Objective: To establish the fingerprint chromatogram of Tongguanteng Injection (caulis Marsdeniae Tenacissimae). Methods: HPLC with ZORBAX SB C 18 column was used, the (a) 0.05% H 3PO 4 H 2O and (b) ACN 0.05% H 3PO 4 H 2O (13∶87) (gradient elution) as a mobile phase and detection wavelength at 254nm. Results: 22 peaks were indicated on the HPLC fingerprint of Tongguanteng Injection. The relative retention time and relative peak area were obtained with itself peak at retention time 48.5 min. Conclusion: The method is simple and accurate with a good reproducibility and can be used as a quality control method for Tongguantent Injection.

Objective To construct humanized monoclonal antibodies against CD 20 and check their affinity to CD 20 antigen and their anti-tumor activity.Methods Based on the computer model , human IgG1 candidates closest to rituximab in crystal structure were selected in the Protein Data Bank ( PDB) .With the selected human IgG 1 candidates as the frame , we modified and transplanted the complementarity determining region ( CDR) of rituximab .First,the target gene fragments were obtained by overlapping PCR.Then, the sequences of the light chains(L) and the heavy chains(H) were inserted in-to the pcDNA3.3 and pOptiVEC vectors.Next, the constructed clones were transfected into 293F cells through transient transfection.After a large-scale cell culture, the mAb was purified by affinity chromatography rProtein A column.The puri-ty and expression level of the humanized antibodies was tested by sodium dodecyl sulfate ( SDS)-polyacrylamide gelelectro-phoresis(PAGE).The affinity of the humanized antibodies to CD20 was assessed with Fortebio assay.Finally, the anti-tumor activity of the constructed antibodies was detected by checking the tumor growth inhibition of the nude mice transplan-ted with tumor .Results Three humanized monoclonal antibodies against CD 20 were expressed and purified successfully . In reducing SDS-PAGE, the antibodies exhibited two bands of approximately 25 ×103 and 55 ×103 , respectively.The band size of the antibodies matched the expected value.Fortebio assay revealed that the humanized antibodies could bind to CD20 with high affinity (rituximab:6.48 ×10 -9mol/L, L4H7:1.91 ×10 -9mol/L, L5H5:7.35 ×10 -10mol/L,and L5H7:1.91 ×10 -9mol/L).The tumor growth inhibition experiment showed that the anti-tumor activity of L5H7 mAb was better than that of rituximab .Conclusion Three humanized monoclonal antibodies against CD 20 have been successfully construc-ted and expressed.L5H7 mAb possesses high affinity for CD20 and a good ability to kill tumor cells.

Biopsy ; Histological Techniques ; instrumentation ; methods ; Humans ; Indicators and Reagents ; Paraffin Embedding ; Ultrasonics

Objective To explore the roles of macrophage inflammatory protein-1?(MIP-1?)in hypoxic-ischemic encephalopathy(HIE)of newborn infarnts.Methods Serum samples were obtained in 24,72 h and 7 d after birth respectively from 34 newborn infants with HIE,and 20 newborn infants without HIE as control group.Enzyme-linked immunosorbent assay(ELISA)method was used to determine the serum concentrations of MIP-1?.Results Levels of MIP-1? in newborn infants with HIE [(12.47?2.51)ng/L]were significantly higher than that of newborn infants without HIE [(8.63?2.63)ng/L](P0.05).Conclusions MIP-1? are involved in HIE of neonates,and the more severe damage,the higher levels in serum,which suggests that,as an inflammatory mediator,the MIP-1? may play an important role in involvement of brain hypoxic-ischemic damage.

Objective:To express rationally engineered antibodies against EGFR and assess their affinity to EGFR and anti-tumor cell migration effect. Methods:L and V_H genes of humanized antibodies against EGFR were designed and synthesized. Genes encoding V_H and C_H were connected and then cloned into a pIRES based bicistronic expression vector. Gene encoding the corresponding L gene was also cloned into the same vector. 293T cells were transfected with the recombinant plasmid and the antibody expression was confirmed by Western blotting. The antibodies were purified by protein A based affinity chromatography. Binding of the humanized antibody to the EGFR was assessed by Surface Plasmon Renainance with Biacore3000, and the biological activity of the humanized antibody was determined by tumor cell invasion test.Results:Three expression vectors were constructed and the humanized anti-EGFR antibodies were expressed and purified successfully. In reducing SDS-PAGE, the antibodies exhibited two bands of approximately 25×10~3and 50×10~3, respectively. Western blot assay showed that the humanized antibodies had recognition specificity to goat-human IgG antiserum. Biacore assay revealed that the humanized antibody C3 binds to EGFR with high affinity(6.13×10~(-10)M). Cell migration test showed that C2,C3 and C5 could suppress growth and migration of tumor cells.Conclusion:Three anti-EGFR humanized antibodies (C2,C3 and C5) have been constructed and expressed successfully, and the C3 antibody retained high affinity for EGFR and showed improved inhibitory effect on tumor cell growth and migration.

Objective:To compare acetyltransferase MORF level in periodontal ligament stem cells( PDLSCs) derived from healthy individuals ( H-PDLSCs) with those derived from the individuals with periodontitis ( P-PDLSCs ) . And to determine the effect of MORF on the osteogenic differentiation potential of PDLSCs. Methods: Human H-PDLSCs and P-PDLSCs were cultured and cloned with limited dilution method. H-PDLSCs were stimulated by LPS, TNF-α, IL-β and the mix of the 3 inflammatory factors to imitate inflammatory environment ( IP-PDLSCs ) . Quantitative RT-PCR and Western Blot were applied to examine different expression of MORF in H-PDLSCs and P-PDLSCs. Western Blot was applied to detect expression of MORF in IP-PDLSCs. Quantitative RT-PCR, Western Blot and alizarin red staining were applied to determine osteogenic differentiation potential of H-PDLSCs with MORF knock-down. Results:Quantitative RT-PCR and Western Blot showed lower expression of MORF in P-PDLSCs compared with H-PDLSCs( P<0. 05). Western Blot revealed lower expression of MORF in IP-PDLSCs. Quantitative RT-PCR, Western Blot and alizarin red stai-ning indicated osteogenic differentiation potential was inhibited in H-PDLSCs with MORF knockdown(P<0. 05). Conclusion: Peri-odontitis can suppress the expression of MORF in PDLSCs and inhibite the osteogenic differentiation potential of PDLSCs.

Objective To screen and identify B cell epitopes in SAG1, SAG2, SAG3, GRA1, GRA6 and P35 antigens of Toxoplasma gondii. Methods The indexes such as hydrophilicity, accessibility, flexibility, secondary structure and polarity of the 6 antigen moleculars above mentioned were analyzed by BioSun system. Two B cell epitopes with high antigenicity from each antigen molecular were selected, and the total twelve pairs of oligonucleotide chains were designed according to the 12 B cell epitopes’ sequence and synthesized, then cloned into plasmid pET-32c. The 12 fragment B cell epitopes were expressed and the expressed fusion proteins were identified with Western blot. Results Twelve B cell epitopes from 6 Toxoplasma antigens (two from each antigen) were predicted and selected. The epitope genes were successfully cloned into pET-32c and expressed. Western blot results showed that 3 of 12 expressed fusion proteins could be recognized by the immunized rabbit sera with soluble antigen of Toxoplasma gondii, but not by the unimmunized rabbit sera Conclusion Three B cell epitopes of Toxoplasma[with potential diagnostic value are obtained.

Objective To develop a scientific evaluation index system and promote the health unit construction.Methods The framework of evaluation index system was developed using the method of Delphi expert consultation and focus group discussion.The weights at all levels of the index were calculated by Delphi expert consultation and the analytic hierarchy process to develop the evaluation index system.Results The response rates of 2 round consultation including 15 experts were both 100. 00%.The authority coefficient was 0. 825 and the coefficient of variation was less than 0. 30.The P values of coordination coefficient were less than 0. 01.Conclusion This evaluation index system covered major factors of the health unit construction,and could be widely used to evaluate the health unit construction.

OBJECTIVETo investigate the expressions of phosphorylated protein kinase B (p-AKT), phosphorylated glycogen synthase kinase 3β (p-GSK3β) and β-catenin proteins and to evaluate their relationship with the clinical pathological characteristics in epithelial tumors of the ovary.

METHODSThe expression of p-AKT, p-GSK3β, and β-catenin was detected with immunohistochemical staining (EnVision method) in 10 cases of benign epithelial neoplasia, 10 cases of borderline epithelial neoplasia and 70 cases of ovarian carcinoma. The relationship of the expression of p-AKT, p-GSK3β and β-catenin with the clinical pathological features was analyzed.

RESULTSThe positive expression rates of p-AKT, p-GSK3β and β-catenin in epithelial ovarian carcinoma were 67.1% (47/70), 60.0% (42/70) and 71.4% (50/70), respectively. Compared to the results of benign and borderline epithelial neoplasia, the expression of the three proteins in carcinoma of the ovary was significantly different (all P < 0.05).Positive correlation was found between p-AKT and p-GSK3β, p-GSK3β and β-catenin, and p-AKT and β-catenin in epithelial ovarian carcinoma (r = 0.546, 0.581, 0.500, respectively; all P < 0.05). Compared to the results of benign and borderline epithelial neoplasia, the expression of p-AKT protein in epithelial ovarian carcinoma was significantly different (all P < 0.05). The expression of p-AKT was correlated with the differentiation of epithelial ovarian carcinoma (P < 0.05), but no relationship was found between its expression and histological classification and FIGO staging (P > 0.05). The expression of p-GSK3β and β-catenin in epithelial ovarian carcinoma were both higher than that in benign and borderline epithelial neoplasia (P < 0.05), and correlated with tumor differentiation and FIGO staging (P < 0.05), but no relationship were found between their expression with histological classification (P > 0.05).

CONCLUSIONSPositive correlations are found between p-AKT, p-GSK3β and β-catenin in epithelial ovarian carcinoma. The activation of β-catenin is possibly correlated with inactivation of p-GSK3β that binds to p-AKT.

Adult ; Aged ; Carcinoma, Endometrioid ; metabolism ; pathology ; Cell Differentiation ; Cystadenocarcinoma, Mucinous ; metabolism ; pathology ; Cystadenocarcinoma, Serous ; metabolism ; pathology ; Cystadenoma, Mucinous ; metabolism ; pathology ; Cystadenoma, Serous ; metabolism ; pathology ; Female ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Humans ; Middle Aged ; Neoplasm Staging ; Ovarian Neoplasms ; metabolism ; pathology ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; beta Catenin ; metabolism

Objective To investigate the pathogenic mutations of BRCA1 and BRCA2 in patients with early-onset breast cancer(≤35 years) and explore the relationships between BRCA1/2 mutations and clinical features.Methods Seventy-four patients with early-onset breast cancer were enrolled,who were treated in Hospital 307 between September 2014 and June 2016.High-throughput sequencing was used to test the 49 exon sequences and adjacent sequences of BRCA1 and BRCA2.χ2 test was used to analyze the distribution of BRCA1/2 pathogenic mutations in each group that was set up according to clinical features.Results Fifteen mutations(20.27%) were identified,including 5(6.76%) in BRCA1 and 10(13.51%) in BRCA2.Eleven new pathogenic mutations were discovered,and BRCA1:c.5470_5477delTGCCCAAT was found in one patient.The frequency of BRCA1/2 mutations in the group with a family history of breast cancer or ovarian cancer was higher than in the group without a family history (40.91% vs 11.54%) (χ2=6.534,P=0.011).Conclusion BRCA1/2 pathogenic mutation is significant for early-onset breast cancer,especially for those with a family history of breast or ovarian cancer.The new mutations may be specific to Chinese people.BRCA1:c.5470_5477delTGCCCAAT may be the ancestor mutation among the Chinese.