Nitrogen metabolism and secondary metabolism regulation of Atropa belladonna by exogenous NO under NaCl stress / 中国中药杂志

Atropa belladonna seedlings were used as experimental materials and cultivated by soil culture method. Different concentrations(0,0.05,0.1,0.2,0.5 mmol·L~(-1))of NO donor sodium nitroprusside(SNP) were sprayed on the leaves. The effects of different concentrations of SNP and different treatment time(4,8,12,16 d) on nitrogen metabolism, secondary metabolite content, precursor content of tropane alkaloid synthesis pathway and expression of key enzyme genes under 100 mmol·L~(-1) NaCl stress were studied. The results showed that with the prolongation of salt stress, the nitrogen metabolism and the accumulation of secondary metabolites of A. belladonna were inhibited to some extent. After treatment with different concentrations of exogenous SNP, the ammonium nitrogen content decreased dramatically, and the contents of nitrate nitrogen, free amino acid, soluble protein and the activities of key enzymes of nitrogen metabolism(NR, GS, GDH) were all greatly improved; the contents of precursor amino acids(ornithine, arginine) and polyamines(Put, Spd, Spm) in the secondary metabolic pathway have increased to varying degrees. The qRT-PCR analysis showed that exogenous SNP treatment can effectively promote the high expression of key enzyme genes PMT, TRⅠ and H6H in the secondary metabolic pathway of A. belladonna, and the production of hyoscyamine and scopolamine were increased notably. In summary, the application of appropriate concentration of SNP can effectively alleviate the inhibition of salt stress on the nitrogen metabolism and secondary metabolism of Atropa belladonna, and enhance its salt tolerance. Overall, 0.1 mmol·L~(-1) and 0.2 mmol·L~(-1) SNP treatment achieved the most remarkable effect.

Age-related Changes in the Global DNA Methylation Profile of Oligodendrocyte Progenitor Cells Derived from Rat Spinal Cords.

Demyelination of axons plays an important role in the pathology of many spinal cord diseases and injuries. Remyelination in demyelinated lesions is primarily performed by oligodendrocyte progenitor cells (OPCs), which generate oligodendrocytes in the developing and mature central nervous system. The efficiency of remyelination decreases with age. Many reports suggest that this decline in remyelination results from impaired OPC recruitment and differentiation during aging. Of the various molecular mechanisms involved in aging, changes in epigenetic modifications have received particular attention. Global DNA methylation is a major epigenetic modification that plays important roles in cellular senescence and organismal aging. Thus, we aimed to evaluate the dynamic changes in the global DNA methylation profiles of OPCs derived from rat spinal cords during the aging process. We separated and cultured OPCs from the spinal cords of neonatal, 4-month-old, and 16-month-old rats and investigated the age-related alterations of genomic DNA methylation levels by using quantitative colorimetric analysis. To determine the potential cause of dynamic changes in global DNA methylation, we further analyzed the activity of DNA methyltransferases (DNMTs) and the expression of DNMT1, DNMT3a, DNMT3b, TET1, TET2, TET3, MBD2, and MeCP2 in the OPCs from each group. Our results showed the genomic DNA methylation level and the activity of DNMTs from OPCs derived from rat spinal cords decreased gradually during aging, and OPCs from 16-month-old rats were characterized by global hypomethylation. During OPC aging, the mRNA and protein expression levels of DNMT3a, DNMT3b, and MeCP2 were significantly elevated; those of DNMT1 were significantly down-regulated; and no significant changes were observed in those for TET1, TET2, TET3, or MBD2. Our results indicated that global DNA hypomethylation in aged OPCs is correlated with DNMT1 downregulation. Together, these data provide important evidence for partly elucidating the mechanism of age-related impaired OPC recruitment and differentiation and assist in the development of new treatments for promoting efficient remyelination.

Fabrication of Isoniazid/Rifampicin/Poly L-lactic Acid Donut-shaped Implants via Three Dimensional Printing Technique.

Objective To investigate the possibility of manufacturing dual-drug loaded isoniazid/rifampicin/poly L-lactic acid (PLLA) implant with donut-shaped structure via three-dimensional (3D) printing technique and study the drug release characteristic and biocompatibility of the implant in vitro.Methods PLLA was crushed into particles with diameters around 75-100 µm.Isoniazid and rifampicin bulk drugs were dissolved into the organic dissolvent respectively to be the binding liquid.The 3D printing machine fabricated the donut-shaped implant via binding the PLLA powder layer by layer.Dynamic socking method was used to study the in vitro release characteristics,and cell culture experiment was used to test the cytocompatibility of the implant.Results PLLA slow-release implants were made by using the PLLA powder as matrix and isoniazid/rifampicin organic solvent as binding liquid through 3D printing.The drugs in the implants distributed in nest under electron microscope.The concentrations of both drugs were still higher than the lowest effective bacteriostasis concentration after release for 32 days.Cytotoxicity and direct contact tests indicated that the implants had rare cytotoxicity and favorable biocompatibility. Conclusion The donut-shaped implants can be successfully fabricated using the 3D printing method,which offers a new method for the manufacturing of topical slow-release anti-tuberculosis drugs.

Versatility of reverse sural fasciocutaneous flap for reconstruction of distal lower limb soft tissue defects.

In this study we present our experiences with the reverse sural fasciocutaneous flap to reconstruct the distal lower limb soft tissue defects caused by traumatic injuries. These flap graftings were carried out from Oct. 2010 to Dec. 2012 in our department. The series consisted of 36 patients, including 21 men and 15 women with an average age of 46.2 years (14-83 years) and with a medium follow-up period of 18 months (12-24 months). Of all the cases of acute trauma, there were 10 cases of trauma of distal tibia, 9 cases of trauma of perimalleolus, and 17 cases of trauma of midfoot and forefoot. Related risk factors in the patients were diabetes (2 cases), advanced age (>65 years, 3 cases) and cigarette smoking (6 cases). The reverse flow sural island flap irrigation depended on lower perforators of the peroneal artery. The fasciocutaneous pedicle was 3-4 cm in width and the anatomical structures consisted of the superficial and deep fascia, the sural nerve, short saphenous vein, superficial sural artery together with an islet of subcutaneous cellular tissue and skin. The most proximal border of the flap was only 1.5 cm away from the popliteal skin crease and the pivot point was 5-7 cm above the tip of the lateral malleolus. All the flaps survived. No arterial crisis occurred in any case. The venous congestion occurred in 2 cases and got better after raising the limbs and bloodletting. Only in an old man, 1.5 cm necrosis of distal margin of his flap occurred and finally healed after continuous dressing change. One-stage skin grafting was performed, and all the donor sites were sutured and successfully healed. It was concluded that the reverse sural fasciocutaneous flap is safe and reliable to extend to the proximal third even near the popliteal skin crease. We also concluded this flap can be safely and efficiently used to treat patients with large and far soft-tissue defects from the distal leg to the forefoot with more versatility and it is easier to reach the recipient sites.

In vivo testing of canine prosthetic femoral components with HA-Ti ladder-type coating on vacuum plasma-sprayed Ti substrate.

The purpose of the present study was to observe the structure and functional change of the bone-coating-prosthesis interface in vivo and to evaluate the histocompatibility of self-made prosthetic femoral components in the body and the degree of their bonding with the surrounding bone tissues as well as their stability. Six mature beagle dogs underwent bilateral hip replacement with prosthetic femur components. Three groups were established in terms of different coating of prothesis (four joints in each group): atmosphere (A) plasma-sprayed pure titanium (Ti) prosthetic joint with hydroxyapatite (HA) coating (HA+Ti+A group); vacuum (V) plasma-sprayed pure Ti prosthetic joint with HA coating (HA+Ti+V group); vacuum plasma-sprayed pure Ti prosthetic joint with Ti-HA stepped coating (Ti+HAG+Ti+V group). The hip joints were functionally evaluated, and subjected to X-ray examination, biomechanics inspection, and histological examination. As a result, X-ray imaging revealed all prosthetic joints were in a good location and no dislocation of joint was found. Shear strength of interface was significantly higher in Ti+HAG+Ti+V group than in HA+Ti+V group (P<0.05) and HA+Ti+A group (P<0.05) at 28th week. Histological examination showed the amount of newborn bone in Ti+HAG+Ti+V group was more than in HA+Ti+V group and HA+Ti+A group after 28 weeks. It was suggested that vacuum plasma-sprayed pure Ti prosthetic joint with TI-HA stepped coating could improve the bonding capacity of bone-prosthesis, enhance the stability of prosthesis, and increase the fixion of prosthetic femoral components because of better bone growth. This new type of biological material in prosthetic femoral components holds promises for application in clinical practice.

Clinical analysis of 73 cases of intraspinal nerve sheath tumor.

Seventy-three patients with spinal nerve sheath tumor who were surgically treated in our hospital during the years 2004-2010 were retrospectively reviewed with respect to recovery of neurological function, recurrence of the tumor and occurrence of kyphotic deformities. Preoperative clinical manifestations, imaging data, surgical records and follow-up results were comprehensively analyzed. The follow-up duration was 12-60 months with an average of 32.0 months. Out of the 73 cases enrolled, 69 had gradual recovery of sensation, motor and sphincter functions 1 week to 3 months after operation. Forty-six cases had incomplete paralysis, whose American Spinal Injury Association (ASIA) grades, however, were gradually increased during the follow-up period, 4 cases had no significant improvement of the clinical symptoms and no change in ASIA grades during the follow-up period. Two cases had postoperative recurrence of the tumor. There were no deaths, no spinal instability, and no kyphotic malformations found in any cases. Our study indicated that complete removal of the tumor is important for good recovery, and an ideal surgical method may reduce the recurrence of the tumor or the occurrence of complications.

Bone induction by biomimetic PLGA-(PEG-ASP)n copolymer loaded with a novel synthetic BMP-2-related peptide in vitro and in vivo.

BMP-2 is one of the most important growth factors of bone regeneration. Polylactide-co-glycolic acid (PLGA), which is used as a biodegradable scaffold for delivering therapeutic agents, has been intensively investigated. In previous studies, we synthesized a novel BMP-2-related peptide (designated P24) and found that it could enhance the osteoblastic differentiation of bone marrow stromal cells (BMSCs). The objective of this study was to construct a biomimetic composite by incorporating P24 into a modified PLGA-(PEG-ASP)n copolymer to promote bone formation. In vitro, our results demonstrated that PLGA-(PEG-ASP)n scaffolds were shown to be an efficient system for sustained release of P24. Significantly more BMSCs attached to the P24/PLGA-(PEG-ASP)n and PLGA-(PEG-ASP)n membranes than to PLGA, and the cells in the two groups subsequently proliferated more vigorously than those in the PLGA group. The expression of osteogenic markers in P24/PLGA-(PEG-ASP)n group was stronger than that in the PLGA-(PEG-ASP)n and PLGA groups. Radiographic and histological examination, Western blotting and RT-PCR showed that P24/PLGA-(PEG-ASP)n scaffold could induce more effective ectopic bone formation in vivo, as compared with PLGA-(PEG-ASP)n or gelatin sponge alone. It is concluded that the PLGA-(PEG-ASP)n copolymer is a good P24 carrier and can serve as a good scaffold for controlled release of P24. This novel P24/PLGA-(PEG-ASP)n composite promises to be an excellent biomaterial for inducing bone regeneration.

[Adhesion, proliferation and osteodifferentiation of bone mesenchymal stem cells on PLGA-[ASP-PEG] tri-bolck polymer scaffolds].

OBJECTIVE: To explore the adhesion,proliferation and osteodifferentiation of bone mesenchymal stem cells (BMSCs)on the prepared lactic acid/glycolic acid/asparagic acid-co-polyethylene glycol(PLGA-[ASP-PEG])tri-block polymer scaffolds. METHODS: Modified PLGA with polyethylene glycol (PEG) and asparagic acid(ASP)that has many liga nds,and then the synthesis PLGA-[ASP-PEG] tri-block polymer material was prepared. BMSCs were cultured in PLGA-[ASP-PEG] polymer material and poly lactic acid-co-glycolic acid(PLGA)were used as control group. Precipitation method, MUT assay and total cellular protein detection were used to test the adhersion and proliferation of BMSCs. After the third generation of BMSCs was cultured on PLGA-[ASP-PEG] tri-block polymer scaffolds for 14 day and 28 day with osteogenic supplements,the osteodifferentiation of MSCs were observed through alkaline phosphatase(ALP) staining and calcium tubercle staining. RESULTS: BMSCs grew adherent to the surface of PLGA-[ASP-PEG] polymer scaffolds and the number of BMSCs was much higher than that of PLGA. The precipitation method suggested that adhesion and proliferation of BMSCs on the surface of PLGA-[ASP-PEG] was much higher than the control group (P < 0.05). MTU assay showed that after BMSCs were cultured for 20 days,the absorbance A of PLGA-[ASP-PEG] polymer scaffolds and PLGA were 1.336 and 0.780 respectively. Total cellular protein could image the adhersion and proliferation of BMSCs indirectly. After BMSCs were cultured for 12 days,the total cellular protein of PLGA-[ASP-PEG] and PLGA were 66.44 microg/pore and 41.23 microg/pore respectively. PLGA-[ASP-PEG] polymer scaffolds had well biocompatibility and cell adhersion. The positive results with ALP staining and calcium tubercle staining in both groups indicated tri-block polymer scaffold and its degradations had no effect on osteodifferentiation. CONCLUSION: PLGA-[ASP-PEG]could improve the adhesion and proliferation of seed cells on bone-matrixmaterial, maintain the morphous of seed cells and had no obvious effect on cell osteodifferentiation.

[Anterior radical debridement and bone grafting with one-stage instrumentation anteriorly or posteriorly for the treatment of thoracic and lumbar spinal tuberculosis].

OBJECTIVE: To evaluate the clinical outcomes of anterior radical debridement and autologous ilium or titanium cage interbody autografting with one-stage instrumentation anteriorly or posteriorly for the treatment of thoracic and lumbar tuberculosis. METHODS: Sixty-eight cases of thoracic and lumbar tuberculosis were surgically treated with anterior radical debridement and autologous ilium or titanium cage interbody autografting with one-stage instrumentation anteriorly or posteriorly from Jan 2001 to Feb 2006. Thirty-nine were male and 29 were female age ranged from 28 to 76 years, (average 36.8 years. The course of illness was from 3 months to 1.5 years average 8 months). Fifty-five of them underwent anterior instrumentations, and the remaining underwent posterior instrumentations. All patients were followed up to determine the stages of intervertebral bony fusion and the corrections of spinal kyphosis and the restoration of neurological deficit RESULTS: The follow-up period ranged from 1.5 years to 5 years (mean 36 months). Sinus formation occurred in 3 cases and healed after continuous dressing changes. The ESR of these patients decreased to normal levels after an average of 3.2 months postoperatively. The functions of feeling, motion and sphincter in 27 cases among all 28 paraplegia cases gradually recovered 24 h to 3 months postoperatively and ASIA grades increased at least one grade at the final follow-up. Only 1 case did not recover at all and ASIA grade did not increase at the final follow-up. Intervertebral bony fusions were all achieved for a mean of 4.8 months (ranged from 3 to 15 months) postoperatively. No internal fixation devices were loose, extracted or broken. Average Cobb angle of kyphotic deformities was 41.2 degree preoperatively and decreased to 13.6 degree at 1 week postoperatively. The average correction was 27.6 degree. The average Cobb angle was 15.8 degree at the final follow-up and the average loss of correction was only 2.2 degree. There were no recurrences in all cases. CONCLUSION: The method of anterior radical debridement and interbody grafting with one-stage instrumentation anteriorly or posteriorly was effective for the treatment of thoracic and lumbar spinal tuberculosis.

[Synthetic RGD-containing peptide K16GRGDSPC affected the adhesion, proliferation and osteogenic differentiation of rabbit bone marrow stromal cells on PLGA-[ASP-PEG] scaffold materials].

OBJECTIVE: To explore the effects of synthetic RGD-containing peptide K16GRGDSPC covalent bonding with PLGA-[ASP-PEG] scaffold materials on the adhesion, proliferation and osteogenic differentiation of rabbit bone marrow stromal cells (BMSCs). METHODS: The peptide was synthesized by solid-phase synthesis method and characterized by mass spectrometry and high pressure liquid chromatography. PLGA-[ASP-PEG] scaffold materials were modified with the peptide by cross linker Sulfo-LC-SPDP and detected by XPS. The BMSCs obtained from rabbit were cultured on PLGA-[ASP-PEG] modified with the peptide and those cultured on unmodified PLGA-[ASP-PEG] were also observed as control group. The adhesion and proliferation behaviors of the cells were analyzed by conventional precipitation method, micropipette aspiration technique, MTT assay and Coomassie Brilliant Blue dyes. The osteogenic differentiation of the cells was showed by the activity of alkaline phosphatase (ALP) assayed by ALP Assay Kit and the mRNA levels of ALP, osteocalcin (OCN), osteopontin (OPN) and collagen I assessed by real-time PCR (RT-PCR). Immunofluorescence stain was also used to detect the expression of core binding factor a1 (Cfba1) which was an osteogenic maker as well. RESULTS: The peptide was successfully manufactured and linked to the surface of the PLGA-[ASP-PEG] by the cross-linker. The abilities of adhesion and proliferation and the expressions of osteogenic makers of the cells were significantly higher than those of control group (P < 0.05). CONCLUSION: RGD-containing peptide K16GRGDSPC could promote the adhesion, proliferation and osteogenic differentiation of BMSCs on the biomimetic bone-matrix materials.

[Wild-type Smad3 gene promotes osteoblastic differentiation of rat bone marrow-derived mesenchymal stem cells in vitro].

OBJECTIVE: To observe the effect and mechanism of wild-type Smad3 gene on the proliferation and osteoblastic differentiation of rat bone marrow-derived mesenchymal stem cell (MSC). METHODS: Rat bone-derived MSC were cultured and transfected with the complexes of pcDNA3.0-Myc-Smad3 or pcDNA3.0-Myc-Smad3DeltaC and LipofectAMINE reagent. Immunofluorescence technique was performed to evaluate the c-Myc expression. The cell proliferation was detected by MTT method. In order to observe the osteoblastic characteristics in stable expression MSC, alkaline phosphatase (ALP) mRNA and core binding factor alpha1 (cbfa1) mRNA were investigated by RT-PCR technique, and ALP activity and mineralization were examined by PNP method and alizarin red staining respectively. PD98059 was used to interdict selectively ERK pathway in order to investigate the effect of ERK pathway on Smad3 regulating MSC. RESULTS: c-Myc protein was expressed in Smad3-MSC and Smad3DeltaC-MSC. The proliferation of Smad3-MSC was slower than that of Smad3DeltaC-MSC or V-MSC. The relative levels of ALP mRNA and cbfa1 mRNA in Smad3-MSC were higher markedly than those in Smad3DeltaC-MSC or V-MSC, as well as ALP activity and mineralization. However, without a significant difference ALP activity and mineralization was after PD98059 treatment (both slightly decreased P > 0.05). CONCLUSION: The wild-type Smad3 gene, which is a crucial component promoting bone formation, can inhibit the proliferation of MSC and promote the osteoblastic differentiation and maturation of MSC independent of ERK pathway.

Effects of TGF beta1 autocrine blockage on osteosarcoma cells.

OBJECTIVE: To evaluate the effects of transforming growth factor beta1 (TGFbeta1) autocrine blockage on proliferation activity and drug sensitivity of osteosarcoma. METHODS; Northern blot, MTT determination, and 3H thymidine incorporation were used to investigate the effects of antisense TGF beta1 gene on osteosarcoma. RESULTS: The proliferation of osteosarcoma cells transfected by antisense TGF beta1 gene was suppressed markedly, and adriamycin sensitivity was significantly increased. CONCLUSION: Blockage of osteosarcoma cells TGF beta1 autocrine loop inhibits cell proliferation and enhances chemotherapy sensitivity.

Effects of basic fibroblast growth factor on biological characteristics of osteoblasts.

OBJECTIVE: To elucidate the effects of exogenous basic fibroblast growth factor (bFGF) on biological characteristics of rat osteoblasts cultured in vitro. METHODS: The osteoblasts isolated from a Sprague-Dawley rat and cultured in vitro were treated with different concentrations of bFGF (5-50 ng/ml) respectively. At 24 hours after treatment, the proliferating cell nuclear antigen was measured with immunocytochemistry, alkaline phosphatase (ALP) activity was determined and the expression of transforming growth factor beta 1 (TGF-beta(1)) was detected to observe the effects of bFGF on growth and differentiation of osteoblasts. RESULTS: bFGF (5-50 ng/ml) could obviously promote the growth of osteoblasts. The intracellular expression of TGF-beta(1) mRNA increased significantly, but the intracellular ALP content decreased. CONCLUSIONS: bFGF can obviously stimulate the proliferation of osteoblasts and promote the synthesis of TGF-beta(1), but cannot promote the differentiation of osteoblasts.

[Study of rat osteoblasts transfected by transforming growth factor-beta 1 gene].

OBJECTIVE: To investigate the effect of transforming growth factor-beta 1 (TGF-beta 1) gene transfer on the biological characteristics of osteoblasts. METHODS: The expression of TGF-beta 1 in the transfected osteoblasts was detected by in situ hybridization and assay of TGF-beta 1 activity in the supernatant (mink lung epithelium cell growth-inhibition test). The effects of gene transfer and supernatant of the transfected osteoblasts on the proliferation and alkaline phosphatase(ALP) activity of osteoblasts were detected by 3H-TdR and MTT. RESULTS: The results of in situ hybridization analysis suggested that the osteoblasts transfected by TGF-beta 1 gene could express TGF-beta 1 obviously. The complex medium, which was the mixture of serum-free DMEM and the activated supernatant according to 1:1, 1:2, 1:4, could inhibit growth of Mv-1-Lu evidently and the ratios of inhibition were 16.3%, 22.7%, 28.2% respectively. TGF-beta 1 gene transfer had no effect on the biological characteristics of osteoblasts, but the activated supernatant of transfected osteoblasts stimulated proliferation and inhibited ALP activity of osteoblasts. CONCLUSION: TGF-beta 1 gene transfer promotes the expression of TGF-beta 1 and the biological characteristics of transfected osteoblasts are stable, which is helpful for gene therapy of bone defects in vivo.

Raman Spectra of Porous beta-TCP Bioceramics Implanted into Rabbit Femur.

The Raman spectra of porous beta-TCP(beta-tricalcium phosphate)bioceramics implanted into rabbits femur the boundary and rabbit femur were studied using Raman spectroscopy(excited with 514.5nm and 623.8nm laser)and near-infrared FT-Raman spectroscopy. The Raman characteristic frequencies were assigned. The advantages and disadvantages of visible and near IR excitation are described. The Raman characteristic frequencies of calcium phosphate collagen protein and lipid(or phospholipid)were shown in the Raman spectra. These results demonstrated that besides calcium phosphate collagen protein and lipids also existed in the implants and the boundary forming components of organic bone tissues. Results indicated that the beta-TCP bioceramic when implanted into rabbit femur was partly dissolved and degraded the new bone tissues were formed on the surface and in pores of implanted bioceramics.

Dispersive X-ray Spectroscopy Analysis of Porous beta-TCP Bioceramics after Implantation into Femur.

In this paper, energy dispersive X-ray spectroscopy and element ratio of implanted bioceramic, interface and rabbit femur when the bioceramic implanted into femur were measured by two different scan electron microscopy (SEM) EDXA modes. The changes of element ratio and components of materials and interface implanted into femur were compared. Thorough studies were made on the transformation of TCP from lifeless materials into living bones after implantation, by using SEM-EDXA, Raman spectra and infrared spectra. These results provided rich evidence for understanding biodegration and bone bonding mechanism of calcium phosphate bioceramics.