Aim To establish a method to quantitatively determine cardiolipin content and investigate effects of age and ischemia on cardiolipin in isolated rat hearts.Methods Male SD rats, 4 months old,12 months old and 24 months old were used throughout the experiment. Each had 24 ones,which were randomly distributed to control,ischemia 30 min and ischemia 40 min groups(n=8). Control group hearts were perfused for a total of 70 min and ischemia groups hearts were perfused for a 20 min equilibration period,and then underwent 30 or 40 min of no-flow ischemia followed by reperfusion for a 20 min period, respectively.Cardiolipin was quantitatively determined by high-performance liquid chromatography following extraction of lipids from subsarcolemmal mitochondria(SSM)and interfibrillar mitochondria(IFM).Results Under the condition of HPLC,the retention time of cardiolipin was 13.092 min. The standard curve was Y^ (?V?s) =20 877 455X(mmol?L-1)-352 028(r=0.9993) with a validated quantitation range of 0.2～3.2 mmol?L-1,and the lowest concentration of detection was 0.005 mmol?L-1 (S/N=3). At the concentration of 0.2,0.8,3.2 mmol?L-1,absolute recovery rates ranged from 0.8505 to 0.9519 and relative recovery rates ranged from 0.9898 to 1.0429. The RSD of the precision within-day and between-day was less than 0.11. Cardiolipin content in SSM of nonischemic group of 24 months old rats was lower than that of both 4 months old and 12 months old, but no differences among them were observed in IFM.In all age rats, compared with control group,cardiolipin contents of SSM from ischemia 30 min and ischemia 40 min groups were lower, and effects of ischemia 40 min are not pronounced; there were no differences in CL contents of IFM among three groups.Conclusions The assay method was shown to be sensitive, suitable and accurate for determination of cardiolipin located in mitochondria from perfused hearts of rats. Myocardial ischemia and older age decreased cardiolipin content in SSM, and had no remarkable effects on cardiolipin in IFM.

The necessity and superiority of the surgical operation on children with floating knee injury and the fracture union and complications were investigated. Twenty-eight children with floating knee injury were subjected to open reduction and internal fixation or external fixator. The patients were followed up for 18 months to 7 years. The curative effectiveness was scored by Karlstrom criteria. The results showed that no nonunion or deformity was found. The affected limb was 1.2 cm to 1.5 cm longer in 2 cases, 0.8 to 1.2 cm shorter in 3 cases than the contralateral. No severe dysfunction of knee joint occurred. The excellent-good rate was 92.8% and the curative rate 71.4% respectively. So for children whose age is older than 5 years, it's a good way to treat the fractures of femur and tibia with open reduction and internal fixation or external fixator. The method can be advantageous for the nursing care, early function recovery, shortening of the hospital stay and avoidance of severe complications.
Femoral Fractures/complications ; Femoral Fractures/*surgery ; Fracture Fixation ; Knee Injuries/classification ; Knee Injuries/etiology ; Knee Injuries/*surgery ; Tibial Fractures/complications ; Tibial Fractures/*surgery ; Treatment Outcome

BACKGROUND:There are several reports about the application of fresh bone marrow aspirate being injected directly to repair partial ligament injury, but the application about fresh bone marrow aspirate directly being planted on scaffolds to build tissue-engineered ligament is rarely mentioned.
OBJECTIVE:To evaluate the feasibility of applying fresh bone marrow aspirate planted directly on scaffolds to construct tissue-engineered ligament
METHODS:We constructed fibroin fiber/smal intestinal submucosa composite scaffold, then planting fresh bone marrow directly to built bone marrow seeding group and planting seed cel s (bone marrow mesenchymal stem cel s) on the scaffold to built cel seeding group. The control group had no treatment. After that, we detected the density of cel adhesion, cel proliferation ability and extracel ular matrix secretion. Then, the composite in the bone marrow seeding group was implanted into the broken anterior cruciate ligament in rabbits, and material biocompatibility in vivo was evaluated after 12 weeks.
RESULTS AND CONCLUSION:After 4 hours of incubation, bone marrow seeding group was significantly higher than the cel seeding group in cel adhesion density and proliferation rate (P<0.05). Bone marrow seeding group and cel seeding group showed higher type I, III col agen secretion compared with the control group (P<0.05), but the col agen secretion of bone marrow seeding group and cel seeding group showed no significant difference. Composite cel scaffold implantation in vivo did not cause fatal immune rejection and severe inflammatory reaction, and no significant ligament regeneration and vascularization occurred. These findings indicate that fresh bone marrow aspirate can be seeded directly on scaffolds to construct tissue-engineered ligament, and the short-term biocompatibility in vivo is good.

To investigate the efficacy of a combination therapy on gluteal muscle contracture, 286 definitely diagnosed patients were subjected to surgical treatment, and then functional exercises and physical therapy. The patients with severe symptoms were asked to have a set of specially-designed functional exercises. All the patients were followed up for 3 to 24 months by hospital visit, correspondence or telephone interview. The effective rate was 100%, and the curative rate was up to 94.6%. Few patients developed complications and relapse was rare. It is concluded that the combination therapy, including surgical removal of diseased tissues, functional exercises and physical therapy, is an effective approach for the treatment of severe juvenile gluteal muscle contracture.
Buttocks ; Exercise Therapy ; Follow-Up Studies ; Muscle Contraction ; Muscle, Skeletal/*pathology ; Patient Compliance ; Physical Therapy Modalities ; Time Factors ; Treatment Outcome

Objective To explore the molecular mechanisms involved in hypokalemic salt-losing tubulopathies ( SLTs) through genetic screening of WNK gene in patients with SLTs. Methods Forty-four kindreds of SLTs were diagnosed Batter's syndrome or Gitelman's syndrome after CLCNKB and SLC12A3 sequencing and analysis, 8 of whose phenotype can not be simply attributed to CLCNKB or SLC12A3 mutations. Primers for PCR-amplified exons of WNK4 and WNK1 gene in genomic DNA were designed, and direct sequencing was performed to analyse the PCR products. Results Two missense mutations of WNK1, Ile~(1172)→ Met (I1172M) and Ser~(2047) → Asn (S2047N), were identified. Both of these 2 mutations segregated with the disease in SLTs kindred. Conclusion Two heterozygote missense mutations of WNK1 gene (I1172 M and S2047N) were found in 8 SLTs kindreds, indicating that WNK1 might be another gene responsible for hypokalemic salt-losing tubulopathies.

0.05).CONCLUSION:The excellent mechanical properties of composite ligament can meet the mechanical requirements of appropriate ligament tissue engineering scaffolds.

To investigate therapeutic efficiency of Ad/CMV- hTGF-beta1 gene for rabbit intervertebral disc degeneration model. 60 Japanese white rabbits were selected to form the 1.5-L6 Anterior-Lateral-Anulus-Fibrosus-Incision-Induced model in order to simulate human intervertebral disc degeneration. 36 rabbits, whose corresponding intervertebral discs were injected with 20 microl (10 x 10(6) pfu) of Ad/CMV- hTGF-beta1 gene, constituted the therapy group, 12 were injected with 20 microl (10 x 10(6) pfu)of Ad/CMV-LacZ gene as comparison group, while 12 were only injected with equivalent capacity of saline for empty comparison group, 3 weeks after injection, examples were taken for investigation of HE staining, MRI, Western Blotting and immunohistochemical research TGF-beta1. Wide distribution of TGF-beta1 was detected by immunohistochemical research in the degenerated annulus fibrosus after injection. Western Blotting research showed significant increase of TGF-beta1 content in intervertebral discs treated with TGF-beta1 gene than comparison groups. MRI signal transformed from low to comparatively high and that intervertebral disc pathological degree improved. Ad/CMV- hTGF-beta1 gene transfection is a potential method to increase TGF-beta1 content and reverse intervertebral disc degeneration.
Adenoviridae/genetics ; Gene Therapy ; Gene Transfer Techniques ; Genetic Vectors ; *Intervertebral Disk/pathology ; Lumbar Vertebrae ; Spinal Diseases/*drug therapy ; Transfection ; Transforming Growth Factor beta/*genetics ; Transforming Growth Factor beta1

To construct the antisense transforming growth factor beta 1 (TGF beta 1) gene and investigate the effect of TGF beta 1 autocrine loop blockage on the proliferation of osteosarcoma cells. TGF beta 1 cDNA was cloned by RT-PCR from human osteosarcoma cells (MG-63) and inserted into pcDNA3 to construct an antisense expression vector, which was dubbed pcDNA3-TGF beta 1(-). MTT was used to detect the proliferation of osteosarcoma cells transfected by antisense TGF beta 1 gene. Our results showed that the proliferation of the transfected osteosarcoma cells was suppressed markedly. It is concluded that TGF beta 1 autocrine loop blockage in osteosarcoma cells could inhibit cell proliferation, which might be helpful for gene therapy of osteosarcoma.
Antisense Elements (Genetics) ; biosynthesis ; genetics ; Bone Neoplasms ; genetics ; metabolism ; pathology ; Cell Division ; Humans ; Osteosarcoma ; genetics ; pathology ; Transcription, Genetic ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Transforming Growth Factor beta1 ; Tumor Cells, Cultured

A novel paper-based analytical device(PAD)was prepared and applied to determine the xanthine oxi-dase(XOD)inhibitory activity of Salvia miltiorrhiza extracts(SME).First,polycaprolactone was 3D printed on filter paper and heated to form hydrophobic barriers.Then the modified paper was cut according to the specific design.Necessary reagents including XOD for the colorimetric assay were immobilized on two separate pieces of paper.By simply adding phosphate buffer,the reaction was performed on the double-layer PAD.Quantitative results were obtained by analyzing the color intensity with the specialized device system(consisting of a smartphone,a detection box and sandwich plates).The 3D-printed detection box was small,with a size of 9.0 cm x 7.0 cm x 11.5 cm.Color component G performed well in terms of linearity and detection limits and thus was identified as the index.The reaction con-ditions were optimized using a definitive screening design.Moreover,a 10％glycerol solution was found to be a suitable stabilizer.When the stabilizer was added,the activity of XOD could be maintained for at least 15 days under 4℃or-20℃storage conditions.The inhibitory activity of SME was investigated and compared to that of allopurinol.The results obtained with the PAD showed agreement with those ob-tained with the microplate method.In conclusion,the proposed PAD method is simple,accurate and has a potential for point-of-care testing.It also holds promise for use in rapid quality testing of medicinal herbs,intermediate products,and preparations of traditional Chinese medicines.

The necessity and superiority of the surgical operation on children with floating knee injury and the fracture union and complications were investigated. Twenty-eight children with floating knee injury were subjected to open reduction and internal fixation or external fixator. The patients were followed up for 18 months to 7 years. The curative effectiveness was scored by Karlstrom criteria. The results showed that no nonunion or deformity was found. The affected limb was 1.2 cm to 1.5 cm longer in 2 cases, 0.8 to 1.2 cm shorter in 3 cases than the contralateral. No severe dysfunction of knee joint occurred. The excellent-good rate was 92.8 % and the curative rate 71.4 % respectively. So for children whose age is older than 5 years, it's a good way to treat the fractures of femur and tibia with open reduction and internal fixation or external fixator. The method can be advantageous for the nursing care, early function recovery, shortening of the hospital stay and avoidance of severe complications.