Construction and identification of PRDX3 eukaryotic expression vector / 军事医学

Objective To construct the eukaryotic expression vector of PRDX3 labeled with FLAG tag and to study its localization in human tongue cancer cell line SCC15.Methods PRDX3 gene was obtained from the breast library by PCR and cloned into PCDH vector to construct PCDH-FLAG-PRDX3.The plasmid was transiently transfected into 293T cells and the expression was detected by Western blot.Subcellular localization was detected by cellular immunofluorescence.Results The result of double digestion and sequencing showed that PCDH-FLAG-PRDX3 eukaryotic expression vector was constructed.The expression of FLAG-PRDX3 in human 293T cells was positively confirmed by Western blotting.In human tongue cancer cell line SCC15, the result of cellular immunofluorescence showed FLAG-PRDX3 was located in the cytoplasm rather than in the nucleus.Conclusion PRDX3 eukaryotic expression vector labeled with FLAG tag is constructed successfully, which is located in cytoplasm in human SCC15 cells.Construction and identification of PRDX3 could shed light on the function and mechanism of PRDX3 in tongue cancer.

Knockdown of PES1 inhibits tongue squamous cell cancer cell growth / 军事医学

Objective To construct PES1 shRNA stable expression cell lines in tongue squamous cell carcinoma ( TSCC) cells and to study the effect of knockdown of PES 1 on the growth of TSCC cells .Methods Recombinant lentivirus carrying PES1 shRNA was packaged and obtained in 293T cells.TSCC cells (Tca8113, SCC6 and SCC15) were infected with the lentivirus and selected for stable cells .PES1 expression was identified by Western blot .The effect of inhibition of PES1 on the growth and cell cycle of TSCC cells was detected by growth curve and flow cytometry .Results TSCC cells stably expressing PES1 shRNA were constructed.Knockdown of PES1 inhibited cell proliferation and induced cell cycle ar-rest at G0/G1 phase.Knockdown of PES1 inhibited expression of cyclin D1 in TSCC cells.Conclusion Inhibition of PES1 results in reduced cell proliferation , cell cycle arrest at G 0/G1 phase and reduction of cyclin D 1 expression in TSCC cells . PES1 may be a target for TSCC gene therapy .

Construction of eukaryotic expression vector of E4F1 and interactions between E4F1 and p53 / 军事医学

Objective To construct eukaryotic expression vector of wild type E 4F1 and the mutant deleting amino acid region 32-81, and to detect the interaction between wild type or mutant E 4F1 and p53 and to study the effect of E4F1 on the expression level of p21.Methods Wild type and mutant sequences of E 4F1 were amplified from the mammary library using standard PCR and recombinant PCR .The sequences were cloned into pXJ 40-MYC vector to generate the MYC-E4F1 and MYC-E4F1(Δ32-81) recombinant plasmids that were transfected into 293T cells and identified by Western blotting . FLAG-p53 and MYC-E4F1 or MYC-E4F1(Δ32-81) were co-transfected into 293T cells and immunoprecipitation assay was performed to detect the interaction of wild type or mutant E 4F1 with p53.Wild type and mutant E4F1 expressing vec-tors were co-transfected into osteosarcoma U2OS cells and the expression of p21was detected.Results Recombinant plas-mids of MYC-E4F1 and MYC-E4F1(Δ32-81) were successfully constructed.Both wild type and mutant E4F1 interacted with p53.Deletion of amino acid region 32-81 of E4F1 increased the interaction .The expression level of p21 was in-creased by wild-type E4F1, but not by mutant E4F1.Conclusion The eukaryotic expression vector of wild type E4F1 and its deletion mutant is successfully constructed .Both of them interact with p53.Deletion of amino acid region 32-81 of E4F1 increases the interaction .This study contributes to further studies on the regulation and mechanism of E 4F1 on p53.

Regulatory effects of nerve growth factor on the differentiation of goat bone marrow mesenchymal stem cells into osteoblasts / 实用口腔医学杂志

Objective: To investigate the effects of nerve growth factor(NGF) on the differentiation of goat bone marrow mesenchymal stem cells (BMSCs) into osteoblasts. Methods: The goat BMSCs were cultured in vitro and the marker proteins on the BMSCs surface were identified by flow cytometry. The third passage of BMSCs were randomly divided into blank control group, osteoblasts control group, NGF group and experimental group. The activities of alkaline phosphatase (ALP) and osteocalcin (0C) were examined and the of von Kossa staining method was used to observe the osteogenic differentiation. Results: CD90 and CD105 were strong positive while the CD34 and CD45 were negatively expressed in BMSCs. The activities of ALP and OC was significantly higher in the experimental group than that in the other three groups(P<0.05). The staining of von Kossa was positive and the black calcium nodules were appeared in the the osteoblasts control group. The number and the area of the calcium nodules were greater in the experimental group. But there were no significant differences of each index between the NGF group and the blank control group. Conclusion: NGF can′t induce goats BMSCs to osteoblasts, but can clearly promote the differentiation of goats BMSCs.

Applied anatomy of superior trapezius myocutaneous and spina scapulae osteomyocutaneous flaps pedicled with transverse cervical artery / 实用口腔医学杂志

Objective:To study the anatomic basis of superior trapezius myocutaneous and spina scapulae osteomyocutaneous flaps pedicled with transverse cervical vessel.Methods:The blood vessels,the size of the superior trapezius muscle and the spina scapulae were dissected and examined in 32 adult corpses.Results:The superior trapezius muscle was in the shape of trapezium.The border length (mm) of A,B,C and D was 174.63,157.18,86.98 and 80.95 in average respectively.The area of the muscle was 126.78 cm2 on the average.The spina scapulae was 131.21 mm in average length.The length (mm) of transverse cervical artery trunk, superficial cervical artery trunk and its ascending artery,spina scapulae branch artery was 42.50,27.80,43.12,28.75 in average,their external diameter(mm) was 2.71,2.39,1.96 and 0.50 respectively.Entering the muscle,the ascending artery had 3～6 branches with the external diameter of 0.5 mm or more.The venous vessels were following the copartner artery.Conclusion:The superior trapezius muscle and the spina scapulae may be made for the myocutaneous and osteomyocutaneous flaps pedicled with transverse cervical artery used in oral and maxillofacial reconstruction.