Objective To explore the survival,migration and differentiation of embryonic stem cells after transplantation into striatum and provide advantageous data for feasibility and safety of therapeutic transplantation. Methods We transplanted embryonic stem cells(undifferentiated ESCs and ESCs that had already developed into the stage of neural progenitor cells respectively) into striatum of the rat.Then differentiated cells were determined by morphological observation,Nissl's staining,TH and BrdU immunocytochemistry. Results We found that after transplanted 4 weeks,partially differentiated ESCs could survive and migrate into the surrounding host tissue.Some of them differentiated into TH-positive cells which had Nissl's bodies in cytoplasm.Whereas undifferentiated ESCs couldn't differentiate into TH-positive cells effectively and have the tendency to form tumor.Conclusion When conducting transplantation experiments of ESCs,it's better for ESCs to be induced into the stage of neural progenitor cells first and then transplanted.;

Here we discuss whether N terminal sequencing is appropriate as one of the conventional control methods for monoclonal antibody products. We determined the N terminal sequences of two monoclonal antibody products targeting two antigens separately with both Edman degradation and mass peptide spectrometry. We also identified the characteristic peptide fragments with mass spectrometry. Furthermore, we analyzed their heterogeneity with ion exchange chromatography, capillary zone electrophoresis and Imaged Capillary Isoelectric Focusing. Edman degradation method showed that the N terminal 15 amino acids of heavy and light chains of the two monoclonal antibodies were identical. Peptide mass spectrometry demonstrated that T1 peptide fragments of heavy and light chains of the two antibodies were also the same. But in contrast, peptide mapping and the three analytical methods for heterogeneity analysis could effectively identify and differentiate the two antibodies. The N terminal sequences of two monoclonal antibodies are identical because the number of framework sequences of humanized or human monoclonal antibodies is relatively limited, so whether N terminal sequencing analysis could be regulated as one of the practical control methods should be carefully discussed. Our work also proves that the above analytical methods could combinatorially applied to the identification of monoclonal antibody products, and are more objective compared to N terminal sequencing.
Amino Acid Sequence ; Antibodies, Monoclonal ; isolation & purification ; Chromatography, Ion Exchange ; Humans ; Isoelectric Focusing ; Mass Spectrometry ; Peptide Mapping ; Peptides ; Sequence Analysis, Protein ; methods

Objective To analyze the differences of size and charge heterogeneities between origi-nal humanized anti-TNF-αantibody and four similar biotherapeutic products ( SBP ) .Methods The size exclusion chromatography ( SEC-HPLC ) and weak cation exchange chromatography ( WCX-HPLC ) were used to analyze the size and charge heterogeneities , respectively.Carboxypeptidase B (CpB) treatment was employed to analyze the source of charge heterogeneity of the antibody products .Results Four SBPs showed the same pattern with the originator in SEC-HPLC, and no significant difference with the percentage of mono-mer was observed .The percentages of the aggregates of SBP-3 and SBP-4 were a little higher than those of the originator .The charge distribution of SBPs was significantly different from the originator ′s, especially in the basic region .The results from the samples treated with CpB indicated that the difference of charge distri -bution in the basic region might be caused by the C-terminal lysine variants .Conclusion Four SBPs showed similar size heterogeneity with the originator , but significant differences with charge heterogeneity were observed among them .The study suggested that more attention should be paid to the charge heterogene -ity analysis of the biosimilar products .

Background and purpose: Ovarian lymphoma (OL) is usually misdiagnosed as ovarian cancer with bulk lymph node invasion (OC-BLN), and vice versa. Therefore, to distinguish these two types of disease, we compared their clinical characteristics in this study. Methods: This study retrospectively reviewed 14 OL and 14 OC-BLN patients from Fudan University Shanghai Cancer Center and Shanghai Eighth People's Hospital. The clinical char-acteristics, image and laboratory examination data were compared. Results: There was no significant difference in age, symptom, fever, weight loss and volume of ascitic fluid between the two groups. Comparing with OC-BLN, OL patients have larger tumor in ovaries [(13.04±5.94) cm vs (7.78±6.38) cm, P=0.033], and higher percentage of solid ovarian tumor (85.71% vs 28.5%, P=0.006). Lactate dehydrogenase(LDH)/CA125 was higher in OL (7.66±8.03) than OC-BLN (0.31±0.27, P=0.009). Using LDH/CA125 to diagnose OL, area under the curve (AUC) was 0.952. When the threshold value was set at 1, the sensitivity and specificity was 91.7% and 100%, respectively. Conclusion: OL and OC-BLN are easily to be misdiagnosed. OL has larger and more solid tumor than OC-BLN. LDH/CA125 can help to distinguish these two diseases and guide clinical decision making.

The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo™ Luciferase Assay System using Jurkat/NFAT-luc+FcγRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y = (A-D)/[1 + (X/C)(B)] + D. The optimized parameters of the method were determined including the antibodies diluted concentration (18,000 ng·mL(-1)), dilution rate (1:5), the ratio of effector cell and target cell (6:1), and induction time (6 h). The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of (44.39±3.93)%, (72.74±2.78)%, (128.28±7.01)% and (168.19±2.70)% respectively, with a variation coefficient of less than 10%, and the recoveries of (88.78±7.85)%, (96.99±3.70)%, (102.63±5.61)% and (112.12±1.80)% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.

To establish a detection method of oncolytic adenovirus/p53 and standard of quality control, human telomerase reverse transcriptase (hTERT) promoter, CMV fusion promoter containing hypoxia reaction element (HRE) and p53 gene were identified by vector DNA restriction enzyme digestion and PCR analysis. The result conformed that all modified regions were in consistent with theoretical ones. Particle number was 2.0 x 10(11) mL(-1) determined by UV (A260). Infectious titer was 5.0 x 10(10) IU mL(-1) analyzed by TCID50. In vitro p53 gene expression in human lung cancer cell H1299 was determined by ELISA, and A450 ratio of nucleoprotein in virus infection group to control group was 5.2. Antitumor potency was evaluated by cytotoxicity assay using human lung cancer cell A549, and the MOI(IC50) of this gene therapy preparation was 1.0. The tumor cells targeted replication ability of recombinant virus was determined by TCID50 titer ratio of filial generation virus between human lung cancer cell A549 and human diploid epidermal fibrolast BJ cells after infected by virus with same MOI. TCID50 titer ratio of tumor cell infection group to normal cell infection control group was 398. The IE-HPLC purity of virus was 99.5%. There was less than 1 copy of wild type adenovirus within 1 x 10(7) VP recombinant virus. Other quality control items were complied with corresponding requirements in the guidance for human somatic cell therapy and gene therapy and Chinese pharmacopeia volume III. The detection method of oncolytic adenovirus/p53 was successfully established for quality control standard. The study also provided reference for quality control of other oncolytic viral vector products.

Humans ; Enhanced Recovery After Surgery ; Stomach Neoplasms/surgery* ; Length of Stay ; Laparoscopy ; Postoperative Complications ; Gastrectomy

Antibiotics, Antineoplastic ; pharmacology ; Breast Neoplasms ; enzymology ; pathology ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Female ; Glucosyltransferases ; biosynthesis ; genetics ; Humans ; Oligodeoxyribonucleotides, Antisense ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transfection

Objective　To study the role of TNFα in the plasm a and skin and IL-1 in the serum in the formation of secondary thrombosis after skin avulsion. Methods　After avulsive flap at size of 12 cm×4 cm was inflicted on the hind legs of pigs, skin specimens and venous blood sam ples were taken at various time points. The contents of TNFα in plasma and skin were determined with radio-immunoassay, and the activity of serum IL-1 wi th 3［H］-TdR. Results　The TNFα contents in the plasma and skin were increased significantly after avulsion（P<0.01），which were (41 5±24) ng/L and (298±18.5) ng/L respectively on the 3rd day after the injury. T he activity of IL-1 in the serum was increased (P<0.05) and was (2.59± 0.85 ) ng/L on day 3. Conclusion　The changes of TNFα contents and I L-1 activity in blood and skin play important roles in the inducetion and aggra vation of secondary tissue necrosis and early thrombosis after skin avulsion.

This paper reports the determination of the drug antibody ratio in an antibody-drug conjugate with two methods, i.e. LC-MS and UV/VIS, and to provide a reliable method to scientifically evaluate and effectively control the drug antibody ratio. Deglycosylated sample was analyzed with C4 column followed by MS, and the number of conjugated drugs in the antibody was determined by the molecular weight increase due to the addition of different number of drugs to the antibody, and then drug antibody ratio was calculated by weighted average of different number of drugs conjugated to the antibody. Optical density at 252 and 280 nm was measured with UV/VIS, and due to the difference of extinction coefficients between the antibody and the drug, the drug antibody ratio was calculated from linear equation with two unknowns. The drug antibody ratio was 3.21 and 3.25 respectively measured by the two methods, and the results were similar with the two methods. Our study indicated that both methods, LC-MS and UV/VIS, could be applied to the analysis of drug antibody ratio of the antibody drug conjugate.