| Heparan sulfate(HS) is a type of linear sulfated glycosaminoglycan consisting of uronic acid--N-acetylglucosamine repeating disaccharide units. HS, the side chain of HS proteoglycans located on cell-membrane and in extracellular matrix, can interact with various types of proteins such as growth factors, chemokines, morphogens and the proteins in extracellular matrix, thereby being involved in major biological processes, including stem cell differentiation, cancer cell invation and mestasis, embryonic development and virus entry. Study on the relationship of HS structural feature and its modifying enzymes will provide theoretical principles and potential new technology routines for regenerative medicine and cancer treatment. Here we focus on the biological functions of cell-surface HS and HS sulfation pattern encoding enzymes—HS sulfotransferases. The main results are listed as follows:1. Rat bone marrow mesenchymal stem cells(BMSCs) were purified by density gradient centrifugation, the expression of mesenchymal stem cell surface stemness marker was analyzed by flow cytometry, an osteogenic differentiation model was established using chemical induction reagents and confirmed via alkaline phosphatase(ALP) activity detection and the expression of the osteogenic differentiation markers Runx2 and Ocn. The expression profiles of HS sulfotransferases in rat BMSCs before and after osteogenic induction were detected by RT PCR and Western blot. We found that cell spheroids were formed in both control and osteogenic culture systems when BMSCs were grown to high confluence. We determined that this type of cell spheroid was a highly calcified nodule by histochemical staining. Among all the sulfotransferases examined, HS6ST3 m RNA and protein were upregulated in these calcified cell spheroids. Relative m RNA expression was increased to 2.13±0.022 from 0.70±0.031(p < 0.05), and relative protein expression was increased to 0.91±0.058 from 0.37±0.024(p<0.05). HS6ST3 knockdown BMSCs were established with RNA interference, relative ALP enzymatic activity was reduced to 1.29±0.146 in HS6ST3 knockdown group from 2.27±0.163 in mock control group during osteogenic differentiation(p<0.05), and the osteogenic differentiation markers Runx2 and Ocn was significantly reduced to 0.26±0.025, 0.42±0.036 from 0.88±0.064 and 0.92±0.080 respectively(p<0.05). These findings suggest that HS6ST3 is a candidate gene involved in BMSCs differentiation.2.Two cell culture models, including myelodysplastic syndrome(MDS) SKM-1 cell line granulocytic differentiation and epithelial-mesenchymal transition(EMT) ofA549 non-small-cell lung cancer cell line, were established using all trans retinoic acid(ATRA) and TGFb1 respectively. Here we found HS 2-O sulfotransferase 1(HS2ST1) was downregulated at both m RNA and protein levels during granulocytic differentiation of SKM-1 leukemia cells. Relative m RNA expression of HS2ST1 was decreased to 0.32±0.015 and 0.27±0.017 from 0.93±0.057(p<0.05), and relative protein expression of HS2ST1 was decreased to 0.72±0.047 and 0.41±0.034 from 2.06±0.161(p<0.05). Both alterations showed an ATRA dose dependent manner. HS(glucosamine) 3-O sulfotransferase 3A(HS3ST3A) was also downregulated at both m RNA and protein levels in epithelial-mesenchymal transition of A549 lung cancer cells. Relative m RNA expression of HS3ST3 A was decreased to 0.92±0.037, 0.67±0.029 and 0.33±0.044 from 1.67±0.062(p<0.05), and relative protein expression of HS3ST3 A was dramatically decreased to 0.28±0.026, 0.08±0.004 and 0.07±0.003 from 0.48±0.031(p<0.05). Both alterations showed a TGF-b1 dose dependent manner. Further, HS3ST3 A was negatively correlated with the in vitro cell metastasis capability of A549 cells confirmed by RNA interference technology and in vitro Matrigel invasion assay. Number of invasion cells in HS3ST3 A knockdown group was 1097±57 compared to 353±31 and 337±27 in mock and negative control group, respectively(p<0.05). Together, these suggest HS2ST1 is associated with the process of granulocytic differentiation of SKM-1 leukemia cells, whereas HS3ST3 A is a candidate gene involved in lung cencer cell metastasis.3.A series of sulfated heparosan polysaccharides derivatives with different sulfation patterns were prepared which are structurally similar to heparan sulfate and their biocompatibility and bioactivity were investigated in stem cell osteogenic and chondrogenic differentiation. Firstly, heparosan, the synthesis precusor of HS, was prepared by high cell-density fermentation and anion exchange chromatography. Subsequently, sulfation groups were added to –NH– and/or –OH of the precursor heparosan and the modi?ed heparosan was qualitatively analyzed by FT-IR, 1H NMR, and 13 C NMR techniques. Cell viability was not signi?cantly affected by the sulfated heparosan polysaccharides. Among these polysaccharides, relative m RNA expression of the chondrogenic differentiation marker COL2A1 during stem cell chondrogenic differentiation was signi?cantly upregulated to 0.52 ± 0.027 in cells treated with the N,O-sulfated heparosan polysaccharide from 0.19 ± 0.017 in control cells treated with PBS(p<0.05), whereas N-sulfated and O-sulfated heparosan had no significant effect. And highly sulfated heparin inhibited the growth of stem cells with an inhibition ratio of 18.3%. These suggest the stem cell chondrogenic promotion bioactivity of HS isaccociated with its sulfation pattern. A cell model was established in which cell-surface HS was specifically removed. We constructed an expression vector of heparinase I with a C-terminal 6×His tag and got a heparinase I with 9-fold higher stability. Further, soluble expression level of heparinase I was enhanced by 23.6% using SUMO-fusion technology. Heparinase I was further purified by cation exchange chromatography and G25 gel permetration and used in stem cell culture. We found that BMSCs had significantly reduced growth ability and significantly smaller calcified nodules upon heparinase I treatment. Although this type of calcified cells had significantly higher ALP activity, overall, it still indicated the BMSCs had lower osteogenic potential upon heparinase I treatment. |
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