Evaluation of anti-HBV effect of recombinant human serum albumin-interferon α-2b fusion protein in vitro / 中国药理学与毒理学杂志

OBJECTlVE To study the anti-HBV activity of prepared recombinant human serum aIbu-min-interferon α-2b fusion protein(HSA-IFNα-2b) in vitro. METHODS HepG2 ceIIs were infected with recombinant adenovirus with green fIuorescence protein and 1.6-foId HBV DNA(AdGFP-HBV). The ex-pression of HBV antigens,HBsAg and HBeAg in cuIture medium was detected by ELISA assay. The tox-icity of HSA-IFNα-2b on HepG2 ceIIs was evaIuated by mTT assay.The reIative expression of HBV RNA in ceIIs and the absoIute quantity of HBV DNA in cuIture supernatant were determined by quantitative PCR assay. The activity of HBV enhancer Ⅰ was detected by DuaI-Reporter gene assay. RESULTS HBV couId repIicate and express in HepG2 ceIIs after infection with AdGFP-HBV. The expression of HBsAg and HBeAg in cuIture serum of HepG2 ceIIs infected with AdGFP-HBV decreased by 51.32%(P﹤0.01)and 50.26%(P﹤0.01),respectiveIy,when HSA-IFNα-2b 500 kU·L-1 was added. The same concentration of HSA-IFNα-2b didn't inhibit the proIiferation of HepG2 ceIIs,but inhibited HBsAg in a concentration-dependent manner. The regression formuIa between HBsAg inhibitory rate(Y)and con-centration of HSA-IFNα-2b(X)was Y=21.11 IgX+11.91(r 2 = 0.954),IC50 = 63.76 kU·L-1 . HBV RNA in ceIIs and HBV DNA in the cuIture serum decreased by 52.83%(P﹤0.01)and 53.07%(P﹤0.01), respectiveIy,when HSA-IFNα-2b 500 kU·L-1 was added. The activity of enhancer Ⅰ decreased by 40.04%(P﹤0.01)when HSA-IFNα-2b 500 kU·L-1 was added. CONCLUSlON The ceII modeI of HBV repIication for evaIuating anti-HBV agents is successfuIIy estabIished. HSA-IFNα-2b exhibits noticeabIe anti-HBV effect invitro.

Construction and validation of a dual-luciferase reporter gene system for screening and evaluating anti-liver fibrosis drugs that inhibit transcription of the gene encoding collagen I, chain a1 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To construct a dual-reporter gene system that will be applicable for use as a tool to screen and evaluate therapeutic drug compounds that inhibit transcription of the gene encoding collagen I, chain at1 (COL1A1).</p><p><b>METHODS</b>The full-length eDNA of transforming growth factor beta1 (TGFbeta1) was cloned by RT-PCR and inserted into two vectors, pcDNA3.1 and pJW4303, for construction of two eukaryotic expression vectors, pcDNA3.1-TGFbeta1 and pJW4303-TGFbeta1.Next, the promoter region of COL1A1, cloned by PCR using human genome DNA as template, was inserted into the vector pGL4.29 to construct the reporter gene vector, pGL4.29-COL1A1 promoter.All three recombinant vectors were verified by restriction enzyme digestion and DNA sequencing.Either the pcDNA3.1-TGFbeta1 or pJW4303-TGFbeta1 vector along with the pGL4.29-COL1A1 promoter vector or a pRL-null, control reporter, vector were co-transfected into the LX-2 human hepatic stellate cells to establish the transcription-activated dualreporter gene system.This system was used as a cell model for screening anti-liver fibrosis compounds that inhibit the transcription of COL1A1.Dexamethasone, a model drug that is known to inhibit the expression of COL1A1, was used as a control to validate the dual-reporter gene system.</p><p><b>RESULTS</b>The two TGFbeta1-expressing vectors and the reporter gene vector containing the promoter region of COL1A1 were successfully constructed.The results of a dual-reporter gene assay showed that TGFbeta1 co-expression increased the activity of the COL1A1 promoter by above 200-fold (t =21.78, P =0.0001), whereas in the absence of TGF31 co-expression the activity was below 2-fold (t =3.396, P =0.0274).The transcriptionactivated dual-reporter gene system was successfully established.The model drug, dexamethasone, effectively inhibited the activity of the COL 1A1 promoter in dose-dependent manner; the activity decreased 29.6% with 10 mumol/L dexamethasone (t =4.140, P =0.0144) and 53.9% with 100 mumol/L (t =6.193, P =0.0035).</p><p><b>CONCLUSION</b>The dual-luciferase reporter system of TGFbeta1 and COL1A1 co-expression developed here can be used as a cell model to screen and evaluate anti-liver fibrosis compounds that inhibit activity of the COL1A1.</p>

Serum Collagen and Arterial Stiffness in Hypertensive Patients / 中华高血压杂志

Objective To investigate the relationship between serum of type Ⅲ collagen and arterial compliance in hypertensive patients.Methods One hundred fifty-eight in-patients of hypertension were enrolled.All subjects underwent laboratory measurements including serum PⅢNP,blood-lipid,glucose and high sensitivity C-reactive protein.Carotid to femoral pulse wave velocity(PWVcf),C1 and C2 were measured by a Complior Colson device and DO-2020.Results(1)PWVcf positively correlated with serum PⅢNP(P