Comparative pharmacokinetic (PK) analysis is often carried out throughout the entire period of drug development, the common approach for the assessment of pharmacokinetics between different treatments requires that the individual PK parameters, which employs estimation of 90% confidence intervals for the ratio of average parameters, such as AUC and Cmax, these 90% confidence intervals then need to be compared with the pre-specified equivalent interval, and last we determine whether the two treatments are equivalent. Unfortunately in many clinical circumstances, some or even all of the individuals can only be sparsely sampled, making the individual evaluation difficult by the conventional non-compartmental analysis. In such cases, nonlinear mixed effect model (NONMEM) could be applied to analyze the sparse data. In this article, we simulated a sparsely sampling design trial based on the dense sampling data from a truly comparative PK study. The sparse data were analyzed with NONMEM method, and the original dense data were analyzed with non-compartment analysis. Although the trial design and analysis methods are different, the 90% confidence intervals for the ratio of PK parameters based on 1000 Bootstrap are very similar, indicated that the analysis based on NONMEM is a reliable method to treat with the sparse data in the comparative pharmacokinetic study.
Area Under Curve ; Confidence Intervals ; Humans ; Nonlinear Dynamics ; Pharmacokinetics ; Sampling Studies

Tri-dimensional poly (DL-lactic-co-glycolic acid) (PLGA) scaffolds were fabricated using a rapid prototyping (RP) technique and the gene of human bone morphogenetic protein 2 (hBMP-2) was transferred into rabbit bone marrow stromal cells (MSCs) via recombinant adeno-associated virus vectors (rAAV-hBMP-2). Thirty-two PLGA scaffolds, size (4 mm X 4 mm X 4 mm), were coated with collagen type I and equally divided into 2 groups. In group A, each scaffold was loaded with 2 X 10(4) hBMP-2 (+) MSCs to establish a hBMP-2 (+) MSCs/PLGA composite. In group B, each scaffold was loaded with 2 X 10(4) hBMP-2 (-) MSCs to establish a hBMP-2 (-) MSCs/PLGA composite. The composites in both groups were cultured for subcutaneous implantation in nude mice. All animals were killed 30 days after implantation and the differentiation of composites was evaluated. As a result, MSCs infected with rAAV-hBMP-2 efficiently expressed hBMP-2 protein. RP-based PLGA scaffolds had ideal microarchitecture. The diameters of macropore and micropore of the scaffolds were 300 microm and 3-5 microm, respectively. At 3-5 days after culture, a number of seeding cells well grew on the scaffolds of both groups. The composites in group A had chondrogenesis ability in vivo and the expression of collagen type II was positive. In group B, however, only polymers and fiber tissues were predominantly found. The percentage of polymer remnant area was significantly lower in group A than in group B (P<0.01). Our results therefore indicate that RP-based PLGA scaffolds efficiently coated with collagen type I have good biocompatibility with hBMP-2 (+) MSCs and the techniques developed in this study may favor cartilage tissue engineering.
Animals ; Biocompatible Materials ; Bone Marrow Cells ; cytology ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; genetics ; Cell Differentiation ; Cells, Cultured ; Chondrogenesis ; Guided Tissue Regeneration ; methods ; Humans ; Implants, Experimental ; Lactic Acid ; Male ; Mice ; Mice, Nude ; Polyglycolic Acid ; Polymers ; Rabbits ; Stromal Cells ; cytology ; Tissue Engineering ; methods ; Transfection ; Transforming Growth Factor beta ; genetics

This study is to investigate if madecassoside can protect against myocardial reperfusion injury in rabbit heart in vivo. The ischemia reperfusion model was established. Left ventricular function and ECG were monitored at the ischemia and reperfusion period. The infarct areas were expressed as percentage. The levels of LDH, CK, MDA and SOD were measured and C-reactive protein (CRP) in serum was measured by ELISA kit. Cardiomyocyte apoptosis were measured by TUNEL staining. A monoclonal rabbit anti-goat Bcl-2 proteins as primary antibody was used for Bcl-2 immunohistochemical staining. Treatment with madecassoside (3.2, 1.6 and 0.8 mg x kg(-1)) i.v. during ischemia reperfusion injury attenuated myocardial damage, that is, characteristic of decreasing infarct size, decreasing LDH and CK release. Activities of SOD were diminished and MDA level increased obviously in control group whereas pretreatment with madecassoside significantly blunted the decrease of SOD activity, markedly reduced the levels of MDA, CRP and cardiomyocyte apoptosis, and upregulated the expression of Bcl-2. Madecassoside has the protective effect against myocardial ischemia reperfusion injury, and effects of anti-lipid peroxidation, enhancement of SOD activity, anti-inflammation and anti-apoptosis.
Animals ; Apoptosis ; drug effects ; C-Reactive Protein ; metabolism ; Cardiotonic Agents ; isolation & purification ; pharmacology ; Centella ; chemistry ; Creatine Kinase ; blood ; Electrocardiography ; L-Lactate Dehydrogenase ; blood ; Lipid Peroxidation ; drug effects ; Male ; Malondialdehyde ; blood ; Myocardial Reperfusion Injury ; metabolism ; pathology ; Myocardium ; metabolism ; pathology ; Myocytes, Cardiac ; pathology ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rabbits ; Random Allocation ; Superoxide Dismutase ; blood ; Triterpenes ; isolation & purification ; pharmacology

This study was aimed to establish the real-time fluorescent quantitative PCR (RT-qPCR) with erythrocyte Kidd blood group gene for detecting the hematopoietic chimera and to investigate the feasibility of this method. The TaqMan MGB probes and special primers were designed on basis of difference of erythrocyte Kidd blood group alleles, the hematopoietic chimerism was detected by RT-qPCR, the DNA chimerism was simulated by means of dilution of multiple proportions, and the sensitivity analysis was performed. The results showed that the RT-qPCR with erythrocyte Kidd blood group gene could effectively distinguish JK*A and JK*B alleles. There was no significant difference between the theoretic value and the practical measured value by this method (P > 0.05). As 156 donor's cells could be discriminated from 10(4) chimeric cells, this method may effectively detect donor's cells with correlation coefficient 0.998. It is concluded that the established RT-qPCR with erythrocyte Kidd blood group gene shows the feasibility for quantitative detection of hematopoietic chimera, and may be used to quantitatively detect chimera in a certain range.
Chimera ; Erythrocytes ; Humans ; Kidd Blood-Group System ; genetics ; Real-Time Polymerase Chain Reaction

The effect of acupuncture on substance withdrawl syndromes and craving relapse prevention of the recent 10 years were reviewed as well as its mechanism. The therapeutic effect and the possible mechanism were analyzed on the basis. From the three aspects of anti protracted abstinence symptoms, craving relapse prevention and mechanism of acupuncture, the development tendency and the prospect of application on drug withdrawl with acupuncture were expected. And it is proposed that clinical observation of acupuncture intervention on craving should be developed, the mechanism of acupuncture impact on cognitive behavior, blocking study and memory processing related to drug addiction should be explored, so as to further give play to the advantages of acupuncture on anti-drug addiction.
Acupuncture Therapy ; Humans ; Substance-Related Disorders ; therapy ; Time Factors

AIMTo establish a sensitive and efficient reporter gene-based screening model for finding agonists of estrogen receptor beta subtype.

METHODSA recombinant vector pTAL-ERE-SEAP was constructed by inserting a synthetic sequence composed of five estrogen responsive elements in front of promoter of pTAL-SEAP vector. pTAL-ERE-SEAP was then transfected into human embryonic kidney (HEK293) cells. G418 (200 microg x mL(-1)) was added to select positive clones that can be induced by E2 to express reporter gene SEAP. The speciality was tested by several ligands of relative nuclear receptors of the same family. The stability of the model, the time-effect relationship, the dose-response relationship, and the immunocytochemistry staining of ERbeta expression after transfection were observed. 2 622 compounds were screened by using this model.

RESULTSStably transfected clones were obtained. The expression levels of reporter gene SEAP of positive clones was induced by E2 in a dose-response and time-effect relationship manners. The Z' factor value was 0.7. The expression levels of dexamethasone and other ligands were low. The result of immunocytochemistry staining showed the expression of ERbeta. E2 had no proliferating effects on stably transfected clones.

CONCLUSIONStably transfected positive clones transfected with recombinant vector pTAL-ERE-SEAP were obtained. The positive clones may be used to screen for agonists of estrogen receptor beta subtype by measurement of luminescent value of expressed SEAP in wells of microlitre plate.

Alkaline Phosphatase ; genetics ; metabolism ; Cell Line ; Estradiol ; pharmacology ; Estrogen Receptor beta ; agonists ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; Genetic Vectors ; Humans ; Immunohistochemistry ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Response Elements ; genetics ; Transfection

OBJECTIVETo investigate the mRNA expression profiles of megakaryocytes (MKs) from human cord blood CD34+ cells ex vivo expanded using Solexa technique.

METHODSCD34+ Cells were isolated using density gradient centrifugation and magnetic activated cell sorting. Cultures were stimulated with recombinant human thrombopoietin (100 ng/ml). After 12 days, the MKs fraction was separated from the non-MKs fraction using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The mRNA expression of MKs and non-MKs was detected by Solexa sequencing.

RESULTSWe obtained 3 773 147 and 3 533 805 Tags from MKs and non-MKs, respectively. The amounts of unambiguous tags were 3 291 132 and 2 967 947 and those of distinct tags were 197 769 and 245 318. The expression of 1161 genes was up-regulated and that of 902 genes down-regulated. The expression of 2717 tags was up-regulated and that of 1519 tags down-regulated.

CONCLUSIONSMKs and non-MKs have remarkably different mRNA expression profiles. The differential gene-encoded products may be involved in cellular development, adhesion, apoptosis metabolism, intra- and intercellular signal transduction, and immune response. Further studies on this topic may clarify the expression mechanism, signal transduction, and regulation mechanisms.

Antigens, CD34 ; Cells, Cultured ; Fetal Blood ; cytology ; Humans ; Megakaryocytes ; cytology ; metabolism ; RNA, Messenger ; genetics ; Transcriptome

This study was purposed to investigate the nucleotide sequences of a novel HLA-B*15:124 allele and its molecular mechanism. The genomic DNA from whole blood was extracted by using commercial DNA extraction kit. The sequences of exon 2, 3 and 4 of HLA-B locus in the proband were amplified by PCR with group-specific primers, the PCR products were purified by enzymes digestion, then exon 2 to 4 of HLA-B locus for both orientations was sequenced. The results showed that 2 HLA-B alleles of proband were gained after amplification and sequencing of group-specific primers, among them one was a B*40:03, another was a novel allele. After BLAST analysis, the novel allele showed nucleotides different from HLA-B*15:52 in exon 3 at nucleotide position 427 A > T and 440 G > T which resulted in amino acid change from Thr to Ser at codon 143 and Trp to Leu at conon 147. It is concluded that a novel HLA-B allele has two different nucleotides. This HLA-B allele is identified and has been officially named B*15:124 by the WHO Nomenclature Committee.
Alleles ; Base Sequence ; Exons ; Female ; HLA-B Antigens ; classification ; genetics ; Humans ; Sequence Analysis, DNA

This study was aimed to analyze the possibility of high resolution matching for human leukocyte antigen (HLA) loci in unrelated donor-recipient pair with low resolution match in HLA-A, -B, -DRB1 loci. Samples were genotyped for HLA-A, -B, -C, -DRB1 and -DQB1 by polymerase chain reaction sequence based typing (PCR-SBT). The results showed that the total number of patients and the donors were 166 and 274. 97 (58.43%) patients were matched for 1 donor and 47 (28.31%) patients were matched for 2 donors at low resolution level; among 274 donor-recipient pairs, HLA-A, -B, -C, -DRB1 and -DQB1 loci matching for 6/10, 7/10, 8/10, 9/10 and 10/10 were 32 (11.68%), 54 (19.71%), 62 (22.63%), 49 (17.88%) and 48 (17.52%) respectively; there were mismatch in HLA-A, -B, -C, -DRB1 and -DQB1 loci, and the most mismatch was in HLA-C locus. The number of alleles of HLA-A, -B, -C, -DRB1 and -DQB1 loci were 23, 46, 21, 30 and 17 respectively in the donors. The alleles number HLA-A, -B, -C, -DRB1 and -DQB1 loci were 20, 40, 22, 29 and 16 respectively in the patients; the haplotype number of HLA loci were 311 in the donors and 224 in the patients. The high frequency of haplotype was A*02:07-B*46:01-C*01:02-DRB1*09:01:02-DQB1*03:03 (5.63% and 6.88%). It is concluded that the probability of high resolution mismatch of HLA loci is high in unrelated donor-recipient pairs with low resolution match in HLA-A, -B, -DRB1 loci.
Alleles ; Gene Frequency ; Genotype ; HLA Antigens ; genetics ; immunology ; HLA-A Antigens ; genetics ; immunology ; HLA-B Antigens ; genetics ; immunology ; HLA-C Antigens ; genetics ; immunology ; HLA-DQ Antigens ; genetics ; immunology ; HLA-DQ beta-Chains ; HLA-DR Antigens ; genetics ; immunology ; HLA-DRB1 Chains ; Haplotypes ; Hematopoietic Stem Cell Transplantation ; methods ; Histocompatibility Testing ; methods ; Humans ; Probability ; Tissue Donors

This study was aimed to discriminate the alleles in the HLA-C*07:01:01G and HLA-C*07:02:01G groups and analyze their associations with HLA-B locus. Samples previously typed as HLA-C*07:01:01G and HLA-C*07:02:01G were collected. The nucleotide sequences in exons 1 to 7 of the HLA-C locus were sequenced by polymerase chain reaction sequence-based typing (PCR-SBT) and HLA-B genotyping was also preformed by PCR-SBT in these samples. The results showed that 4 samples (30.8%) were confirmed as HLA-C*07:01:01 and 9 samples (69.2%) were HLA-C*07:06 among 13 samples previously typed as HLA-C*07:01:01G. Linkage disequilibrium (LD) analysis showed that HLA-C*07:06 allele was strongly related with HLA-B*44:03. All samples were typed as C*07:02:01 among 102 individuals previously typed as C*07:02:01G. LD analysis showed that C*07:02:01 was strongly related with HLA-B*51:01, B*46:01, B*39:01, B*40:01, B*38:02, B*15:02 alleles. It is concluded that HLA-C*07:01:01 and HLA-C*07:06 alleles are confirmed in the HLA-C*07:01:01G group and HLA-C*07:02:01 is a preferred allele in the HLA-C*07:02:01G.
Alleles ; Base Sequence ; Exons ; HLA-B Antigens ; genetics ; HLA-C Antigens ; genetics ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA