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.

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.

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. [

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.

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

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.

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.

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.

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.

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.