for the cells with nuclei and (or) cytoplasm stained pink; b positive for the cells stained the bright red; while middle positive for the cells stained between the both. The image analysis software （Image pro plus 4.5） was applied in the gland tissue and interstitial tissue in each slice to calculate the average integrated optical density for the expression of c-FLIP. Results In the experimental group, there were all b positive expressions of c-FLIP in the interstitial tissue at all the time points, mainly expressed in the cytoplasm of fibroblast and very little or almost no expression in the glandular tissue. Positive expression of c-FLIP in the interstitial tissue was significantly ber than that of gland tissue (Plt;0.05); There were no significant differences among each time point in either the interstitial tissue or gland tissue (Pgt;0.05). In the sham operation group, there were all weak positive expressions of c-FLIP in the interstitial tissue and gland tissue at all the time points and was no significant difference (Pgt;0.05), no difference between each phase (Pgt;0.05). The expression of c-FLIP in the experimental group was significantly higher than that in the sham operation group in the interstitial tissue at all the time (Plt;0.05), while no significant that in the gland tissue (Pgt;0.05). Conclusion After bile duct injury, the expression of c-FLIP in anastomotic interstitial tissue is sustainable, by which the continuing obstruction effect to apoptosis may have a close relationship with the formation of biliary benign stricture.
Objective To be expressed human vascular endothelial growth factor (VEGF) recombinant protein in Escherichia Coli in high level. Methods VEGF was amplified from human fetal brain cDNA library, the amplified fragment was inserted into M13mP18 and confirmed to be VEGF165cDNA by restriction mapping and DNA sequencing, then it was combined with an expression vector PRL621. This recombinant plasmid overexpressed a 20kd recombinant protein in E.Coli(TG1), the protein was isolated and purifed from E.Coli, and initially renatured. Results The overexpressed recombinant protein was 35% of the total cell protein, the sequence of its first 15-N terminal amino acid was identrical to that of the human natural VEGF protein, Chorioallantoic membrane(CAM) assay showed that the rhVEGF promated new capillary vessels formation. Conclusion The genetic engineering Escherichia Coli can express human vascular endothelial growth factor in high level.
Objective To observe the effects of vascular endothelial growth factor antisense oligonucleotide (VEGF-ASODN) on expression of vascular endothelial growth factor (VEGF) and growth in gastric cancer cells. Methods The VEGF-ASODN was synthesized artificially with phosphorothioic acid. After transfecting with VEGF-ASODN in gastric cancer cells SGC-7901, the initial copy number of mRNA was detected by real-time RT-PCR, and the quantity of VEGF protein in both cell and supernatant were detected by ELISA. The levels of expression of survivin protein in cells were measured by Western blot. FCM and MTT method were used to detect cellular apoptosis and the activity of cells, respectively. The effect of transfection on the growth of cells was evaluated by growth curve. Results The copy number of VEGR mRNA, protein levels of VEGF in the cells and in culture fluid all decreased when the concentration of transfected VEGF-ASODN increased, as well as the levels of survivin protein （P＜0.05）. The ratio of apoptosis increased, the activity of cells also decreased as the concentration of transfected VEGF-ASODN increased （P＜0.05）. Conclusion Transfection with VEGF-ASODN in gastric cancer cells SGC-7901 can inhibit the expressions of VEGF and survivin remarkably. It can enhance cellular apoptosis and suppress growth of cells.
To explore the expression of Wnt-1 during the process of inducing neural stem cells (NSCs) into neurons by using all-trans-retinoic acid (ATRA) in vitro and the effect of Wnt-1 on NSCs differentiation. Methods NSCs isolated from cerebral cortex of SD rat embryo (12-16 days’ gestation) were cultured. The concentration of cells at passage 3 were adjusted to 1 × 106 cells /mL and treated with ATRA at 0.5, 1.0, 5.0 and 10.0 μmol/L, respectively. Differentiation ratio of NSCsinto neurons in each group was detected by double-labelling immunofluorescence technique and flow cytometry, and 1.0 μmol/ L was selected as the best concentration for ATRA to promote NSCs differentiation. In experimental group, NSCs at passage 3 were cultured with ATRA at 1.0 μmol/L in vitro, and expression of Wnt-1 was detected by immunocytochemistry staining, realtime flurescent quantitive PCR and Western blot at 3, 5, 7 and 9 days after culture, respectively. The cells at passage 3 receiving no ATRA served as control group. Results Immunocytochemistry staining: in the control group, there was l ittle Wnt-1 protein expression; in the experimental group, peak expression of Wnt-1 and numerous positive cells occurred at 3 days after culture, the positive expression of Wnt-1 was still evident at 5 days after culture, and there was significant difference between two groups in integrated absorbance (IA) value at 3 and 5 days after culture(P lt; 0.05), obvious decrease of positive expression of Wnt-1 was evident, and no significant difference was evident between two groups in IA value at 7 and 9 days (P gt; 0.05). Real-time fluorescence quantitative PCR: the relative expression of Wnt-1 mRNA in the control group was 0.021 7 ± 0.072 1; the relative expression of Wnt-1 mRNA in the experimental group at 3, 5, 7 and 9 days was 0.512 2 ± 0.280 0, 0.216 4 ± 0.887 0, 0.038 5 ± 0.299 4 and 0.035 5 ± 0.309 5, respectively, indicating the value decreased over time, and there were significant difference between two groups at 3 and 5 days (P lt; 0.05), and no significant difference at 7 and 9 days (P gt; 0.05) . Western blot detection: specific and visible staining band was noted; in the control group, Wnt-1 protein expression was 0.005 1 ± 0.558 3; in the experimental group, Wnt-1 protein expression at 3, 5, 7 and 9 days was 0.451 7 ± 0.071 3, 0.311 7 ± 0.080 5, 0.007 3 ± 0.052 7 and 0.004 7 ± 0.931 4, respectively, suggesting the value decreased over time; there were significant differences between two groups at 3 and 5 days (P lt; 0.05), and no significant differences at 7 and 9 days (P gt; 0.05). Conclusion With the induction of ATRA at 1.0 μmol/L, Wnt-1 and NSCs differentiation in early stage are positively correlated. Its possible mechanism may rely on the activation of such signals as classic Wnt-1 signal pathway, indicating Wnt-1 relates to the differentation of NSCs into neurons.