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Wet age-related macular degeneration (AMD),which is the leading cause of visual impairment in elderly people,significantly affects quality of life of millions worldwide.Currently,anti-VEGF is the first-line therapy for wAMD,bringing encouraging results in improving the vision.However,not all of the patients will response to this therapy,moreover,anti-VEGF may be associated with several issues,such as multiple injections,systemic adverse effects,delay or lack of response.With a deep understanding of the mechanism of wAMD,there are rapid developments of new approaches to more effective therapy.Ophthalmologists should pay attention to the advantages and disadvantages of these anti-VEGF drugs as well as the current advances of anti-VEGF drugs in order to provide better treating strategies for wAMD patients better.
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OBJECTIVE: To synthesize immunomagnetic nanoparticles with uniform particle size, strong superamagnetism as well as strong immune activity which can be specifically and sensitively combined with circulating tumor cells in peripheral blood of patients with breast cancer.METHODS: Superparamagnetic oxide iron nanoparticles containing active carboxyl groups (SMNP-COOH) were synthesized by polyol methods, thermogravimetric analysis was used to determine the amount of carboxyl groups on the surface of SMNP-COOH, while the content of iron was determined by o-phenanthroline. Mediated by 1-ethyl-3,3-dimethylaminopropyl carbodiimide(EDC) and N-hydroxysuccinimide (NHS), immunomagnetic nanoparticles(IMNP) against human breast carcinoma cell line were constructed by binding the monoclonal antibodies against hMAM with SMNP-COOH. X-Ray diffraction was used to confirm their synthesis,meanwhile,transmission electron microscope (TEM), dynamic light scattering (DLS), and vibrating sample magnetometry (VSM) were applied to characterize their physicochemical properties. The conjugation amount of the antibodies and the activity of IMNPs were evaluated by enzyme linked immunosorbent assay (ELISA).RESULTS: X-Ray diffraction showed that the chracteristic peaks of the crystalline powder of SMNP-COOH and IMNP agreed with the Fe3O4 standard. The concentration of iron in SMMP-COOH and IMNP were 0.205 and 0.164 mol•L-1, respectively.TEM showed that both synthesized SMNP-COOH and IMNP were almost spherical or ellipsoidal. The sizes of SMNP-COOH and IMNP were (13.7±3.6) and (15.4±4.5) nm, respectively. Dynamic light scattering(DLS) demonstrated the intensity particle size and polydispersity index (PDI) of SMNP-COOH and IMNP were 23.4 nm and 0.303, and 71.2 nm and 0.175,respectively. VSM results showed that both SMNP-COOH and IMNP had strong superamagnetism, and the saturation magnetization of SMNP-COOH and IMNP were 71.37 and 67.68 emu•g-1Fe, respectively, which confirmed antibody binding may reduce the magnetism of SMNP-COOH. The ELISA results showed the conjugation amount of antibody was about 93 μg on 1 mg SMNP-COOH by covalent bond. The obtained immunomagnetic nanoparticles (IMNP) which were bound with the hMAM monoclonal antibodies could specifically and sensitively combine with breast cancer cell line MDA-MB-415.CONCLUSION: IMNP with strong superparamagnetic property,excellent stability and perfect antibody activity were successfully synthesized, which demonstrate the potential to magnetically separate circulating tumor cells in peripheral blood from patients with breast cancer, thus providing a favorable weapon to accurately detect CTCs in breast tumor patients.
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Objective To study the clonal heterogeneity in differentiation potential of immortalized mesenchymal stem cells in vitro and in rive.Methods The monoclonal cell lines were performed with limiting dilution cloning,and were induced to adipocytic,asteogenic and neuronal differentiation in vitro.After transplanted the monoclonal cell lines into SCID mice,the xenotransplants were removed and evaluated by immunohistochemistry.Results From the parental HMSC-TERT,32 single-cell derived clones were established,of which the differentiation properties varied considerably in vitro.The cells grow in different plating densities during expansion in culture:HMSC-TERT-2 expressed more strongly in LCA,GFAP and vimemin;HMSC-TERT-C19 expressed more strongly in keratinose than HMSC-TERT-2,HMSC-IERT-20,and MSC-H;HMSC-TERT-C2 expressed more strongly in actin than HMSC-TERT-2.Conclusions The HMSC-TERT monoclone cells are heterogeneity in differentiation petential in vitro and in vivo,suggesting that standard in vitro culture and in vivo inoculate procedure phy an important role in the clinical application of stem cells.
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To isolate and culture the purified monoclonal neural stem cells from the cerebral cortex of new born mice, new-born mice cerebral cortex was isolated and dissociated to single-cell suspension by mechanical trituration. The dissociated single cells were cultured in serum-free medium. After the formation of neurospheres, single-cell clone culture was performed by limiting dilution and the proliferated single-cell clones were harvested for subculture. Immunocytochemistry was used to detect the specific marker of neuroepithelial stem cells (Nestin) of the primary and monoclonal neurospheres. In the differentiated cells we detected the specific antigen of NF-200 and GFAP. Our results showed that the primary neurospheres expressed Nestin antigen positively. By limiting dilution, we cultured the cell lines from single-cell clone and the monoclonal neurospheres expressed Nestin and had capabilities of self-renewal, proliferation and the potentiality of differentiation into neurons and glial cells. It is concluded that monoclonal neural stem cells which have the ability of proliferation and multi-directional differentiation can be isolated and cultured from the cerebral cortex of new-born mice by limiting dilution.
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AIM: To detect sequence and mutation of K-ras oncogene in tissue and stool DNA of patients with colorectal cancer in order to provide a method of noninvasive and simple colorectal cancer diagnosis. METHODS: DNA was separated and purified from colorectal cancer tissue or stool of patient with colorectal cancer, then the K-ras gene was amplified by PCR and PCR products were cloned, the K-ras gene was sequenced, and the mutation was identified. The expression of color/colorectal cancer antigen was inspected by immunohistochemical technique. Stool sample of patient with colorectal cancer was detected with enzyme-linked immunosorbent assay (ELISA). RESULTS: K-ras gene sequence of the stool was completely same as that of the tissue of the patient;K-ras mutation was detected in one case. There was relativity between the mutation of K-ras gene and the pathology type of colorectal cancer and the expression level of colorectal cancer antigen in stool sample. CONCLUSION: It is feasible that colorectal tumors can be detected by a noninvasive method based on the molecular pathogenesis of the disease. Detecting K-ras gene mutations of stool DNA can provide bases for the screening, early detection, and prognosis to patients with colorectal cancer.
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Objective To study the effect of optimized media and clones on growth and anthraquinone yield in hairy root cultures of Rheum palmatum.Methods The four monoclones and four media were optimized statistically for studying the effect on the biomass accumulation and five anthraquinones yield in hairy root cultures of R.palmatum.Results Significant differences in biomass were shown among various clones and media,in which monoclone DH7a was the highest and WP medium was the best.Chrysophanol and physcion were predominant among five free anthraquinones in hairy root cultures of R.palmatum.Notable differences of total five free anthraquinones yield were observed among different clones, in which DH7a was the highest.Reciprocity analysis showed that the highest yield of five free anthraquinones is in the combinations of DH7a with B5 medium.DH5a less and DH5a the least with WP medium,DH7a and B5 medium were predominent for the yield of above five free anthraquinones with exception of DH5c which has no significant difference in four media.Conclusion The study lays the foundation for mass production of anthraquinone by hairy root culture of R.palmatum.
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A one - dose 5 - 1 - 6 monoclonal antibody injected intravenously can induce atransient massive proteinuria in rat. Comparison between Radix Bupleuritreating group and PBS control group 1, 3, 5, 8 days after injection showed significant difference. Under immuno -flourescence exam, the granular precipitation along the glomerular capillaries in experimental group decreased distinctively, yet the effect of Chailing Decoction on proteinuria can not be proved by oral infusion, suggesting that Radix Bupleuri may change the permeability of glomerular capillary in rat through certain mechanism.
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Objective To study the immuno-protective effect against Schistosoma japonicum challenge of the positive monoclonal phages which were screened from the 12 mers-phage random peptide library by the new model rabbit serum. Methods The new model was established by injecting the Schistosoma japonicum infection rabbits with inhibitor of phenol oxidose. Positive clones immunoscreened with the new model rabbit sera were absorbed by SEA immune rabbit sera, and 14 clones selected randomly from them were compared and their antigenic ability was identified by ELISA. The two best positive monoclonal phages (No.8, No.13) recognized by the new model rabbit sera were selected to immunize Kunming mice by subcutaneously injecting 1?10~15 pfu positive phages at 0, 2nd, 4th week respectively. After 4 weeks of the last immunity,each mouse was challenged with 40?1 S.japonicum cercariae. All mice were sacrificed after 42 days and the reduction rates of adult worms and the liver eggs were investigated. Results The positive phage clones after immune absorption were weakly recognized by the SEA immune rabbit sera. The 14 monoclonal phages were recognized by the rabbit sera of the new model and the normal model. Especially No.8, No.13 were strongly recognized by the rabbit sera of the new model,while weakly recognized by the SEA immune rabbit sera. The reduction rates of adult worms and liver eggs induced by the monoclonal phage No.13, the monoclonal phage No.8 and the original peptide library were 35.81% and 63.32%, 32.09% and 52.02%, 14.90% and 30.64%, respectively. Conclusion Most clones of simulated epitopes of SEA can be removed by absorbing positive clones with SEA immune rabbit serum .The 14 monoclonal phages from the new model contain the simulated S.japonicum epitope. The two monoclonal phages have higher reduction rates of adult worms and eggs than original 12 mers-phage random peptide library and the positive polyclonal phages.[
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Objective:To construct a monoclone cell line expressing M-HBsAg.Methods:Transfected SP2/0 with pRc/CMV-S 2S by Calcium Phosphate Method,selected the cells by G418,detected HBsAg by Dot-blot、ELISA and Western-blot hybridization.Results:By selection of G418,G418 resistent cells were obtained;tests of Western-blot hybridization and ELISA demonstrated their expression of aimed protein.Conclusion:Monoclone Cell Lines of SP2/0-S 2S were obtained.
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Objective To determine the optimal condition for labeling rat bone marrow mesenchymal stem cells(MSCs) with green fluorescent protein(GFP)infection by lentiviral vector so as to establish a subgroup of MSCs which have a high level of GFP expression.Methods MSCs were infected with GFP by lentiviral vector for 12h or 24h at different MOI(25,50,100,200 and 400).The infection efficiency and fluorescence intensity were analyzed by inverted fluorescent microscopy and flow cytometry.The effect of infection at different MOI on proliferation of MSCs was evaluated by MTT.Based on those mentioned above,we could determine the optimal condition for infection.Then single cell clones of MSCs labeled with GFP under optimal condition were selected by using cloning rings.Results The efficiency of infection for 24h at MOI 100,200 and 400 was 88.94%,99.65% and 99.42%,respectively.The infection had no significant effect on the proliferation of MSCs infected at MOI 100 or 200,compared with MSCs without infection.However,those MSCs infected at MOI 400 proliferated slowly.The rate of GFP expression on single-cell clone of MSCs infected for 24h at MOI 200 was 99.95%,and their fluorescence intensity was strong and uniform.Conclusion Infection for 24h at MOI 200 is optimal for labeling rat bone marrow MSCs with GFP by a lentiviral vector.A subgroup of MSCs which have a stably high level of GFP expression can be obtained by single cell cloning technique.