Background and purpose:Bispecific antibody is a special pattern of genetic engineering antibody.It possesses 2 antigen binding sites, one directed at the effecter cells and the other at the target cells which are always the tumor cells.This enables the effecter cells to recruit to the targets efficiently and exert cytotoxic effect.The experiment was aimed to construct the expression vector of anti-CD3?anti-CD19 bispecific antibody, purification and analyzing the activity of the diabody.Methods:Applyed PCR and overlap PCR to construct the expression vector of anti-CD3?anti-CD19 bispecific antibodies, transformed it into E.coli 16C9 for expression.Purified the diabody by His-tag affinity chromatography and confirmed by 12% SDS-PAGE and Western blot.Antigen binding activity of the diabody was analyzed by FACS.Results:The DNA sequence of the expression vector containing anti-CD3?anti-CD19 fragment was comfirmed.The yield of purified diabody was up to 5 mg/L.The molecular weight of diabody was correctly indicated by SDS-PAGE and Western blot.The diabody could specifically bind to CD19+ Raji cells and CD3+ Jurkat cells while competitively inhibit binding of HIT3a and HIT19a to the cells.Conclusion:Successfully constructed the expression vector of anti-CD3?anti-CD19 bispecific antibodies, the diabody acquired by the method could be produced with high level expression and also had high specific affinity with CD19+ Raji cells and CD3+ Jurkat cells.

Aim To study the mechanism of synergistic antitumor of EBB and doxorubicin in doxorubicin-resistant MCF-7/ADR breast carcinoma cells.Methods The antitumor activity of doxorubiein alone and its combination with EBB were measured by MTT assay in MCF-7/ADR and MCF-7cells. The rate of doxorubicin-induced apoptosis and the protein and mRNA levels of P-glycoprotein(P-gp) were determined in MCF-7/ADR treated with EBB by flow cytometry (FACS), respectively.Laser scanning confocal microscopy was used to detect the intracellular accumulation of drug in EBB-treated MCF-7 and MCF-7/ADR cells.Results EBB had antitumor effects for MCF-7 and MCF-7/ADR.It could potentiate the antitumor effect of dororubicin with CDI of 0.73 and 0.49 for MCF-7 and MCF-7/ADR,respectively.EBB and doxorubicin acted synergistically in elevating apoptosis of MCF-7/ADR and downregulating the expression of P-gp in a dose-dependent manner in MCF-7/ADR.EBB restored the intracellular accumulation of doxorubicin in MCF-7/ADR cells in a dose-dependent manner.After pretreatment with EBB for 24 h and 48 h,the intracellular accumulation of doxorubicin and Rh123 was obviousely restored in MCF-7/ADR cells compared with control in a time-dependent manner.Conclusion EBB is a potential agent which has strong inhibitory effect on both multidrug resistant cells and their parental cells.EBB can significantly potentiate the antitumor effects of dororubicin in MCF-7/ADR cells by blocking the function of P-glycoprotein and inhibiting the expression of P-glycoprotein.

Objective The purpose of this study was to investigate the value of serum galactomannan (GM) in the diagnosis of invasive pulmonary aspergillosis (IPA) in patients with chronic obstructive pulmonary disease (COPD).Methods We enrolled 60 COPD patients in the study, including 19 IPA and 41 non-IPA cases.We examined serum GM of the patients by ELISA, evaluate the value of serum GM test for the diagnosis of IPA in patients with COPD, and compared the GM values before and after treatment.Results With 0.5 as the positive cutoff value, the sensitivity, specificity, positive predictive value, and negative predictive value of serum GM were 57.9%, 95.3%, 84.6%, and 83.0%, respectively, with a high specificity and a low sensitivity.The 7 IPA cases showed a significantly decreased GM value after treatment as compared with the baseline (0.30±0.21 vs 1.48±1.37, P=0.004).Conclusion The serum GM test has a limited value in the diagnosis of IPA in patients with COPD, but dynamic monitoring of the changes of the serum GM value may help evaluate the patient's condition.

Background and purpose:Multidrug resistance(MDR)is one of the major causes of progressive breast cancer chemotherapy failure.One of the major mechanisms of MDR is the overexpression of P-glycoprotein (P-gp).Therefore,the identification of novel agents which can inhibit the drug transporter function of P-gp or its expression is of utmost interest in cancer research.The aim of this study was to explore the antitumor and reversal effect of PHⅡ-7,natural products from traditional Chinese medicine(TCM).Methods:The cytotoxicity of PHⅡ7 alone and combined application of PHⅡ-7 and adriamycin(ADR)on breast cancer cells were determined using MTT assay.Annexin V–FITC/PI apoptosis detection kit was used to observe the apoptosis-inducing effect of PHⅡ-7 in MCF-7 and MCF-7/ADR cells.The effect of PHⅡ-7 on mdr1 mRNA was determined by reverse transcription PCR and real time PCR,flow cytometer was used to measure the intracellular ADR accumulation.Results:PHⅡ-7 alone inhibited cell growth of MCF-7 and MCF-7/ADR cells with the IC 50 (6.07?0.85),(5.51?1.22)?mol/L,respectively when combined with ADR,PHⅡ-7 enhanced the cytotoxicity of ADR toward MCF-7/ADR cells.In addition,PHⅡ-7 induced apoptosis both on MCF-7 and MCF-7/ADR cells;PHⅡ-7 reversed the drug resistance to ADR in MCF-7/ADR cells by inhibiting mdr1 mRNA transcription and increasing the intracellular ADR accumulation.Conclusion:PHⅡ-7 displayed significant anti-proliferative and apoptosis-inducing effect on sensitive and multidrug resistant breast cells in vitro.PHⅡ-7 reversed effectively MDR by blocking the drugs to be pumped out by inhibiting P-gp expression and function pathway.

Objective:To prepare and characterize specific and discrepant mouse hybridoma antibodies on membrane of HL60 and HL60/ADR cell lines.Methods:BALB/c mice were immunized by subtractive immunization induced Cp(Cyclophosphamide).McAbs were prepared by hybridoma technique,screened and detected by FACS and LSCM.Results:51 candidates and discrepant antibodies were found,and one of them (5F6) was purified and identified.Conclusion:Combination of SI with discrepant screening method should facilitate the preparing and identifying discrepant McAbs for identifying antibodies that can distinguish the differences in proteins expressed in HL60 and HL60/ADR,which is a significative and potential method in the research and target therapy associated drug-resistance.

Objective:To analyze the effect of the multipoint tiny incision with continuous buried suture method in double eyelidplasty for patients with mild or moderate dermatolysis.Methods:Totally, 1 161 patients from May 2008 to February 2019 underwent the double-eyelid surgical procedure with the multipoint tiny incision with continuous buried suture method. 1 148 patients were female and 13 patients were male, ranging from 25 to 50 years old. All the patients had normal single-eyelid or inborn narrow double-eyelid without blepharoptosis and they were performed double eyelidplasty for the first time. During the operation, moderate orbicularis oculi muscle and orbital septum fat were cut down from the four or five tiny incisions which were all 2 mm long. Skin levator fixation was performed through continuous buring suture between subcutaneous and the levator aponeurosis. Postoperative follow-up time points were 3 months, 1 year and 5 years.Results:There were 10 patients that showed slight asymmetry, 8 patients' eyes showed shallow creases and 5 patients' double eyelids narrowed. These 23 patients were operated for the second time. Besides that, all the creases were symmetry, parallel and natural. Notched scars did not occur, and patients' satisfaction was achieved in the rest of the cases.Conclusions:Our double eyelid procedure creates natural and beautiful double eyelids without depressed scars. The blepharoplasty of multipoint tiny incision with continuous buried suture method can produce a desired aesthetic effect and it lasts for a long time.

Objective To evaluate the dosimetric effect of three-dimensional(3D)printed headrests made of different materials in radiotherapy for head and neck tumor,and to evaluate whether the existence of customized 3D-printed headrest can be neglected during planning phase by comparing differences in target area doses,homogeneity index(HI),conformity index(CI),monitor units(MU),and organ-at-risk(OAR)dose.Methods Ten patients with head and neck tumors,including 5 cases with nasopharyngeal carcinoma and 5 with other head and neck tumors,were enrolled.The headrest contours were outlined using Monaco treatment planning system,and with the same calculation parameters,treatment plans were generated for scenarios without a headrest,with a standard headrest,and with 3D-printed headrests made of 10%filled polylactic acid and thermoplastic polyurethane.The target area doses,OAR dose,MU,and other results were recorded and subjected to statistical analysis.Results No significant differences were observed among the 4 groups(ignoring headrest,standard headrest,3D-printed headrests with 10%filled polylactic acid and thermoplastic polyurethane)in parameters such as D95,D5,Dmean,HI,CI,MU,and OAR dose(the maximum dose to the spinal cord)(P>0.05).Considering the presence of the headrest,significant differences were found in CI for nasopharyngeal carcinoma and other head and neck tumors(P<0.05).Conclusion During the planning phase,the existence of customized 3D-printed headrest can be ignored,but it should be noted that the presence of a headrest may reduce the MU in radiotherapy plans.When considering the headrest,the average CI of patients with other head and neck tumors is significantly higher than that with nasopharyngeal carcinoma.