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Circulating tumor DNAs (ctDNAs) are DNA fragments released from tumor cells into bloodstream, containing genetic mutations and epigenetic variations related to cancer. DNA methylation variation is a kind of epigenetic variation which happens in early carcinogenesis and dynamically changes with cancer development. Liquid biopsy of ctDNA methylation has the advantages of non-invasiveness, target molecule stableness, considerable cost-effectiveness, high diagnostic performance and wide application expansion, detection of whose level is conducive to early diagnosis and prognostic evaluation of cancer. The pre-analytical procedures and methylation detection methodologies significantly influence after test results, and should be standardized to obtain high quality results. Up to now, a large amount of literature covering the utility of ctDNA methylation in cancer diagnosis and prognosis have been published. It is believed that in the near future, the detection process of ctDNA methylation would be standardized, and the large-scale clinical application of ctDNA methylation as a liquid biopsy project would be promoted.
ABSTRACT
In recent years , the potential clinical value of circulating tumor cells ( CTCs ) is more obvious, and a myriad of detection methods for CTCs have been developed .Among them, a series of microfluidic devices are particularly promising as they uniquely offer micro-scale analytical systems that are highlighted by low consumption of samples and reagents , high flexibility to accommodate other cutting-edge technologies , precise and well-defined flow behaviors , and automation capability , presenting themselves significant advantages over the conventional larger systems .Here, a brief review of the recent advances in the CTC detection by microfluidic devices and their challenges in clinical application will be given .
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Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and high mortality.Early diagnosis is the key for HCC treatment.DNA methylation is one of the important mechanisms of epigenetic genetics.To investigate the mechanism of DNA methylation can provide a theoretical basis for the diagnosis of HCC,and the methylation level of the specific genes can also be used as the biomarkers of HCC for the early diagnosis and prognosis evaluation.
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BACKGROUND:General y considered bone marrow cells obtained by adherent method are mesenchymal stem cells, and they can differentiate into osteoblasts, adipocytes and chondrocytes. OBJECTIVE:To explore the differentiation capacity of bone marrow adherent cells into osteoclasts. METHODS:Primary mouse mesenchymal stem cells were obtained using adherent method, and bone marrow cells were obtained through adherence 1-5 days. Both of them were cultured with normal medium and inducing medium containing m-csf and RANKL. After 9 days, cells were stained by alkaline phosphatase and tartrate-resistant acid phosphatase. The passage 2 mesenchymal stem cells were divided into four groups, which were induced by mock, m-csf, RANKL and m-csf+RANKL, respectively. After 9 days of culture, the cells were stained by alkaline phosphatase and tartrate-resistant acid phosphatase. RESULTS AND CONCLUSION:Primary culture of adhered cells was uniform. Alkaline phosphatase and tartrate-resistant acid phosphatase staining of primary and passaged bone marrow adherent cells induced with m-csf+RANKL were positive. This result showed that there were cells that could be induced into osteoclasts in the primary and passaged bone marrow adherent cells. Alkaline phosphatase and tartrate-resistant acid phosphatase staining showed differences of adherent cells at different times after the induction, indicating that adherent cells at different times had different differentiation capacity.
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BACKGROUND: The transcription factor Runx2 is the key factor that regulates osteogenic differention and bone development. It has been reported that the C2C12 mesenchymal cells can be induced to differentiate into osteoblasts by Runx2 overexpression, but the molecular mechanism of induction is stil largely unclear. OBJECTIVE: To investigate the role of the members of the miR-376 family during Runx2-induced osteogenic differentiation in C2C12 cells. METHODS: The expression of the members of the miR-376 family was detected by real-time quantitative PCR at different time points using C2C12/Runx2Dox sub-line with conditional Runx2 expression. In miR-376b-3p-transfected C2C12/Runx2Dox cells, the expression of osteoblast markers, such as alkaline phosphatase and osteocalcin, was detected by real-time quantitative PCR, and the alkaline phosphatase activity was also examined by alkaline phosphatase staining. The putative miR-376b-3p targets were commonly predicted by online tools (miRanda, miRWalk and TargetScan). The functional classification of these putative targets was performed by DAVID Bioinformatics Resources database. RESULTS AND CONCLUSION: The expression of miR-376b-3p was significantly increased during Runx2-induced osteogenic differentiation of C2C12 cells, but the expression of other members was not changed. Transfection of miR-376b-3p mimic upregulated alkaline phosphatase expression, but had no effect on osteocalcin expression. The alkaline phosphatase activity was also increased by transfection of miR-376b-3p. The functional classification of miR-376b-3p putative targets showed that miR-376b-3p is involved in the skeleton development, indicating the role of miR-376b-3p in osteoblast differentiation. Taken together, these results suggest that Runx2 promotes early osteogenic differentiation in C2C12 cells by regulating the expression of genes related to osteogenic differentiation through upregulation of miR-376b-3p.
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Coding sequences of BMP-2 and BMP-7 were amplified using PCR and ligated with a DNA sequence encoding a flexible peptide (Gly4Ser)5. The fusion gene was inserted into plasmid pIRESneo3. The expression level of BMP2/7 heterodimers in the transfected CHO-K1 cells was 230.75+/-13.34 ng/mL. Culture medium of stably tansfected clone pool was collected as conditional medium to treat osteoblast MC3T3 cells. Staining of Alkalin phosphatase and Alizarin red demonstrated that the conditional medium significantly promoted osteogenic differentiation to a higher extent than BMP-2 homodimers expressed in either CHO-K1 cells or E. coli. Transcriptional levels of Osteogenic phenotype-related molecular markers such as OC, ALP, Runx2 and Osx were increased (P<0.05), and BMP/Smad signal activities were significantly enhanced by BMP-2/7 heterodimers, comparing with BMP-2 homodimers (P<0.05). The results demonstrate that BMP-2/7 heterodimers expressed in CHO-K1 cells have potent ability to stimulate osteogenic differentiation.
Subject(s)
Animals , Cricetinae , Humans , Mice , 3T3 Cells , Artificial Gene Fusion , Bone Morphogenetic Protein 2 , Genetics , Bone Morphogenetic Protein 7 , Genetics , CHO Cells , Cell Differentiation , Cloning, Molecular , Cricetulus , Dimerization , Escherichia coli , Genetics , Metabolism , Gene Fusion , Genetics , Osteoblasts , Cell Biology , Osteogenesis , TransfectionABSTRACT
AIM: To obtain a high and stable expression analog of human basic fibroblast growth factor by genetic engineering. METHODS: The cysteins 78 and 96 of natural hbFGF polypeptide was substituted with serines by means of site-directed mutagenesis. Using pET- 3c as vector, the mutated polynucleotide was cloned and then transferred into BL21 (DE3)plysS. After induction by IPTG, the analog was obtained and analyzed by SDS - PAGE. RESULTS: After purification the form of soluble mutant increased remarkably but the forms of dimmer and higher multimer were reduced greatly to no more than 8% of the total recombinant protein. By MTT assay, the analog showed the same biological activity. This new analog represented a desirable complementation for native hbFGF to develop pharmaceutical drug in clinical use. CONCLUSION: Substitution of certain amino acids of polypeptide without altering native protein' s bioactivity to get the analog is an effective means to increase stability of foreign protein and its solubility in E. coli.
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AIM: To get high biological activity of recombinant human bone morphogenetic protein -2 (rhBMP-2) expressed in Escherichia coli by the methods of refolding. METHODS: The rhBMP-2,expressed in Escherichia coli, was washed by Triton X- 100 and further purified by DEAE chromatography.The inclusion bodies were resolved in 8 mol/L urea, and were refolded and dimerized in the redox systems (reduced and oxidized glutathione). Finally, a one - step purification procedure based on the heparin affinity chromatography was implemented. The biological activity of purified rhBMP - 2 were tested by induction of the alkaline phosphatase activity in C2C12 cells. RESULTS: The rhBMP - 2 was expressed in Escherichia coli in a non - active aggregated form of inclusion bodies using a temperature - inducible expression system. The high - purified rhBMP - 2 was obtained in the form of inclusion bodies by several purification courses. The rhBMP - 2 was refolded and dimerized in the redox systems (reduced and oxidized glutathione) and a one - step purification procedure based on the heparin affinity chromatography was implemented to isolate the rhBMP - 2 dimers and monomers. The purified rhBMP - 2 dimers showed the higher biological activity than the commercial rhBMP - 2. CONCLUSIONS: The method achieved the refolding of rhBMP - 2 would be applied to the whole TGF - β superfamily because the BMP - 2 belongs to the superfamily. Meanwhile, the inexpensive,high yield rhBMP - 2 is suitable for clinic application.
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AIM: To get high biological activity of recombinant human bone morphogenetic protein-2 (rhBMP-2) expressed in Escherichia coli by the methods of refolding. METHODS: The rhBMP-2, expressed in Escherichia coli, was washed by Triton X-100 and further purified by DEAE chromatography. The inclusion bodies were resolved in 8 mol/L urea,and were refolded and dimerized in the redox systems (reduced and oxidized glutathione). Finally, a one-step purification procedure based on the heparin affinity chromatography was implemented. The biological activity of purified rhBMP-2 were tested by induction of the alkaline phosphatase activity in C2C12 cells. RESULTS: The rhBMP-2 was expressed in Escherichia coli in a non-active aggregated form of inclusion bodies using a temperature-inducible expression system. The high-purified rhBMP-2 was obtained in the form of inclusion bodies by several purification courses. The rhBMP-2 was refolded and dimerized in the redox systems (reduced and oxidized glutathione) and a one-step purification procedure based on the heparin affinity chromatography was implemented to isolate the rhBMP-2 dimers and monomers. The purified rhBMP-2 dimers showed the higher biological activity than the commercial rhBMP-2. CONCLUSIONS: The method achieved the refolding of rhBMP-2 would be applied to the whole TGF-? superfamily because the BMP-2 belongs to the superfamily. Meanwhile, the inexpensive, high yield rhBMP-2 is suitable for clinic application.
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AIM: To screen the cytotoxicity of degradable calcium metaphosphate (dCMP) compared with hydroxyapatite (HA). The proliferation and differentiation abilities of human marrow mesenchymal stem cells (MSC) were used to exhibit the cytotoxicity. METHODS: The cell morphology of MSC was analysed after direct contact with dCMP at different time points by scanning electron microscopy analysis. The degradation products of dCMP and HA were analysed with inductively coupled plasma torch and ion chromatography. The cytotoxic effect of degradation products of dCMP was evaluated by FACS, quantitative assay of ALP and ARS, respectively. RESULTS: dCMP enhanced the proliferation of MSC, but didn't interfere the osteogenic differentiation process of MSC and its mineralization. HA inhibited the proliferation of MSC and the mineralization of osteogenic differentiated MSC, while it did not interfere the osteogenic differentiation process of MSC. CONCLUSION: dCMP had a better cytocompatibility with MSC than HA, which might allow for its use as skeleton scaffolds.
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AIM: To further investigate the role of PKARⅠ? in the growth-promoting effects of Shuanglong-Jiegu pill (SLJGP), a Chinese medicine, on cultured osteoblasts. METHODS: pcDNA-antiPKARⅠ?, a recombinant expressing the antisense sequence of PKARⅠ?, was constructed and transformed HFOB1.19 by lipofectin. MTT was undertaken to assess the cell growth with the treatment of high dosage of SLJGP containing serum. RESULTS: Antisense gene blocked the growth-promoting effects of SLJGP containing serum on HFOB1.19. CONCLUSION: The function of SLJGP is closely related to cAMP-dependent protein kinase A. [
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AIM: To study the expression of murine interleukin-12(mIL-12) gene in B16F10 melanoma cells. METHODS: mIL-12 gene was inserted into pcDNA3.1 eukaryotic expression vector by DNA recombination and then transfected into B16F10 melanoma cells by electroporation. The integration and expression of mIL-12 gene in B16F10 melanoma cells were detected by PCR,RT-PCR and Western blot after positive cell clones had been screened. RESULTS: mIL-12 gene was confirmed to have been transfected and expressed in B16F10 cells on three levels of DNA, mRNA and protein. CONCLUSION: mIL-12 gene can successfully be transfected and expressed in B16F10 melanoma cells in vitro , which lays a foundation for the further investigation of mIL-12 gene tumor vaccine.
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To improve the expression level of non fusion hbFGF in E coli , the coding sequence of human bFGF gene, which had been cloned from primarily cultured human fibroblast, was mutated according to the principle of lowering the GC content and increasing the codon preference After being ligated into pET 3c and transformed into BL21(DE3), the recombinant induced by IPTG Expression level was up to 30% of the total bacterial protein The result indicated that optimizing of the TIR would promote the expression level of recombinant protein
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AIM: To construct retroviral expression vector pLXmIL-12SN containing mouse IL-12 (mIL-12) gene and detect the gene expression in B16F10 cells. METHODS: Vector pGCp35-IRES-p40SN was digested to obtain p35-IRES-p40 (mIL-12 gene fragment) and retroviral vector pLXSN was digested by restriction enzyme, and then they were linked by ligase. The recombinant vector that mIL-12 gene fragment had correctly been inserted into pLXSN was identified by sequencing. The recombination vector with mIL-12 gene was transfected into B16F10 cell and was detected by RT-PCR. RESULTS: Expression vector pLXmIL-12SN was successfully constructed. mIL-12 gene was confirmed to express in B16F10 cells at the level of mRNA. CONCLUSION: Acquired vector pLXmIL-12SN and confirmed mIL-12 gene expression lays a foundation for mIL-12 gene therapy to eye diseases.
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AIM: To construct engineered E.coli strains which can express nattokinase with fibrinolysis activity using gene engineering technology. METHODS: The pro-nattokinase (pro-NK) gene was amplified by PCR and inserted into expression vector pET3c. The recombined plasmid pENK which expressed the fusion protein of pro-NK and 22 amino acid peptide was then transferred into lysogenic host strains BL21(DE3)pLysS - and BL21(DE3)pLysS +. Both SDS-PAGE and the fibrin plate assay were used to examine the expression and the activity of the target protein. RESULTS: SDS-PAGE assay showed the fused gene encoding 42 kD fusion protein was expressed in both expression strains pENK-(DE3)pLysS - and pENK-(DE3)pLysS +, and the fibrin plate assay indicated that the expression product had fibrinolysis activity. pENK-(DE3)pLysS - exhibited the basal expression of the target gene, while fusion protein was only induced by IPTG in pENK-(DE3)pLysS +. Basal expression of the fused toxic gene in pENK-(DE3)pLysS - led to bacteriolysis and hollow lawns. CONCLUSION: A pro-NK fusion protein with fibrinolysis activity is successfully expressed in E.coli , which lay a foundation for the exploitation of nattokinase.
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AIM: To investigate the condition necessary for high-level expression of basic fibroblast growth factor (hbFGF) in Escherichia coli, a dicistron system was constructed. METHODS: The phage T7 gene ?10 N-terminal sequence, present in the expression plasmid pET-3c, was incorporated into the first cistron, the coding region for bFGF was incorporated into the second, and both were under the control of a transcription promotor recognized by the bacteriophage T7 RNA polymerase. RESULTS: Recombinants were transformed into BL21(DE3) and induced by IPTG for expression, accumulation of up to 20% of the total cell protein of bFGF were obtained, showing bioactivity indistinguishable from standard protein. CONCLUSION: It suggested that such dicistron system was effective for enhancing hbFGF expression.
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AIM: To construct a recombinant hEGF-hbFGF(78-154aa)fusion protein, which not only has the heparin-binding ability, but also promotes the growth of the cells, and to express the fusion protein in E. coli expression system with high expression level.METHODS: hEGF gene was joined with 231 bp fragment coding hbFGF(78-154aa) and expressed in E. coli. The fusion protein was purified using affinity chromatography of heparin-Hyper D and analyzed with western blot. The pI value and the biological activity were both assayed.RESULTS: The fusion protein was expressed in a high expression level of about 30% of the total cell protein, as estimated by SDS-PAGE. Western analysis results showed that the antigenicity of fusion protein was similar to hEGF. Fusion protein could not only bind heparin but also promote the growth of 3T3 cell. The pI value of fusion protein was 5.2.CONCLUSION: The recombinant hEGF-hbFGF(78-154aa) fusion protein possessed the characteristics of both hEGF and hbFGF. This new-designed protein would become a good object for the research on the relationship between the structure and the function of the growth factor.