Objective:To prepare the monoclonal antibodies against chicken CARP.Methods:The chicken CARP cDNA encoding the N-terminal 110aa length of encoding region was cloned into prokaryotic expression vector pET28b.The BALB/c mice were immunized with purified the recombinant protein,spleen cells of mouse were fused with SP2/0,and the positive clones were screened by indirect ELISA.The properties of McAb were analyzed by ELISA and Western blot.Results:Two hybridomas were proved to have the capability of stably secreting McAb against chicken CARP,designated as 4A8 and 4E6,the subtype of both was IgG1,and the light chain was ?.The titers of ascites were 3.2?104 and 1.28?105 respectively.The Western blot results suggested that they could recognize chicken CARP protein.Conclusion:Two murine hybridomas secreting anti-chicken CARP McAbs have been successfully obtained.

Objective:To construct lentiviral vector which can overexpression miR-137 and produce lentivirus by lentivirus packaging system, and to explore its infection efficiency and expression in HEK293T cells.Methods: miR-137 sequence was chemically synthesized and cloned into lentiviral vector GV209, and the recombinant plasmid containing human miR-137 was obtained and identified.Then miR-137 recombinant plasmid together with two helper plasmids were transfected into HEK293T cells using Lipofectamine 2000.After the HEK293T cells were infected in multiplicity of infection(MOI) 40 for 48 h, the expression of green fluorescent protein (GFP) was observed by fluorescence microscope and the expression level of miR-137 was detected by fluorescence quantitative PCR.Results:The sequencing results showed that the inserted gene sequence was completely consistent with the published human miR-137 gene sequence in GenBank.The GFP was observed in the HEK293T cells infected with miR-137 overexpression lentivirus under fluorescence microscope.The fluorescence quantitative PCR results showed that the expression level of miR-137 in the cells infected with overexpression lentivirus was 12.74 times higher than that in the control cells.Conclusion:The lentivirus containing miR-137 gene is successful packaged, and it could efficiently infect the HEK293T cells.

Objective To explore the effect of inhibiting placental growth factor ( PIGF ) by small interfering RNA ( siRNA) on migration, invasion and chemoresistance of human pancreatic cancer cell line PANC1.Methods Three specific siRNAs targeting PIGF (siRNA-PIGF) were designed.PANC1 cells were transfected with siRNA-PIGF by liposome transfection using untransfected cells as blank controls and nonspecific siRNA ( siRNA-NC) transfected cells as negative controls .The PIGF mRNA and protein expression was examined by real-time RT-PCR and ELISA.MTT method was used to assess the inhibition rate of chemotherapeutic reagents on cell proliferation .The abilities of migration and invasion were evaluated by Transwell assay.Results The inhibition rate of PIGF mRNA in PANC1 cells transfected by 3 siRNA-PIGF were (64.38 ±8.92)%, (70.48 ±7.72)% and (81.25 ±6.02)%, which was lowest in siRNA-PIGF-3 transfected cells.The expression of PIGF mRNA in PANC1 cells were decreased by (63.72 ±8.20)%at 24 h after siRNA-PIGF transfection compared with siRNA-NC transfected cells;and the level of PIGF protein in the supernatant of cultured PANC1 cells was lowered by (42.92 ±1.34)% compared with siRNA-NC transfected cells and by (46.25 ±3.64)% compared with untransfected cells at 48h after transfection, which all had significant difference .There was no statistical difference between untransfected and siRNA-NC transfected cells.After 3 ng/L gemicitabine treatment , the inhibition rate of cell proliferation in siRNA-PIGF group was even higher than that in siRNA-NC and untransfected group [(44.35 ±5.05)% vs(34.29 ±3.60)% and (31.01 ±1.08)%;both P<0.05], and no significant difference was not observed after 5-FU and adriamycin treatment.In migration and invasion assay , the number of transmembrane cells from siRNA-PIGF group was 38.1%and 28.2%of that from siRNA-NC group and 40.8% and 36.2% of that from untransfected group , which had statistical difference (all P<0.05).Conclusions PIGF silencing could significantly suppress the migration and invasion of PANC 1 cells and improve the sensitivity to gemicitabine .

BACKGROUND:Osteoarthritis, a degenerative joint disease, is not only a result from the breakdown of joint cartilage and underlying bone, but also an imbalance of bone remodeling and crosstalk among tissues in the joints. OBJECTIVE: To review the effect of bone-cartilage crosstalk in the progression of osteoarthritis and its new treatment strategy. METHODS: A computer-based search of PubMed and CNKI databases was performed for relevant literatures about the relationship between the progress of osteoarthritis and the bone-cartilage crosstalk published from 2007 to 2017. The keywords were cartilage, interaction, osteoarthritis, pathogenesis, cytokines, signaling pathway in English and Chinese, respectively. The relationship between the progress of osteoarthritis and the bone-cartilage crosstalk was summarized in views of cytokines, signaling pathway, and new treatment strategy. RESULTS AND CONCLUSION: Totally 169 articles were retrieved, and finally 54 eligible papers were enrolled based on the inclusion and exclusion criteria. There is a close physical association between subchondral bone and cartilage, and the bone-cartilage interface is a functioning synergistic unit. Increased vascularization, micro-crack formation and abnormal bone remodeling may accelerate the molecules transporting from cartilage to bone in osteoarthritis. Therefore, the bone-cartilage crosstalk plays a pivotal role in the occurrence and development of osteoarthritis.