Objective To study the effect of fascial flap with vessels inducing the vascularization of uncel-lular tissue engingeering complex and the regenration of bone on the repair of bone defect, so as to provide the basis for the clinical application. Methods An animal model of bone defect on adult Newzland rabbits'right radial bone was established .and autologous red bone marrow were taken out and mixed into uncellulax tissue engineering comple-xes with OAM which contained BMP. The experiment animals were divided into two groups : experiment group and control group( n = 12 for each ). The control group was only implanted with complexes, meanwhile, the experiment group had fascial flap with vessels. By microsurgery technology,a non-named fascial flap with vessels was prepared, which belonged to capillary net,around the bone defect,and let it wrap tissue engineering complex,fill up bone de-fect. In a certian time, radiograph(X-ray) and light density measure was conducted, gross morphology and histological inspection was exmained. Bone shape measurement analysis and image of vessel analysis were conducted. All the sta-tistics were analyzed by the SPSS 11.5 software. Results Because of mechanically preventing fiber connective tis-sues and surrounding soft tissues from entering the areas of bone defect by fascial flap, it can keep bone defect having a relative stable environment ;The subfascial space itself, and also the shape and mass of filled-in subject had the de-cisive effect on the results of the regeneration of the bone; Owing to the establishment of blood supply during the con-structing tissue engineering complex. The experiment group was obviously superior to the control group. Compared with control group,the absor bance obviously increased in experiment group [(0. 732 ± 0. 021 ) vs (0. 651± 0.018)] (P < 0. 001 ) four weeks after the operation; also the bone trabecular body was significantly increased [(2.32±2.57)% vs(19.37±3.52)% ,(8.37±3.52)% vs(30.24±3.42)% ,(28.57±2.98)% vs(58.76± 4.62)% ,(47.24±3.42)% vs(88.72±5.84)%] ,and capillary area [(5.04±1.62)% vs(17.53±2.86)%, (10.37 ±2.96)% vs(35.24±1. 13)%,(18.20±2. 12)% vs(48.76±4. 62)%,(17.82 ±2. 74)% vs (57.72 ±5.84)%] (P <0.05) at each time period(4 weeks,8 weeks,12 weeks,and 16 weeks after operation). Despite of growth of implant's internal vessel, the number and speed of forming bone trabecula and cartilaginous tis-sue, even developing of mature bone structure, recreating of diaphysis structure, reconstructing of marrow cavity, ab-sorbing and decomposing of implant, the experiment group was obviously superior to the control group. Conclusions The induction of fascial flap with vessels shows double effects, one of which is the vascularization of uncellular tis-sue engineering complex and the other is membrane guided bone regeneration, So the method has a wonderful effect on the repair of bone defect.

Objective To study the influence of autoblood cardioplegia on ATPase in neonatus myocardium with congenital heart disease and approach the mechanism of self-blood cardioplegia in protecting the myocardium in neonatus.Methods There were 30 cases of neonatus with congenital heart disease with body weight less than 8 kg,including 2 cases of ventricular septal defect(VSD),11 of VSD with severe pulmonary hypertension(PH),9 cases of USD with ASD,2 cases of atrial septal defect (ASD),6 of VSD and FPO.30 neonatus were divided into autoblood cardioplegic solution group(group A,n=10),allograft blood cardioplegic solution group (group B,n=10)and crystalloid cardioplegic solution group(group C,n=10).The biopsies were taken from right atrium just before arrested and after heart self-recovery to measure ATPase.Results Comparing with preoperative one,Na+-K+-ATPase creased obviously after operation in group A,B ,C (P＜0.05 ).There had no significant difference among the three groups before operation (P＞0.05).After operation,myocardial cell's Na+-K+-ATPase,Ca2+-ATPase and Ca2+Mg2+-ATPase in group A were decreased obviously as compared with that in group B and C (P＜0.05).Conclusion There is slight influence of autobloed cardioplegia on ATPase in neonatus with congenital heart disease,which can give a good protection to the myocardium in neonatus.

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 .

Objective To compare the capability of capillary electrophoresis-sodium dodecyl sul-fate ( CE-SDS) and size exclusion-high performance liquid chromatography ( SE-HPLC) for analysis of size heterogeneity of monoclonal antibody products .Methods The size heterogeneity of one humanized anti-VEGF monoclonal antibody was analyzed by using non-reduced and reduced CE-SDS, and conventional , de-natured and denatured reduced SE-HPLC.Results The percentage of aggregates detected by non-reduced CE-SDS (0.82%±0.01%) was equal to that by using denatured SE-HPLC (1.05%±0.02%), but it was significantly lower than that by using conventional SE-HPLC analysis (5.08%±0.10%).With regard to fragments analyzed with non-reduced antibodies, its percentage was (7.12±0.04)% measured by non-re-duced CE-SDS analysis that was significantly higher than that by conventional SE -HPLC analysis (0.02%± 0.01%) and denatured SE-HPLC analysis (0.62%±0.01%).Using reduced antibodies , the percentage of fragments was (3.19±0.50)%tested by reduced CE-SDS analysis that was significantly higher than that by using denatured reduced SE-HPLC analysis (0.07%±0.01%).Conclusion Conventional SE-HPLC was more objective than CE-SDS for content analysis of aggregates , as both the covalent and non-covalent forms of aggregates could be detected .Non-reduced CE-SDS could demonstrate the content of clips , while reduced CE-SDS showed the degraded fragments .Therefore, CE-SDS had an advantage over conventional SE-HPLC for content analysis of fragments .The use of the two analytical methods in combination provided solid techni-cal supports for the quality control of size heterogeneity of monoclonal antibodies .

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.

AIM: To establish human umbilical vein endothelial cells (HUVECs) to express green fluorescent protein (GFP), and to study the suppression of GFP by siRNA in HUVECs. METHODS: Using lipofectamine 2000 to transform plasmid pN_3-EGFP encoding GFP into HUVECs. The HUVEC containing pN_3-EGFP, named HUVEC-GFP, was screened and selected by antibiotic G418. Using in vitro transcription T7 kit, GFPsiRNA targeting GFP mRNA and control-siRNA used as control were synthesized. The siRNAs were transfected into HUVEC-GFP with oligofectamine. 48 h later, the expression levels of GFP protein and mRNA in HUVEC-GFP were determined. RESULTS: The HUVEC-GFP was screened to express GFP in the presence of G418. The agarose gel electrophoresis analysis showed that the siRNAs prepared were integrated. 48 h after transfection with siRNAs, compared to control group, the level of GFP fluorescence was obviously decreased in the HUVEC-GFP transfected with GFPsiRNA. The results of RT-PCR detection showed that GFP mRNA expression was obviously suppressed by GFPsiRNA at the rate of 40%, and no obvious suppression of GFP mRNA expression was found in the HUVEC-GFP transfected with control siRNA. CONCLUSION: The siRNA targeting GFP mRNA, synthesized in vitro, efficiently suppresses the GFP expression in HUVECs.

Objective To investigate the relation between 25-hydroxyvitamin D3[25(OH)D3] and coronary artery disease. Methods Three hundred and ten patients with selective coronary angiogram (CAG) were enrolled in this study and they were divided into two groups: non-coronary artery stenosis group with 76 patients and coronary artery stenosis group with 234 patients. The degree of coronary artery stenosis was evaluated by the international general Gensini integration system. The levels of fasting plasma glucose (FPG), total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL-C), low density lipoprotein (LDL-C) were detected by automatic biochemistry analyzer. The level of 25(OH)D3 was detected by tandem mass spectrometry. The relationship of Gensini integration scores and risk factors were analyzed. Logistic regression analysis was used in multicity factors analysis. Results The levels of age, Gensini integration scores, 25(OH)D3, FPG and LDL-C in non-coronary artery stenosis group and coronary artery stenosis group had significant differences (P<0.05). The number of coronary stenosis and Gensini integration scores in 25(OH) D3 deficiency group were significantly higher than those in non-25 (OH)D3 deficiency group (P<0.01). Logistic regression analysis showed that age, FPG and 25(OH)D3 levels were risk factors for coronary artery stenosis (P<0.01 or<0.05), and the level of 25(OH)D3 had negative correlation with coronary artery stenosis (B =- 0.100), and it was a protection factor (OR =0.904, 95%CI:0.911-0.983, P=0.000). Conclusions 25(OH)D3 deficiency is one of the risk factor of coronary artery disease.

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.

OBJECTIVETo investigate the effect of parathyroid hormone (1-34) (PTH) on tumor growth in a mouse model of lung cancer with bone metastasis.

METHODSMouse models of proximal tibial bone metastasis of lung cancer were established in 30 female BALB%c mice. The mouse models were randomly divided into 3 groups and received injections with 40 mg%kg PTH (1-34), equal amount of solvent (blank control), or cyclophosphamide (positive control). Body weight of the mice was measured every 2 days and the right hind limb tumor growth was observed. The mice were sacrificed after 28 days for X-ray and CT examinations to observe the tumor shape, size, tibial bone density, and tumor volume. HE staining and immunohistochemistry were performed to observe the tumor morphology and pathological type, and serum concentration of serum alkaline phosphatase (ALP) was detected.

RESULTSThe body weight change curves did not show significant difference between PTH (1-34) group and the blank control group (P>0.05). In both PTH (1-34) group and the blank control group, X-ray and micro-CT revealed significant bone defects, and in cyclophosphamide group the bone cortex was basically intact with only mild bone destruction. The tumor volume was similar between PTH (1-34) group and the blank control group (P>0.05), but significantly smaller in cyclophosphamide group (P<0.05). The bone density in PTH (1-34) group was significantly greater than that in the blank control group, but lower than that in cyclophosphamide group (P<0.05). Pathological examination revealed mainly osteolytic lesions mixed with bone destruction, which was severer in PTH (1-34) group and blank control group with obvious tumor cell filling of the defects; immunohistochemistry identified the tumors as adenomas. ALP activity was higher in PTH (1-34) group than in the other two group and differed significantly between the 3 groups (P<0.05).

CONCLUSIONIntermittent small-dose injections of parathyroid hormone PTH (1-34) does not promote bone metastatic tumor growth in mice and increases the bone quantity around the metastatic lesions.

Animals ; Bone Density ; Bone Neoplasms ; secondary ; Disease Models, Animal ; Female ; Lung Neoplasms ; pathology ; Mice ; Mice, Inbred BALB C ; Parathyroid Hormone ; pharmacology ; Tibia ; pathology