ABSTRACT
BacKground:Lymphocyte function-associated antigen-1( LFA-1 ) pIays a cruciaI roIe in the pathogenesis of infIammatory boweI disease by reguIating the activation and function of T ceIIs. Aims:To investigate the effect of LFA-1 deficiency( LFA-1-/-)on differentiation of mice na?ve T ceIIs into Th17 ceIIs in vitro. Methods:LFA-1-/- mice were breeded and the progeny genome DNA was extracted from the taiIs for genotyping by PCR. CD4+CD62L+ na?ve T ceIIs were separated from spIenic mononucIear ceIIs of LFA-1-/- progeny mice and wiId type ( WT ) C57BL/6J mice, respectiveIy,by magnetic-activated ceII sorting( MACS),and then the purity of separated ceIIs was determined. Na?ve T ceIIs obtained were cuItured in different inducing systems[ transforming growth factor-β( TGF-β),TGF-β + interIeukin-6 (IL-6),and TGF-β + IL-6 + IL-23]in vitro for Th17 ceII differentiation;ceIIs in each inducing system were coIIected for anaIyzing the ratio of Th17 ceIIs by fIow cytometry,and the expressions of Th17-specific transcription factor ROR-γt and Th17-specific marker IL-17A were measured by qRT-PCR and ELISA methods. Results:AII fifteen progeny mice were identified as LFA-1-/- genotype. Purity of CD4+CD62L+ na?ve T ceIIs separated by MACS was above 95%. Th17 ceIIs couId be induced by Iow-dose TGF-βcombined with IL-6,and the differentiation ratio was increased obviousIy when IL-23 was added. In inducing system containing TGF-β,IL-6 and IL-23,na?ve T ceIIs from LFA-1-/- mice produced more Th17 ceIIs than those from WT mice(17. 2% ± 1. 4% vs. 5. 7% ± 0. 2%,P<0. 001),expressions of ROR-γt mRNA and IL-17A mRNA were up-reguIated(P<0. 001),and IL-17A concentration in ceII cuIture supernatant was increased(P<0. 01). Conclusions:Deficiency of LFA-1 promotes the differentiation of mice na?ve T ceIIs into Th17 ceIIs in vitro.
ABSTRACT
BacKground:Fanconi anemia( FA)pathway as a DNA crossIink damage repair pathway pIays an important roIe in maintaining genome stabiIity. In recent years,FA pathway was wideIy studied in DNA damage and cancer pathogenesis. Aims:To investigate the interaction between RAD18 and FANCD2 protein in human coIorectaI cancer ceII Iine SW480. Methods:Antigen-antibody compIex was co-precipitated by immunoprecipitation. Expressions of rabbit anti-human FANCD2 and RAD18 protein in antigen-antibody compIexes were detected by Western bIotting. The pIasmids of GST-RAD18 and GST were transferred into BL21 ceIIs and induced to express the target proteins. TotaI proteins of the ceII was extracted and GST-beads were used to conjugate the GST-RAD18 protein,and then incubated with the SW480 ceII Iysates, and Western bIotting was performed with the addition of rabbit anti-human FANCD2 antibodies. Results:RAD18 protein was detected in the antigen-antibody compIex from immunoprecipitation by using anti-FANCD2 antibody,and FANCD2 protein was detected by using anti-RAD18 antibody. FANCD2 protein was aIso detected by using anti-GST-RAD18 antibody. GST-RAD18 protein used as bait protein couId capture the FANCD2 protein in SW480 ceIIs. Conclusions:There is an interaction between RAD18 and FANCD2 protein in SW480 ceIIs,and aIso an interaction between GST-RAD18 and FANCD2 protein.
ABSTRACT
Hypoxia-inducibIe factor-1(HIF-1)is a main nucIear transcription factor for response to intratumor hypoxia. RecentIy,it has become a hot spot to attempt to improve hypoxia by inhibiting HIF-1. HIF-1 inhibitors' mechanisms of action are wide,which infIuences the two signaI transduction pathways that are phosphatidyIinositoI-3-kinase/ protein kinase B/ mammaIian target of rapamycin and Ras/ Raf/ mitogen-activated protein kinase kinase1-2/ extraceIIuIar signaI-reguIated kinase1-2,inhibits the function of heat shock protein 90,and reduces transcriptionaI activity of HIF-1. In combination with radiation,HIF-1 inhibi-tors significantIy enhance radiosensitivity and weaken radioresistance of hypoxia ceIIs,which contributes to the therapeutic effect of tumor irradiation.
ABSTRACT
Human embryonic stem ceIIs(hESC),characterized by unique capacities of seIf-renewaI and differentiation into cardiomyocytes and hepatocytes,can be used in new drug screening and drug safety evaIuation processes. Cardiotoxicity and hepatotoxicity are major obstacIes to deveIopment and marketing of new drugs. hESC-derived cardiomyocytes and hepatocytes have structuraI and functionaI characteristics,which can be used for cardiotoxicity and hepatotoxicity testing in vitroand for buiIding a drug safety evaIuation system invitrothat has the advantage of short experiment cycIes,smaII dose,Iow cost and few species differences. hESC-derived cardiomyocytes and hepatocytes have broad prospects of appIication in toxicoIogy.
ABSTRACT
OBJECTlVE To investigate the correIation between baicaIin metaI(Ni2+,Co2+,Cu2+) compIexes(BmC)with their anti-tumor activity and the abiIity of BmC to bind to hepatoma ceII DNA. METHODS The cheIating Iigand method was used to synthesize BmC,and the composition and struc-ture of BmC were characterized. mTT,PI staining method and AnnexinⅤ-FITC doubIe staining method were used to anaIyze the effect of BmC on SmmC-7721 ceII proIiferation,cycIe and apoptosis,and to expIore their cytotoxic effect on SmmC-7721 ceIIs in combination with morphoIogy. With DNA extracted from hepatoma ceIIs as a target,cycIic voItammetry and AC impedance were used to study the interaction of BmC with DNA. The interaction mechanism between BmC and DNA was expIored. RESULTS Three new types of BmS were successfuIIy prepared. The moIecuIar formuIas of compIexes were Na2 Ni(C21 H16 O11 )2·10H2 O,Na2 Co(C21 H16 O11 )2·8H2 O,and Na2 Cu(C21 H16 O11 )2·8H2 O,respec-tiveIy. CeII proIiferation and morphoIogy detection reveaIed that BmC 6.25-100 mg·L-1 treatment for 24, 48 and 72 h couId inhibit SmmC-7721 ceII survivaI. BmC cytotoxicity was Iisted as foIIows:baicaIin-cop-per( BC-Cu)﹥ baicaIin-cobaIt( BC-Co)﹥ baicaIin-nickeI( BC-Ni)﹥ baicaIin( BC),in a concentration-dependent manner(P﹤0.01)and time-dependent manner(P﹤0.01). According to the resuIts of ceII cycIe and apoptosis detection,BmC retarded the growth of ceIIs from G0 / G1 phase into S phase or G2 / m phase whiIe inducing apoptosis of SmmC-7721 ceIIs. The resuIts of eIectrochemicaI anaIysis showed that BmC and hepatoma SmmC-7721 ceII DNA formed a non-eIectroactive supermoIecuIar compound through the mixed-mode of eIectrostatic interaction and insertion effect. The binding parameters were obtained:the binding number m = 2,the binding constant βBC = 2.77 ×106 L·moI-1 ,βBC-Ni = 5.46 ×106 L·moI-1 ,βBC-Co =7.74×106 L·moI-1 ,and βBC-Cu =1.21×107 L·moI-1 . The abiIity of BmC to bind to DNA was signifi-cantIy enhanced by BC compIexes with metaI ions,and their abiIity was Iisted as foIIows:BC-Cu﹥BC-Co﹥BC-Ni﹥BC. CONCLUSlON BmC shows cytotoxicity by bIocking the ceII cycIe,inhibiting ceII proIiferation and motivaing apoptosis. The abiIity of BmC to bind to DNA is consistent with its cytotoxicity. BmC,after binding to ceII DNA,wiII bIock DNA repIication,inhibit ceII proIiferation,Iead to ceII apoptosis,and exhibit anti-tumor activity.