【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 study the effect of knockdown of signal transducer and activator of transcription 3 (STAT3) expression by short hairpin RNA (shRNA) on proliferation, apoptosis and invasion of human gastric cancer cell line MKN-45 in vitro . Methods Specific shRNA plasmids to STAT3 were constructed, and then transfected into MKN-45 cells by lipofectamine methods. Cells were divided into three groups: control group, psiRNA-H1 transfected group as negative group and psiRNA-H1/STAT3 transfected group. Semi-quantitative RT-PCR and Western blotting were used to detect the expression of STAT3 mRNA and protein, respectively. Proliferation and apoptosis of the transfected cells were observed by methyl thiazolyl tetrazolium (MTT) method and flow cytometry (FCM), respectively. The invasion of the transfected MKN-45 cells was measured by Boyden chamber. Results Compared with the negative control cells, semi-quantitative RT-PCR and Western blotting showed that the expressions of STAT3 mRNA and protein were down-regulated in the psiRNA-H1/STAT3 transfected group ( P ＜ 0.05) . The subcloned recombinant plasmid expressing shRNA effectively inhibited MKN-45 cell growth and proliferation while empty plasmid had no such specific effect. Cell apoptosis rate increased significantly in psiRNA-H1/STAT3 transfected group （ P ＜ 0.01）, and the invasion of MKN-45 cells was efficiently inhabited in psiRNA-H1/STAT3 transfected group as compared with control group and psiRNA-H1 transfected group( P ＜ 0.01).Conclusion Recombinant plasmid psiRNA-H1/STAT3 shRNA significantly inhibits the proliferation and invasion of MKN-45 cells and promotes their apoptosis.
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 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.
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.
The expression and rearrangement of bcl-2 gene in 64 cases with colorectal carcinoma were studied by immunohistochemical technique and semi-nest PCR respectively. The results showed the abnormal changes of the expression and rearrangement of bcl-2 gene had emerged in the early stage of colorectal carcinoma. The tumors with the expression of bcl-2 were associated with a higher incidence of metastasis to lymphatic node. The rearragement of bcl-2 was significantly higher in late-stage than that in early-stage. These suggest that bcl-2 gene involves in the regulation of the development of colorectal carcinoma. The state of the changes of bcl-2 gene in colorectal carcinoma may predict the therapeutic effect and prognosis of colorectal carcinoma.
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 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 explore the expression of the vascular cell adhesion molecule 1 (VCAM1) in the acellular dermal matrix grafting in pigs. Methods Experimental models were established with 15 Inbred Strain mini pigs, 6 full-thichness skin defect wounds, 6 cm × 6 cm in size, were produced on both-side backs of the each pig, and then the pigs were randomly divided into 3 groups. In Group A (n=5, control) the thin auto-skintransplantation alone was made; in Group B (n=5), the grafting was performed in the acellular allo-dermal matrix combined with the thin auto-skins; in Group C (n=5), the grafting was performed in the acellular xeno-dermal matrixcombined with the thin auto-skins. The areas of the wounds were measured and the survival condition of the grafted skins was observed at 3, 9, 21 and 30 days after the grafting. The histological samples were harvested from the grafting area at 3, 6, 9, 12, 21 and 30 days after the procedure. The flow cytometry was employed to analyze the changes in the VCAM1 level in the sample at different time points after the grafting. Results In the 3 groups, the transplanted skin base was easily separated at 3 days after transplantation; the areas of the wound healing accounted for 94%±12%，92%±9%, and 91%±11%, respectively, at 21 days; good wound healing was achieved at 30 days. At 9 and 12 days after transplantation, there was an evidentlyincreased level of the VCAM-1 expression in the tissue samples in the composite skin grafting groups. Compared with the control group, the difference was significant (Plt;0.05); however, the VCAM-1 expression at 3 days was not statistically different between the composite skin grafting groups and the control group after transplantation. In contrast, the level of the VCAM-1 expression was significantly higher at 6 days in the control group than in the composite skin grafting groups (Plt;0.05). The levels of the VCAM-1 expression were significantly lower at 30 days than at 3 days after transplantation in all the 3 groups (plt;0.01). Conclusion The highest level of the VCAM-1 expression can be delayed in the composite skin grafting when compared with that in the thin auto-skins alone, which implies that the VCAM-1 expression may be correlated with angiogenesis and composite skin survival. The VCAM-1 expression is not different between the acellular allo-dermal matrix composite skin grafting groups and the acellular xeno-dermal matrix group.
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.
