Songhuman
my postgraduate paper Abstract Objective: Acquisition and culturing of human nucleus pulposus cells in vitro and studying of its biological character;To investigate the mechanism and signaling pathways of TLR3 mediates human nucleus pulposus cells expressing chemokine CXCL10. Methods: (1) Digest and isolate human nucleus pulposus cells by trypsin and type II collagenase methods. (2) Immuncytochemistry of human nucleus pulposus cells via type II collagen . (3) Determine the doubling time of human nucleus pulposus cells by CCK-8 kit。 (4) Flow cytometry detects the proliferation rate of human nucleus pulposus cells in different passages and normoxia or hypoxic environment. (5) Detect the senescence of human nucleus pulposus cells by β-Galactosidase staining kit. (6) Cell viability detection by CCK-8 kit after different centrifugal force and time or continuous culture cell to 24-48hr (7) Testing different mRNA expression of chemokines of human nucleus pulposus cells after Poly(I:C) stimulation by real-time PCR. Testing CXCL10 mRNA expression in human nucleus pulposus cells of different specimens. Detection of CXCL10 mRNA expression in human nucleus pulposus cells by stimulation of various TLR ligands. Dose and time effect of CXCL10 mRNA expression in human nucleus pulposus cells by stimulation of Poly(I:C). (8) Human nucleus pulposus cell activity detection after Poly(I:C) stimulation by CCK-8 kit. (9) HCQ,a TLR3 specific blocker,and siRNA interference act on human nucleus pulposus cells,testing CXCL10 mRNA expression level after Poly(I:C) stimulation. (10) Testing CXCL10 mRNA expression level stimulated by Poly(I:C) through real-time PCR after cycloheximide,a inhibitor of protein synthesis,pretreated human nucleus pulposus cells. (11) Testing CXCL10 mRNA expression level,in small molecule inhibitors of signal pathway pretreatment human nucleus pulposus cells after stimulation of Poly(I:C), by real-time PCR. (12) Determine activation of subunit p65 subjected NF-κB after Poly (I:C) stimulated human nucleus pulposus cells pretreated in NF-κB signal pathway small molecule inhibitor.
Apr 2, 2015 7:25 AM