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.
OBJECTIVE To determine the effect of basic fibroblast growth factor (bFGF) on the biological behaviour of ovarian epithelial neoplasm. METHODS Ten cases of normal ovarian tissues and eighty cases of ovarian epithelial tumor tissues were detected by immunohistochemical methods. Mias-2000 Picture Analysis System was used to study the relationship of bFGF expression intensity and microvessel count, FIGO stage, pathological grade and classification of ovarian epithelial neoplasm. RESULTS 1. Expression of bFGF was mainly in cytoplasm and nucleus in several cells of borderline and malignant tumor. 2. The expression intensity of bFGF was closely related to the malignant degree of ovarian epithelial neoplasm. The density of bFGF expression was (3.35 +/- 3.52)% in normal ovarian epithelium, (19.25 +/- 21.73)% in benign tumor, (33.78 +/- 10.86)% in borderline tumor and (48.18 +/- 12.93)% in malignant tumor. The results indicated that bFGF might play an important role in carcinogenesis of ovarian epithelial neoplasm. 3. The expression intensity of bFGF was increased with the FIGO stage of ovarian tumor. 4. The expression intensity of bFGF was increased accompanying with the decrease of differentiation degree in ovian neoplasm. 5. In borderline tumor, expression intensity of bFGF in serous cystadenoma was significantly higher than in mucinous cystadenoma, which indicated bFGF might be an important factor in canceration of ovarian epithelial neoplasm. CONCLUSION bFGF may play important roles in carcinogenesis, development, invasion and metastasis of ovarian epithelial neoplasm.
OBJECTIVE: To explore the expressive characteristics of epidermal growth factor (EGF) and its receptor (EGFR) in tissues of fetal and adult intestines. METHODS: The expression intensity and distribution of EGF and EGFR were detected with pathological and immunohistochemical methods in 6 specimens of adult (16-54 years) intestines and 18 specimens of fetal intestines with different gestational ages (13-31 weeks). RESULTS: Positive protein particles of EGF and EGFR could be detected in tissues of fetal and adult intestines. The protein expressions of EGF and EGFR were elevated progressively with the gestational age. EGF was mainly located in the cytoplasm and extracellular matrix of intestinal villus cells, endothelial cells and tunica serosa epithelial cells, while EGFR chiefly distributed in the cellular membrane of these cells. CONCLUSION: The endogenous EGF and EGFR might be involved in the intestinal development at embryonic stage, in the structural and functional maintenance at adult stage, and in the wound healing after injury.
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 observe the expression of heat shock protein 70 (HSP70) in human liver after hepatic transplantation, and to study its correlation with the occurrence and progression of acute allograft rejection.Methods Fifteen biopsy specimen of allograft liver after transplantation were collected and divided into three groups according to their pathological changes: control group (no rejection), mild acute rejection group, and moderate/serious acute rejection group. The expressions of HSP70 in grafts were detected by using immunohistochemical method and imaging analysis. Results HSP70 was expressed in all 3 groups, and appeared mainly in hepatocellular cytoplasm. The immunohistochemical imaging analysis of HSP70 showed: integral optical density (IOD) which was 30.99±11.14 in the control group was lower than that in the mild acute rejection group (68.84±21.37) and that in the moderate/serious acute rejection group (71.82±19.99), P＜0.01； and the IOD in the moderate/serious acute rejection group was higher than that in the mild acute rejection group (P＜0.05). Conclusion HSP70 plays a role in cellular protection for allograft liver, and the continuously increasing expression of HSP70 in graft maybe closely relates to the occurrence and progression of acute allograft rejection.
Objective To investigate the changes in the expression level of PDGF in the bone callus of rats with femoral fracture and brain injury to explore the effect of brain injury on the fracture heal ing and the related mechanism. Methods Sixty-four 12-week-old SD rats weighing (356 ± 25) g were randomly divided into 8 groups with 8 rats in each. The rats in groups A1, B1, C1 and D1 had a femoral fracture and a brain injury for 1, 2, 3 and 4 weeks, respectively; the rats in groups A2, B2, C2 and D2 had a mere fracture without a brain injury for 1, 2, 3 and 4 weeks, respectively. After the CR films were taken, the bone callus was obtained 1, 2, 3 and 4 weeks after operation, respectively. Then, the bone callus and its histology were examined by HE staining, the expressions and changes in the level of PDGF were examined by the immunohistochemical staining, and the level of PDGF mRNA was measured by in situ hybridization. Results The CR films showed that the callus formation in the A1-D1 groups was earl ier and greater than that in the A2-D2 groups at the same time point. The HE staining indicated that more fibroblasts and early-stage chondrocytes were found in group A1; some fibroblasts in the fracture interspace and few early-stage chondrocytes were found in group A2; some newly-formed trabecular bones were found at the end of the fracture in group B1; but no trabecular bone formation was found in group B2; woven bone formation and a few chondrocytes between trabecular bones in the fracture interspace were found in group C1; only a few trabecular bones in the fracture interspace were found in group C2；woven bones turned to lamellar bones in group D1；and more immature trabecular bones in the fracture interspace were found in group D2. The positive expression of PDGF and PDGF mRNA was b in the cytoplasms of fibroblasts, mesenchymal cells, vascular endothel ial cells, early-stage chondrocytes, osteoblasts and osteoclasts. The percentage of the positive cells for PDGF and PDGF mRNA in the callus was significantly higher in groups A1-D1 than in groups A2-D2 at the same time point (P lt; 0.05). Conclusion Brain injury can promote the fracture heal ing process, which is probably related to an increase in the expression level of PDGF after the brain injury.
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.
ObjectiveTo detect the expression of motilin in gastric cancer tissues and to explore the relationship between motilin protein expression and clinicopathologic characteristics of gastric cancer. MethodsThe immunohistochemical staining was used to detect the expression of motilin protein in gastric cancer, paracancerous tissues, and normal gastric mucosa tissues. The relationship between motilin protein expression and clinicopathologic characteristics of gastric cancer was analyzed. ResultsThe expression of motilin protein in gastric cancer tissues (1 206.43±631.67) was significantly higher than that in normal gastric mucosa tissues and paracancerous tissues, respectively (Plt;0.01). The difference of motilin protein expression between normal gastric mucosa tissues and paracancerous tissues was not significant (Pgt;0.05). The expression of motilin protein in gastric cancer was correlated with the site of tumor, differentiation degree, and lymph node metastasis (Plt;0.05). ConclusionMotilin may participate in the carcinogenesis of gastric cancer, and correlated with the invasion and metastasis of gastric cancer.
【摘要】 目的 觀察不同種培養基中重組人色素上皮衍生因子(rPEDF)融合蛋白的表達。 方法 將前期研究已構建的pET28aPEDF原核表達重組體轉化E.coli BL21大腸桿菌表達宿主菌,酶切鑒定陽性菌落后,分別在M9和LB培養基中用異丙基βD硫代半乳糖(IPTG,IsopropylbetaDthiogalactoside)誘導表達,SDSPAGE電泳檢測表達的PEDF蛋白, 美國ImagePro Plus 分析系統進行蛋白定量分析。結果 LB和M9培養基中均獲得相對分子質量約54×103的rPEDF融合蛋白。但LB培養基獲得的是rPEDF融合蛋白的包涵體,目的蛋白占總蛋白含量為21046%,M9培養基獲得的是可溶性的rPEDF的融合蛋白,目的蛋白占總蛋白含量的1231%。結論 不同種培養基中均有rPEDF 融合蛋白的表達。【Abstract】 Objective To observe the express of recombinant pigment epithelial derivative facto (rPEDF) in the different medium. Methods The pET28aPEDF was transformed into E.coli BL21. After the colonies were positive identification which were induced by IsopropylbetaDthiogalactoside in medium M9 and LB. The PEDF protein were detected by SDSPAGE and analyzed by American ImagePro Plus system. Results LB and M9 medium obtained the relative molecular mass about 54×103 rPEDF fusion protein. But LB medium obtained the inclusion bodys of rPEDF fusion protein，the purpose protein account for 21.046%；LB medium obtained the soluble rPEDF fusion protein，the purpose protein account for 12.31%. Conclusion The rPEDF protein was expressed in the different medium.
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.