ABSTRACT
Objective:To study and promote the quality standard for Shuangbai Quyu powder. Methods:A microscopic identifi-cation method was performed to identify Phellodendri amurensis cortex, Rhei radix et rhizoma, Platycladi cacumen and Andrographis herba. TLC was performed to identify Phellodendri amurensis cortex, Andrographis herba and Rhei radix et rhizoma. HPLC was used to determine the content of berberine hydrochloride in Phellodendri amurensis cortex. and the lower limit was determined. Results: The microscopic identification method was simple. The spots in TLC were distinct without any interference from the negative coutrol. Ber-berine hydrochloride had a good linear range of 0. 010-0. 400 μg(r=0. 999 9). The average recovery was 99. 51%, and RSD was 0. 31% (n=6). Conclusion:The method can be used for the quality control of Shuangbai Quyu powder effectively,which is beneficial to the quality standard promotion of hospital preparations.
ABSTRACT
BACKGROUND: Immunoloregulation of mesenchymal stem cells (MSCs) is commonly approved. Previous studies have confirmed the ability of Flk-1~+ bone marrow MSCs (BMSCs) to inhibit T/B lymphocyte proliferation in vitro. OBJECTIVE: To investigate the therapeutic effect of Flk-1~+ BMSCs in collagen-induced arthritis mice.METHODS: A total of 18 healthy male DBA-1(H-2K~q) mice aged 10 weeks were randomly divided into 3 groups. All the mice were injected at the base of the tail with bovine type II collagen (CII), and received a booster injection of CII on day 21 to establish the CIA mice model. DBA-1(H-2K~q)mouse Flk-1~+ BMSCs were isolated in vitro by the density gradient centrifugation and adherence screening. Following initial immunity, mice in the cell transplantation group were infused with Flk-1~+ BMSCs (1-2)×106 cells/mouse via the caudal vein. Mice in the cell transplantation group were injected with the same volume of Flk-1~+ BMSCs during booster. Mice in the model control group were injected with an equal volume of saline 0 or 21 days following initial immunity. Following initial immunity and booster immunization, claw pad thickening and clinical score were observed, changes of joint pathology and dynamic changes in serum factor mass concentration were determined in mice. RESULTS AND CONCLUSION: Compared with the model control group, no significant difference in claw pad thickening and mean clinical score was detected in the cell transplantation group following initial immunity (P > 0.05), with the presence of obvious damage to synovial membrane and inflammatory cell infiltration. Mass concentration of each serum cell factor was similar. The claw pad was significantly thickened (P < 0.01), mean clinical score reached 3.35 points, with severe damage to synovial membrane, proliferation of blood capillary in the cell transplantation group following booster immunization. Interleukin-6 levels were greatly increased at day 28 following initial immunity (P < 0.1), but decreased at day 35 following initial immunity (P < 0.1). Results indicated that in the collagen-induced arthritis mouse models, Flk-1~+ BMSC transplantation did not obtain prospective therapeutic efficacy, but aggravation of arthritis was observed in the cell transplantation group following booster immunization. Upregulation of interleukin-6 concentration could aggravate the behavior symptom of rheumatoid arthritis mice.
ABSTRACT
To construct a scFv antibody mini-library by phage display technique from the spleen cells of BALB/c mice immunized with extracellular domain of N-cadherin (N-cad), CD34 and AC133, the extracellular domain genes of N-cad, CD34 and AC133 were cloned into a phagemid pSEX81 respectively, and were then displayed on the phagemid in the form of fusion protein with p III protein. After being effectively immunized with the fusion protein, the spleen of the mice were harvested, and total RNA were extracted from the spleen. The cDNA of VH and VL genes were amplified by RT-PCR, and a scFv-phage display antibody library was constructed with the amplified V-genes. The content, multiplicity and expressing potential of the library were examined. As a result, we had produced a scFv library containing 1.4 x 10(6) individual clones which showed different patterns after being digested with restriction endoneuclease Mua I. The surface display expression of the library was also verified. It indicated that the capacity and diversity of the library was sufficient for screening specific scFv antibody against N-cad, CD34 and AC133. The library will be useful for isolating corresponding specific scFv antibodies.
Subject(s)
Animals , Female , Mice , AC133 Antigen , Antibodies , Genetics , Antigens, CD , Allergy and Immunology , Antigens, CD34 , Allergy and Immunology , Base Sequence , Cadherins , Allergy and Immunology , Glycoproteins , Allergy and Immunology , Immunoglobulin Fragments , Genetics , Immunoglobulin Variable Region , Genetics , Mice, Inbred BALB C , Molecular Sequence Data , Peptide Library , Peptides , Allergy and ImmunologyABSTRACT
AIM: To clone the extracellular domain of N-cadherin cDNA, and to observe the antigenicity of the expressed protein. METHODS: Total RNA was extracted from CD34+ cells separated from fetal liver and bone marrow cells. The extracellular domain of N-cad cDNA was amplified with RT-PCR and inserted into a vector pOPE101-215. The recombinant pOPE-N-cad was expressed with IPTG induction. Then, mice were immunized with the protein. RESULTS: The extracellular domain of N-cad cDNA from CD34+ cells was identified by DNA sequencing. The recombinant pOPE-N-cad in host XL1-blue expressed a 70 kD protein after induced with IPTG, and anti-N-cad antibody was detected in serum of the immunized mice after 5 times injection of the recombinant N-cad protein. CONCLUSION: CD34+ cells bore N-cad gene and the recombinant protein of the extracellular domain of N-cad cDNA shows good immunogenicity.