BACKGROUND:Construction of vascularized bone substitutes to mimic free vascularized fibular grafting in treating large bone defects stil remains chal enges to researchers during the past years.
OBJECTIVE:To design and construct a new vascularized tissue-engineered bone graft using pre-differentiated adipose derived stem cells, fibrin sealant and porous calcium phosphate cement scaffold.
METHODS:Adipose derived stem cells isolated from rats were directly differentiated to endothelial cells and then incorporated in fibrin sealant and porous calcium phosphate cement scaffolds in vitro. Subsequently, the different composites of the three groups including vascularized tissue-engineered bone scaffold (group A), fibrin sealant and porous calcium phosphate cement scaffold (group B) and porous calcium phosphate cement scaffold (group C) were directly embedded into the quadriceps of the rats. Histological quantitative analysis and western blot assay were conducted 2 and 4 weeks after implantation.
RESULTS AND CONCLUSION:The pre-differentiated adipose derived stem cells were demonstrated in good condition after 7 days co-culturing with the fibrin sealant and porous calcium phosphate cement scaffold. The results of in vivo experiments showed that the scaffolds were in-grown together with fibrous connective tissues and blood vessels. Newly formed vessels and immature capil aries were observed in the group A. The vessel density, vessel diameter and vascular endothelial growth factor C expression in the group A were significantly higher than those in the groups B and C. Our findings demonstrated that compared with simply fibrin sealant and porous calcium phosphate cement scaffold, the combination of pre-differentiated adipose derived stem cells (endothelial differentiation) and fibrin sealant can achieve rapid angiogenesis of porous calcium phosphate cement scaffold.

AIM: To investigate the effects of Chinese medicine, Jinan injection, on ultrastructure and mitochondria in cultured lung cancer cell lines. METHODS: The cultured lung cancer cell lines PG and PAa were used and divided into 4 groups: control (C), cisplatin (DDP), Jinan (JA) and Jinan in combination with cisplatin (DJ), respectively. The changes of morphology and mitochondria membrane potential, intracellular Ca~(2+) and pH in every group were observed by inverted microscope and electronic microscope as well as by using flow cytometry, staining by rhodamine, Fluo-3 and BCECF, respectively. RESULTS: Degeneration cells showed chromatin condensation and peripheral congregation. In cytoplasm autophage lysosome increased and myelinoid body was seen easily. In mitochondria structure, where the space between the inner and outer membranes of these organelles expanded as the matrix was compressed. The electron-dense or swelled was observed as vacuole degeneration and its matrix showed electron-lucent. Compared to control, mitochondria membrane potential increased in every group after 24 h and 48 h treatment. DDP increased intracellular calcium ion in PG cells, however, in PAa cells, JA and DJ decreased it. Intracellular pH got lower at 24 h and higher at 48 h in PG and PAa cells. There were significance in every group vs control in PG and PAa by statistic t-test (P

Objective To establish a method for detecting HBV cccDNA in hepatocytes of chronic hepatitis B patients.Method 21 liver biopsies from the hepatic operation patients in the hospital of jiangsu province,concluding 19 HBV chronic infected patients (10 HBeAg positive patients and 9 HBeAg negative patients) and 4 uninfected patients,HBV DNA(+) serum of hepatitis B patients was thought as rcDNA.To use proteinase K to release HBV cccDNA and genomic DNA,then divide the cell lysis solution into two parts,one for detecting HBV cccDNA,the other for detecting the number of ?-Globin as internal control. Nucleic acid for detecting HBV cccDNA extracted by phenol-chloroform was digested by plasmid-safe ATP dependent DNase which was applied to digest the single strand DNA in rcDNA and ssDNA,then was quantitated by the primers spanning across the nick and SYBR Green Ⅰ dye.The specifity of PCR production was confirmed by the sequence analysis and rcDNA comparison.The significance of the difference of HBV cccDNA level between HBeAg(+) and HBeAg(-) group was analyzed by two group t test.Results The agarose gelelectrophoresis showed the molecular weight of the PCR production was about 350bp.The coincidence rate of PCR production and goal fragement was nearly 99% by sequence analysis.The result of PCR detection of rcDNA group was negative.The positive rate of HBV cccDNA of liver biopsies of HBeAg (+) patients detected by this method was 100%,the level of HBV cccDNA in the liver biopsies of HBeAg (+) patients was higher than HBeAb(+) patients.Conclusions The specificity of the method is proved by agarose electrophoresis,gene sequencing of the PCR product and rcDNA comparison.The quantitative method that use SYBR Green Ⅰ dye and ?-Globin as internal control is more specific,sensitive and economical,and more suitable for clinical purpose.

Interleukin-1 receptor antagonist (IL-1ra), a member of IL-1 family, is a naturally occurring IL-1 inhibitor as "receptor antagonist", which blocks biological responses mediated by IL-1. Recombinant human IL-1ra (rhIL-1ra, Kineret) was introduced in clinical trials involving patients with RA. Between 2001 to approximately 2002, rhIL-1 ra was approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicine Procedure. Unfortunately, 10,000 to 100,000-fold excess amounts of IL-1ra are needed to relieve disease because minimal IL-1 can induce complete biological responses, and the dosage of 100 to approximately 150mg/day in a RA patient is so big that it greatly influence patients' physical, psychological and economical situation. In this study, IL-1ra mutants were established by site-specific mutagenesis to improve its stability. The sites of mutagenesis included R6 K7-AA,R93 K94-AA and K97 R98-AA. IL-1ra and its mutants were expressed in E. coli BL21 (DE3) using pTIG-Trx expressing system with the induction of IPTG. The recombinant proteins were purified by Ni2+ chelate chromatography and Sephadex G75 gel filtration chromatography. The activity of mutants is as high as IL-1ra. We characterized the pharmacokinetic profile of IL-1ra and its mutants. The third mutant's half life is 2.26 times than wt IL-1ra. The study has provided some approaches and experience for further research to improve the metabolism stability of IL-1ra.
Animals ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; pharmacokinetics ; Mutagenesis, Site-Directed ; methods ; Mutant Proteins ; biosynthesis ; pharmacokinetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacokinetics

OBJECTIVETo observe the effect and safety of a comprehensive protocol of integrated Chinese and Western medicine (ICWM) in treating acute intracerebral hemorrhage (AICH).

METHODSA multi-center, single blinded, random and control clinical trial was adopted with 404 patients with AICH, they were divided into the treatment group (199 cases) treated with ICWM protocol, and the control group (205 cases) treated with Western medicine plus placebo, and AICH related indexes were assessed after treatment.

RESULTSCompared with the control group, the neurological deficit score (National Institute of Health Stroke Scale, NIHSS) determined on the 28th and the 90th day after treatment was ameliorated in the treatment group (P < 0.01). Moreover, the 90th day assessment showed that the mortality and severe disability rate was lower (BI < 75) (P <0.05), the complete rehabilitation and mild disability rate (BI > or = 95, P < 0.05), and the sociability rate (Functional Assessment Questionnaire, FAQ) were higher (P < 0.01) in the treatment group than those in the control group. The incidence of adverse reaction was 5.53%, manifested as non-infective diarrhea.

CONCLUSIONThe ICWM protocol used in this study may improve neural function and quality of life of AICH patients, and reduce the mortality and severe disability rate in those after 90 days' treatment, with only non-infective diarrhea as the adverse reaction.

Adult ; Aged ; Aged, 80 and over ; Cerebral Hemorrhage ; drug therapy ; surgery ; Combined Modality Therapy ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Phytotherapy ; Single-Blind Method

In the last several years, traditional Chinese medicine (TCM) has made much progress in the treatment of neurological diseases. The living space of TCM in neurological diseases lies in refractory diseases, aging and chronic diseases caused by multiple factors as well as sub-health state and chronic fatigue state. The effect model of TCM mainly consists of whole effect, self-organization, self-stable model, holographic effect and butterfly effect. The effective point of TCM in neurological diseases lies mainly in end-points and health-related events. Moreover, TCM has advantages in the evaluation of symptoms, syndrome and quality of life (QOL). Some key indexes should be included when evaluating the efficacy of TCM in neurological diseases. Meanwhile, the advantages of TCM such as end-points, health-related events and QOL should be highlighted. Multi-subject researching methods could be adopted to make a comprehensive evaluation of subjective and objective indexes. The clinical evidence on the TCM efficacy evaluation may come from RCTs, and other types of designs can also be considered.
Aging ; Humans ; Medicine, Chinese Traditional ; Nervous System Diseases ; drug therapy ; psychology ; Quality of Life

DNA barcoding is a rapidly developing frontier technology in the world and will be useful in promoting the quality control and standardization of traditional Chinese medicine. Until now, many studies concerning DNA barcoding have focused on leaf samples but rarely on Chinese herbal medicine. There are three issues involved in DNA barcoding for traditional Chinese medicinal materials: (1) the extraction methods for total DNA of the rhizomes of the medicinal materials; (2) intra-specific variation among samples from different places of origin; (3) accuracy and stability of this method. In this study, Gentianae Macrophyllae Radix was used to verify the stability and accuracy of DNA barcoding technology. Five regions (ITS2, psbA-trnH, matK, rbcL, and ITS) were tested for their ability to identify 86 samples of Gentianae Macrophyllae Radix and their adulterants. After improving the DNA extraction method, genomic DNA from all samples was successfully obtained. To evaluate each barcode's utility for species authentication, PCR amplification efficiency, genetic divergence, and species authentication were assessed. Among all tested regions only ITS2 locus showed 100% of PCR amplification and identification efficiencies. Based on the established method, we successfully identified two samples of Gentianae Macrophyllae Radix bought in pharmacy to the original species.

Objective To investigate the current situation of the prevention and contml of iodine deficiency disorders(IDD)in Hubei Province,SO as to provide a policy.making basis for controlling work of IDD. Methods Using the method of proportional population sampling(PPS),30 eounties were seiected in Hubei Province.In each selected county,1 primary school was chosen.In every primary school,40 pupil8 aged 8～10 years were selected to examine thyroid size,intelligent quotient(IQ),and salt iodine contents at their home.In the selected pupils,2 boys and 2 girls were chosen to determine their urinary iodine contents in each age grouD.Twenty pupils in the above school and 5 housewives ileal"to this school were tested in health education questionnaire. Results The median of salt iodine was 30.1 mg/kg,and the rate of comsuming qualified iodized salt was 96.2% (1154/1200).The rates ofchild goiter were 6.5%(78/1200)by palpation and 4.1%(49/1200)by B ultrasound.The median of urinary iodine Was 358.4μg/L and mean of the IQ was 105.3±14.4.The rate of qualified scores of both students and housewives Was 25.5%(153/600)、90.7%(136/150).Conclusions The current Bituation of iedine nutrition is good.The goal of eliminating IDD has been achieved in Hubei Province.

OBJECTIVETo study the dynamic expression of protease-activated receptor-1(PAR-1) after acute intracerebral hemorrhage (ICH) and the influence of Naomai capsule (NMC II) on the expression in rats.

METHOD72 rats were randomly divided into 9 groups (n = 8 in each group). They were normal group, ICH model groups at 6, 24 h, 3, 7 d and NMC II groups at 6, 24 h, 3, 7 d. ICH models were induced by collagenase type VII-S. Immunohistochemical method was used to detect PAR-1 protein and RT-PCR technique was used to detect PAR-1mRNA in brain tissue around the haematoma at different groups.

RESULTPAR-1 protein and mRNA were mild positive in normal group. In model groups, intensity of PAR-1 expression started to enhance at 6 h, and enhanced more at 24 h. PAR-1 expression reached the peak at 3 d and began to descend. At 7 d the decent was obvious. At 6, 24 h, 3, 7 d time point. The PAR-1 protein positive cell number and PAR-1 mRNA absorbance ratio in ICH model and NMC II groups were significantly higher than those in normal group (P < 0.05 or P < 0.01). The PAR-1 protein positive cell number and PAR-1mRNA absorbance ratio in NMC II group were significantly lower than those in ICH model group (P < 0.05 or P < 0.01).

CONCLUSIONAfter ICH, PAR-1 is continuously activated because of the simulation of thrombin. Function of thrombin after ICH maybe mediated by PAR-1; NMC II may inhibit the expression of PAR-1. This may be one of the main therapeutics mechanisms of NMC II.

Acute Disease ; Animals ; Capsules ; Cerebral Hemorrhage ; metabolism ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Male ; Materia Medica ; administration & dosage ; pharmacology ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Wistar ; Receptor, PAR-1 ; biosynthesis ; genetics

OBJECTIVETo discuss the clinical effects of open reduction and internal fixation (ORIF) for treatment of patients with Lisfranc injury combined the second metatarsal base comminuted fracture.

METHODSFrom March 2007 to June 2012, 7 patients with Lisfranc injury combined the second metatarsal base comminuted fracture were treated including 5 males and 2 female aged from 22 to 51 years old (means 42 years), 4 of sprain and 3 of traffic injury. According Myerson classification, there was 1 case of type A, 3 of type B and 3 of type C. Kirschner wire was used to fix Lisfranc ligament placing from the medial cuneiform bone to the second metatarsal base during the operation. After the operation American Orthopaedic Foot and Ankle Society (AOFAS) criteria system were applied to evaluate the foot and ankle function. Preoperative and postoperative AP, lateral and oblique X-ray and CT scan were collected for radiographic evaluation.

RESULTSAll patients were followed up from 12 to 20 months (16.8 months in average). According to AOFAS criteria system, 3 cases were excellent result,3 good, 1 fair. All the wounds were primary healing without skin necrosis, infection, Kirschner loose,broken, or other complications.

CONCLUSIONKirschner wire had good clinical efficacy for fixing Lisfranc ligament injury with the second metatarsal base comminuted fracture, and could avoid arthrodesis.

Adult ; Bone Wires ; Female ; Humans ; Male ; Metatarsal Bones ; injuries ; surgery ; Middle Aged ; Tarsal Joints ; injuries ; surgery ; Wound Healing